Microfluidic generation of biofunctionalized giant unilamellar vesicles for targeted cargo delivery

Abstract

The present invention describes a method for preparing monodisperse cell-targeting giant unilamellar vesicles based on the symmetric partitioning of parent polymer-shell-stabilized giant unilamellar vesicles into smaller ones with diameters ranging from 1 μm to 10 μm using a microfluidic partitioning device. The method of the present invention enables the preparation of differentially charged giant unilamellar vesicles, as well as bioligand-conjugated and PEG-conjugated giant unilamellar vesicles, which are useful for targeted cell delivery with high efficiency and specificity. A further advantage of the present invention is that the giant unilamellar vesicles can deliver large cargoes, such as drug-releasing porous microparticles, large amounts of in vivo imaging probes, viruses, or promising DNA origami robots. [Figure 26]

Description

Detailed Description of the Invention

[0001] The present invention relates to a method for preparing monodisperse cell-targeting giant unilamellar vesicles based on the symmetric partitioning of parent polymer-shell-stabilized giant unilamellar vesicles into smaller polymer-shell-stabilized giant unilamellar vesicles with diameters of 1 μm to 10 μm using a microfluidic partitioning device.

[0002] The method of the present invention allows the preparation of differently charged giant unilamellar vesicles, as well as bioligand-conjugated and PEG-conjugated giant unilamellar vesicles, which are useful for target cell delivery with high efficiency and specificity. A further advantage of the present invention is that the giant unilamellar vesicles can deliver large cargoes, such as drug-releasing porous microparticles, large amounts of in vivo imaging probes, viruses, or promising DNA origami robots.

[0003] [Background of the invention] Lipid-based small unilamellar vesicles (SUVs) have diameters less than 100 nm and have been extensively studied as delivery systems in the food, cosmetic, and pharmaceutical industries. Specifically, they have been implemented to function as nanosensors for monitoring food quality during storage, as vehicles for transdermal delivery of cosmetic agents, and more recently as carrier systems for targeted drug therapy. Such liposomal delivery systems offer improved control over drug pharmacokinetics and pharmacodynamics, reducing toxicity and adverse side effects, enhancing targeted delivery to specific tissues, and potentially increasing compound circulation time.

[0004] Despite the beneficial impact of delivery systems based on small unilamellar vesicles, the road to their wide applicability is complicated. The main problems are: First, the total amount of pharmaceutically active compound that can be encapsulated in a single liposome is very small and may not reach the desired therapeutic dose at the destination.

[0005] Second, methods known in the art have very low liposomal encapsulation efficiency, and methods involving active loading approaches are expensive and time-consuming.

[0006] Third, there is still no efficient method to load large cargos, such as nanoparticles or supramolecular DNA complexes, into small liposomes for therapeutic approaches.

[0007] Therefore, for drug delivery applications, giant unilamellar vesicles (GUVs) with diameters comprised between 1 and 10 µm offer great potential, as they can carry large amounts of active compounds as well as particles up to a few micrometers in size.

[0008] Despite the growing demand for biomedical and synthetic biology applications based on giant unilamellar vesicles, the possibility of delivering large, complex cargoes into the intracellular space in giant unilamellar vesicles, or in general, the ability of giant unilamellar vesicles to interact with living cells, has only superficially been explored.

[0009] Microfluidic approaches offer the ability to generate monodisperse giant unilamellar vesicles at various contents in a high-throughput manner (Stein H. et al., 2017, "Production of isolated giant unilamellar vesicles under high salt concentrations," Frontiers in Physiology).

[0010] Funakoshi et al. introduced jetting as a microfluidic method for the formation of giant unilamellar vesicles (Funakoshi et al., 2007, "Formation of giant lipid vesicle-like compartments from a planar lipid membrane by a pulsed jet flow," J. Am. Chem. Soc.). Here, buffer solution is projected onto a preassembled lipid bilayer at the water-oil interface using a micronozzle or micropipette, and giant unilamellar vesicles are formed from this bilayer. The size, inner and outer solutions, and lipid composition of the inner and outer leaflets can be tailored. However, a residual amount of oil required for bilayer formation at the interface with the dissolved lipid reservoir is retained in the hydrophobic core of the membrane. This can lead to changes in the mechanical properties and diffusion behavior within and across the membrane.

[0011] In 2008, Shum et al. developed another microfluidic approach, the so-called double emulsion method, to generate monodisperse giant unilamellar vesicles at the water-oil-water interface (Shum et al., 2008, Double emulsion templated monodisperse phospholipid vesicles). Here, water-in-oil-in-water droplets are formed, and unilamellar vesicles form from the double emulsion droplets as the solvent contained in the oil phase evaporates. However, even here, some residual solvent may remain trapped within the bilayer, making the unilamellar vesicles unsuitable for diffusion studies or domain formation. This method has been improved to form ultrathin-shell double emulsion vesicles with minimal residual solvent, thus enabling the formation of microdomains (Stein et al., 2007). Following this method, Stein et al. used a microfluidic capillary device to generate giant unilamellar vesicles with diameters greater than 200 μm.

[0012] U.S. Patent Application Publication No. 2007 / 264320A1 discloses a method for forming monodisperse vesicles for drug delivery using a microfluidic device having a main microfluidic channel connected to first and second reactant inlet channels. The first inlet channel is used to deliver cationic lipids to the main channel, while the second channel is used to deliver nucleic acids. A droplet generation zone is provided in the main channel at the intersection of the first and second carrier channels containing a hydrophobic fluid. The cationic lipids, nucleic acids, and hydrophobic fluid are then pumped through the device. In the droplet generation zone, shear forces from the hydrophobic fluid pinch off the droplets. As the droplets move through the mixing region, the cationic lipids and nucleic acids mix within the generated droplets. Multiple split channels may be connected to the device's outlet to generate smaller monodisperse droplets with picoliter volumes.

[0013] A scientific paper by Bucher P et al. (Langmuir 1998, 14, 2712-2721) describes an experimental procedure for the preparation of thin-walled giant phosphatidylcholine vesicles, which are useful for microinjection. In these microinjection experiments, target vesicles (typically about 50-100 μm in diameter) are punctured with a microneedle, and an aqueous solution is injected into the vesicle's internal volume. The method used for giant vesicle preparation is a modification of the so-called electroformation method.

[0014] The assembly of giant unilamellar vesicles from small unilamellar vesicle precursors within microfluidic water-in-oil droplet structures provides excellent control over giant unilamellar vesicle formation and high production rates.

[0015] This approach has previously been used to generate cell-sized or even larger giant unilamellar vesicles with diameters up to 50 μm (Spatz et al., WO 2018 / 228894 A1). However, giant unilamellar vesicles the same size as or larger than typical cells cannot be used for drug delivery applications. The formation of polymer-shell-stabilized unilamellar vesicles with sizes between 1 μm and 10 μm involves advanced lithography procedures and complex channel structures. These drawbacks typically limit the reliable high-throughput production of polymer-shell-stabilized unilamellar vesicles with sizes between 1 μm and 10 μm.

[0016] Therefore, an object of the present invention is to provide giant unilamellar vesicles with an optimal size comprised between 1 μm and 10 μm, with controlled lipid composition for drug delivery applications, and optionally with surface bioligands that enable cell-specific drug delivery. Importantly, the giant unilamellar vesicles disclosed herein can encapsulate complex cargo, such as viral particles. A high level of control over lipid and luminal composition is possible by generating the giant unilamellar vesicles within a polymer shell that stabilizes the inner unilamellar vesicle.

[0017] The object of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures and examples of the present application.

[0018] [Brief description of the invention] To overcome the limitations of the prior art, the present invention provides a method for producing polymer-shell-stabilized unilamellar vesicles with diameters between 1 μm and 10 μm, which can be used for efficient and specific delivery of advanced cargo. This is achieved by developing a microfluidic device containing a multi-Y-shaped droplet splitting unit, also called a "splitting zone" (splitting zone (7) in Figure 1 and Figure 26), which generates small polymer-shell-stabilized unilamellar vesicles with diameters less than 10 μm, starting from parent polymer-shell-stabilized vesicles with diameters preferably greater than 10 μm.

[0019] The presented technique is based on three steps: 1) Well-controlled assembly of polymer-shell-stabilized giant unilamellar vesicles, including the use of functionalized lipids, fine-tuning of charge-mediated cellular interactions, reducing nonspecific interactions, lysosomal escape mechanisms for efficient cytoplasmic release, and robust cargo loading; 2) Mechanical partitioning of polymer-shell-stabilized giant unilamellar vesicles. 3) Release of segmented polymer shell-stabilized giant unilamellar vesicles into a physiological environment.

[0020] The first technical advantage of the method of the present invention is the high-throughput generation by mechanical partitioning of large quantities of polymer-shell-stabilized unilamellar vesicles with final diameters of 1 μm to 10 μm (Figures 2 and 3), which can be functionalized to interact with target cells with high specificity for advanced cargo delivery.

[0021] A second technical advantage of the present invention is that it provides giant unilamellar vesicles with highly efficient cargo loading and unprecedentedly controlled biological and physicochemical properties, which are essential for highly specific drug delivery applications.

[0022] A third technical advantage is that the biofunctionalized giant unilamellar vesicles of the present invention enable targeted transport of their cargo, avoiding degradation and immune recognition of the cargo within the vesicle lumen. Once at their destination, the giant unilamellar vesicle cargo can be exported intracellularly via the developed lysosomal escape mechanism.

[0023] A fourth technical advantage of the present invention is that the giant unilamellar vesicles of the present invention can encapsulate and deliver complex, robust, and high-molecular-weight cargoes, such as drug-releasing porous microparticles, large amounts of in vivo imaging probes, gigabase-long DNA, viruses, or promising DNA origami robots.

[0024] A fifth technical advantage of the present invention is that the giant unilamellar vesicles of the present invention are produced by symmetric division such that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%.

[0025] Specifically, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of 1 to 10 μm, preferably greater than 1 μm and less than 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100%.

[0026] According to a preferred embodiment of the present invention, the polymer shell-stabilized unilamellar vesicles provided in step a) are prepared by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an aqueous phase comprising at least one lipid, and an oil phase comprising a surfactant of formula (I):

[0027] [ka]

[0028] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, and wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); is obtained by

[0029] According to one embodiment of the present invention, the polymer shell-stabilized unilamellar vesicles provided in step a) can be prepared by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); is obtained by

[0030] According to one aspect of the present invention, step d) comprises removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell.

[0031] According to one embodiment of the present invention, the method for preparing monodisperse cell-targeting giant unilamellar vesicles comprises, after step d), the following steps: e) purifying the giant unilamellar vesicles from step d) by centrifugation; Further includes:

[0032] According to a further aspect of the invention, the aqueous phase in step a') is neutral lipids selected from the group comprising ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, diacylglycerols, phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, lysophosphatidylethanolamines, lysoethanolamines, inverted headgroup lipids, sphingosines, sterol-modified phospholipids, ether ester lipids, diether lipids, vinyl ethers (plasmalogens); an anionic lipid selected from the group comprising phosphatidic acid, lysophosphatidic acid derivatives, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, and bis(monoacylglycero)phosphate derivatives; a cationic lipid selected from the group comprising dioleyl-N,N-dimethylammonium chloride; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; N,N-distearyl-N,N-dimethylammonium bromide; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol; 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; dioctadecylamidoglycylcarboxyspermine; N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride and 1,2-dioleoyl-3-dimethylammonium-propane, a pH-sensitive lipid selected from the group comprising the lipids N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-sn-glycero-3-succinate, 1,2-dioleoyl-sn-glycero-3-succinate, N-palmitoylhomocysteine, photoswitchable lipids, Acylglycine derivatives, prenol derivatives, prostaglandin derivatives, glycosylated diacylglycerols, eicosanoid derivatives, (palmitoyloxy)octadecanoic acid derivatives, diacetylene derivatives, diphytanoyl derivatives, fluorinated lipids, brominated lipids, lipopolysaccharides, one of the foregoing lipids conjugated to a functionalized ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid-nickel, polyethylene glycol, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, folate, square, galloyl, glycan, thiol, arginylglycylaspartic acid, fluorophore, magnetic resonance imaging agent, chelator, and one of the aforementioned lipids bound to polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol; The lipid composition comprises at least one lipid selected from the group comprising:

[0033] According to a further embodiment of the invention, the aqueous phase in step a') comprises at least one anionic lipid, at least one neutral lipid, and optionally one neutral lipid functionalized with a fluorescent dye molecule.

[0034] According to a further embodiment of the invention, the aqueous phase in step a') comprises at least one cationic lipid, at least one neutral lipid, and optionally one neutral lipid functionalized with a fluorescent dye molecule.

[0035] According to a further aspect of the invention, the aqueous phase in step a') comprises at least one lipid functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, thiol, wherein the method optionally comprises, after step d), the following step: d') binding the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles; Includes:

[0036] According to a further embodiment of the invention, the aqueous phase in step a') comprises at least one lipid coupled to polyethylene glycol having a molecular weight comprised between 350 g / mol and 50,000 g / mol.

[0037] According to a further embodiment of the invention, the aqueous phase in step a') comprises at least one pH-sensitive lipid in a molar percentage comprised between 20% and 80%, or the aqueous phase in step a') further comprises polyethyleneimine in a concentration comprised between 2 μg / ml and 100 μg / ml.

[0038] According to a further aspect of the present invention, the aqueous phase in step a') further comprises at least one agent selected from the group comprising drug-releasing porous particles, molecular imaging agents, diagnostic agents, therapeutic agents, proteins or protein fragments, polypeptides, peptides, enzymes or enzyme fragments, nucleic acids, oligonucleotides, polynucleotides, potential DNA origami robots, small molecule drugs, virus particles, virus-like particles, microbial antigens, steroids, proteoglycans, lipids, monosaccharides, oligosaccharides, polysaccharides, magnetic particles, nanorods, carbon nanotubes, dentritosomes, polymersomes, metal nanoparticles, and combinations or conjugates thereof.

[0039] According to a further aspect of the present invention, the amphiphilic copolymer in step b) consists of (i) a triblock copolymer comprising two perfluorinated polymer end blocks and one polyether glycol block, or (ii) a diblock copolymer comprising one perfluorinated polymer end block and a polyether glycol block, wherein the triblock copolymer or diblock copolymer is folded such that the perfluorinated polymer end blocks are located on the outside of the polymer shell and the polyether glycol block is located on the inside of the polymer shell.

[0040] According to a further aspect of the present invention, step b) comprises mechanically splitting the polymer shell-stabilized giant unilamellar vesicle into two smaller polymer shell-stabilized giant unilamellar vesicles using a microfluidic device comprising a multi-Y-junction splitting zone (7) comprising at least one Y-junction, wherein the Y-junction consists of one inlet channel and two outlet channels, and step c) comprises repeating step b) by using four or more successive generations of Y-junctions, wherein the inlet channel of each Y-junction consists of the outlet channel of the previous Y-junction.

[0041] The present invention is further directed to a microfluidic device for preparing polymer-shell-stabilized giant unilamellar vesicles having a diameter of 1 μm to 10 μm, or greater than 1 μm and less than 10 μm, wherein the diameter is measured by confocal microscopy, the device comprising a multi-Y-shaped splitting zone (7) and a flow control system, wherein the multi-Y-shaped splitting zone (7) comprises the sequential generation of one or more Y-junctions, wherein each Y-junction consists of one inlet channel and two outlet channels, wherein the inlet channel of each Y-junction consists of the outlet channel of the previous junction, a stabilization plane (8) for stabilizing the split polymer-shell-stabilized giant unilamellar vesicles, one outlet channel (9) for directing the split polymer-shell-stabilized giant unilamellar vesicles to the outlet (10), and one outlet (10) through which the split polymer-shell-stabilized giant unilamellar vesicles exit the microfluidic device.

[0042] In a further embodiment, the microfluidic device for preparing polymer shell-stabilized giant unilamellar vesicles further comprises a parent polymer shell-stabilized giant unilamellar vesicle production zone arranged upstream of the splitting zone, the production zone comprising one oil phase inlet (1) for introducing an oil phase into the microfluidic device, optionally one oil phase filter structure (2), one or more aqueous phase inlets (3) for introducing an aqueous phase into the microfluidic device, optionally one aqueous phase filter structure (4), one junction (5) of the aqueous phase inlets (3) if there are two or more aqueous phase inlets (3), and a flow-focusing junction (6) consisting of a horizontal inlet channel and two vertical inlet channels, wherein the three inlet channels converge into an outlet channel through a narrow opening, and wherein the outlet channel is connected to the splitting zone, and wherein the parent polymer shell-stabilized giant unilamellar vesicles have a diameter between 1 μm and 100 μm.

[0043] definition Liposomes are composed of a lipid bilayer that separates an aqueous interior compartment from a bulk aqueous phase. Liposomes are spherical vesicles with at least one lipid bilayer. The main types of liposomes are multilamellar vesicles (MLVs, with several lamellar lipid bilayers), small unilamellar vesicles (SUVs, with one lipid bilayer), large unilamellar vesicles (LUVs), and giant unilamellar vesicles (GUVs).

[0044] Small unilamellar vesicles, large unilamellar vesicles, and giant unilamellar vesicles are usually spherical, typically with diameters of 25 nm to 50 nm for small unilamellar vesicles, 50 nm to over 1,000 nm for large unilamellar vesicles, and 1 μm to over 1,000 μm for giant unilamellar vesicles. However, the chemical and mechanical instability of unsaturated fatty acids, especially polyvalent cations, under high ionic strength conditions and their sensitivity to pH changes are considered major challenges in utilizing these vesicles. Furthermore, inserting molecules into these vesicles presents particular challenges given the vesicle's impermeability and mechanical instability.

[0045] The term "vesicle" refers to a non-natural or synthetic membranous, usually fluid-filled pouch resulting from the supramolecular assembly of lipids, including but not limited to phospholipids. The interior contents of a phospholipid vesicle are separated from the external environment by at least one phospholipid bilayer. A phospholipid bilayer is a two-molecule-thick sheet of lipids oriented so that the hydrophilic phosphate heads face "outward" toward the solution on either side of the bilayer and the hydrophobic tails face "inward" toward the core of the bilayer. This results in two "leaflets," each a monolayer of phospholipids. A "unilamellar vesicle" refers to a vesicle containing only one phospholipid bilayer. A "multilamellar vesicle" refers to a vesicle containing multiple phospholipid bilayers. A "symmetric bilayer" is defined as a bilayer with two leaflets of the same composition, while an "asymmetric bilayer" is defined as a bilayer with two leaflets of different composition.

[0046] The vesicles of the present invention can have a monolayer bilayer comprising lipids and components integrated by the bilayer. The integrated components can be proteins or fragments thereof, peptides, polypeptides, enzymes or fragments thereof. Exemplary proteins are ATPases, integrins, and alpha-hemolysins. An exemplary polypeptide is the antibiotic gramicidin.

[0047] An "aqueous droplet" is a droplet that contains water or a dispersion of any substance in water. Furthermore, an aqueous droplet is a droplet that consists of water or a dispersion of any substance in water. More specifically, an aqueous droplet in the sense of the present invention is a droplet that consists of water containing a salt and at least one lipid.

[0048] According to the present invention, "polymer shell-stabilized unilamellar vesicles" comprise a polymer shell surrounding "aqueous droplets" and forming so-called "water-in-oil droplets" or "water-in-oil emulsions." Preferably, the droplets are at least substantially ellipsoidal, or at least substantially spherical. More preferably, the parent polymer shell-stabilized giant unilamellar vesicles provided in step a) have an outer diameter of at least 10 μm, preferably at least 15 μm, preferably at least 20 μm, preferably at least 30 μm, preferably at least 40 μm, preferably at least 80 μm.

[0049] In the present invention, the term "water-in-oil droplets" is used synonymously with "water-in-oil emulsions" and "polymer shell-stabilized unilamellar vesicles." A "giant unilamellar vesicle," according to the present invention, is a unilamellar vesicle, preferably spherical, and having a diameter comprised between 1 μm and 100 μm. The partitioned giant unilamellar vesicles formed using the disclosed methods include release from the polymer shell and have an outer diameter of less than 1 μm, preferably less than 2 μm, preferably less than 3 μm, preferably less than 4 μm, preferably less than 5 μm, preferably less than 6 μm, preferably less than 7 μm, preferably less than 8 μm, preferably less than 9 μm, preferably less than 10 μm.

[0050] In other words, the divided giant unilamellar vesicles of the present invention produced after mechanical division and release preferably have a diameter of 1 μm to 20 μm. In some embodiments, the divided giant unilamellar vesicles can have a diameter of 2 μm to 20 μm. In other embodiments, the divided giant unilamellar vesicles can have a diameter of 3 μm to 20 μm. In other embodiments, the divided giant unilamellar vesicles can have a diameter of 4 μm to 20 μm. In other embodiments, the divided giant unilamellar vesicles can have a diameter of 5 μm to 20 μm. In preferred embodiments, the divided giant unilamellar vesicles of the present invention can have a diameter of 1 μm to 10 μm, preferably a diameter of 2 μm to 10 μm, preferably a diameter of 3 μm to 10 μm, preferably a diameter of 4 μm to 10 μm, and preferably a diameter of 5 μm to 10 μm. In further embodiments, the divided giant unilamellar vesicles of the present invention can have a diameter of 1 μm to 15 μm, preferably 2 μm to 15 μm, preferably 3 μm to 15 μm, preferably 4 μm to 15 μm, preferably 5 μm to 15 μm, hi more preferred embodiments, the divided giant unilamellar vesicles of the present invention can have a diameter of 1 μm to 5 μm, preferably 2 μm to 5 μm.

[0051] The diameter of the giant unilamellar vesicles is preferably measured by confocal microscopy as described in the methods. Alternatively, the distribution size of the original lipid preparation, eg, SUVs or liposomes, is preferably measured by dynamic light scattering as described in the methods.

[0052] The polymer shell-stabilized divided giant unilamellar vesicles formed using the disclosed methods have an outer diameter of less than 1.5 μm, preferably less than 2 μm, preferably less than 3 μm, preferably less than 4 μm, preferably less than 5 μm, preferably less than 6 μm, preferably less than 7 μm, preferably less than 8 μm, preferably less than 9 μm, preferably less than 10 μm.

[0053] In other words, the polymer shell-stabilized giant unilamellar vesicles of the present invention produced after mechanical partitioning preferably have a diameter of 1.5 μm to 23 μm. In some embodiments, the polymer shell-stabilized giant unilamellar vesicles can have a diameter of 2 μm to 23 μm. In other embodiments, the polymer shell-stabilized giant unilamellar vesicles can have a diameter of 3 μm to 23 μm. In other embodiments, the polymer shell-stabilized giant unilamellar vesicles can have a diameter of 4 μm to 23 μm. In other embodiments, the polymer shell-stabilized giant unilamellar vesicles can have a diameter of 5 μm to 23 μm. In a preferred embodiment, the polymer shell-stabilized giant unilamellar vesicles of the present invention can have a diameter of 1.5 μm to 12 μm, preferably a diameter of 2 μm to 12 μm, preferably a diameter of 3 μm to 12 μm, preferably a diameter of 4 μm to 12 μm, and preferably a diameter of 5 μm to 12 μm. In a further embodiment, the polymer shell-stabilized giant unilamellar vesicle of the present invention can have a diameter of 1.5 μm to 16 μm, preferably 2 μm to 16 μm, preferably 3 μm to 16 μm, preferably 4 μm to 16 μm, preferably 5 μm to 16 μm, hi a more preferred embodiment, the polymer shell-stabilized giant unilamellar vesicle of the present invention can have a diameter of 1.5 μm to 6 μm, preferably 2.5 μm to 6 μm.

[0054] According to a particularly preferred embodiment of the present invention, a dispersion is provided in steps a) to c), in which droplets (polymer shell-stabilized vesicles) are dispersed in an oil phase. In this embodiment, the giant unilamellar vesicles are stabilized not only by the polymer shell but also by the outer oil phase, so that the chemical and mechanical stability of the polymer shell-stabilized giant unilamellar vesicles is dramatically improved compared to the respective giant unilamellar vesicles without a polymer shell.

[0055] In a preferred embodiment, during step d), the polymer shell is removed from the giant unilamellar vesicles, which are then transferred from the oil phase to the aqueous phase. The terms "cellular delivery," "cellular targeting," "delivery," "cell-type specific delivery," or "targeted delivery" are used interchangeably herein and refer to the delivery of specific drugs or compounds or macromolecules to intracellular or extracellular regions, tissues, or cells in vivo or in vitro by the giant unilamellar vesicles of the present invention.

[0056] When delivery occurs in the extracellular space of a cell, delivery allows the drug or compound or macromolecule to be carried a sufficiently short distance to the target cell and interact with said cell, thereby enabling the desired effect on said cell.

[0057] The drug or compound or macromolecule can be present on the surface of the giant unilamellar vesicle or in the lumen of the giant unilamellar vesicle. The terms "protein" and "peptide" are used interchangeably herein and refer to a polymer of amino acid residues. These terms apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. As used herein, the terms encompass any length of amino acid chain, including full-length proteins, and fragments thereof, in which the amino acid residues are linked by covalent peptide bonds.

[0058] The terms "nucleic acid," "oligonucleotide," and "polynucleotide" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise specified, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0059] The term "production buffer" or "intraluminal buffer" refers to the aqueous solution used to disperse lipids and, optionally, nucleic acid molecules or other cargo to be encapsulated in the giant unilamellar vesicles. This solution is then incorporated into the lumen of the formed giant unilamellar vesicles, and is therefore also referred to as the "intraluminal buffer." Preferred production buffers are PBS, water, and suitable cell culture media, such as DMEM.

[0060] According to one embodiment of the present invention, the production buffer preferably comprises an aqueous phase containing at least one lipid and a "cation." A preferred "cation" is Mg 2+ , Ca 2+ However, the present invention is not limited with respect to the type of suitable cation.

[0061] However, when using positively charged surfactants, such as surfactants of formula (I) as claimed in claim 2, the presence of cations in the aqueous phase is not necessary. The term "release buffer" refers to an aqueous solution that is added to the dispersion of giant unilamellar vesicles after destabilization to release the giant unilamellar vesicles into the aqueous solution.Preferred release buffers are PBS, water, and suitable cell culture media such as DMEM.Preferably, the release buffer is the same as the production buffer.

[0062] "Aqueous mixture" or "aqueous phase" or "water phase" refers to a solution or suspension having lipids or other molecules substantially in water. Aqueous mixtures are immiscible with oily mixtures in the present invention.

[0063] "Oil mixture" or "oil phase" refers to a solution or suspension of lipids or other molecules in a perfluorinated water-immiscible solvent. Exemplary water-immiscible solvents, referred to as "oils," suitable for preparing certain lipid membranes and vesicles in the present invention include HFE-7000 (1,1,1,2,2,3,3-heptafluoro-3-methoxy-propane), methyl nonafluoro-n-butyl ether, HFE-7100 (a mixture of the isomers [methyl nonafluoroisobutyl ether and methyl nonafluorobutyl ether] 1,1,1,2,2,3,3,4,4-nonafluoro-4-methoxy-butane and 1,1,1,2,3,3-hexafluoro-3-methoxy-2-(trifluoromethyl)propane), Examples of suitable solvents include, but are not limited to, HFE-7200 (a mixture of isomers 1-ethoxy-1,1,2,2,3,3,4,4,4-nonafluorobutane and 1-ethoxy-1,1,2,3,3,3-hexafluoro-2-(trifluoromethyl)propane), HFE-7300 (1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane), and HFE-7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)hexane), FC-40. Those skilled in the art will appreciate that other solvents are useful in the oil blends of the present invention.

[0064] The "destabilizer" is a demulsifier surfactant that can destabilize the structure of the polymer shell surrounding the giant unilamellar vesicle, allowing the giant unilamellar vesicle to be released in an aqueous release buffer. Exemplary suitable demulsifiers are 1H,1H-perfluoro-1-pentanol, 1H,1H-perfluoro-1-octanol, 1H,1H,8H-perfluoro-1-octanol, and 1H,1H,2H,2H-perfluoro-1-octanol.

[0065] The "polymer shell" of the giant unilamellar vesicle is made of an amphiphilic copolymer with a lipophilic end located on the outside of the polymer shell and a hydrophilic end located on the inside of the polymer shell, the structure of which is described in detail in the paragraph above.

[0066] The terms "monodisperse" or "monodispersed" refer to vesicles of uniform size in a dispersed phase, where the vesicles exist in a non-aggregated and discrete state. The dispersed phase according to the present invention is preferably an oil phase for polymer-shell-stabilized giant unilamellar vesicles.

[0067] "Uniformly sized" vesicles means that the polymer shell-stabilized giant unilamellar vesicles obtained after partitioning and the giant unilamellar vesicles released from the polymer shell exhibit a size coefficient of variation of less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%.

[0068] The terms "symmetric division," "symmetrically dividing," and "symmetrically splitting" mean that the polymer-shell-stabilized giant unilamellar vesicles obtained after division and the giant unilamellar vesicles released from the polymer shell have similar lipid compositions, expressed as a mole percent of a reference lipid, such as the original lipid mixture used to generate the parent polymer-shell-stabilized giant unilamellar vesicles. In other words, the giant unilamellar vesicles obtained according to the method of the present invention have a lipid composition, expressed as a mole percent of a reference lipid, similar to the lipid composition of the aqueous phase in step a') of the method. "Similar" lipid composition means that the mole percent change between the original aqueous phase and the divided giant unilamellar vesicles is less than 5%, preferably less than 4%, or less than 3%.

[0069] More specifically, the mole percent change is calculated as follows: [(mole percent of reference lipid) 最終 - (mole percent of reference lipid) 最初] / (mole percent of reference lipid) 最初 ] * 100.

[0070] The mole percent change is also referred to as the "ratio change" in, for example, Table 3. The term "symmetric division" also refers to the fact that the divided giant unilamellar vesicles have a similar intraluminal content as the mother or parent giant unilamellar vesicles. The symmetric division of the luminal contents can be observed by incorporating fluorescent molecules into the initial aqueous phase in step a'), which are then encapsulated into the mother or parent polymer-shell-stabilized giant unilamellar vesicles. Therefore, the coefficient of variation of the intraluminal content is calculated based on the coefficient of variation of the fluorescence intensity in the divided vesicles, expressed as standard deviation / mean fluorescence. × It is calculated as 100. The coefficient of variation is preferably lower than 20%.

[0071] The term "loading efficiency" refers to the ability of divided giant unilamellar vesicles to encapsulate a cargo of interest with high efficiency. "Loading efficiency" is calculated as the ratio of the amount encapsulated to the amount taken up in the encapsulation process.

[0072] Due to the high loading efficiencies achieved in giant unilamellar vesicles and / or by the methods of the present invention, the cargo-to-lipid ratio of the encapsulated giant unilamellar vesicles, calculated based on the amount of cargo and giant unilamellar vesicles incorporated into the loading step, is greater than 60%, greater than 70%, greater than 80%, greater than 90%, and typically greater than 95% cargo-to-lipid ratio ("input" ratio). In practice, substantially 100% (quantitative) encapsulation is common.

[0073] The cargo-to-lipid ratio in a giant unilamellar vesicle can be characterized in terms of weight ratio (weight of cargo per weight or moles of giant unilamellar vesicle lipid) or molar ratio (moles of cargo per weight or moles of giant unilamellar vesicle lipid). The weight ratio of cargo in giant unilamellar vesicles of the present invention is typically 0.05 mg, 0.1 mg, 0.2 mg, 0.35 mg, 0.5 mg, or at least 0.65 mg of cargo per mg of lipid. In terms of molar ratio, the cargo-to-lipid ratio of the present invention is at least about 0.02 moles to about 5 moles, preferably at least 0.1 moles to about 2 moles, and more preferably about 0.15 moles to about 1.5 moles of cargo per mole of giant unilamellar vesicle lipid.

[0074] An "interface" is defined as an area of ​​contact between two or more objects that have a distinct boundary. In some cases, an interface is a small area of ​​contact between closely opposed compartments that results from the exclusion of the surrounding medium.

[0075] The term "cell targeting" refers to a vesicle that shows tropism for specific cells. Cell targeting vesicles can selectively interact with target cells and can be taken up by target cells through endocytosis (negatively charged vesicles) or membrane fusion (positively charged vesicles).Therefore, cell targeting vesicles can selectively deliver drugs to the intended target of the cell targeting vesicle, while avoiding the undesirable effects of off-targets that are not specific cells.

[0076] The cell targeting properties are influenced by the specific composition of the giant unilamellar vesicles, specifically by the content of positively or negatively charged lipids; the content of pegylated lipids; and binding to specific ligands / bioligands, such as membrane receptors, antigens, antibodies, glycans, transmembrane proteins, such as integrins, fibronectin, tetraspanins, etc.

[0077] [Description of the Invention] The present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter between 1 and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0078] More specifically, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles having a diameter of at least 40 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0079] Even more specifically, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles having a diameter of at least 20 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0080] According to one embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles having a diameter of at least 10 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0081] According to another embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles having a diameter of at least 8 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0082] According to a preferred embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) Providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 10 μm by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an aqueous phase comprising at least one lipid, and an oil phase comprising a surfactant of formula (I):

[0083] [ka]

[0084] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, where Symmetrical division means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0085] According to another embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) Providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 10 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and an oil phase containing an amphiphilic copolymer to form polymer shell-stabilized giant unilamellar vesicles, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing by contrast means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0086] [Release of partitioned giant unilamellar vesicles from polymer shells] The present inventors have demonstrated that the addition of a demulsifier to polymer-shell-stabilized giant unilamellar vesicles collected after mechanical partitioning can efficiently release the giant unilamellar vesicles from the polymer shell. The demulsifier destabilizes the structure of the surrounding polymer shell, thus enabling the giant unilamellar vesicles to be released from the polymer shell into an aqueous buffer solution, also referred to as a "release buffer."

[0087] This procedure allows the release of giant unilamellar vesicles from the polymer shell with a high efficiency of up to 50%, which means that one out of every two giant unilamellar vesicles is released into the aqueous phase (Figure 4), approximately 4 × 10 6 This means that the production rate of giant unilamellar vesicles reaches 10 ...

[0088] The demulsifier is selected from the group consisting of 1H,1H,2H,2H-perfluoro-1-octanol, 1H,1H-perfluoro-1-pentanol, 1H,1H-perfluoro-1-octanol, 1H,1H,8H-perfluoro-1-octanol, and preferably a short-chain perfluorinated aliphatic carbon chain.

[0089] The demulsifier is preferentially added in a ratio ranging from 1:1 to 10:1 with the intraluminal buffer (also called the production buffer). Thus, according to one embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, and Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0090] Therefore, the present invention is also directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an aqueous phase comprising at least one lipid, and an oil phase comprising a surfactant of formula (I):

[0091] [ka]

[0092] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of less than 10 μm; d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, and Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0093] Therefore, the present invention is also directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing at least one lipid, an aqueous phase containing a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of less than 10 μm; d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a demulsifier; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0094] According to a preferred embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell and is selected from the group comprising 1H,1H,2H,2H-perfluoro-1-octanol, 1H,1H-perfluoro-1-pentanol, 1H,1H-perfluoro-1-octanol, 1H,1H,8H-perfluoro-1-octanol; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0095] Therefore, the present invention is also directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an aqueous phase comprising at least one lipid, and an oil phase comprising a structure of formula (I):

[0096] [ka]

[0097] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell and is selected from the group comprising 1H,1H,2H,2H-perfluoro-1-octanol, 1H,1H-perfluoro-1-pentanol, 1H,1H-perfluoro-1-octanol, 1H,1H,8H-perfluoro-1-octanol; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0098] Furthermore, the present invention is also directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the steps of: a) providing polymer-shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell and is selected from the group comprising 1H,1H,2H,2H-perfluoro-1-octanol, 1H,1H-perfluoro-1-pentanol, 1H,1H-perfluoro-1-octanol, 1H,1H,8H-perfluoro-1-octanol; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0099] Thus, according to optional step d), the polymer shell and the oil phase are removed from the polymer-shell-stabilized giant unilamellar vesicles. After the incorporation of at least one drug and one or more proteins or fragments thereof into the polymer-shell-stabilized giant unilamellar vesicles and after mechanical partitioning, which represents a step requiring the mechanical stability provided by the polymer shell, the polymer shell is no longer needed, and it is actually preferable to carry out step d) so as to obtain giant unilamellar vesicles in the aqueous phase.

[0100] The removal step, which is carried out by adding a destabilizer demulsifier, is described in the Methods section and in Example 1. The polymer shell and oil phase may also be removed from the polymer shell-stabilized giant unilamellar vesicles during step d) by other techniques, such as using a microfluidic device, as described, for example, in International Patent Application WO201822894A1, Example 5.

[0101] [Purification of divided giant unilamellar vesicles] The divided giant unilamellar vesicles are usually centrifuged after release from the polymer shell to allow purification from vesicles of undesired size and other impurities.

[0102] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing a polymer shell-stabilized giant monolayer with a diameter comprised between 1 μm and 100 μm; b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0103] According to a more preferred embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an aqueous phase comprising at least one lipid, and an oil phase comprising a structure of formula (I):

[0104] [ka]

[0105] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0106] According to a preferred embodiment, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0107] Centrifugation can be carried out for a time period comprised between 5 and 60 minutes and at an acceleration rate comprised between 800 and 100,000 g, depending on the size of the target divided GUVs. For GUVs with a diameter comprised between 1 and 3 μm, centrifugation can be preferentially carried out at an acceleration rate comprised between 10,000 and 30,000 g, and for a time period comprised between 10 and 60 minutes.

[0108] For giant unilamellar vesicles with a diameter comprised between 3 and 6 μm, centrifugation is preferentially carried out at an acceleration comprised between 5,000 and 20,000 g and for a time comprised between 10 and 60 minutes.

[0109] For giant unilamellar vesicles with a diameter comprised between 6 and 10 μm, centrifugation can be preferentially carried out at an acceleration comprised between 2,000 and 10,000 g and for a time comprised between 10 and 60 minutes.

[0110] [Lipid composition of giant unilamellar vesicles] The giant unilamellar vesicle can contain one or more lipids. Preferably, the giant unilamellar vesicle of the present invention contains at least two lipids, preferably at least three lipids, more preferably at least four lipids. The lipids can be isolated from naturally occurring sources, or the lipids can be synthesized separately from any naturally occurring source.

[0111] The present invention is not particularly limited with respect to the chemical nature of the at least one lipid contained in the aqueous phase in step a'), and therefore the chemical nature of the at least one lipid contained in the inner space of the polymer-shell-stabilized giant unilamellar vesicle, as long as it can form a lipid bilayer. Good results are achieved, in particular, with phospholipids, specifically with lipids selected from the group consisting of phosphocholine, phosphocholine derivatives, phosphoethanolamine, phosphoethanolamine derivatives, phosphatidylcholine, phosphatidylcholine derivatives, phosphatidylglycerol, phosphatidylglycerol derivatives, and any combination of two or more of the aforementioned lipids.

[0112] At least one of the lipids is an amphipathic lipid, which is defined as having a hydrophilic and a hydrophobic portion, typically a hydrophilic head and a hydrophobic tail. The hydrophobic portion typically orients toward the hydrophobic phase, for example, within the bilayer, while the hydrophilic portion typically orients toward the aqueous phase, for example, outside the bilayer and possibly between adjacent bilayer surfaces. The hydrophilic portion may contain polar or charged groups, such as carbohydrates, phosphate, carboxyl, sulfato, amino, sulfhydryl, nitro, hydroxy, and other similar groups. The hydrophobic portion may contain apolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic lipids include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0113] Typically, lipid is phospholipid.Phospholipid includes but is not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and their derivatives.It should be understood that other lipid membrane components can also be used, such as cholesterol, sphingomyelin, cardiolipin, etc.

[0114] Lipids can be "uncharged lipids" or "charged lipids." "Uncharged lipids" refer to lipids that do not have any charged or ionizable groups, such as, for example, phosphate or choline groups. Examples of uncharged lipids include, but are not limited to, diacylglycerols and prostaglandins.

[0115] "Charged lipids" include neutrally charged, i.e., zwitterionic, cationic, and anionic lipids. Generally, lipids with a positive or negative charge exhibit low solubility in the oil phase.

[0116] Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH. "Zwitterionic lipids" have both positively charged and ionizable groups, such as amino and choline groups, which have a net positive charge, and negatively charged and ionizable groups, such as phosphate, sulfate, and carboxylate. Examples of zwitterionic lipids include, but are not limited to, phosphorylcholine and phosphorylethanolamine.

[0117] "Anionic lipids" are lipids that are negatively charged at physiological pH. "Cationic lipids" are lipids that are positively charged at physiological pH. Further suitable lipid is pH-sensitive lipid. " pH-sensitive " lipid refers to lipid whose ability to form and / or maintain lipid bilayer is at least partially dependent on the pH of surrounding environment.The liposome and unilamellar vesicle containing this lipid are destabilized under the acidic conditions of endocytosis pathway.Therefore, encapsulated components are delivered to intracellular biological environment through destabilization or fusion with endosomal membrane.

[0118] Specific examples of lipids suitable for synthesizing giant unilamellar vesicles according to the methods disclosed herein are listed in Table 1. Preferably, the lipid is biodegradable to allow release of the encapsulated agent in vivo and / or in vitro. Biodegradable lipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphocholine (dioleoyl-phosphocholine, DOPC), anionic 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol) (dioleoyl-phosphoglycerol, DOPG), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (distearoyl-phosphoethanolamine, DSPE).

[0119] Functionalized Lipids According to an embodiment of the present invention, at least one lipid contained in the aqueous phase of step a') is a lipid functionalized with a functionalizing ligand and / or polyethylene glycol. Specific examples of suitable functionalizing moieties, reactive ligands, and containing functionalized lipids are listed in Table 2.

[0120] [Table 1-1]

[0121] [Table 1-2]

[0122] [Table 1-3]

[0123] [Table 1-4]

[0124] [Table 1-5]

[0125] [Table 1-6]

[0126] [Table 1-7]

[0127] [Table 1-8]

[0128] [Table 1-9]

[0129] Table 1-10

[0130] Table 1-11

[0131] Table 1-12

[0132] Table 1-13

[0133] Table 1-14

[0134] Table 1-15

[0135] Table 1-16

[0136] Table 2-1

[0137] Table 2-2

[0138] Table 2-3

[0139] Table 2-4

[0140] Table 2-5

[0141] Table 2-6

[0142] Table 2-7

[0143] Table 2-8

[0144] Table 2-9

[0145] Table 2-10

[0146] Preferably, the functionalizing ligand is selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyls, cyanuric acid, folic acid, square, galloyl, thiol, arginylglycylaspartic acid, fluorescent dye molecules, magnetic resonance imaging reagents, and chelating agents. Sulfhydryls, also known as thiols, are present in proteins in the side chain of cysteine ​​(Cys, C) amino acids. Sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinyl sulfones, pyridyl disulfides, TNB-thiols, and disulfide reducing agents.

[0147] Various lipids that can be provided with thioether bonds include maleimides, aromatic maleimides, such as N-[4-(p-maleimidophenyl)-butyryl] (MPB) or 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC) groups. The maleimide functional group of MCC, which contains an aliphatic cyclohexane ring, is more stable to hydrolysis in aqueous reaction environments than the aromatic phenyl group of MPB.

[0148] The carbohydrate is selected from the group consisting of β-galactose, α-mannose, β-mannose, and α-fucose. It has been shown that the carbohydrate can be bound to cholesterol and incorporated into liposomes, and in vitro results have shown that sugar-bound liposomes are efficiently recognized by cells that overexpress carbohydrate-binding receptors on their cell surface (Rajabi and Mousa, 2016, Current Pharmaceutical Biotechnology, 17, 8).

[0149] SNAP-tag is a self-labeling protein tag that is commercially available in a variety of expression vectors. SNAP-tag is a 182-residue polypeptide (19.4 kDa) that can be fused to any protein of interest and then specifically and covalently labeled with an appropriate ligand, such as a fluorescent dye.

[0150] Functionalized and non-functionalized lipids are available from a number of commercial sources, including Avanti Polar Lipids (Alabaster, Alabama).

[0151] In some embodiments, the molar percentage (mol%) of cationic lipid typically comprises 0% to 10%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60% of the total lipid present in the vesicle.

[0152] In some embodiments, the molar percentage (mol%) of anionic lipids typically comprises 0% to 10%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, or 20% to 60% of the total lipids present in the vesicle.

[0153] In some embodiments, the molar percentage (mol%) of neutral lipids typically comprises 49% to 99%, 49% to 89%, 49% to 79%, 49% to 69%, 59% to 99%, 59% to 89%, 59% to 79%, 59% to 69% of the total lipids present in the vesicle.

[0154] According to a preferred aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a structure of formula (I) to form a polymer shell-stabilized giant unilamellar vesicle:

[0155] [ka]

[0156] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * Calculated as 100, and wherein the at least one lipid in step a') is neutral lipids selected from the group comprising ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, diacylglycerols, phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, lysophosphatidylethanolamines, lysoethanolamines, inverted headgroup lipids, sphingosines, sterol-modified phospholipids, ether ester lipids, diether lipids, vinyl ethers (plasmalogens); an anionic lipid selected from the group comprising phosphatidic acid, lysophosphatidic acid derivatives, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, and bis(monoacylglycero)phosphate derivatives; a cationic lipid selected from the group comprising dioleyl-N,N-dimethylammonium chloride; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; N,N-distearyl-N,N-dimethylammonium bromide; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol; 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; dioctadecylamidoglycylcarboxyspermine; N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride and 1,2-dioleoyl-3-dimethylammonium-propane, a pH-sensitive lipid selected from the group comprising the lipids N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-sn-glycero-3-succinate, 1,2-dioleoyl-sn-glycero-3-succinate, N-palmitoylhomocysteine, photoswitchable lipids, Acylglycine derivatives, prenol derivatives, prostaglandin derivatives, glycosylated diacylglycerols, eicosanoid derivatives, (palmitoyloxy)octadecanoic acid derivatives, diacetylene derivatives, diphytanoyl derivatives, fluorinated lipids, brominated lipids, lipopolysaccharides, one of the foregoing lipids conjugated to a functionalized ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, folate, square, galloyl, glycan, thiol, arginylglycylaspartic acid, fluorophore, magnetic resonance imaging agent, chelator, and one of the aforementioned lipids bound to polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol; is selected from the group comprising:

[0157] According to a particular aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and an oil phase containing an amphiphilic copolymer to form polymer shell-stabilized giant unilamellar vesicles, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * Calculated as 100, and wherein the at least one lipid in step a') is: neutral lipids selected from the group comprising ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, diacylglycerols, phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, lysophosphatidylethanolamines, lysoethanolamines, inverted headgroup lipids, sphingosines, sterol-modified phospholipids, ether ester lipids, diether lipids, vinyl ethers (plasmalogens); an anionic lipid selected from the group comprising phosphatidic acid, lysophosphatidic acid derivatives, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, and bis(monoacylglycero)phosphate derivatives; a cationic lipid selected from the group comprising dioleyl-N,N-dimethylammonium chloride; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; N,N-distearyl-N,N-dimethylammonium bromide; N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol; 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; dioctadecylamidoglycylcarboxyspermine; N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride and 1,2-dioleoyl-3-dimethylammonium-propane, a pH-sensitive lipid selected from the group comprising the lipids N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-sn-glycero-3-succinate, 1,2-dioleoyl-sn-glycero-3-succinate, N-palmitoylhomocysteine; photoswitchable lipids, Acylglycine derivatives, prenol derivatives, prostaglandin derivatives, glycosylated diacylglycerols, eicosanoid derivatives, (palmitoyloxy)octadecanoic acid derivatives, diacetylene derivatives, diphytanoyl derivatives, fluorinated lipids, brominated lipids, lipopolysaccharides, one of the foregoing lipids conjugated to a functionalized ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, folate, square, galloyl, glycan, thiol, arginylglycylaspartic acid, fluorophore, magnetic resonance imaging agent, chelator, and one of the aforementioned lipids bound to polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol; is selected from the group comprising:

[0158] [Controlling attractive and repulsive GUV-cell interactions for targeted delivery] Here, we develop a PEG-based passivation strategy aimed at modulating specific or nonspecific interactions between cells and giant unilamellar vesicles to promote ligand-based specific attractive interactions while suppressing charge-mediated nonspecific interactions (Figures 15-19).

[0159] Specifically, we demonstrated that the zeta potential of both negatively and positively charged vesicles can be reduced by increasing the length of the PEG chain and the rate of PEGylation.

[0160] Polyethylene glycol can be conjugated to any lipid, and preferentially to phosphatidylethanolamine, or to ceramide using standard coupling reactions known and used by those skilled in the art. Additionally, preformed polyethylene glycol-phosphatidylethanolamine conjugates are commercially available from Avanti Polar Lipids (Alabaster, Alabama).

[0161] Polyethylene glycols of various molecular weights can be used to form the bilayer stabilizing component of the present invention. Polyethylene glycols of various numbers of ethylene glycol units (and therefore molecular weights) are commercially available from many different sources, or alternatively, can be synthesized using standard polymerization techniques well known to those skilled in the art. PEG molecules suitable within the scope of the present invention have molecular weights falling within the range of 350 to 50,000 g / mol, corresponding to PEG 350 to PEG 50,000. For example, the following PEG molecules can be used: PEG 200, PEG 350, PEG 550, PEG 750, PEG 1000, PEG 2000, PEG 3000, PEG 5000, PEG 20,000, and PEG 50,000.

[0162] Preferred PEGylated lipids for the methods described in this invention are: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 18:1PEG350PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 18:1PEG750PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 18:1PEG1000PE.

[0163] The giant unilamellar vesicles contain PEGylated lipids in a molar concentration of at most 5%, preferentially at most 10%, preferentially at most 20%, preferentially at most 50%, preferentially at most 60%, preferentially at most 70%, preferentially at most 80%. It is particularly preferred if the PEGylated lipids are present in a molar concentration of 50%.

[0164] Thus, according to a particular aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a structure of formula (I) to form a polymer shell-stabilized giant unilamellar vesicle:

[0165] [ka]

[0166] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein at least one lipid in step a') is bound to a polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol.

[0167] According to another particular aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein at least one lipid in step a') is bound to a polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol.

[0168] [Fine-tuning charge-mediated GUV-cell interactions] The present inventors have demonstrated that the charge of giant unilamellar vesicles can be finely tuned between very high positive and very low negative charges by adjusting the respective lipid formulations. The generation of giant unilamellar vesicles with different charges has great technical advantages because it allows for the control of the interaction of giant unilamellar vesicles with target cells.

[0169] We were able to show that giant unilamellar vesicles containing various amounts of negatively charged lipids, such as 1,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol), have a negative zeta potential and are taken up by cells present in the cytoplasm by endocytosis (Figures 7, 8, 9).

[0170] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one anionic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0171] A preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one anionic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0172] [ka]

[0173] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0174] A list of suitable anionic and neutral lipids is provided in Table 1. Table 2 provides a list of suitable fluorescent dyes. Therefore, a preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one anionic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the anionic lipid is selected from the group comprising phosphatidic acid, lysophosphatidic acid, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, bis(monoacylglycero)phosphate; wherein the neutral lipid is selected from the group comprising ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, diacylglycerol, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine, phosphatidylethanolamine, and lysoethanolamine; and wherein the fluorophore is selected from the group including Lissamine rhodamine B sulfonyl, Atto 488, Alexa Fluor 488, Alexa Fluor 647, fluorescein, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), Cy5, Cy5.5, Cy7, Topfluor® Alexa Fluor 488, Topfluor® Alexa Fluor 594.

[0175] A more preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one anionic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0176] [ka]

[0177] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the anionic lipid is selected from the group comprising phosphatidic acid, lysophosphatidic acid, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, bis(monoacylglycero)phosphate; wherein the neutral lipid is selected from the group comprising ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, diacylglycerol, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine, phosphatidylethanolamine, and lysoethanolamine; wherein the fluorophore is selected from the group including Lissamine rhodamine B sulfonyl, Atto 488, Alexa Fluor 488, Alexa Fluor 647, fluorescein, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), Cy5, Cy5.5, Cy7, Topfluor® Alexa Fluor 488, Topfluor® Alexa Fluor 594.

[0178] In some embodiments, the molar percentage (mol%) of anionic lipids typically comprises 0% to 10%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, or 20% to 60% of the total lipids present in the vesicle.

[0179] In some embodiments, the molar percentage (mol%) of neutral lipids typically comprises 49% to 99%, 49% to 89%, 49% to 79%, 49% to 69%, 59% to 99%, 59% to 89%, 59% to 79%, 59% to 69% of the total lipids present in the vesicle.

[0180] In some embodiments, the molar percentage (mol%) of neutral lipids functionalized with fluorescent dye molecules typically comprises 0.1% to 5%, 0.5% to 5%, 1% to 5%, 1% to 4%, or 1% to 3% of the total lipids present in the vesicle.

[0181] Specifically, good results have been obtained when the molar percentage of anionic lipids is comprised between 20% and 50% and the molar percentage of neutral lipids is comprised between 49% and 99%.

[0182] In contrast, giant unilamellar vesicles containing various amounts of positively charged lipids, such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), have a positive zeta potential, and their interaction with cells depends on the amplitude of the zeta potential of the giant unilamellar vesicles: vesicles with a zeta potential value of approximately 2 mV adhere to the membrane of target cells, while vesicles with a zeta potential value greater than 2 mV fuse with the membrane of target cells (Figures 7 and 8).

[0183] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one cationic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0184] A more preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one cationic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0185] [ka]

[0186] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0187] A list of suitable cationic and neutral lipids is given in Table 1. Table 2 reports a list of suitable fluorescent dyes. Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one cationic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, cationic lipids include dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol); N-(1,2-dimyristyloxyprop-3yl)-N,N-dimethyl-hydroxyethylammonium bromide (DMRIE); DOTMA and cationic liposomes comprising 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE); cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE; cationic lipids comprising a solution of dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol; N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride (DODMA) and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), wherein the neutral lipid is selected from the group comprising ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, diacylglycerol, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine, phosphatidylethanolamine, and lysoethanolamine; wherein the fluorophore is selected from the group including Lissamine rhodamine B sulfonyl, Atto 488, Alexa Fluor 488, Alexa Fluor 647, fluorescein, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), Cy5, Cy5.5, Cy7, Topfluor® Alexa Fluor 488, Topfluor® Alexa Fluor 594.

[0188] A more preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one cationic lipid, at least one neutral lipid, optionally one neutral lipid functionalized with a fluorescent dye molecule, and a cation, and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0189] [ka]

[0190] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, cationic lipids include dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3β-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol); N-(1,2-dimyristyloxyprop-3yl)-N,N-dimethyl-hydroxyethylammonium bromide (DMRIE); DOTMA and cationic liposomes comprising 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE); cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and DOPE; cationic lipids comprising a solution of dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol; N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride (DODMA) and 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), wherein the neutral lipid is selected from the group comprising ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, diacylglycerol, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine, phosphatidylethanolamine, and lysoethanolamine; wherein the fluorophore is selected from the group including Lissamine rhodamine B sulfonyl, Atto 488, Alexa Fluor 488, Alexa Fluor 647, fluorescein, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), Cy5, Cy5.5, Cy7, Topfluor® Alexa Fluor 488, Topfluor® Alexa Fluor 594.

[0191] In some embodiments, the molar percentage (mol%) of cationic lipid typically comprises 0% to 10%, 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60% of the total lipid present in the vesicle.

[0192] In some embodiments, the molar percentage (mol%) of neutral lipids typically comprises 49% to 99%, 49% to 89%, 49% to 79%, 49% to 69%, 59% to 99%, 59% to 89%, 59% to 79%, 59% to 69% of the total lipids present in the vesicle.

[0193] In some embodiments, the molar percentage (mol%) of neutral lipids functionalized with fluorescent dye molecules typically comprises 0.1% to 5%, 0.5% to 5%, 1% to 5%, 1% to 4%, or 1% to 3% of the total lipids present in the vesicle.

[0194] In particular, good results have been obtained when the molar percentage of cationic lipids is comprised between 1% and 51% and the molar percentage of neutral lipids is comprised between 49% and 99%.

[0195] [Biofunctionalization of giant unilamellar vesicles] The present inventors have also developed a toolbox of strategies for bioorthogonal functionalization of giant unilamellar vesicles with specific target biomolecules to provide cell-type-specific delivery of therapeutic compounds or macromolecules. Cellular delivery refers to delivery of a compound to the extracellular space of a target cell or within a target cell. In fact, in some embodiments, a compound in a giant unilamellar vesicle can exert its effect on a target cell even when delivered near or to the extracellular surface of the cell. This can be achieved, for example, by presenting a ligand on the vesicle surface to a protein receptor in the outer cell membrane. Therefore, when delivery occurs in the extracellular space of a cell, the compound or macromolecule is taken to the target cell at a distance short enough to allow the compound to interact with the cell and thus have the desired effect on the cell.

[0196] To illustrate the diversity of biofunctionalization possibilities of giant unilamellar vesicles, we prepared giant unilamellar vesicles using biotinylated lipids for binding to streptavidin-tagged proteins and NTA-Ni for binding to histidine-tagged proteins. 2+ The functionalized lipids were generated by microfluidic droplet partitioning using DOPE lipids containing primary ammines for attachment to N-hydroxysuccinimide (NHS) functionalized molecules (Figures 10-14).

[0197] Suitable functional ligands are selected from the group consisting of biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric acid, square, galloyl, and thiol. As shown in Table 2, these functional ligands react with specific moieties with high affinity, such as the ligand biotin reacting with the moiety streptavidin or avidin. Therefore, macromolecules of interest can be bound to the released giant unilamellar vesicle surface by utilizing the high-affinity interaction between the functional ligand and the respective reactive moiety.

[0198] Table 2 lists functionalized ligands that can be bound to lipids, their functions, and interacting moieties. Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is a solution of a perfluorinated water-immiscible solvent, wherein the at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, and thiol; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') binding the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles, and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0199] A preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an oil phase comprising at least one lipid and a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0200] [ka]

[0201] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; wherein at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, thiol, and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') binding the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles, and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0202] The "macromolecules" attached to the giant unilamellar vesicles can be carbohydrates, nucleic acids, proteins, protein fragments, polypeptides, cell receptors, imaging probes, nanoparticles.

[0203] Preferably, the protein is selected from the group comprising antibodies, antigen-binding fragments, cell receptors, ligands, antigens, cytokines, cytokine receptors, glycoproteins, enzymes.

[0204] As used herein, the term "fragment" or "active fragment" of a protein refers to a fragment of that protein that retains the ability to be incorporated into or specifically bound to a giant unilamellar vesicle. The term "fragment" or "active fragment" of a protein also refers to a fragment of that protein that retains the ability to perform its function in a target cell.

[0205] For example, in the case of a membrane protein, a protein fragment refers to the cytoplasmic domain, the transmembrane domain, or the extracellular domain of said protein. For example, in the case of an enzyme, a protein fragment refers to the catalytic domain of the enzyme.

[0206] For example, in the case of antibodies, a protein fragment refers to a fragment of an antibody that retains the ability to specifically bind to an antigen. An antibody or antigen-binding fragment can be derived from a natural source or partially or wholly synthetically produced. In some embodiments, the antibody is a monoclonal antibody. In some of these embodiments, the monoclonal antibody is an IgG antibody. In certain embodiments, the monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4. In some other embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antibody fragment, also referred to as an antigen-binding fragment, is selected from an antigen-binding fragment (Fab), Fab', and F(ab')2, F(ab)2, variable fragment (Fv), and Fd fragment. In certain embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv) or (ScFv)2 fragment. In certain other embodiments, the antibody or antigen-binding fragment is a single-domain antibody. In some embodiments, the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

[0207] For example, in the case of a protein antigen, a protein fragment refers to a fragment of the antigen that retains its ability to induce an immune response in humans or animals and / or to be specifically recognized by an antibody.

[0208] As used herein, the term protein, or fragment thereof, includes "variants" of a protein, or fragment thereof, and refers to proteins or fragments that share a particular amino acid sequence identity with a reference protein or fragment when aligned by methods known in the art. Variants of a protein, or variants of a fragment thereof, can include substitutions, insertions, deletions, frameshifts, or rearrangements in other proteins. In some embodiments, variants share at least 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with the native protein or fragment thereof.

[0209] Any recitation of a protein provided herein encompasses functional variants of the protein. The term "functional variant" of a protein refers to a variant of the protein that retains the ability to be incorporated into or specifically targeted to giant unilamellar vesicles.

[0210] The percentage of "sequence identity" is determined by comparing two optimally aligned protein or polypeptide sequences over a "comparison window" spanning the entire length of the reference sequence. As used herein, the "comparison window" refers to the optimal alignment between the reference sequence and the variant sequence after the two sequences have been optimally aligned, where the variant nucleic acid or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) of 20% or less, typically 5-15%, or 10-12%, compared to the reference sequence for optimal alignment (not including additions or deletions). The percentage of identity is calculated by determining the number of positions where identical amino acid residues occur in both sequences, dividing the number of matching positions by the total number of positions (i.e., the full length of amino acids or nucleotides) in the reference sequence, and multiplying the result by 100% to obtain the percentage of sequence identity. Two protein or polypeptide sequences are said to be "identical" if the nucleotide or amino acid sequence in the two sequences is the same when optimally aligned as described above.

[0211] The percentage of "sequence identity" can be determined in the comparison window defined above with the aid of blastp using the "BLAST2 Sequences" tool available on the NCBI website (Tatusova A. et al., FEMS Microbiol Lett. 1999, 174:247-250).

[0212] Alternatively, the variant sequence can be any amino acid sequence resulting from substitutions allowed at any number of positions in the parent sequence according to the following formula: Ser substituted by Ser, Thr, Gly, and Asn; Arg substituted by any one of Arg, His, Gin, Lys, and Glu; Leu substituted by any one of Leu, Ile, Phe, Tyr, Met, and Val; Pro substituted by any one of Pro, Gly, Ala, and Thr; Thr replaced by any one of Thr, Pro, Ser, Ala, Gly, His, and Gin; Ala substituted by any one of Ala, Gly, Thr, and Pro; Val substituted by any one of Val, Met, Tyr, Phe, Ile, and Leu; Gly substituted by any one of Gly, Ala, Thr, Pro, and Ser; Ile substituted by any one of Ile, Met, Tyr, Phe, Val, and Leu; Phe substituted by any one of Phe, Trp, Met, Tyr, Lie, Val, and Leu; Tyr substituted by any one of Tyr, Trp, Met, Phe, Ile, Val, and Leu; His substituted by any one of His, Glu, Lys, Gin, Thr, and Arg; Gln substituted by any one of Gin, Glu, Lys, Asn, His, Thr, and Arg; Asn substituted by any one of Asn, Glu, Asp, Gin, and Ser; Lys substituted by any one of Lys, Glu, Gln, His, and Arg; Asp substituted by any one of Asp, Glu, and Asn; Glu substituted by any one of Glu, Asp, Lys, Asn, Gln, His, and Arg; Met substituted by any one of Met, Phe, Ile, Val, Leu, and Tyr.

[0213] Preferably, the imaging probe is selected from the group comprising fluorescent dyes, colorimetric dyes. Preferably, the carbohydrate is selected from the group comprising polyglycans, lactose, sucrose, β-galactose, α-mannose, β-mannose, and α-fucose.

[0214] Preferably, the nucleic acid is selected from the group comprising cDNA, genomic DNA, left-handed DNA and RNA, naturally or non-naturally occurring DNA including peptide nucleic acid (PNA), nuclear DNA, mitochondrial DNA, RNA including mRNA, rRNA, tRNA, oligonucleotides, triple helix forming molecules, immunostimulatory nucleic acids such as immunostimulatory CpG nucleic acids, small interfering RNA (siRNA) or microRNA (miRNA) used to regulate gene expression, antisense oligonucleotides used to regulate gene expression, aptamers, ribozymes, genes or gene fragments, regulatory sequences.

[0215] Specific examples of suitable macromolecules are bovine serum albumin, poly-L-lysine, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), wheat germ agglutinin (WGA), anti-cadherin, anti-α4 integrin, interleukin-2 (IL2), insulin, bradykinin, tat (HIV)-GFP, cysteine, CD95L, e-cadherin, fibronectin, anti-CD3 antibody, gold nanoparticles, arginylglycylaspartic acid (RGD), neural cell adhesion molecule (NrCAM), or a fragment of any of the foregoing.

[0216] In slightly different terms, embodiments of the present invention are directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is composed of a solution of a perfluorinated water-immiscible solvent, and wherein the at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, and thiol; and a'') optionally incorporating one or more proteins or activated fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') coupling the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or activated fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles, and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0217] More slightly rephrased, a preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeted giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an oil phase comprising at least one lipid and a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0218] [ka]

[0219] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, wherein at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, thiol, and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') binding the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or active fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles, and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0220] In slightly different terms, embodiments of the present invention are directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is composed of a solution of a perfluorinated water-immiscible solvent, and wherein the at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, and thiol; and a'') optionally incorporating one or more proteins or activated fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') coupling the giant unilamellar vesicle with at least one macromolecule comprising at least one moiety reactive with one of said functional ligands, wherein the macromolecule is selected from the group comprising carbohydrates, nucleic acids, proteins or activated fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles; and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * Calculated as 100, and Here, the protein or active fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the native protein or with a fragment thereof.

[0221] More slightly rephrased, a preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeted giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') To form polymer-shell stabilized giant unilamellar vesicles, Mixing an oil phase comprising at least one lipid and a surfactant of formula (I):

[0222] [ka]

[0223] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, wherein at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, thiol, and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') binding the giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or active fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles, and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or active fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the native protein or with a fragment thereof.

[0224] A further preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and a cation, and an oil phase comprising an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent, and wherein the at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, and thiol; and a'') optionally incorporating one or more proteins or activated fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') coupling the giant unilamellar vesicle with at least one polymer comprising at least one moiety reactive with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles; and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or active fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the native protein or with a fragment thereof.

[0225] An even more preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an oil phase comprising at least one lipid and a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0226] [ka]

[0227] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, wherein at least one lipid is functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, square, galloyl, thiol, and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); d') coupling the giant unilamellar vesicle with at least one polymer comprising at least one moiety reactive with one of said functionalized ligands, wherein the polymer is selected from the group comprising carbohydrates, nucleic acids, proteins or active fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles; and e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or active fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the native protein or with a fragment thereof.

[0228] [Protein incorporation] In addition to the protein biofunctionalization of the released giant unilamellar vesicles reported above, one or more proteins or fragments thereof may be incorporated into the parent polymer shell-stabilized giant unilamellar vesicles before partitioning in step a'').

[0229] The proteins may be provided in a buffer-soluble form, or they may already be incorporated into the walls of small protein liposomes, thus forming proteoliposomes, i.e., vesicles preferably having at least one lipid bilayer, into which one or more proteins are inserted. These protein liposomes fuse with giant unilamellar vesicles provided within a polymer shell.

[0230] For example, step a") may be carried out by electro-microfluidically incorporating one or more proteins or fragments thereof into the polymer-shell-stabilized giant unilamellar vesicles provided in step a') using an injector, preferably a pico-injector as described in WO2018228894A1. More preferably, the one or more proteins or fragments thereof are incorporated into the polymer-shell-stabilized giant unilamellar vesicles in this embodiment by injecting them using a pico-injector in the form of respective proteoliposomes.

[0231] During step a''), a transmembrane protein or a fragment thereof and / or a cytoskeletal protein or a fragment thereof may be incorporated into the lipid bilayer and / or into the interior space of the polymer shell-stabilized giant unilamellar vesicle, respectively.

[0232] The present invention is not limited with respect to the type of protein incorporated into the polymer-shell-stabilized giant unilamellar vesicle. For example, proteins selected from the group including receptors, ATP synthases, polymerases, actin, tubulin, antibodies, integrins, ribosome-associated proteins, nuclear-associated proteins, signal transduction proteins, immunologically related proteins, antibodies, various ion-pump proteins, adhesion-associated proteins, and any combination of two or more of the aforementioned proteins are suitable for incorporation into the polymer-shell-stabilized giant unilamellar vesicles in step a').

[0233] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0234] A preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an oil phase comprising at least one lipid and a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0235] [ka]

[0236] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0237] In slightly different terms, embodiments of the present invention are directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0238] More slightly rephrased, a preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeted giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an oil phase comprising at least one lipid and a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0239] [ka]

[0240] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0241] A further embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the steps of: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the naturally occurring protein or with a fragment thereof.

[0242] An even more preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0243] [ka]

[0244] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the naturally occurring protein or with a fragment thereof.

[0245] In other words, a further embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the naturally occurring protein or with a fragment thereof.

[0246] In other words, a preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0247] [ka]

[0248] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') incorporating one or more proteins or active fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); e) purifying the giant unilamellar vesicles by centrifugation; Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, Here, the protein or fragment thereof shares at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with the naturally occurring protein or with a fragment thereof.

[0249] [Lysosomal escape of giant unilamellar vesicle cargo (Carago) for efficient cytoplasmic delivery] Three main strategies can be used to promote endosomal escape of liposomes: 1 For example, "pH-sensitive lipids" such as 1,2-distearoyl-3-dimethylammonium-propane (DOBAQ). The "proton sponge mechanism" involves cationic polymers containing protonatable nitrogen atoms (e.g., polyethyleneimine (PEI)), which act as "proton sponges." They attract protons within the endosome, leading to the diffusion of more protons, along with chloride ions, into the endosome. When the osmotic pressure becomes high enough to disrupt the endosome, the DNA particles are released into the cytosol. 3 pH-sensitive fusogenic peptides, used by bacteria to facilitate the escape of giant unilamellar vesicle cargo (cargo) from lysosomes into host cells, as these peptides can disrupt endosomal membranes and affect release.

[0250] Importantly, we have optimized the lysosomal escape mechanism to efficiently release giant unilamellar vesicle cargo (carag) into cells, avoiding lysosomal degradation (Figures 20-23). ​​This strategy represents a crucial requirement for giant unilamellar vesicles to deliver therapeutic agents, such as pharmacological compounds that target cytoplasmic components. Specifically, we demonstrated that PEI-loaded giant unilamellar vesicles accumulate in cells and are protected from degradation, potentially because PEI can act in the giant unilamellar vesicle lumen as a potential pH buffer, preventing acidic degradation and mature lysosome formation (Figures 21 and 23). For approaches requiring stable integration of giant unilamellar vesicles and giant unilamellar vesicle cargo into cells, this may represent a promising implementation mechanism. Separately, giant unilamellar vesicles containing N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ) in the lipid bilayer were not only protected from lysosomal degradation but also successfully released the giant unilamellar vesicle cargo into the cytoplasm (Figure 22).

[0251] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the aqueous phase in step a') comprises at least one pH-sensitive lipid in a molar percentage comprised between 20% and 80%; or wherein the aqueous phase in step a') further comprises poly-ethylene-imine at a concentration comprised between 2 μg / ml and 100 μg / ml.

[0252] A preferred embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0253] [ka]

[0254] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the aqueous phase in step a') comprises at least one pH-sensitive lipid in a molar percentage comprised between 20% and 80%; or wherein the aqueous phase in step a') further comprises poly-ethylene-imine at a concentration comprised between 2 μg / ml and 100 μg / ml.

[0255] Targeted delivery of large, rugged cargo Furthermore, we have been able to encapsulate sophisticated drug delivery cargoes into giant unilamellar vesicles, opening the way to more efficient drug administration that was not possible with conventional delivery methods based on small unilamellar vesicles.

[0256] The present invention contemplates encapsulation of a cargo of interest in the giant unilamellar vesicles of the present invention to enable delivery of the giant unilamellar vesicles of the present invention to a region, tissue, or cell in vivo or in vitro.

[0257] For example, we were able to load purified baculovirus (BV), which is considered a promising future candidate for transduction and handling of large amounts of genetic material in genome engineering approaches. We successfully assembled and released BV-loaded giant unilamellar vesicles (Figure 25). After formation, the BV-loaded DOBAQ-containing giant unilamellar vesicles were incubated with rat embryonic fibroblast REF52 cells. We observed the cellular uptake of the giant unilamellar vesicles and the release of BV (stained with Hoechst 33342) into the cytoplasm. This was accompanied by the expression of the mitochondrial-targeted dsRed protein encoded by BV, indicating successful giant unilamellar vesicle-based transduction in mammalian cells using BV-carrier giant unilamellar vesicles. These results highlight the advantages of giant unilamellar vesicle-based drug delivery for more efficient drug administration of high-level cargo.

[0258] As used herein, cargo or freight may be a substance selected from the group comprising drug-releasing porous particles, molecular imaging agents, diagnostic agents, therapeutic agents, proteins or fragments thereof, polypeptides, peptides, enzymes or fragments thereof, nucleic acids, oligonucleotides, polynucleotides, potential DNA origami robots, small molecule drugs, virus particles, virus-like particles, microbial antigens, steroids, proteoglycans, lipids, (poly)carbohydrate monosaccharides, oligosaccharides, polysaccharides, magnetic particles, nanorods, carbon nanotubes, dentritosomes, polymersomes, metal nanoparticles, and combinations or complexes thereof.

[0259] The encapsulated proteins or fragments thereof can be single- or multi-chain proteins and peptides. Examples include antibodies, single-chain antibodies, antibody fragments, enzymes, cofactors, receptors, ligands, transcription factors and other regulatory factors, antigens, cytokines, chemokines, etc. These protein-based agents may be naturally occurring or non-naturally occurring, but can be synthesized within the subject, for example, by using genetically engineered cells.

[0260] "Diagnostic agent" refers to an agent capable of diagnosing a condition or disease. Diagnostic agents include, but are not limited to, molecular imaging probes such as chromophores, fluorophores, and radiolabels.

[0261] "Therapeutic agent" refers to an agent that can treat and / or ameliorate a condition or disease. Therapeutic agents include, but are not limited to, compounds, drugs, peptides, oligonucleotides, DNA, antibodies, etc.

[0262] Therapeutic or diagnostic agents can be encapsulated in the giant unilamellar vesicles of the present invention, including, but not limited to, imaging agents, immunomodulatory agents such as immunostimulatory agents and immunoinhibitors, antigens, adjuvants, cytokines, chemokines, anti-cancer agents, anti-infective agents, nucleic acids, antibodies or fragments thereof, fusion proteins such as cytokine-antibody fusion proteins, Fc-fusion proteins, etc.

[0263] As used herein, a molecular "imaging agent" is an agent that directly or indirectly emits a signal, thereby enabling its detection in vivo. Imaging agents include contrast agents and radioactive materials that can be detected using medical imaging techniques such as nuclear medicine scans and magnetic resonance imaging (MRI). Molecular contrast agents for magnetic resonance imaging (MRI) include Gd(DOTA), iron oxide, or gold nanoparticles; contrast agents for nuclear medicine include 201T1, the gamma radionuclide 99mTc; contrast agents for positron emission tomography (PET) include positron-emitting isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluorine, copper-64, gadoamide, and radioisotopes of Pb(II) such as 203Pb, and 11In, fluorescent dyes or dye-conjugated nanoparticles. Other contrast agents are fluorescent dyes and colorimetric dyes.

[0264] "Microbial antigens" are antigens derived from microbial species, such as, but not limited to, bacteria, viruses, fungi, parasites, and mycobacteria species. Thus, microbial antigens include bacterial antigens, viral antigens, fungal antigens, parasite antigens, and mycobacteria antigens. Examples of bacteria, viruses, fungi, parasites, and mycobacteria species are provided herein. Microbial antigens may be part of a microbial species or may be the whole microorganism.

[0265] "Nucleic acid agents" that can be delivered to a subject according to the present invention include naturally or non-naturally occurring DNA, left-handed DNA and RNA, peptide nucleic acids (PNAs), including cDNA, genomic DNA, nuclear DNA, mitochondrial DNA, RNA, mRNA, rRNA, tRNA, oligonucleotides, triple helix-forming molecules, immunostimulatory nucleic acids such as immunostimulatory CpG nucleic acids, small interfering RNA (siRNA) or microRNA (miRNA) used to regulate gene expression, antisense oligonucleotides used to regulate gene expression, aptamers, ribozymes, genes or gene fragments, regulatory sequences, potential DNA origami robots, including analogs, derivatives, and combinations thereof.

[0266] An aqueous solution containing a cargo agent is typically mixed with the aqueous phase in step a') to obtain giant unilamellar vesicles containing said cargo.

[0267] Accordingly, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the aqueous phase in step a') further comprises at least one substance selected from the group comprising drug-releasing porous particles, molecular contrast agents, diagnostic agents, therapeutic agents, proteins or fragments thereof, polypeptides, peptides, enzymes, nucleic acids, oligonucleotides, polynucleotides, potential DNA origami robots, small molecule drugs, virus particles, virus-like particles, microbial antigens, steroids, proteoglycans, lipids, monosaccharides, oligosaccharides, polysaccharides, magnetic particles, nanorods, carbon nanotubes, dentritosomes, polymersomes, metal nanoparticles, and combinations or complexes thereof.

[0268] Preferably, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0269] [ka]

[0270] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450, and wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100, wherein the aqueous phase in step a') further comprises at least one substance selected from the group comprising drug-releasing porous particles, molecular contrast agents, diagnostic agents, therapeutic agents, proteins or fragments thereof, polypeptides, peptides, enzymes, nucleic acids, oligonucleotides, polynucleotides, potential DNA origami robots, small molecule drugs, virus particles, virus-like particles, microbial antigens, steroids, proteoglycans, lipids, monosaccharides, oligosaccharides, polysaccharides, magnetic particles, nanorods, carbon nanotubes, dentritosomes, polymersomes, metal nanoparticles, and combinations or complexes thereof.

[0271] [Suitable copolymers for stabilizing giant unilamellar vesicles] To allow good dispersion of the polymer-shell-stabilized vesicles in the oil phase and to allow good dispersion of the lipid-containing aqueous phase within the polymer shell of the vesicle, the polymer shell is preferably made of a positively charged surfactant having a hydrophobic end located on the outside of the polymer shell and a hydrophilic end located on the inside of the polymer shell. Particularly preferred is a positively charged surfactant of formula (I):

[0272] [ka]

[0273] During the ceremony, m is an integer between 5 and 150, n is an integer between 5 and 450 inclusive.

[0274] In some embodiments, m in formula (I) is an integer comprised between 5 and 10, preferably between 5 and 20, more preferably between 5 and 30, more preferably between 5 and 40, more preferably between 5 and 50, more preferably between 5 and 60, more preferably between 5 and 70, more preferably between 5 and 80, more preferably between 5 and 90, more preferably between 5 and 100, more preferably between 5 and 110, more preferably between 5 and 120, more preferably between 5 and 130, and most preferably between 5 and 150.

[0275] In some other embodiments, n in formula (I) is an integer comprised between 5 and 10, preferably between 5 and 20, more preferably between 5 and 30, more preferably between 5 and 40, more preferably between 5 and 50, more preferably between 5 and 100, more preferably between 5 and 150, more preferably between 5 and 200, more preferably between 5 and 250, more preferably between 5 and 300, more preferably between 5 and 350, more preferably between 5 and 400, and most preferably between 5 and 450.

[0276] This positively charged surfactant in formula (I) has the distinct advantage of enabling the formation of positive charges at the periphery of the polymer shell, and thus polymer-shell-stabilized giant unilamellar vesicles can be generated without magnesium through direct lipid-surfactant interaction (Figures 28 and 29).

[0277] Accordingly, an embodiment of the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase comprising at least one lipid and an oil phase comprising a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles:

[0278] [ka]

[0279] wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent, a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0280] According to another embodiment of the present invention, the polymer shell is made of an amphiphilic copolymer having a hydrophobic end disposed on the exterior of the polymer shell and a hydrophilic end disposed on the interior of the polymer shell.

[0281] This may be achieved by forming the polymer shell of the droplets from a diblock or triblock copolymer. In this case, good results are obtained when the polymer shell of the droplet is made of a diblock copolymer containing a hydrophobic block disposed on the outside of the polymer shell and a hydrophilic block disposed on the inside of the polymer shell. The hydrophobic block may be, for example, but not limited to, a member selected from the group consisting of perfluorinated polymers such as perfluorinated polyethers, perfluorinated polystyrenes, or poly(olefin oxides) such as poly(propylene oxide), while the hydrophilic block may be selected from, for example, polyether glycols, polyether amines, polyacrylate acids, polymethyl acrylate acids, or poly[poly(ethylene glycol) methyl ether methacrylate]. Similarly, good results are obtained when the polymer shell of the droplet is made of a triblock copolymer containing two hydrophobic perfluorinated polymer end blocks and a hydrophilic polyether glycol block between them, where the triblock copolymer is folded, so that the hydrophobic perfluorinated polymer block is disposed on the outside of the polymer shell and the hydrophilic polyether glycol block is disposed on the inside of the polymer shell. Examples of the hydrophobic block and the hydrophilic block are the same as those described above.

[0282] Preferably, the perfluorinated polymer block is a perfluorinated polyether block (PFPE), more preferably a perfluorinated polyether block having a weight average molecular weight of 1,000 to 10,000 g / mol. Similarly, the polyether glycol (PEG) and polyetheramine (JEFFAMINE) blocks preferably have a weight average molecular weight of 100 to 50,000 g / mol. More specifically, a suitable example of each copolymer is PFPE-carboxylic acid (Krytox™, MW 2500 or 7000 g / mol), and a suitable example of each diblock copolymer is PFPE (7000 g / mol)-PEG (1400 g / mol), PFPE (7000 g / mol)-PEG (600 g / mol), PFPE (25 ... FPE (4000 g / mol)-PEG (600 g / mol), PFPE (4000 g / mol)-PEG (1400 g / mol), PFPE (2000 g / mol)-PEG (600 g / mol), PFPE (7000 g / mol)-JEFFAMINE (600 g / mol), PFPE (7000 g / mol)-JEFFAMINE (900 g / mol), PFPE ( 2500g / mol)-JEFFAMINE (600g / mol), PFPE (2500g / mol)-JEFFAMINE (900g / mol), PFPE (4000g / mol)-JEFFAMINE (900g / mol), PFPE (2500g / mol)-JEFFAMINE (600g / mol), PFPE (2000g / mol)-JEFFAMINE(600 g / mol), PFPE(2000 g / mol)-JEFFAMINE(900 g / mol), and suitable examples of each triblock copolymer are PFPE(7000 g / mol)-PEG(1400 g / mol)-PFPE(7000 g / mol), PFPE(7000 g / mol)-PEG(600 g / mol)-PFPE(7000 g / mol),PFPE(4000g / mol)-PEG(1400g / mol)-PFPE(4000g / mol)PFPE(2500g / mol)-PEG(600g / mol)-PFPE(2500g / mol), PFPE(2000g / mol)-PEG(600g / mol)-PFPE(2000g / mol), PFPE(7000g / mol)- JEFFAMINE(900g / mol)-PFPE(7000g / mol)PFPE(7000g / mol)-JEFFAMINE(600g / mol)-PFPE(7000g / mol), PFPE(4000g / mol)-JEFFAMINE(900g / mol)-PFPE(4000g / mol), PFPE(4000 g / mol)-JEFFAMINE(600 g / mol)-PFPE(4000 g / mol), PFPE(2500 g / mol)-JEFFAMINE(900 g / mol)-PFPE(2500 g / mol), PFPE(2500 g / mol)-JEFFAMINE(600 g / mol)-PFPE(2500 g / mol), PFPE(2000 g / mol)-JEFFAMINE(900 g / mol)-PFPE(2000 g / mol), and PFPE(2000 g / mol)-JEFFAMINE(600 g / mol)-PFPE(2000 g / mol). Molecular weights are determined by gel permeation chromatography using polystyrene standards.

[0283] The general formula for Krytox™ is:

[0284] [ka]

[0285] where n is an integer between 10 and 60 inclusive. In some embodiments, the amphiphilic copolymer has formula (VIII):

[0286] [ka] is a diblock copolymer of

[0287] During the ceremony, m is an integer between 5 and 150, inclusive; n is an integer between 5 and 450 inclusive.

[0288] In some embodiments, the amphiphilic copolymer has formula (IX):

[0289] [ka] is a triblock copolymer of

[0290] During the ceremony, m is an integer between 5 and 150, inclusive; n is an integer between 5 and 450 inclusive.

[0291] In some embodiments, the amphiphilic copolymer is a triblock copolymer of formula (IX). According to a further aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, wherein the amphiphilic copolymer in step a') consists of (i) a triblock copolymer comprising two perfluorinated polymer end blocks and one polyether glycol block, or (ii) a diblock copolymer comprising one perfluorinated polymer end block and one polyether glycol block, wherein the triblock copolymer or diblock copolymer is folded such that the perfluorinated polymer end blocks are disposed on the outside of the polymer shell and the polyether glycol block is disposed on the inside of the polymer shell; Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0292] According to a further aspect, the present invention is directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer-shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, wherein the amphiphilic copolymer in step a') consists of (i) a triblock copolymer comprising two hydrophobic, oleophobic perfluorinated polymer end blocks and one hydrophilic polyether glycol block, or (ii) a diblock copolymer comprising one hydrophobic and oleophobic perfluorinated polymer end block and one hydrophilic polyether glycol block, wherein the triblock copolymer or diblock copolymer is folded such that the perfluorinated polymer end blocks are disposed on the outside of the polymer shell and the polyether glycol block is disposed on the inside of the polymer shell; Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * It is calculated as 100.

[0293] [Synthesis of giant unilamellar vesicles stabilized by parent polymer shells] The parent polymer shell-stabilized giant unilamellar vesicles can be synthesized using methods known in the prior art (Gopfrich et al., 2018, "Mastering Complexity: Towards Bottom-up Construction of Multifunctional Eukaryotic Synthetic Cells"; Haller et al., 2018, "Charge-controlled microfluidic formation of lipid based single- and multicompartment systems"; Gopfrich et al., 2019, "One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells", Weiss et al. 2017 Nature Materials).

[0294] The at least one lipid contained in the aqueous phase in step a') can be provided as an aqueous dispersion of small, large, or giant unilamellar lipid vesicles, although it is preferred if the at least one lipid is provided as small or large well-dispersed unilamellar vesicles.

[0295] In this regard, good results are obtained when at least one lipid is contained in the aqueous phase in step a') in the form of small or large unilamellar lipid vesicles, which are formed, for example, by dissolving the lipid in a solvent such as chloroform, drying the resulting mixture under an inert gas atmosphere, resuspending the dried lipid in an aqueous buffer, vortexing the mixture, and homogenizing the vesicle size by extruding the resulting mixture through a filter. For example, the filter can be a polycarbonate filter with a pore size of 50 nm. Alternatively, in the case of proteoliposomes, large unilamellar lipid vesicles can be formed by removing the detergent.

[0296] Alternatively, at least one lipid is included in the aqueous phase in step a') in the form of small, large, or giant unilamellar lipid vesicles, which are formed by an electroforming process, preferably by a process comprising dissolving the lipid in a solvent such as chloroform, spreading the resulting mixture over two indium oxide coated glasses, evaporating the solvent, filling the space between the two glasses with water, and applying an alternating potential of 0.1 to 10 volts and 1 to 100 Hz for 0.1 to 10 hours.

[0297] The present invention is not limited by the technique for mixing an aqueous phase containing at least one lipid and an oil phase containing an amphiphilic copolymer to obtain parent polymer shell-stabilized giant unilamellar vesicles in step a). For example, any suitable microfluidics or other technique for water-in-oil emulsions may be used. However, according to one particularly preferred embodiment of the present invention, mixing an aqueous phase containing at least one lipid and an oil phase containing an amphiphilic copolymer during step a) is carried out using a flow-focusing microfluidics device, as described in WO 2018 / 228894 A1. In this technique, the two phases, i.e., the continuous phase and the dispersed phase, meet at a flow-focusing junction. The flow-focusing junction consists of three inlet channels that converge through narrow openings into the main or outlet channels, respectively. For example, two of the three inlet channels are vertically oriented, where both vertical inlet channels converge at a flow-focusing junction, one coming from above the flow-focusing junction and the other coming from below. Furthermore, a third inlet channel is horizontally oriented and intersects with the other two inlet channels at a flow-focusing junction coming from the left side. The main or outlet channels are also horizontally oriented, each starting from a narrow opening opposite the end of the horizontal inlet channel. During operation, a continuous oil phase containing an amphiphilic copolymer dispersed or dissolved in oil (which later forms a polymer shell) flows through the two vertical inlet channels, where both continuous oil phase substreams converge at a flow-focusing junction. A dispersed aqueous phase containing lipids flows through the horizontal inlet channel and is squeezed at the flow-focusing junction by the oil phase flowing through the two vertical inlet channels.Both phases pass through small openings located downstream of the three inlet channels, where the dispersed phase stream narrows and breaks into droplets of the lipid-containing aqueous phase. The droplets are then coated with lipophilic copolymers to form polymer shells with the lipophilic or hydrophobic end of the copolymer facing the outer shell toward the continuous oil phase and the hydrophilic end of the copolymer facing the inner shell toward the dispersed lipid-containing aqueous phase. The droplet size can be adjusted by the flow rates, flow ratios, and channel geometry of the two phases. Alternatively, the continuous oil phase containing the amphiphilic copolymer can be mixed with the dispersed lipid-containing aqueous phase to form a polymer shell dispersed or dissolved in oil using other flow microfluidics techniques, such as a T-flow-focusing junction.

[0298] [Microfluidic Device] Certain embodiments of the present invention are directed to a method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the following steps: a) providing polymer shell stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm: b) Mechanically symmetrically splitting a polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device containing multiple Y-shaped splitting zones (7); c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of between 1 μm and 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; where the diameter is measured by confocal microscopy, Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence * Calculated as 100, and wherein step b) comprises mechanically symmetrically dividing the polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles using a microfluidic device comprising a multi-Y-junction splitting zone (7) comprising at least one Y-junction, wherein said Y-junction consists of one inlet channel and two outlet channels; and wherein step c) comprises repeating step b) by using four or more successive generations of Y-junctions, wherein the inlet channel of each Y-junction consists of the outlet channel of the previous Y-junction.

[0299] Another embodiment of the present invention is a microfluidic device for preparing polymer shell-stabilized giant unilamellar vesicles having a diameter between 1 μm and 10 μm, wherein said diameter is measured by confocal microscopy and comprises: a multi-Y-shaped split zone (7) and a flow control system, wherein the multi-Y-shaped split zone (7) comprises a succession of one or more Y-shaped junctions, wherein each Y-shaped junction consists of one inlet channel and two outlet channels, wherein the inlet channel of each Y-shaped junction consists of the outlet channel of the previous junction; stabilizing planes to stabilize segmented polymer shell-stabilized giant unilamellar vesicles (8); one exit channel (9) that directs the divided polymer shell-stabilized giant unilamellar vesicles to the exit (10); and The divided polymer shell-stabilized giant unilamellar vesicles include one outlet (10) through which they exit the microfluidic device.

[0300] In a preferred embodiment of the present invention, the microfluidic device further comprises a production zone of parent polymer shell-stabilized giant unilamellar vesicles located upstream of the parting zone, the production zone comprising an oil phase inlet (1) for introducing an oil phase into the microfluidic device, and optionally an oil phase filter structure (2); one or more aqueous phase inlets (3) for introducing an aqueous phase into the microfluidic device, optionally one aqueous phase filter structure (4); When there are two or more aqueous phase inlets (3), a junction (5) of one of the aqueous phase inlets (3), a flow-focusing junction (6) consisting of a horizontal inlet channel and two vertical inlet channels, where the three inlet channels converge into an outlet channel through a narrow opening, and where the outlet channel is connected to a dividing zone; wherein the parent polymer shell-stabilized giant unilamellar vesicles have a diameter between 1 μm and 100 μm.

[0301] Accordingly, a preferred embodiment of the present invention is directed to a microfluidic device for preparing polymer shell-stabilized giant unilamellar vesicles having a diameter between 1 μm and 10 μm, wherein said diameter is measured by confocal microscopy, comprising: a multi-Y-shaped split zone (7) and a flow control system, wherein the multi-Y-shaped split zone (7) comprises a succession of one or more Y-shaped junctions, wherein each Y-shaped junction consists of one inlet channel and two outlet channels, wherein the inlet channel of each Y-shaped junction consists of the outlet channel of the previous junction; stabilizing planes to stabilize segmented polymer shell-stabilized giant unilamellar vesicles (8); One exit channel (9) directs the divided polymer-shell-stabilized giant unilamellar vesicles to the exit (10); One outlet (10) through which the segmented polymer shell-stabilized giant unilamellar vesicles exit the microfluidic device; and further comprising a parent polymer shell-stabilized giant unilamellar vesicle production zone disposed upstream of the partitioning zone, said production zone comprising: One oil phase inlet (1) for introducing an oil phase into the microfluidic device, and optionally one oil phase filter structure (2); one or more aqueous phase inlets (3) for introducing an aqueous phase into the microfluidic device, optionally one aqueous phase filter structure (4); If there are two or more aqueous phase inlets (3), a junction (5) of one of the aqueous phase inlets (3), and a flow-focusing junction (6) consisting of a horizontal inlet channel and two vertical inlet channels; wherein the three inlet channels converge into an outlet channel through a narrow opening, and wherein the outlet channel is connected to a dividing zone; wherein the parent polymer shell-stabilized giant unilamellar vesicles have a diameter between 1 μm and 100 μm.

[0302] The microfluidic device for preparing polymer shell-stabilized giant unilamellar vesicles described above and illustrated in Figure 26 is therefore very different from prior art microfluidic devices, and in particular from those described in patent application US2000264320A1.

[0303] First, the microfluidic device of US20070264320A1 includes a mixing region downstream of the droplet generation zone, which includes sawtooth-shaped channels configured to induce convection within the droplets. The mixing region functions to mix aqueous droplets containing cationic lipids and nucleic acids by passing them through the mixing region in a single direction. Thus, the mixing generates aqueous droplets containing monodisperse lipoplex assemblies, i.e., complexes formed between cationic lipids and nucleic acids after mixing and incubation.

[0304] Such a mixed region is not required for the present invention. Furthermore, the microfluidic device of US20070264320A1 requires the presence of multiple aqueous inlets to separately introduce DNA and liposomes. In contrast, the microfluidic device described herein can function with a single inlet.

[0305] Furthermore, the microfluidic devices described herein have an advantageous structure compared to that of US20070264320A1, since they can also include filtration structures (2) and (4) for filtering out larger contaminant particles from the oil phase or the aqueous phase, respectively. Importantly, the microfluidic devices described herein also include a planar stabilization region (8), where the split droplets can be stabilized in the planar stabilization region (8) by the addition of a surfactant, such as that included in the oil phase in step a'). [Drawing Description] [Brief explanation of the drawings]

[0306] [Figure 1] Figure 1 shows a phase-contrast microscope image demonstrating a microfluidic device for mechanically dividing a parent polymer-shell-stabilized giant unilamellar vesicle into five, allowing the formation of 32 daughter polymer-shell-stabilized giant unilamellar vesicles from a single parent vesicle with a diameter of 60 µm. The inset shows the time course of mechanical division of a droplet at a Y-junction from a high-magnification recording in bright field. The scale bar is 300 µm; the scale bar in the inset is 60 µm. [Figure 2] Figure 2 shows single-plane fluorescence confocal microscopy images of mechanically divided vesicles loaded with AlexaFluor 405 (top left), vesicles labeled with 1 mol% LissRhod PE (top right), green fluorescent protein (bottom left), and 1 μm fluorescent polystyrene beads (bottom right). The upper right corner shows the coefficient of variation (CV) of the mean fluorescence intensity of the vesicles (n = 665 single droplets). The scale bar is 40 μm. [Figure 3] Figure 3 shows single-plane fluorescence confocal microscopy images of giant unilamellar vesicles (20 mol% egg PG, 79 mol% egg PC, and 1 mol% LissRhod PE) obtained by mechanical fragmentation and released into PBS. The scale bar is 25 µm. [Figure 4]Optimization of the intraluminal lipid concentration in small unilamellar vesicles for the production of polymer-shell-stabilized giant unilamellar vesicles by mechanical fractionation. Polymer-shell-stabilized vesicles containing lipid concentrations of 12 mM, 6 mM, 3 mM, 1.5 mM, and 0.75 mM were mechanically fractionated, and the number of giant unilamellar vesicles before and after release of the polymer shell was counted to obtain the release efficiency. [Figure 5] Figure 1 shows an assessment of the mechanical stability of giant unilamellar vesicles kept at 4°C without mechanical agitation and giant unilamellar vesicles shaken at 800 rpm / 37°C. Results are shown as mean values ​​with SD from three technical replicates. [Figure 6] Figure 6 shows a representative phase-contrast image of mechanically disrupted giant unilamellar vesicles incubated with rat embryonic fibroblast REF52 cells. The white arrow indicates a single giant unilamellar vesicle. The scale bar is 10 μm. [Figure 7] Figure 7 shows the attraction quantification (zeta potential shown on the x-axis) of differently charged giant unilamellar vesicles using cell lines of endothelial (MDCK), epithelial (A431D and A431), and adrenal (PC12) origin after 24 h of incubation (results shown as mean values ​​normalized to a −31 mV mean and SD from three technical replicates). [Figure 8] Figure 8 shows representative single-plane fluorescence confocal microscopy images and a schematic diagram of charge-mediated interactions between giant unilamellar vesicles and A431D cells after 24 h of incubation and several washing steps. Nuclei (first column) were stained with Hoechst 33342, cell membranes (second column) were stained with WGA-AlexaFluor 488, and giant unilamellar vesicles (third column) were visualized by the fluorescent signal of LissRhod-PE lipids incorporated into the giant unilamellar vesicles. The charge of each giant unilamellar vesicle is indicated on the left side of the image. The scale bar is 20 μm. [Figure 9]Figure 9 shows transmission electron microscopy (TEM) of internalized giant unilamellar vesicles. (a) Horizontal TEM overview of a REF52 cell layer incubated with negatively charged giant unilamellar vesicles for 16 hours. The arrows point to giant unilamellar vesicles within the cytoplasm. The scale bar is 2.5 μm. (b) High-magnification horizontal TEM view of giant unilamellar vesicles internalized by the cells. The black arrow points to the giant unilamellar vesicle membrane, and the cyan arrow points to the endosomal membrane. The scale bar is 200 nm. [Figure 10] Figure 10 illustrates a strategy for functionalizing giant unilamellar vesicles. (a) Schematic and single-plane fluorescence confocal microscopy images of triply functionalized giant unilamellar vesicles generated by a segmented microfluidic device. Fluorescence corresponds to biotin-conjugated Atto425-streptavidin, NTA2+-conjugated His-tagged GFP, and ammine-conjugated NHS-Alexa647. (b and c) Schematic and microscopy images of consecutive giant unilamellar vesicles functionalized with 50 nm gold nanoparticles in (b) and with antibodies in (c). The right panels in (b) and (c) show phase-contrast images of giant unilamellar vesicles linked to gold nanoparticles (indicated by white arrows) and fluorescence confocal images of AlexaFluor488-linked anti-CD3 IgG immobilized on NTA2+-functionalized lipids via His-tagged protein G, respectively. Scale bars are 1 μm and 2 μm in (b) and (c), respectively. [Figure 11] Figure 11 shows the formation of interaction areas between anti-CD3-functionalized giant unilamellar vesicles and CD3+ Jurkat T cells after 24 hours of incubation. The cell membrane was stained with WGA-AlexaFluor 488 (top left image), giant unilamellar vesicles were visualized by incorporating LissRhod-PE fluorescent lipid (bottom left image), and the core of the complex was stained with Hoechst 33342 (right image). Arrows indicate the site of lipid clustering at the giant unilamellar vesicle-cell interface. The right image shows bright field. The scale bar is 6 μm. [Figure 12] Figure 12 shows the attraction analysis of giant unilamellar vesicles functionalized with increasing concentrations of RGD peptide (1-10%) and incubated with various cell lines. The results presented are normalized to the attraction of naive giant unilamellar vesicles without RGD and are shown as the mean ± SD from three technical replicates. The attraction of RGD-functionalized giant unilamellar vesicles to suspension Jurkat cells was assessed by flow cytometry. [Figure 13] Representative fluorescence confocal microscopy images of fluorescently labeled giant unilamellar vesicles (LissRhod-PE) decorated with 2 mol% RGD ligand and incubated with membrane stain (WGA-AlexaFluor 488) A431D cells are shown. The inset shows a magnified image with accumulation of giant unilamellar vesicles at the cell periphery and in the perinuclear region observed after 24 h of incubation. The scale bar is 60 μm. [Figure 14] Figure 14 shows a comparison of cell interactions with giant unilamellar vesicles with or without NrCAM on the cell surface. The attractive force values ​​for naive and recombinant NrCAM-functionalized uncharged giant unilamellar vesicles incubated with SH-SY5Y cells for 24 hours are shown. It should be noted that functionalization of nonspecific lipid-based interactions to be stronger than specific ligand-receptor-based attraction fails to achieve a significant increase in attractive force. [Figure 15] Figure 15 shows PEGylation of giant unilamellar vesicles for giant unilamellar vesicle-cell repulsion. (a) Grayscale representation of the zeta potential of small unilamellar vesicles and giant unilamellar vesicles decorated with various amounts of PEG containing different amounts of positively (DOTAP) or negatively (DOPG) charged lipids and increasing molecular weight. (b) Attraction analysis of the giant unilamellar vesicles from (a) using six different cell lines. [Figure 16]Figure 16 shows the modulation of attractive and repulsive giant unilamellar vesicle-cell interactions. Heat maps of the attraction analysis between A431D cells and PEG-functionalized giant unilamellar vesicles containing 15 mol% positively charged lipids are shown. [Figure 17] Figure 17 shows representative bright-field (row 1) and fluorescent (row 2) confocal microscopy images of A431 cells interacting with naive negatively charged giant unilamellar vesicles (top row) or 50 mol% PEG1000-functionalized negatively charged giant unilamellar vesicles (bottom row). Naive giant unilamellar vesicles directly contact the cells, whereas PEGylated giant unilamellar vesicles accumulate in the intercellular space (blended image, row 3) and form a contact inhibition zone with the cells (row 4, enlarged from the area shown in the blended image). The white dotted line indicates the perimeter of the cell cluster estimated from the bright-field images. The scale bar is 15 μm. [Figure 18] Figure 18 shows the attractive force of biofunctionalized giant unilamellar vesicles. a) The attractive force values ​​of giant unilamellar vesicles decorated with 15 different peptides and proteins (bound to the giant unilamellar vesicle surface via 1 mol% NHS lipid) for six cell lines are shown. All values ​​were normalized to the attractive force of BSA-bound giant unilamellar vesicles. b) The attractive force values ​​of nonspecific (i.e., BSA, poly-L-lysine (PLL), WGA, and tat-peptide) and specific (i.e., anti-cadherin, recombinant cadherin, and bradykinin) protein-biofunctionalized giant unilamellar vesicles for human fibroblast BJ cells, neuroblastoma SH-SY5Y cells, endothelial MDCK cells, epithelial A431 cells, dexamethasone-treated epithelial-transformed A431 cells, and HeLa cells are shown. All values ​​were normalized to the attractive force of BSA-bound giant unilamellar vesicles. [Figure 19]Figure 19 shows a schematic diagram (upper panel) of PEGylated giant unilamellar vesicles biofunctionalized with NrCAM in coculture with astrocytes and neurons differing in their expression of the axonin-1 receptor. The lower panel shows representative single-plane confocal microscopy images of astrocyte-neuron cocultures after 24 hours of incubation with NrCAM-PEGylated giant unilamellar vesicles. Hs683 astrocytes and SH-SY5Y neurons were stained with CellTracker green and blue, respectively. Giant unilamellar vesicles were visualized by incorporation of LissRhod-PE fluorescent lipid. The scale bar is 50 μm. [Figure 20] Figure 20 shows the evaluation of the mechanisms of vesicle lysosomal degradation and lysosomal escape to enable intracellular giant unilamellar vesicle cargo release. (a) Representative fluorescence confocal microscopy images of REF52 cells loaded with endosome-encapsulated negatively charged giant unilamellar vesicles are shown. Nuclei (top left image) were stained with Hoechst 33342, endosomes (top middle image) were labeled by staining with WGA-AlexaFluor 488 for 24 hours, giant unilamellar vesicles (bottom left image) were visualized by incorporating LissRhod-PE fluorescent lipid, and the cytoplasm (bottom middle image) was stained with CellTracker Blue. The combined image (right image) shows giant unilamellar vesicles present in the cytoplasm and encapsulated in endosomal vesicles. The scale bar is 10 μm. (b) Representative fluorescence microscopy images of REF52 cells showing colocalization of giant unilamellar vesicles with the lysosomal compartment after 24 h of incubation. Lysosomes (left panel) were stained with LysoTracker Green, and giant unilamellar vesicles (middle panel) were visualized by incorporation of LissRhod-PE fluorescent lipid. The right panel shows a bright-field (BF) image. The scale bar is 10 μm. [Figure 21]Figure 21 shows representative fluorescence confocal microscopy images of poly-ethyleneimine (PEI)-, GALA peptide-, and DOBAQ-mediated lysosomal escape for HPTS-loaded giant unilamellar vesicles incubated with REF52 cells for 24 hours. Scale bar: 50 μm. [Figure 22] Figure 22 shows dynamic light scattering measurements of the zeta potential of giant unilamellar vesicles containing 60 mol% of the pH-sensitive lipid DOBAQ or giant unilamellar vesicles with pH-sensitive lipids at different pHs. Note the transition from negative to positive zeta potential for DOBAQ-containing giant unilamellar vesicles as the pH decreases. Results are shown as the mean and SD values ​​from three technical replicates. [Figure 23] Figure 23 shows quantification of intracellular giant unilamellar vesicle degradation. A431D cells were incubated with giant unilamellar vesicles, and the giant unilamellar vesicles were quantified over time. While "empty" giant unilamellar vesicles show rapid degradation after uptake, PEI-loaded giant unilamellar vesicles show progressive intracellular accumulation, indicating successful escape from lysosomal degradation. [Figure 24] Figure 24 shows representative fluorescence confocal and bright-field images of primary mouse hippocampal neurons (right panel), primary mouse hippocampal neurons incubated with giant unilamellar vesicles (left panel, visualized by the incorporation of 1 mol% LissRhod PE lipid), and primary mouse hippocampal neurons incubated with giant unilamellar vesicles loaded with HPTS (middle panel). Scale bar is 30 μm. [Figure 25]Figure 25 demonstrates robust cargo delivery by giant unilamellar vesicles. (a) Representative bright-field (left panel) and corresponding confocal microscopy (right panel) images of baculovirus-loaded giant unilamellar vesicles are shown. The scale bar is 10 μm. (b) Maximum z-projection of fluorescence confocal microscopy images (top right image) of REF52 cells (nuclei stained with Hoechst 33342, top left image), REF52 cells incubated with baculovirus for 24 hours (bottom left image, due to oversaturation of the Hoechst 33342 channel), and REF52 cells incubated with baculovirus containing DOBAQ-carried giant unilamellar vesicles for 24 hours are shown. Note the expression of mitochondrial-targeted dsRed (bottom right image). The scale bar is 25 μm. [Figure 26]A microfluidic device is shown, including a production zone for parent polymer-shell-stabilized vesicles and a division unit for producing smaller giant unilamellar vesicles with diameters less than 10 μm. (1) The oil-phase inlet for introducing the oil phase into the device is shown. (2) An optional oil filter structure for retaining and filtering large contaminants such as dust. (3) The aqueous inlet for introducing the aqueous phase into the device is shown. Optionally, two or more single inlets can be used (here, two separate inlets). (4) An optional aqueous filter structure for retaining and filtering large contaminants such as dust. (5) The aqueous inlet junction is shown for multiple aqueous inlets. This structure is not required when separate aqueous inlets are mixed and only one aqueous inlet is used. (6) The flow-focusing junction (an example of a "production zone," the region where parent polymer-shell-stabilized giant unilamellar vesicles are formed) is shown. (7) The division structure is shown, demonstrating the continuous mechanical division of parent droplets (the "division unit"). (8): Depicts the stabilization plane, a large area where the split droplets can be stabilized and mixed, where the droplets are surrounded by excess oil, providing enough surfactant to stabilize the smaller droplets (splitting leads to an increased surface area and therefore requires more surfactant). (9): Depicts the exit channel, which allows the droplets to flow to the outlet. (10): Depicts the outlet, where the split, stabilized polymer shell-stabilized giant unilamellar vesicles exit the device. [Figure 27] Figure 1 shows an SUV dilution series for total lipid quantification of GUV solutions. An example curve obtained using SUVs containing 1 mol% LissRhod PE lipids at eight different concentrations is shown. The dotted line is an exponential fit to the equation shown on the graph. For example, GUVs generated using these SUVs had a fluorescence signal intensity of 5888, corresponding to a concentration of 72 µM. [Figure 28A] Figure 1 shows the formation of polymer shell-stabilized GUVs by using negatively charged surfactants (A) or positively charged surfactants (B). [Figure 28B] Figure 1 shows the formation of polymer shell-stabilized GUVs by using negatively charged surfactants (A) or positively charged surfactants (B). [Figure 29] Fluorescence micrographs of polymer-shell-stabilized GUVs (A) produced by using a positively charged surfactant of formula (I) and GUVs (B) produced by using the positively charged surfactant after release from the polymer shell. The scale bar in (A) is 100 μm, and the scale bar in B is 10 μm. DETAILED DESCRIPTION OF THE INVENTION

[0307] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes for said practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the scope of the invention.

[0308] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this specification. Accordingly, this specification is to be construed as illustrative only, and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as exemplary embodiments. Elements and materials may be substituted for those shown and described herein, parts and steps may be reversed, and certain features of the invention may be utilized independently, all as will become apparent to those skilled in the art after having the benefit of this specification of the invention. Changes may be made in the elements described herein without departing from the scope of the invention, as set forth in the following claims.

[0309] [Example] material Egg PG L-α-phosphatidylglycerol (egg, chicken), egg PC L-α-phosphatidylcholine (egg, chicken), 18:1 DOPG 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 18:1 DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholesterol, 18:1 DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, LissRhod PE 1,2-dioleoyl-sn-glycero-3-phospho Ethanolamine-N-(Lissamine rhodamine B sulfonyl), 18:1 DGS-NTA(Ni) 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid) succinyl] (nickel salt), 18:1 DOTAP 1,2-dioleoyl-3-trimethylammonium-propane, 18:1 to 12:0 Biotin PE 1-oleoyl-2-(12-biotinyl-(aminododecanoate)) Distearoyl))-sn-glycerol-3-phosphoethanolamine, DSPE-RGD1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-(cisarginylglycyl aspartate-maleimidomethyl)-cyclohexane-carboxamide], 18:1PEG350PE1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350], 18:1PEG750PE1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750], 18:1PEG1000PE1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], and extrusion sets with 50 nm pore size polycarbonate filter membranes were purchased from Avanti, USA. All lipids were purchased from Avanti Polar Lipids. All lipids were stored in chloroform at −20° C. and used without further purification.DyLight 405 NHS ester, AlexaFluor 647-NHS, anti-CD3 (16-0038-81), anti-CD3-Alexa488 (53-0037-42), hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), Hoechst 33342, CellTracker blue CMAC dye, CellTracker green CMFDA dye, LysoTracker green DND-26 dye, wheat germ agglutinin (WGA)-AlexaFluor conjugate, Dulbecco's modified ELISA DMEM medium (DMEM) high glucose, 1:1 DMEM:F12, RPIM-1640, FluoroBrite DMEM (high glucose), heat-inactivated fetal bovine serum, penicillin-streptomycin (10,000 U / mL), GlutaMax supplement, L-glutamine (200 mM), trypsin-EDTA (0.05%) with phenol red, phosphate-buffered saline, basic fibroblast growth factor (amino acids 10–155), epidermal growth factor, and AlexaFluor 405 dye were purchased from Thermo Fisher Scientific, Germany. NHS palmitic acid N-hydroxysuccinimide ester, 97% L-cysteine, 50 nm Au nanoparticles, heat-inactivated horse serum, wheat germ agglutinin (Wheat Germ Agglutinin), 1H,1H,2H,2H-perfluoro-1-octanol (PFO) demulsifier, bradykinin, polyethyleneimine (branched, Mw ~25,000), Atto 425-biotin, human interleukin-2, recombinant insulin, bovine plasma-derived fibronectin, poly-L-lysine, and DOBAQN-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium were purchased from Sigma-Aldrich, Germany. Polydimethylsiloxane (PDMS) Sylgard 184 was purchased from Dow Corning.Protein G His-tag was purchased from BioVision, USA. Bovine albumin fraction V (BSA) was purchased from Carl Roth, Germany. His-tagged NrCAM8425-NR-050 and human recombinant cadherin were purchased from R&D Systems, USA. Fluoresbrite YG particles 1.00 μm were purchased from Polysciences Europe, Germany. Perfluoropolyether-polyethylene glycol (PFPE-PEG) block-copolymer fluorosurfactant was purchased from Ran Biotechnologies, USA. Anti-VE-cadherin and anti-alpha4-integrin (CD49d) antibodies were purchased from Santa Cruz (Sc-28644) and Millipore (MAB1383). Recombinant human CD95L was purchased from BioLegend, USA. A431, Hela, Hs683, SH-SY5Y, and Jurkat cell lines were obtained from ATCC, USA. The REF52 cell line was a generous gift from Professor Benjamin Geiger (Weizmann Institute Rechovot). PC12 cells were a generous gift from Amin Rustom (Institute for Neurobiology, Heidelberg). Primary mouse hippocampal neurons were obtained from the Interdisciplinary Center for Neurosciences, Institute for Neurobiology, Heidelberg, Germany.Purified baculovirus was obtained from Martin Pelosse (Commissariat a l'energie atomique et aux energies alternatives, France) and produced as described in Mansouri et al., 2016, Highly efficient baculovirus-mediated multigene delivery in primary cells, Nature Communications. Tat-HIV-GFP peptide was received as a generous gift from Rudiger Arnold (Life Science Lab, German Cancer Research Center).

[0310] [Microfluidic-based production of giant unilamellar vesicles] To produce polymer-shell-stabilized giant unilamellar vesicles (also known as "water-in-oil droplets" or "droplets" only), a solution of small unilamellar vesicles was prepared with the lipid composition shown in Table 4. Briefly, lipids dissolved in chloroform were mixed at the appropriate ratio in a glass vial and dried under a gentle stream of nitrogen. The dried lipid film was rehydrated for 30 minutes in the production buffer shown in Table 4 to a final lipid concentration of 3 mM. The solution was then shaken at 600 rpm or higher for 5 minutes. The resulting liposome solution was extruded at least nine times through a 50 nm pore size polycarbonate filter. The small unilamellar vesicle solution was stored at 4°C for up to 3 days or used immediately to produce polymer-shell-stabilized giant unilamellar vesicles.

[0311] A droplet-based microfluidic mechanical partitioning device was fabricated using poly(dimethylsiloxane) (PDMS). The complete device was fabricated using photolithography and soft lithography methods as previously described (Soft Lithography, Xia Y and Whitesides, GM, 1998). Flow rate was controlled by an Elveflow OB1 MK3 microfluidic flow control system. Unless otherwise noted, small unilamellar vesicle solutions were diluted to a final lipid concentration of 1.5 mM to form giant unilamellar vesicles within microfluidic droplets. For droplet formation, the small unilamellar vesicle solution was introduced into the aqueous channels of the microfluidic device. Negatively charged giant unilamellar vesicles were formed using 1.25 mM PFPE (7000 g / mol)-PEG (1500 g / mol)-PFPE (7000 g / mol) triblock surfactant dissolved in FC-40. Positively charged giant unilamellar vesicles were formed using a 0.5% PEG-based fluorosurfactant (catalog number: 008-FluoroSurfactant-1G, RAN Biotechnologies) diluted in FC-40. For the formation of giant unilamellar vesicles containing DOBAQ lipids for lysosomal escape, 1.25 mM PFPE (2500 g / mol)-PEG (600 g / mol)-PFPE (2500 g / mol) triblock surfactant diluted in FC-40 was used. A ratio of approximately 1:4 water to oil phase was used. Droplets were formed at the flow-focusing junction of the splitter and collected in a microtube from the outlet of the microfluidic chip. After collection, the polymer-shell-stabilized giant unilamellar vesicles were equilibrated at 4°C for a minimum of 2 h before release.

[0312] [Release of giant unilamellar vesicles from polymer shells] For release of the polymer-shell-stabilized giant unilamellar vesicles into aqueous buffer, following the formation of the polymer-shell-stabilized giant unilamellar vesicles, the excess oil phase was removed from the microtube, and the polymer-shell-stabilized giant unilamellar vesicle layer was mixed with the destabilizing agent PFO and added to the aqueous "intraluminal" buffer (PBS, water, or DMEM) in a 1:1 ratio. The release buffer was then added to the collected polymer-shell-stabilized giant unilamellar vesicles as a single droplet or layer, in an additional 1:1 ratio relative to the aqueous "intraluminal" buffer. Each separated layer was mixed by gentle rotation in the collection tube.

[0313] After equilibration for 30 min, the aqueous phase containing the giant unilamellar vesicles was transferred to a 2 ml microtube.

[0314] Finally, release buffer was added in a 1:1 ratio with respect to the aqueous production buffer, and the released giant unilamellar vesicles were centrifuged at >10,000 g for 15 min. The supernatant was discarded, and the precipitate was suspended to the desired concentration.

[0315] The release buffer is preferably the same buffer used as the production buffer (also called the "intraluminal" buffer) that is encapsulated by the polymer shell-stabilized giant unilamellar vesicles.

[0316] Table 6 reports the lipid composition, functionalization and buffers used in the examples of the present invention.

[0317] [Dynamic Light Scattering] The zeta potentials of giant unilamellar vesicles and original small unilamellar vesicles were measured using a Malvern Zetasizer Nano ZS system at a total lipid concentration of 15 μM in PBS. The equilibration time was set to 600 s at 25 °C, followed by three replicate measurements for each sample at a scattering angle of 173° using in-build automatic run-number selection. The material refractive index was set to 1.4231, and the solvent properties were set to η = 0.8882, n = 1.33, and ε = 79.0. For the zeta potential measurement of giant unilamellar vesicles, the equilibration time and number of individual measurements were set to 120 s and two replicates, respectively. The zeta potential of giant unilamellar vesicles containing DOBAQ was evaluated by diluting the giant unilamellar vesicles to a final lipid concentration of 15 μM with PBS solution adjusted to the desired pH using 4 N NaOH or 10% HCl. All zeta potential measurements were performed at least twice.

[0318] Furthermore, the distribution size of the original lipid formulations, such as small unilamellar vesicles or liposomes, was measured by dynamic light scattering. Analysis of the hydrodynamic radius of small unilamellar vesicles was measured using a Malvern Zetasizer Nano ZS system. Samples were diluted to a final lipid concentration of 15 μM with PBS filtered through a 0.22 μm filter. The temperature equilibration time was set to 300 s at 25°C. Three individual measurements were performed for each sample at a scattering angle of 173° based on the built-in automatic run-number selection. The material refractive index was set to 1.4233, and the solvent properties were set to η = 0.8882, n = 1.33, and ε = 79.0.

[0319] [Evaluation of droplet uniformity] To assess the transmission heterogeneity of intraluminal droplet contents, water-in-oil droplets were generated at the flow-focusing junction of the droplet splitter. The droplets contained 10 mM MgCl, 1 mM AlexaFluor 405, 1 μM His-tagged GFP, 1.08 × 10 9 A PBS aqueous phase containing Fluoresbrite YG microspheres (diameter = 1.00 μm) was used. Droplets were collected and the mean droplet fluorescence intensity for all fluorophores was measured from single-plane fluorescence confocal images using global threshold segmentation and the particle analyzer tool in ImageJ software.

[0320] [Quantitative mass spectrometry (MS)] For quantitative mass analysis of the lipid content of intact small unilamellar vesicles and giant unilamellar vesicles, giant unilamellar vesicles were generated from small unilamellar vesicles composed of 33 mol% DOTAP, 33 mol% DOPE, 33 mol% DOPC, and 1 mol% LissRhod PE, along with 0.5 wt% RAN Biotechnologies PEG-based fluorosurfactant diluted in FC-40. Relative quantitative mass analysis was performed using a Sciex QTRAP 4500 mass spectrometer coupled to a Shimadzu Nexera HPLC system. The instrument was controlled via Sciex Analyst 1.7 software. Samples were diluted 1:1000 with MeOH, followed by fractionation using a Supelco Titan C18 column (0.21 x 10 cm, 1.9 macro) operated at 45 °C. The isocratic method featured a flow rate of 0.5 ml / min using a solution of 10 mM NH4Ac in 98% MeOH. MS experiments were performed in multiple reaction monitoring (MRM) mode using the following instrument settings: curtain gas 35 psi, ionization voltage 5500 V, nebulizer gas 30 psi, heater gas 60 psi, heater temperature 180 °C, and CAD gas set to 9. The following compound-specific parameters were used:

[0321] [Table 3]

[0322] Data analysis was performed using SciexAnalyst 1.7 and MultiQuant 3.0.2 software. Calculated concentrations were normalized using a small unilamellar vesicle sample with the following initial lipid ratio: DOPE33 / DOTAP33 / DOPC33 / Liss Rhod PE1.

[0323] Quantification of release efficiency and stability The release efficiency and mechanical stability of the giant unilamellar vesicles after agitation were assessed by manually counting the polymer-shell-stabilized giant unilamellar vesicles and the released giant unilamellar vesicles using a Neubauer chamber attached to a fluorescence microscope. The total lipid concentration in the released and purified giant unilamellar vesicles was quantified by measuring the giant unilamellar vesicle solution fluorescence (Figure 27). The respective fluorescence was normalized to a standard small unilamellar vesicle dilution curve (fitted to a one-phase exponential decay) with known concentrations and equal ratios of fluorescently labeled lipids. For incubation of the giant unilamellar vesicles with cell lines, total lipid concentrations ranging from 1.5 μM to 50 μM were used.

[0324] [Quantification of lysosomal degradation] To quantify lysosomal degradation of giant unilamellar vesicles, A431D monolayers were incubated with fluorescently labeled giant unilamellar vesicles and monitored by live-cell fluorescence time-lapse microscopy. The total number of giant unilamellar vesicles within the field of view was counted for each time frame using global threshold segmentation and the ImageJ build-in particle analyzer plug-in.

[0325] [Cell culture] Fibroblast REF52 cells, endothelial MDCK cells, epithelial A431 and A431D cells, cervical cancer HeLa cells, and astrocytic Hs683 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 g / L glucose, 1% L-glutamine, 1% penicillin / streptomycin, and 10% fetal bovine serum. Neuroblastoma SH-SY5Y cells were cultured in a 1:1 mixture of F12:DMEM supplemented with 1% L-glutamine, 1% penicillin / streptomycin, and 10% fetal bovine serum. Human T lymphocyte Jurkat cells were cultured in RPMI-1640 medium supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. Adrenal PC12 cells were cultured in RPMI-1640 medium supplemented with 1% L-glutamine, 1% penicillin / streptomycin, 5% fetal bovine serum, and 10% heat-inactivated horse serum. Cell cultures were routinely grown at 37°C and a 5% CO atmosphere and passaged at approximately 80% confluency using 0.05% trypsin / EDTA treatment. Jurkat cells were passaged by diluting 1 ml of Jurkat culture into 4 ml of fresh medium.

[0326] "Cell staining" Live cells were stained with Hoechst 33342 at a final concentration of 5 μg / ml to visualize cell nuclei. Cytoplasm was stained with CellTracker Blue CMAC dye and CellTracker Green CMFDA dye according to the manufacturer's instructions. Lysosomes were stained with LysoTracker Green DND-26 dye according to the manufacturer's instructions. Cell membranes were stained with wheat germ agglutinin (WGA)-AlexaFluor conjugate. Cells were grown in fully supplemented growth medium for 10 minutes at room temperature. WGA conjugate was added to a final concentration of 50 μg / ml to stain the membrane. To reduce endocytic dye uptake, stained cells were handled for imaging at room temperature. WGA is known to also stain endosome- and Golgi-associated vesicles. To stain for endosomal giant unilamellar vesicle uptake, cells incubated with giant unilamellar vesicles were incubated with a WGA-AlexaFluor conjugate at a final concentration of 5 μg / ml for 24 hours.

[0327] [Confocal microscopy and live-cell imaging] For fluorescence confocal microscopy, cell lines were cultured on 8-well NuncLabTeK glass-bottom culture slides filled with at least 400 μl of medium. Confocal microscopy was performed using a laser scanning microscope LSM800 (Carl Zeiss AG). Images were acquired using a 20× (Plan-Apochromat 20× / 0.8M27 objective, Carl Zeiss AG) and a 63× immersion oil objective (Plan-Apochromat 63× / 1.40 Oil DIC, Carl Zeiss AG). Images were analyzed using ImageJ (NIH), and vesicle diameter was also measured. Briefly, global intensity-based thresholding was performed on confocal microscopy images, followed by watershed separation of the top particles. Automated particle area measurements were performed on binary images using a built-in particle analyzer. Image brightness and contrast adjustments or background corrections were always performed on the entire image, with special care taken to avoid obscuring or removing any information from the original image. A 2-pixel median filter was applied to images with speckled noise signals. To fix cells before confocal microscopy analysis, cell cultures were washed twice with PBS and subsequently fixed with 2–4% PFA for at least 20 min. For time-lapse live-cell imaging, a Leica DMi8 inverted fluorescence microscope equipped with an sCMOS camera and a 10x HC PL Fluotar (NA 0.32, PH1) objective was used. Cells were cultured in 8-well NuncLabTeK glass-bottom culture slides in FluoroBrite DMEM (high glucose) medium supplemented with GlutaMAX, 10% FBS, and 1% Pen / Strep.

[0328] [Transmission electron microscopy] REF52 cells incubated with giant unilamellar vesicles for 16 hours were fixed with 2.5% glutaraldehyde in 0.1 M Na3PO4 for 30 minutes at room temperature. The cells were further fixed overnight with 0.4% uranyl acetate. Subsequently, the fixed cells were dehydrated using a series of 50%, 60%, 70%, 80%, 90%, and 100% ethanol and embedded in resin overnight at 60°C. Ultrathin sections at 85 nm were prepared and contrasted with lead acetate or osmium tetroxide. A Zeiss EM10CR transmission electron microscope was used for imaging. Image contrast, brightness, and clarity were adjusted as needed using built-in ImageJ plug-ins.

[0329] [Flow cytometry] For flow cytometry analysis of the attraction between RGD-functionalized giant unilamellar vesicles and Jurkat cells, rhodamine B-giant unilamellar vesicles with various RGD densities (0, 1, 2, and 10 mol%, see Table 6) were incubated with Jurkat cells for 24 hours. Subsequently, the cells were centrifuged at 250 g for 5 minutes, and the supernatant containing unbound or uninternalized giant unilamellar vesicles was discarded. The cells were resuspended in fresh medium, and for each condition, intracellular giant unilamellar vesicle fluorescence was quantified using a BD LSR Fortessa Cell Analyzer (BD Biosciences) using the blue laser line of the PE channel (λ<em>max = 575). From the scatter parameter acquisition, gates were set to distinguish Jurkat cells, debris, and possible aggregates. Subsequently, singlet cells were identified from the Jurkat population as described above. Finally, based on this gating strategy, the fluorescence intensity associated with the cell periphery was recorded and quantified.

[0330] [Gravity analysis] Cells were seeded in triplicate in 100 μl of the corresponding growth medium in 96-flat-bottom well plates and incubated for 24 hours to form confluent monolayers. Giant unilamellar vesicles (labeled with LissRhod PE lipids) were added to a final lipid concentration of 1.5 μM and incubated for 24 hours. Fluorescence in each well was measured using an Infinite M200 Tecan plate reader controlled by Tecan iControl software with in-built gain optimization and excitation / emission settings adjusted to 550 / 585 nm, respectively. Subsequently, wells were washed three times with 100 μl of PBS using a multichannel pipette, and residual fluorescence was measured again. Post-wash fluorescence intensity was normalized to the pre-wash intensity to account for any variation in sample preparation. All samples were measured in triplicate in four separate locations / wells to account for variations in cell monolayer density. For comparison of specific attractive forces in biofunctionalized giant unilamellar vesicles, all attractive force values ​​were normalized to the attractive forces of BSA-functionalized giant unilamellar vesicles and each cell line, referencing all attractive force values ​​for each cell type to a common moderately nonreactive protein.

[0331] Anti-CD3-functionalized giant unilamellar vesicles and Jurkat cells with a total lipid concentration of 3 μM were incubated for 24 h for fluorescence confocal microscopy of contact force formation. Prior to imaging, cells were stained with Hoechst 33342 and WGA-AlexaFluor 647 as described above.

[0332] Quantification of preferential giant unilamellar vesicle uptake in co-culture. To quantify preferential giant unilamellar vesicle uptake in SH-SY5Y / Hs683 coculture experiments, SH-SY5Y and Hs683 cells were separately stained with CellTracker Blue CMAC (and CellTracker Green CMFDA), respectively. Cells were co-seeded for 24 hours in a 10:1 SH-SY5Y:Hs683 ratio in a 1:1 mixture of F12:DMEM supplemented with 1% L-glutamine, 1% penicillin / streptomycin, and 10% fetal bovine serum (FBS) with giant unilamellar vesicles composed of 20 mol% PEG750PE, 20 mol% egg PC, 58 mol% egg PC, 1 mol% LissRhod PE, and 1 mol% palmitic acid NHS, conjugated with 1.5 μM His-tagged recombinant NrCAM. The cell cultures were then washed three times with PBS and fixed with 4% PFA for 20 minutes, followed by fluorescence confocal microscopy analysis using appropriate laser excitation. The total area of ​​SH-SY5Y and Hs683 cells within the field of view was calculated from single-plane confocal images. The total number of giant unilamellar vesicles in each region was determined by global threshold segmentation and then normalized to the total cell area. For example, the total area of ​​SH-SY5Y and Hs683 cells shown in Figure 19 was 20,083.37 μm, respectively. 2 and 40741.67 μm 2 They are 1491 (=0.0742) giant unilamellar vesicles / μm 2 ) and 578 (=0.0141 giant unilamellar vesicles / μm 2 ) giant unilamellar vesicles, which corresponds to a 523% increase in SH-SY5Y cells.

[0333] [Baculovirus encapsulation] For intracellular delivery of baculovirus (BV) encoding the mitochondrial-targeted dsRed (Discosoma Red) fluorescent protein, a solution containing BV was mixed with the small unilamellar vesicle solution at a ratio of 1:100. Importantly, because low manganese concentrations inhibited the formation and release rate of polymer-shell-stabilized giant unilamellar vesicles, PBS with 60 mM MgCl2 was used for polymer-shell-stabilized giant unilamellar vesicle production. The released and purified giant unilamellar vesicles containing baculovirus were then incubated with REF52 cells for 24 hours and subsequently stained with 8 μg / ml Hoechst 33342. Mitochondrial-targeted dsRed protein expression and baculovirus localization were assessed by fluorescence confocal microscopy. To visualize intracellular baculovirus, baculovirus-DNA staining was imaged by overexposing the Hoechst 33342 channel.

[0334] [Functionalization and PEGylation of Giant Unilamellar Vesicles] Functionalization of giant unilamellar vesicles was always performed on released giant unilamellar vesicles. Unless otherwise specified, functionalization of giant unilamellar vesicles was performed in PBS in the dark on a horizontal shaker at room temperature. After functionalization, the giant unilamellar vesicles were centrifuged at >10,000 g for at least 15 minutes and resuspended in PBS. For NHS-based coupling reactions, giant unilamellar vesicles were kept at 4°C as long as possible and released from the droplets within 1 hour of formation. The NHS coupling reaction was performed for at least 3 hours. For NHS- and NTA-based functionalization, the respective proteins and peptides were added in a 2- to 5-fold excess relative to the total functionalized lipid, as calculated from the total lipid concentration. For example, fluorescence quantification indicated a total lipid concentration of 150 μM (corresponding to approximately 10% release efficiency). Giant unilamellar vesicles were generated from small unilamellar vesicles with 1 mol% palmitic acid-NHS lipids and a minimum of 1.5 μM of the protein to be conjugated (approximately 50% of the NHS-conjugated lipids reside within the inner membrane leaflet and are not accessible for coupling). For WGA coupling, the lectin was conjugated to giant unilamellar vesicles at a molar concentration 10 times lower than that of the proposed 5 mol% NHS ligand. Functionalization of giant unilamellar vesicles with RGD peptides was performed by introducing the desired amount of DSPE-RGD into the lipid mixture for small unilamellar vesicle generation.

[0335] For sequential functionalization of giant unilamellar vesicles with gold nanoparticles (AuNPs), giant unilamellar vesicles containing 1 mol% NHS were incubated with 3 μM L-cysteine ​​for 6 hours. Subsequently, 50 nm Au nanoparticles were added to a final concentration of 10 μg / ml and shaken at 300 rpm overnight. Functionalization of giant unilamellar vesicles with IgG antibodies was performed by incubating 3 μM His-tagged protein G with giant unilamellar vesicles containing 1 mol% 18:1 DGS-NTA(Ni) lipids for 1 hour. Subsequently, the respective IgGs dissolved in 1% BSA to a final concentration of 3 μM were added to the mixture and incubated for 1 hour. To avoid cross-reactivity, whenever multiple functionalizations based on different coupling reactions were performed, as in the case of triple functionalization, the proteins to be conjugated via NHS were first incubated with giant unilamellar vesicles before other couplings, e.g., biotin-streptavidin coupling, were performed.

[0336] PEGylation of giant unilamellar vesicles with poly-ethylene-glycol polymers was carried out by introducing the desired amount of PEG350-PE, PEG750-PE, or PEG1000-PE into the lipid mixture for the generation of initial small unilamellar vesicles.

[0337] [Lysosomal escape mechanism] For all tested lysosomal escape approaches, polymer-shell-stabilized giant unilamellar vesicles were generated in PBS + 10 mM MgCl2 + 50 mM 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) using 1.25 mM triblock PFPE-PEG-PFPE2500-600-2500 surfactant dissolved in FC-40. Released giant unilamellar vesicles were purified by centrifugation and incubated with REF52 cells for 24 h. Subsequently, intracellular HPTS fluorescence distribution was assessed by fluorescence confocal microscopy (excitation 460 nm, emission 510 nm). When comparing between different approaches, imaging parameters were kept constant. For analysis of lysosomal escape via the poly-ethyleneimine (PEI) proton sponge mechanism, giant unilamellar vesicles composed of 20 mol% egg PG, 79 mol% egg PC, and 1 mol% LissRhod PE were loaded with 44 μg / ml polyethyleneimine during droplet generation. For analysis of lysosomal escape via DOBAQ-mediated intralysosomal fusion, giant unilamellar vesicles composed of 1 mol% LissRhod PE, 60 mol% DOBAQ, 20 mol% egg PG, and 19 mol% egg PC were generated.

[0338] [Synthesis of surfactant of formula (I)] The method described in this section refers to the synthesis of one surfactant using PEG600 and PFPE with a specific molecular weight and can be applied to the synthesis of surfactants with other molecular weights according to formula (I), where m is an integer comprised between 5 and 150, and n is an integer comprised between 5 and 450.

[0339] PFPE-PEG600-aminium derivatives are surfactants of formula (I) that consist of three moieties: (a) a PFPE block that is immiscible in water but miscible in fluorinated oils, (b) a PEG block that is immiscible in fluorinated oils but miscible in water, and (c) an aminium ion terminus that induces a positive charge at the water-oil interface.

[0340] These three moieties are linked using the click reaction first described by Sharpless KB et al., Angew. Chem. Int. Ed., 40:2004-2021. The mild conditions required for this reaction type allow for high yields and reduce side reactions. To do this, PFPE acid is converted to a propargyl derivative by activating the carboxylic acid followed by amide formation with propargylamine (Formula (II)). Both hydroxyl groups of PEG 600 must be converted to azide groups. This can be achieved by tosylation (Formula (III)) followed by displacement with sodium azide (Formula (IV)). For the aminium ion moiety, bromoacetyl bromide is used as the starting material and is first functionalized with an alkyne (Formula (V)), followed by the formation of the aminium ion in the second step (Formula (VI)). In the final two steps, all three moieties are clicked together (Formulas (VI) and (I)).

[0341] [Synthesis of propargyl PFPE amide (II)]

[0342] [ka]

[0343] The synthesis is based on the synthesis published by Scanga R. et al., RSC Adv. 2018, 8:12960-12974. PFPE acid (Krytox FSH, 10 mmol, 70 g) was placed in a flame-dried three-neck flask and dissolved in 125 mL HFE7100 under internal air. After the polymer was completely dissolved, oxalyl chloride (30 mmol, 2.6 mL) was added. The mixture was refluxed for 18 hours. With stirring and heat, the solvent was removed under vacuum. The crude PFPE acid chloride was redissolved in HFE7100 and filtered under inert conditions. The filtrate was added to a flame-dried three-neck flask equipped with a dropping funnel under an inert atmosphere. Tetrahydrofuran (30 mL), propargylamine (10.5 mmol, 666 μL), and triethylamine (15 mmol, 2.1 mL) were added to the dropping funnel. The mixture was added dropwise to the PFPE acid chloride solution and stirred for 18 hours. The crude product was then filtered through Celite, and the solvent was removed under reduced pressure. The product was obtained as a yellow oil (9.33 mmol, 65.7 g, 93.3%).

[0344] [Synthesis of PEG600 ditosylate (III)]

[0345] [ka]

[0346] The synthesis is based on the synthesis published by Scanga R. et al., RSC Adv. 2018, 8:12960-12974. Sodium hydroxide (0.4 mol, 16 g) was dissolved in water (125 mL) under ice cooling and then cooled to 0°C. PEG 600 (0.1 mol, 60 g) was dissolved in tetrahydrofuran (240 mL) and added dropwise to the sodium hydroxide solution through a dropping funnel, taking care not to allow the temperature to rise above 5°C. The reaction mixture was then allowed to reach room temperature and stirred for 1 hour. The mixture was again cooled to 0°C. p-Toluenesulfonyl chloride (0.23 mol, 43.8 g) was dissolved in tetrahydrofuran (185 mL) and added dropwise to the cooled reaction mixture through a dropping funnel. The temperature was not allowed to rise above 5°C during this process. The reaction mixture was stirred for 18 hours without further cooling. The resulting emulsion was separated, and the solvent was removed from the organic layer. The crude product was redissolved in 900 mL of ethyl acetate and washed twice with water and once with saturated sodium chloride solution. The purified solution was then stirred with magnesium sulfate for 1 hour, filtered, and the solvent removed under reduced pressure to give the product as a white amorphous material (78.4 mmol, 71.3 g, 78.4%).

[0347] [Synthesis of PEG600 diazide (IV)]

[0348] [ka]

[0349] The synthesis is based on the synthesis published by Scanga R. et al., RSC Adv. 2018, 8:12960-12974. PEG 600 ditosylate (III, 10 mmol, 9.1 g) and sodium azide (22 mmol, 1.43 g) were dissolved in dimethylformamide (25 mL) and stirred at room temperature for 90 minutes. The mixture was then stirred at 50° C. for 18 hours. The suspension was then filtered, and the solvent was removed from the filtrate under reduced pressure. The resulting oil was mixed with ethyl acetate (175 mL), sonicated for 20 minutes, and filtered again. The filtrate was washed twice with water and once with saturated sodium chloride solution. The purified solution was stirred with magnesium sulfate for 1 hour, filtered, and the solvent was removed under reduced pressure. The product was obtained as a white amorphous material (8.65 mmol, 5.62 g, 86.5%).

[0350] [Synthesis of 2-bromo-N-(prop-2-yn-1-yl)acetamide (V)]

[0351] [ka]

[0352] The synthesis is based on the synthesis patented by Aulakh, V.S. et al., US, Published Patent Application No. 20150266867, September 24, 2015.

[0353] Bromoacetyl bromide (10 mmol, 877 μL) and triethylamine (10 mmol, 1.45 mL) were dissolved in dichloromethane (18 mL) in a flame-dried flask and cooled to 0°C. A solution of propargylamine (10 mmol, 641 μL) in dichloromethane (9 mL) was added dropwise, and the mixture was stirred at 0°C for 2 h. The resulting suspension was filtered, and the solvent of the filtrate was removed under reduced pressure. The crude product was further purified by gradient flash column chromatography (100% hexane to 50% ethyl acetate in hexane). The product was obtained as a white powder (6.92 mmol, 1.22 g, 69.2%).

[0354] [Synthesis of N,N,N-trimethyl-2-oxo-2-(prop-2-yn-1-ylamino)ethan-1-aminium(VI)]

[0355] [ka]

[0356] The synthesis is based on the synthesis patented by Aulakh, V.S. et al., US, Published Patent Application No. 20150266867, September 24, 2015.

[0357] 2-Bromo-N-(prop-2-yn-1-yl)acetamide (5 mmol, 882 mg) was dissolved in acetonitrile (6 mL). Trimethylamine solution (4.2 M in ethanol, 25 mmol, 6 mL) was added, and the mixture was stirred for 20 hours. The solvent was then removed under reduced pressure, and the product was obtained as a white powder by precipitation with diethyl ether (4.78 mmol, 1.12 g, 95.6%). For molecular weight calculations, bromine ions were included as counterions.

[0358] [Synthesis of azido-PEG600-aminium derivative (VII)]

[0359] [ka]

[0360] PEG 600 diazide (IV, 5 mmol, 3.25 g), N,N,N-trimethyl-2-oxo-2-(prop-2-yn-1-ylamino)ethan-1-aminium (VI, 5 mmol, 1.17 g), copper(II) sulfate pentahydrate (0.5 mmol, 125 mg), (+)-sodium ascorbate (0.2 mmol, 200 mg), and neocuproine (0.16 mmol, 165 mg) were dissolved in water (30 mL). The solution was stirred first at room temperature for 1 hour and then at 50 °C for 48 hours. The solution was then lyophilized. The crude product was suspended in ethanol, filtered, and the solvent was removed under reduced pressure. The solid material was redissolved in water, and the remaining starting material was removed by extraction three times with dichloromethane. After lyophilization of the aqueous phase, the resulting product consisted of a monofunctionalized PEG600 derivative (azido-PEG600-aminium derivative) and a bifunctionalized PEG600 derivative (PEG600 diaminium derivative) in an estimated ratio of 2:1. The bifunctionalized PEG600 derivatives were unable to interact in subsequent reactions and could be easily separated later. Therefore, no further purification steps were required. The product mixture was obtained as a yellowish amorphous material (3.38 mmol, 2.93 g, 67.6%; calculated for the desired product was 2.25 mmol, 1.81 g, 45%). For molecular weight calculations, bromine ions were included as counterions.

[0361] [Synthesis of PFPE-PEG600-aminium derivative (I)]

[0362] [ka]

[0363] The synthesis is based on the synthesis published by Scanga R. et al., RSC Adv. 2018, 8:12960-12974. Propargyl PFPE amide (II, 1 mmol, 7.04 g) was dissolved in HFE 7100 (6 mL). Azido-PEG600-aminium derivative (VII, 1.5 mmol, 1.3 g), copper(II) sulfate pentahydrate (0.1 mmol, 25 mg), and neocuproine (0.16 mmol, 33 mg) were dissolved in methanol (3 mL). Sodium (+)-ascorbate (0.2 mmol, 40 mg) was dissolved in water (3 mL). All three solutions were combined and stirred at room temperature for 1 h, followed by stirring at 50 °C for 60 h. Methanol (12 mL) was then added while stirring. The emulsion was destabilized and slowly separated. If no separation could be observed, add more methanol in 1 mL steps until the phases began to separate. Both phases were separated. To the fluorinated oil phase, HFE 7100 (6 mL) was added, and the resulting separated aqueous phase was discarded. The solution was dried over magnesium sulfate for 1 hour, filtered, and the solvent was removed under reduced pressure. The product was obtained as a viscous yellowish oil (0.88 mmol, 6.93 g, 88.3%). For molecular weight calculations, bromine ions were included as counterions.

[0364] Example 1. Microfluidic mechanical partitioning in polymer-shell-stabilized giant unilamellar vesicles. Polymer-shell-stabilized giant unilamellar vesicles ("water-in-oil droplets") with sizes less than 5 μm were obtained using a self-developed droplet-based microfluidic device consisting of a flow-focusing junction for water-in-oil droplet generation and a multi-Y-shaped microfluidic droplet splitting unit (Figure 1). After generation of water-in-oil droplets with a diameter of 60 μm, the splitting unit design enabled high-throughput mechanical droplet splitting in up to five consecutive splitting steps, producing droplets with a final diameter of 2.90 μm ± 0.45 μm (n = 202), as measured by confocal microscopy (Figure 1).

[0365] To evaluate the heterogeneity of the transmission of intraluminal contents from mother droplets to daughter droplets, we used fluorescence confocal microscopy to analyze the fluorescent content encapsulated in the droplets and the respective signal intensity distributions between droplets before and after division (Figure 2). The results revealed only slight droplet-to-droplet variation in the signal intensity of green fluorescent protein (green fluorescent protein) (CV = 18.5% before division, n = 29, and CV = 14.8% after division, n = 665), and 100 nm fluorescently labeled small unilamellar vesicles composed of 20 mol% egg PG, 79 mol% egg PC, and 1 mol% LissRhod PE (CV = 68.0% before division, n = 29, and CV = 15.4% after division, n = 665). Based on this, it can be concluded that the luminal composition of the split droplets is similar to that of the mother droplets, which suggests that the mechanical splitting approach is feasible for high production rates (e.g., 2.5 × 10 at the focusing T-junction, respectively). 5 Droplets / min, 8x10 after 5 mechanical splits 6 We demonstrate that this method is suitable for the controlled production of small water-in-oil droplets with defined and tunable compositions (droplets / min). Importantly, a peripheral distribution of lipid fluorescence in the divided droplets is observed, suggesting successful mechanical division of polymer-shell-stabilized giant unilamellar vesicles.

[0366] [Release of giant unilamellar vesicles from polymer shells] After adding a destabilizing surfactant to the collected, mechanically divided, polymer-shelled giant unilamellar vesicles (see Methods), we were able to release large amounts of giant unilamellar vesicles (diameter = 1.400 μm ± 0.202 μm, n = 122) into the aqueous phase (Figure 3). A concentration of 1.5 mM was found to be the optimal required concentration for the initial solution of small unilamellar vesicles, achieving a release efficiency of over 50% of giant unilamellar vesicles, which corresponds to successful giant unilamellar vesicle release from the droplets approximately every 1 second (Figure 4). Thus, using the optimized conditions, this method can release approximately 4 x 10 6Allows for giant unilamellar vesicle generation rates of giant unilamellar vesicles / min.

[0367] [Symmetric droplet division] Full control over the physicochemical and biological properties of giant unilamellar vesicles is a crucial prerequisite for biomedical and synthetic biology applications. Therefore, mass spectrometry (see the Methods section) was used to quantitatively evaluate the lipid composition of the formed polymer-shell-stabilized giant unilamellar vesicles. The results revealed that the lipid ratio of the split giant unilamellar vesicles was similar to that of the initial small unilamellar vesicles used during the generation of the parent polymer-shell-stabilized giant unilamellar vesicles, and thus, no changes in lipid ratio occurred during microfluidic processing and mechanical droplet splitting (Table 3 below).

[0368] [Table 4]

[0369] [Mechanical stability]

[0370] Furthermore, a basic evaluation of the mechanical stability of the formed giant unilamellar vesicles revealed that approximately 90% of the giant unilamellar vesicles survived incubation at 37°C and mechanical agitation at 800 rpm on a horizontal shaker for 24 hours, suggesting that the formed giant unilamellar vesicles can withstand considerable mechanical stress and may therefore be robust enough for drug delivery applications (Figure 5).

[0371] [Release of giant unilamellar vesicles and their maintenance under physiological conditions] To successfully bind giant unilamellar vesicles to live cells, it is crucial that they can be generated and maintained under physiological buffer conditions. Therefore, the release efficiency of mechanically fragmented giant unilamellar vesicles in serum-supplemented medium was systematically evaluated using vesicles filled with serum-supplemented cell culture medium. Similar to PBS and water, a release efficiency of up to 45% was obtained in cell culture medium (Table 4 below). After release, the giant unilamellar vesicles were incubated with rat embryonic fibroblast (REF52) cells in cell culture. Importantly, time-lapse microscopy analysis showed that the giant unilamellar vesicles remained stable over a 20-hour incubation period (Figure 6).

[0372] [Table 5]

[0373] Example 2. Fine-tuning charge-mediated giant unilamellar vesicles - cell interactions

[0374] Charge-mediated interactions, when precisely controlled, can serve as a powerful tool for directing interactions between cells and giant unilamellar vesicles. Therefore, we systematically investigated the in vitro interaction spectra of differently charged giant unilamellar vesicles with various cell lines. To this end, we generated partitioned giant unilamellar vesicles using varying amounts of positively (DOTAP) and negatively (DOPG) charged lipids, and measured their respective zeta potentials by dynamic light scattering after release from the polymer shell (Table 5, below). The results showed that the charge of the giant unilamellar vesicles could be fine-tuned between highly positive and highly negative by adjusting the respective lipid formulations (Table 5, below).

[0375] [Table 6]

[0376] To quantify the interaction between cells and giant unilamellar vesicles, we performed a plate reader-based attraction assay and tested endothelial (MDCK), epithelial (A431D and A431), and adrenal (PC12) cell lines to determine their interaction with each giant unilamellar vesicle. These cell lines were selected to encompass a broad spectrum of possible target tissues with distinct surface expression patterns. For all tested cell lines, a strong correlation was revealed between the charge of the giant unilamellar vesicles and the strength of cell attraction, with more highly charged giant unilamellar vesicles exhibiting increased attraction compared to less charged and uncharged giant unilamellar vesicles (Figure 7). For example, in the case of A431D cells, a model cell line frequently used in cancer research, giant unilamellar vesicles with a zeta potential of -31 mV exhibited approximately 100-fold higher attraction when compared to giant unilamellar vesicles with a zeta potential of +2 mV. At the same time, giant unilamellar vesicles with a zeta potential of +28 mV exhibited approximately 50-fold increased attraction compared to giant unilamellar vesicles with a zeta potential of +2 mV. However, because this quantitative analysis cannot distinguish between various types of interactions (e.g., uptake, attachment, fusion, or phagocytosis), the qualitative characteristics of the interaction between giant unilamellar vesicles and cells were further investigated by fluorescence confocal microscopy. As shown in Figure 8, three different types of interactions could be induced between giant unilamellar vesicles and A431D cells. Endocytosis and attachment were observed primarily for negatively charged and neutral giant unilamellar vesicles, respectively. In the case of positively charged giant unilamellar vesicles, colocalization of lipid fluorescence with cell membrane staining (often accompanied by pathological cell morphology) was observed, indicating fusion between both membranes. To ensure that negatively charged giant unilamellar vesicles were actually taken up by cells, two additional analyses were performed. First, we stained the cytoplasm and performed z-resolved confocal fluorescence microscopy of the internalized fluorescently labeled giant unilamellar vesicles (data not shown). Second, cells incubated with each giant unilamellar vesicle were fixed and analyzed by transmission electron microscopy (Figure 9). Both evaluations demonstrated that the giant unilamellar vesicles were indeed taken up by the cells and present in their cytoplasm.Taken together, these results reveal a significant effect of giant unilamellar vesicle charge on the properties of giant unilamellar vesicle-cell interactions and highlight the power of the method developed for giant unilamellar vesicle charge control.

[0377] Example 3. Biofunctionalization of giant unilamellar vesicles formed by a segmented microfluidic device. Despite the fact that charge-mediated cellular uptake in giant unilamellar vesicles is an efficient process, giant unilamellar vesicles are unable to provide cell-type-specific delivery of therapeutic compounds. Therefore, we aimed to establish ligand-directed uptake of giant unilamellar vesicles by developing a toolbox of strategies for bio-orthogonal functionalization of the giant unilamellar vesicle surface with ligands that target specific moieties and cell types. To exemplarily demonstrate the versatility of the biofunctionalization possibilities of giant unilamellar vesicles, we prepared giant unilamellar vesicles with biotinylated lipids for binding streptavidin-tagged proteins, nitrilotriacetic acid (NTA-Ni) for binding histidine-tagged proteins, and nitrilotriacetic acid (NTA-Ni) for binding histidine-tagged proteins. 2+ The giant unilamellar vesicles were generated by microfluidic mechanical partitioning using a DOPE lipid containing a primary ammine for conjugation to N-hydroxysuccinimide (NHS)-functionalized molecules, and an N-hydroxysuccinimide (DOPE) lipid containing a primary ammine for conjugation to N-hydroxysuccinimide (NHS)-functionalized molecules. Triple orthogonal functionalization of the released giant unilamellar vesicles was achieved by adding streptavidin-functionalized Atto 425, histidine-tagged green fluorescent protein, and NHS-functionalized Alexa 647 (Figure 10a). Furthermore, a more complex sequential functionalization strategy was tested. To this end, cysteine-functionalized gold nanoparticles were immobilized on the giant unilamellar vesicle lipid via NHS chemistry (Figure 10b). Furthermore, a multistep approach was tested to conjugate immunoglobulins (e.g., anti-CD3) via NTA-immobilized histidine-tagged protein G (Figure 10c).

[0378] In particular, because antibodies offer excellent selectivity for specific cell surface antigens, antibody-based targeting has previously been shown to significantly enhance specific small unilamellar vesicle delivery to defined cell subsets. To evaluate the functionality of anti-CD3-coated giant unilamellar vesicles, we coated anti-CD3-coated giant unilamellar vesicles with CD3. + The giant unilamellar vesicles were incubated with Jurkat cells (see Methods section). When analyzed by confocal microscopy, the formation of attachment sites between the giant unilamellar vesicles and the cells was observed, reminiscent of a "minimal" immunological synapse (Figure 11), indicating successful giant unilamellar vesicle-cell binding. In contrast, giant unilamellar vesicles without anti-CD3 coating did not exhibit this complex interaction structure.

[0379] [RGD-mediated endocytosis] To systematically evaluate the feasibility of applying attractive receptor-specific giant unilamellar vesicle-cell interactions for targeted giant unilamellar vesicle delivery, negatively charged, RGD-biofunctionalized giant unilamellar vesicles were generated using various ligand densities. RGD was used for giant unilamellar vesicle biofunctionalization because integrin receptor-based endocytosis has previously been tested to enhance liposomal drug delivery through the ability of integrin proteins to function as natural intracellular signaling molecules for the initiation of endocytic events (see Methods). Therefore, RGD giant unilamellar vesicles were conjugated with an adherent cell line expressing RGD-binding integrin receptors, and their attractive force was measured. As a control, the same cells were incubated with naive, non-biofunctionalized giant unilamellar vesicles. For all tested cell lines (Figure 12), peripheral RGD ligand density correlated with GUV attraction, revealing that application of 10 mol% RGD ligand decoration could increase GUV-cell binding by approximately 10%. In the case of nonadherent Jurkat T cells, which express high levels of α4β1 integrin, 10 mol% RGD coating increased GUV-cell binding by as much as 15-fold (as measured by fluorescence flow cytometry). Further analysis of the interaction properties of fluorescently labeled RGD-functionalized GUVs by confocal microscopy revealed that, when interacting with A431D cells, GUVs accumulated primarily at the cell periphery, in areas of highest integrin density, and in the perinuclear region, suggesting RGD-integrin-mediated endocytic GUV uptake by the cells (Figure 13).

[0380] Example 4. PEG-based passivation strategies to modulate attractive and repulsive giant unilamellar vesicle-cell interactions to enhance targeted delivery. Although biofunctionalization of giant unilamellar vesicles with anti-CD3 and RGD ligands successfully increased giant unilamellar vesicle-cell interactions, charge-driven and other nonspecific attractive forces at the giant unilamellar vesicle-cell interface may be sufficiently high to prevent ligand-based cell-type specific engagement. For example, when uncharged giant unilamellar vesicles functionalized with NrCAM protein were incubated with SH-SY5Y neuroblastoma cells (NrCAM-positive), the measured attractive forces were comparable to those of unfunctionalized and uncharged giant unilamellar vesicles (Figure 14b). This indicates a significant level of nonspecific lipid-cell interactions. Giant unilamellar vesicles coated with polyethylene glycol (PEG) were synthesized to block these electrostatic and nonspecific interactions. First, the blocking ability of PEG at different concentrations and molecular weights was tested for giant unilamellar vesicles of different charges. To this end, giant unilamellar vesicles were generated using negatively and positively charged lipids at lipid ratios between 15 and 50 mol%. The zeta potential of each small unilamellar vesicle prior to the generation of droplet-stabilized giant unilamellar vesicles was examined by testing lipids conjugated to PEG350, PEG750, or PEG1000 at ratios of 5, 10, 20, and 50 mol%, and measuring the zeta potential of the released giant unilamellar vesicles (Figure 15a). The results revealed that the Z potential of both negatively and positively charged vesicles decreased with increasing PEG chain length and PEGylation rate. Furthermore, we tested whether PEGylation of giant unilamellar vesicles could be used to block the surface charge of the giant unilamellar vesicles and thus introduce repulsive behavior between the giant unilamellar vesicles and cells. To this end, PEGylated giant unilamellar vesicles were incubated with six different cell lines established from different tissues, and the attraction force of each was measured (Figure 15b). The results showed that for all tested cell lines, PEGylation of giant unilamellar vesicles essentially reduced the charge-mediated attraction between the giant unilamellar vesicles and cells, and this effect was more pronounced with higher PEGylation rates and longer PEG lengths.For example, when giant unilamellar vesicles interacted with A431D cancer cells, giant unilamellar vesicles with 5 mol% PEG350 exhibited nearly 50% more attraction than giant unilamellar vesicles with 50 mol% PEG350 (Figure 16). Consistently, confocal microscopy analysis showed that naive negatively charged giant unilamellar vesicles were typically localized within or on cells, with only small fragments observed in single cells or cell clusters (Figure 17, upper panel). In contrast, PEGylated giant unilamellar vesicles were observed to accumulate primarily in the intercellular space, forming contact inhibition zones between giant unilamellar vesicle accumulations and individual cells (Figure 17, lower panel). Presumably, this property may be due to repulsive giant unilamellar vesicle-cell interactions.

[0381] In conclusion, we were able to develop a complementary strategy for the modulation of specific attractive interactions between cells and giant unilamellar vesicles to promote ligand-based specific interactions by using a PEG-based passivation strategy to suppress charge-mediated interactions.

[0382] Example 5 Combination of PEGylation and Ligand-Directed Cellular Interactions After establishing approaches to control and fine-tune the attractive and repulsive interactions between cells and giant unilamellar vesicles, we combined both approaches and subsequently screened for cell-type selectivity for giant unilamellar vesicle targeting. To this end, we generated giant unilamellar vesicles containing 20 mol% negatively charged DOPG lipids for ligand immobilization, 59 mol% neutral DOPC lipids, 20 mol% PEG750-conjugated lipids, and 1 mol% NHS-conjugated lipids. With this lipid composition, the net strength of the ligand-receptor interaction between the giant unilamellar vesicles and target cells must be strong enough to overcome PEG-mediated repulsion, ultimately enabling cell-type-specific endocytosis induced by the negative charge of the giant unilamellar vesicles. To assess specificity, 15 different functionalized giant unilamellar vesicles were first screened. Each vesicle was expected to be either specific for a given cell type (e.g., anti-cadherin antibody or bradykinin) or nonspecific (e.g., poly-L-lysine) by measuring their respective attraction values ​​for six different carcinogenic cell lines. Each carcinogenic cell could resemble a potentially interesting target for giant unilamellar vesicle-based tumor therapy. To reference all attraction values ​​for each cell type to a common, moderately non-reactive protein, all values ​​were normalized to the attraction of BSA-conjugated giant unilamellar vesicles (Figure 18a). Figure 18b shows a summary of the attraction between the six different functionalized giant unilamellar vesicles and six cell lines of different origins. Giant unilamellar vesicles coated with peptides and proteins that do not bind to cells in a specific manner exhibit high attraction to essentially all cell types, since nonspecific attraction can overcome the repulsive PEG barrier in all cases. Examples include poly-L-lysine, which interacts primarily based on electrostatic interactions; wheat germ agglutinin (WGA), which binds to the cellular glycocalyx; or the HIV-derived tat-peptide, an arginine-rich peptide that penetrates cell membranes primarily based on hydrophobic interactions.However, when targeting more specific receptors, such as the bradykinin-specific G-protein-coupled receptor, which is abundantly expressed on endothelial cells, functionalization of giant unilamellar vesicles with the vasodilator bradykinin achieved specific attraction to endothelial cells. This attraction increased by up to 40% compared to other cell lines. When targeting cadherin proteins, similar results could be obtained by coating with recombinant cadherin or anti-cadherin antibodies. This comparison demonstrates that fine-tuning the attractive interaction, as well as the surface charge and PEG-based blocking of ligands, are well-suited strategies for controlling giant unilamellar vesicle-cell interactions.

[0383] Finally, to assess whether preferential attraction can induce cell-type-specific uptake under matched conditions in a more complex, multicellular environment, the giant unilamellar vesicle targeting approach was tested in coculture experiments. To this end, astrocyte (Hs683) and neuronal (SH-SY5Y) model cell lines were selected because these cell types grow and interact closely in the mammalian brain. Furthermore, achieving preferential giant unilamellar vesicle uptake by neurons is desirable when developing therapeutic strategies for neuroblastoma or neurodegenerative diseases. To test preferential attraction, giant unilamellar vesicles were prepared using 20 mol% PEG750, 20 mol% egg PG, 58 mol% egg PC, 1 mol% LissRhod PE, and 1 mol% 18:1 DGS-NTA(Ni) lipids and functionalized with His-tagged neural cell adhesion molecule NrCAM (extracellular domain aa 20-630), which binds to axonin-1 on neuronal membranes (Figure 19). After co-culture of giant unilamellar vesicles with the two cell types for 24 h, confocal microscopy was used to analyze and quantify the attraction of giant unilamellar vesicles to each cell type from separate images (see Methods). Results revealed that neurons contained up to 520% ​​more giant unilamellar vesicles compared to astrocytes, highlighting the importance of fine-tuning attractive and repulsive interactions for targeted delivery of giant unilamellar vesicles in complex environments.

[0384] Example 6. Lysosomal escape of giant unilamellar vesicles for efficient cytoplasmic cargo delivery. To investigate the intracellular uptake mechanism of GUVs, negatively charged GUVs were incubated with REF52 cells stained for endosomal vesicles and cytoplasm. Confocal microscopy of each culture revealed that the internalized GUVs were surrounded by endosomal membranes (Figure 20a). This observation confirmed that GUVs entered cells via endocytic pathways, such as micropinocytosis or phagocytosis, excluding other uptake mechanisms, such as direct penetration of GUVs or phagocytosis alone. However, after uptake, a progressive loss of GUV fluorescence was observed over time, and after 24 hours of incubation, GUVs colocalized with lysosomes (identified by staining with LysoTracker Green DND-26) (Figure 20b). This observation may be due to lysosomal degradation of the vesicles, a process frequently observed in delivery methods based on small unilamellar vesicles. Furthermore, because many pharmacological compounds target cytoplasmic components, an efficient lysosomal escape mechanism that allows the release of giant unilamellar vesicle cargo into cells and prevents lysosomal degradation is a crucial requirement for such applications.

[0385] To avoid this degradation, we evaluated two independent lysosomal escape mechanisms (see Methods), both of which are based on suppressing the rapid pH drop that occurs during endosome-lysosome fusion: 1) lysosomal escape via the proton sponge mechanism by incorporating high-molecular-weight polyethyleneimine (PEI) into giant unilamellar vesicles; and 2) lysosomal escape via intralysosomal fusion by incorporating the pH-sensitive lipid DOBAQ into the giant unilamellar vesicle membrane. The retention, degradation, and release of giant unilamellar vesicle cargo were illustratively assessed by loading giant unilamellar vesicles with the membrane-impermeable dye HPTS and observing its intracellular fluorescence distribution after 24 hours of incubation with A431D cells (Figure 21). HPTS is a highly water-soluble pH indicator with a pKa of approximately 7.3 in aqueous buffer. In the case of PEI-loaded giant unilamellar vesicles, HPTS fluorescence was detected exclusively in a punctuated form, colocalizing with the giant unilamellar vesicle fluorescence within the cells. This indicates HPTS retention within the giant unilamellar vesicles, and therefore endosomal or lysosomal entrapment, suggesting unsuccessful cytoplasmic cargo release. However, in the case of giant unilamellar vesicles containing 60 mol% DOBAQ, HPTS fluorescence was found to be distributed throughout the cell body, demonstrating successful HPTS release from the giant unilamellar vesicles into the cytoplasm. Indeed, DOBAQ-containing giant unilamellar vesicles exhibited a polarity switch in the vesicle Z potential at low pH, as assessed by dynamic light scattering (Figure 22). Notably, by tracking the total number of intracellular giant unilamellar vesicles over time, we found that PEI-loaded giant unilamellar vesicles accumulated intracellularly and did not undergo degradation (Figure 23). This suggests that even in the absence of cargo release, PEI-loaded giant unilamellar vesicles are protected from degradation because PEI potentially acts as a strong pH buffer in the lumen of the giant unilamellar vesicles, preventing acidic degradation and the formation of mature lysosomes.For approaches requiring stable cellular uptake of giant unilamellar vesicles and giant unilamellar vesicle cargo, this may represent a promising mechanism of implementation.

[0386] The dynamics of endocytic and lysosomal activity can differ significantly between transformed and non-transformed cells. Therefore, we aimed to test our approach in primary cells. To this end, we used primary hippocampal neurons cultured in vitro, which represent a medically relevant cell type. Indeed, these cells represent an important target in many therapeutic procedures for neurodegeneration or neuronal tumors. When HPTS was loaded and combined with negatively charged giant unilamellar vesicles containing DOBAQ, widespread uptake of the giant unilamellar vesicles into neurons was observed after 24 hours of incubation (Figure 24). To enhance the attraction of the giant unilamellar vesicles to the sialic acid-containing glycocalyx, the giant unilamellar vesicles were functionalized with wheat germ agglutinin (WGA). Their accumulation in the perinuclear region suggests uptake into the cell's intracellular transport machinery. Importantly, we observed widespread distribution of HPTS fluorescence within the neuronal soma and dendrites. This observation demonstrates that DOBAQ-based lysosomal escape of giant unilamellar vesicle cargo is both functional and compatible with primary cells.

[0387] Example 7. Targeted delivery of large, robust cargo To finally test the cargo capacity of microfluidically formed giant unilamellar ve...

Claims

1. 1. A method for preparing monodisperse cell-targeting giant unilamellar vesicles, comprising the steps of: a) providing polymer shell-stabilized giant unilamellar vesicles with a diameter comprised between 1 μm and 100 μm; b) mechanically symmetrically dividing the polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles without harming the giant unilamellar vesicle by using a microfluidic device; c) repeating step b) and mechanically symmetrically dividing the smaller polymer-shell-stabilized giant unilamellar vesicles provided in step b) until the polymer-shell-stabilized giant unilamellar vesicles reach a desired diameter of 1 to 10 μm; and d) optionally removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c); Including, wherein the microfluidic device is A multi-Y-split zone (7) and a flow control system, wherein said multi-Y-split zone (7) comprises a succession of one or more Y-junctions, wherein each Y-junction consists of one inlet channel and two outlet channels, wherein said inlet channel of each Y-junction consists of said outlet channel of the previous junction; stabilizing planes for stabilizing segmented polymer shell-stabilized giant unilamellar vesicles (8); one outlet channel (9) that directs the divided polymer shell-stabilized giant unilamellar vesicles to an outlet (10); and One outlet (10) through which the divided polymer shell-stabilized giant unilamellar vesicles exit the microfluidic device. Including, Here, monodisperse means that the vesicles are of uniform size and exhibit a size variation coefficient of less than 16%; wherein the diameter is measured by confocal microscopy; Here, dividing symmetrically means that the mole percentage of smaller vesicles changes by less than 5% and the luminal content of the vesicles changes by less than 20%, where the change in luminal content is calculated as: standard deviation / mean fluorescence ＊ Calculated as 100, method.

2. The polymer shell-stabilized unilamellar vesicles provided in step a) can be prepared by the following steps: a') mixing an aqueous phase containing at least one lipid and an oil phase containing a surfactant of formula (I) to form polymer shell-stabilized giant unilamellar vesicles: 【Chemistry 1】 wherein m is comprised between 5 and 150, and wherein n is comprised between 5 and 450; wherein the oil phase consists of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); The method of claim 1, obtained by

3. The polymer shell-stabilized unilamellar vesicles provided in step a) can be prepared by the following steps: a') mixing an aqueous phase containing at least one lipid and a cation, and an oil phase containing an amphiphilic copolymer to form a polymer shell-stabilized giant unilamellar vesicle, wherein the oil phase is comprised of a solution of a perfluorinated water-immiscible solvent; and a'') optionally incorporating one or more proteins or fragments thereof into the polymer shell-stabilized giant unilamellar vesicles provided in step a'); The method of claim 1, obtained by

4. 4. The method according to claim 1, wherein step d) comprises removing the polymer shell from the polymer shell-stabilized giant unilamellar vesicles obtained in step c) by adding a destabilizing agent, wherein the destabilizing agent is a demulsifier surfactant capable of destabilizing the structure of the polymer shell.

5. Step d) is followed by the following step: e) purifying the giant unilamellar vesicles by centrifugation; The method of any one of claims 1 to 4, further comprising:

6. The aqueous phase in step a') comprises: neutral lipids selected from the group comprising ceramides, sphingomyelins, cephalins, cholesterol, cerebrosides, diacylglycerols, phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, lysophosphatidylethanolamines, lysoethanolamines, reverse head group lipids, sphingosines, sterol-modified phospholipids, ether ester lipids, diether lipids, vinyl ethers (plasmalogens); an anionic lipid selected from the group comprising phosphatidic acid, lysophosphatidic acid derivatives, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine, lysophosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, cardiolipin, and bis(monoacylglycero)phosphate derivatives; a cationic lipid selected from the group comprising dioleyl-N,N-dimethylammonium chloride, N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, N,N-distearyl-N,N-dimethylammonium bromide, N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride, 3β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, dioctadecylamidoglycylcarboxyspermine, N-(2,3-dioleyloxy)propyl)-N,N-dimethylammonium chloride and 1,2-dioleoyl-3-dimethylammonium-propane; a pH-sensitive lipid selected from the group comprising the lipids N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 1,2-distearoyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-sn-glycero-3-succinate, 1,2-dioleoyl-sn-glycero-3-succinate, N-palmitoylhomocysteine, photoswitchable lipids, Acylglycine derivatives, prenol derivatives, prostaglandin derivatives, glycosylated diacylglycerols, eicosanoid derivatives, (palmitoyloxy)octadecanoic acid derivatives, diacetylene derivatives, diphytanoyl derivatives, fluorinated lipids, brominated lipids, lipopolysaccharides, one of the foregoing lipids conjugated to a functionalized ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, folate, squara, galloyl, glycan, thiol, arginylglycylaspartic acid, fluorophore, magnetic resonance imaging reagent, chelator, and one of the aforementioned lipids bound to polyethylene glycol having a molecular weight comprised between 350 and 50,000 g / mol; 4. The method of claim 2 or claim 3, further comprising at least one lipid selected from the group comprising:

7. 4. The method of claim 2 or 3, wherein the aqueous phase in step a') comprises at least one anionic lipid, at least one neutral lipid, and optionally one neutral lipid functionalized with a fluorescent dye molecule.

8. 4. The method of claim 2 or 3, wherein the aqueous phase in step a') comprises at least one cationic lipid, at least one neutral lipid, and optionally one neutral lipid functionalized with a fluorescent dye molecule.

9. the aqueous phase in step a') comprises at least one lipid functionalized with a functionalizing ligand selected from biotin, N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, nitrilotriacetic acid (NTA)-nickel, amine, carboxylic acid, maleimide, dithiopyridinyl, pyridyl disulfide, pyridyl dithiopropionate, N-benzylguanine, carboxyacyl, cyanuric, squaric, galloyl, thiol, wherein the method optionally comprises, after step d), the following step: d') binding said giant unilamellar vesicles with at least one polymer comprising at least one moiety that reacts with one of said functionalized ligands, said polymer being selected from the group comprising carbohydrates, nucleic acids, proteins or fragments thereof, polypeptides, cell receptors, imaging probes, nanoparticles; The method of claim 2 or claim 3, comprising:

10. 4. The method according to claim 2 or claim 3, wherein the aqueous phase in step a') comprises at least one lipid conjugated to polyethylene glycol having a molecular weight comprised between 350 g / mol and 50,000 g / mol.

11. said aqueous phase in step a') comprises at least one pH-sensitive lipid in a molar percentage comprised between 20% and 80%, or The method according to claim 2 or claim 3, wherein the aqueous phase in step a') further comprises poly-ethylene-imine at a concentration comprised between 2 μg / ml and 100 μg / ml.

12. 4. The method of claim 2 or claim 3, wherein the aqueous phase in step a') further comprises at least one agent selected from the group comprising drug-releasing porous particles, molecular imaging agents, diagnostic agents, therapeutic agents, proteins or protein fragments, polypeptides, peptides, enzymes or enzyme fragments, nucleic acids, oligonucleotides, polynucleotides, potential DNA origami robots, small molecule drugs, virus particles, virus-like particles, microbial antigens, steroids, proteoglycans, lipids, monosaccharides, oligosaccharides, polysaccharides, magnetic particles, nanorods, carbon nanotubes, dentritosomes, polymersomes, metal nanoparticles, and combinations or conjugates thereof.

13. 4. The method of claim 3, wherein the amphiphilic copolymer in step a') consists of (i) a triblock copolymer comprising two perfluorinated polymer end blocks and one polyether glycol block, or (ii) a diblock copolymer comprising one perfluorinated polymer end block and a polyether glycol block, wherein the triblock copolymer or the diblock copolymer is folded such that the perfluorinated polymer end blocks are disposed on the outside of the polymer shell and the polyether glycol block is disposed on the inside of the polymer shell.

14. 14. The method of claim 1, wherein step b) comprises mechanically splitting the polymer-shell-stabilized giant unilamellar vesicle into two smaller polymer-shell-stabilized giant unilamellar vesicles using a microfluidic device comprising a multi-Y-junction splitting zone (7) comprising at least one Y-junction, wherein the Y-junction consists of one inlet channel and two outlet channels, and wherein step c) comprises repeating step b) by using four or more successive generations of Y-junctions, wherein the inlet channel of each Y-junction consists of the outlet channel of the previous Y-junction.

15. The microfluidic device further comprises a parent polymer shell-stabilized giant unilamellar vesicle production zone disposed upstream of the parting zone, the production zone comprising: one oil phase inlet (1) for introducing an oil phase into the microfluidic device; Optionally, one oil phase filter structure (2), one or more aqueous phase inlets (3) for introducing an aqueous phase into said microfluidic device; Optionally, one aqueous phase filter structure (4), If there are two or more aqueous phase inlets (3), a junction (5) of one of the aqueous phase inlets (3); and a flow-focusing junction (6) consisting of a horizontal inlet channel and two vertical inlet channels, wherein the three inlet channels converge through narrow openings into an outlet channel, wherein the outlet channel is connected to the dividing zone; wherein the parent polymer shell-stabilized giant unilamellar vesicle has a diameter between 1 μm and 100 μm. The method according to any one of claims 1 to 14.

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