Polymersomes functionalized with multiple ligands

Abstract

The present invention relates to nanoparticles or microparticles for binding to cell surfaces, said nanoparticles or microparticles comprising (i) a plurality of different ligand species on their outer surface capable of binding to different receptor species on the cell surface, and (ii) polymer brushes on their outer surface. The present invention further relates to pharmaceutical compositions comprising a plurality of nanoparticles or microparticles of the invention, medical uses of such nanoparticles or microparticles, and vaccines comprising such nanoparticles or microparticles.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to nanoparticles or microparticles for binding to cell surfaces, said nanoparticles or microparticles comprising (i) a plurality of different ligand species on their outer surface capable of binding to different receptor species on the cell surface, and (ii) polymer brushes on their outer surface. The present invention further relates to pharmaceutical compositions comprising a plurality of nanoparticles or microparticles of the invention, medical uses of such nanoparticles or microparticles, and vaccines comprising such nanoparticles or microparticles. [Background technology]

[0002] Background of the Invention A key feature of effective small molecule therapeutics is the ability of a drug to interact as selectively as possible with its biological target. Indeed, most drug discovery tools have been refined to identify molecules that bind with the highest affinity. Current drug discovery is highly sophisticated, and the emergence of "-omic" technologies (e.g., genomics, transcriptomics, proteomics, and metabolomics) has led to the possibility of personalized therapy (Aronson and Rehm, Nature, 2015, 526 (7573), 336-342). The ability to effectively deliver drugs to their site of action in vivo has also improved significantly in recent years. Therefore, the high selectivity of active molecules can now be combined with molecularly engineered carriers, such as nanoparticles and microparticles, that possess the necessary properties to navigate biological environments (Cheng et al., Science, 2012, 338 (6109), 903-910). A key element of such nanomedicine efforts is the introduction of ligands that enable targeting and selectivity to guide the carrier across biological barriers. This technology has the potential to deliver drugs to target biomacromolecules that are inaccessible by simple passive diffusion, such as intracellularly (Akinc and Battaglia, Cold Harb Spring Perspect Biol, 2013, 5(11), a016980) or to the central nervous system (Fullstone et al., Int Rev Neurobiol, 2016, 130, 41-72).

[0003] Today, our ability to create ligands, whether for drug delivery or simple targeting, has advanced significantly and can be extended to almost any biological entity. However, in most diseases (most notably cancer), dysfunction is associated with endogenous receptors that are expressed by both healthy and diseased cells. Such promiscuity is the primary reason why many drugs are associated with unwanted side effects and fail to progress through the clinical development pipeline. This is also why drug carriers often fail to deliver drugs to the desired site of action, instead being adsorbed by the immune system. In such situations, there is a need to improve our ability to target drugs to their intended target sites. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, it would be desirable to provide pharmaceuticals that deliver therapeutic agents to a target locus more effectively, allowing for high selectivity for one specific target site, especially in highly congested environments where nonspecific interactions are difficult to overcome. This would allow for the same or better therapeutic results to be achieved using lower dosages of the drug and / or reduced unwanted side effects. The present invention addresses these challenges and provides improved pharmaceuticals for therapeutic delivery of active agents to their intended site of action. [Means for solving the problem]

[0005] Summary of the Invention The present invention addresses these problems by providing multiple nanoparticles or microparticles for binding to cell surfaces, i.e., nanoparticles or microparticles comprising (i) multiple different ligand species on their outer surface capable of binding to different receptor species on the cell surface, and (ii) polymer brushes on their outer surface. Surprisingly, the inventors have discovered that nanoparticle or microparticle-cell binding via multiple different ligand species results in improved selectivity in the delivery of drug cargo carried within the nanoparticle or microparticle and reduces undesired off-target binding. A further advantage of the present invention is that by using multiple nanoparticles or microparticles, cell surface receptors on target cells (e.g., cancer cells) cannot easily mutate over time in a way that prevents effective nanoparticle or microparticle binding. This is particularly beneficial because the treatment course of some diseases can last for a significant period of time (i.e., months or years). Furthermore, the optimal number of different ligand species on the surface of the nanoparticle or microparticle can be predicted to enable optimal binding to the target cell surface.

[0006] Thus, in one aspect, the present invention provides nanoparticles or microparticles for binding to cell surfaces, the nanoparticles or microparticles comprising at least a first ligand species on their outer surface and at least a second ligand species on their outer surface, the first ligand species capable of binding to a first receptor species on the cell surface, the second ligand species capable of binding to a second receptor species on the cell surface, and the nanoparticles or microparticles comprising 2 to 1000 of the first ligand species and 2 to 1000 of the second ligand species. The nanoparticles or microparticles are typically polymersomes, liposomes, synthosomes, or micelles, and typically comprise polymer brushes on their outer surface.

[0007] In a further aspect, the present invention provides a pharmaceutical composition comprising a plurality of nanoparticles or microparticles of the present invention and one or more pharmaceutically acceptable excipients or diluents.

[0008] In yet another aspect, the present invention provides a nanoparticle or microparticle of the present invention, or a pharmaceutical composition of the present invention, for use in treating a disease (eg, cancer).

[0009] In yet another aspect, the present invention provides a method of treating cancer in a human patient, said method comprising administering to a patient in need thereof a nanoparticle or microparticle of the invention, or a pharmaceutical composition of the invention.

[0010] In yet another aspect, the present invention provides the use of a nanoparticle or microparticle of the present invention, or a pharmaceutical composition of the present invention, for the manufacture of a medicament for treating cancer in a patient.

[0011] In yet another aspect, the present invention provides a vaccine comprising the nanoparticles or microparticles of the present invention and an antigen. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1: Theory of superselectivity. Schematic showing the multiplexed ligand strategy on a polymersome scaffold (a) and the ligand-receptor affinity curves of high affinity monovalent, low affinity monovalent, and multivalent ligands on a polymersome scaffold (b). [Figure 2] Figure 2: Examples of polymer brushes. Schematics of glycocalyx syndecan 4 and LRP1 receptor. Both proteins were reconstructed at atomic resolution using computational methods and minimized for brush conformations. The inset shows a detail of the terminal portion of LRP1 next to four heparan sulfate chains (a). Schematic of a POEGMA-PDPA polymersome modified with the Angiopep peptide. The polymersome was reconstructed using a minimized atomic model of a single block assembled into a 50 nm vesicle. The inset detail shows that the peptide is well embedded in the polyethylene oxide (PEO) brush. [Figure 3]Figure 3: Characterization of polymersomes. Particle size distribution measured by dynamic light scattering for POEGMA-PDPA / Angiopep (a), POEGMA-PDPA / PMPC (b), and POEGMA-PDPA / PMPC+Angiopep (c) polymersomes. Representative transmission electron micrographs of POEGMA-PDPA / Angiopep (25 ligands) (d), and POEGMA-PDPA / PMPC (1000 ligands) (e) formulations. [Figure 4] Figure 4: Selective cellular uptake. Example of Angiopep-polymersome binding to brain endothelial cells after 1 hour of incubation (a). Cellular DNA is stained with DAPI dye (blue), and polymersomes are labeled with Cy5 dye (red). Mean fluorescence per cell measured after 1 hour of incubation of polymersomes with brain endothelial cells (line A), lymphocytes, and macrophages (line B) as a function of the number of ligands for Angiopep peptide (b) and PMPC chains (c). Line C shows the selectivity index calculated using brain endothelial cells as targets and macrophages as sentinel cells. [Figure 5] Figure 5: Verification of superselectivity for brain endothelial cells. Plot showing binding of multiplexed polymersomes modified with both Angiopep peptide and PMPC chains to brain endothelial cells. [Figure 6] Figure 6: Repulsive steric potentials. Schematic of bidding of multivalent POEGMA-PDPA polymersomes modified with the Angiopep peptide and targeting LRP1 (a), PMPC chains and targeting the SRB1 receptor (b), and both ligands and targeting both receptors (c). Detail of the interactions between Angiopep and LRP1 (d) and between PMPC and SRB1 (e), modified with both PEO and a glycocalyx brush. Corresponding repulsive steric potentials exerted upon LRP1 insertion into the PEO brush (f) and polymersome insertion into the glycocalyx brush (g). These are calculated as a function of the polymersome radius, R, and the insertion parameter of the PEO chains, δP, and the insertion parameter of the glycocalyx heparan sulfate chains, δG, respectively. [Figure 7] Figure 7: Targeting of antigen-presenting cells. Spider plot showing the expression of SRB1, TFRC, LRP1, and mannose receptor in healthy and tumor tissues. [Figure 8] Figure 8: Neutrophil targeting. Spider plot showing expression of SRB1, FCAR, and SLFN12L receptors in healthy and inflamed tissues. [Figure 9] Figure 9: Targeting tumor cells. Spider plot showing expression of SRB1, EGFR, and TFRC receptors in healthy liver (a), colorectum (b), and female reproductive tract (c) along with three different cancers related to liver (a), colorectum (b), and cervix (c). Note that only tumor tissues show moderate / high levels of expression of all three receptors, while healthy tissues express only one or two receptor species at moderate levels. [Figure 10] Figure 10: Systemic (a) and somatic brain and endothelial (b) expression levels of LRP-1, GLUT1, EGFR, and PDGFR-α receptors, which can be used to generate a superselectivity spider plot (c) showing the combination of receptor densities to achieve targeting of only BECs and glioma cells. [Figure 11] Figure 11: Adsorption probability as a function of the number of ligands; comparison of theoretical and experimental data. The theoretical curve was calculated using equation (2) described in Example 7, using the (double) Poisson mean for both the number of receptors and the number of ligands, and the mean value (grafting density σ = 1) was used as the fitting parameter. Error bars were calculated as the average of three independent measurements. DETAILED DESCRIPTION OF THE INVENTION

[0013] Detailed Description Nanoparticles and Microparticles The nanoparticles and microparticles of the present invention can be any nanoparticle or microparticle suitable for delivering a drug cargo to a target site of action in vivo. As defined herein, a "nanoparticle" is any particle between 1 and 100 nm in size. As defined herein, a "microparticle" is any particle between 0.1 and 100 μm in size. Nanoparticles or microparticles suitable for use in the present invention include polymersomes, liposomes, synthosomes, latex, micelles, nanocrystals, quantum dots, metal nanoparticles, oxide nanoparticles, silica nanoparticles, protein cages, nano- and microgels, dendrimers, virus-like particles, proteins, polymers, or any other colloidal material within the above-mentioned size ranges. However, typically, the nanoparticles or microparticles for use in the present invention are polymersomes, liposomes, synthosomes, or micelles. Typically, the nanoparticles or microparticles are self-assembled structures.

[0014] The nanoparticles and microparticles of the present invention can be of any feasible shape, for example, substantially spherical, ellipsoidal, cylindrical, or bilayered, but typically are substantially spherical. Typical (maximum) diameters of nanoparticles or microparticles of the present invention are in the range of 50 to 5,000 nm. More typically, the diameters are in the range of 50 to 1,000 nm. Typically, nanoparticles or microparticles of the present invention have a number-average diameter of less than 300 nm, preferably less than 250 nm, and most preferably less than 200 nm or 150 nm. In one aspect, the nanoparticles or microparticles of the present invention are nanoparticles. Alternatively, the nanoparticles or microparticles of the present invention are microparticles. Typically, particle size is measured using transmission electron microscopy (TEM). Typically, particle size distribution is measured using dynamic light scattering (DLS).

[0015] Preferably, the nanoparticle or microparticle of the present invention is a polymersome.Polymersome is a synthetic vesicle formed by amphiphilic block copolymer.Examples of polymersome are described in US 2010 / 0003336 A1, WO 2017 / 144849, WO 2017 / 158382, WO 2017 / 199023 and WO 2017 / 191444 (each of which is incorporated herein by reference in its entirety).In the past 15 years, they have attracted great research attention as a versatile carrier due to their colloidal stability, adjustable membrane properties and ability to encapsulate or integrate other molecules (for one representative review article, see J Control Release 2012 161(2) 473-83, the content of which is incorporated herein by reference in its entirety).

[0016] Polymersomes are typically self-assembled structures. Polymersomes typically comprise amphiphilic block copolymers, i.e., block copolymers comprising a hydrophilic block and a hydrophobic block. For example, a polymersome may comprise at least two such amphiphilic block copolymers that are different from one another.

[0017] Such copolymers can mimic biological phospholipids. Because the molecular weight of these polymers is much higher than that of naturally occurring phospholipid surfactants, they can assemble into more entangled membranes (J. Am. Chem. Soc. 2005, 127, 8757, the contents of which are incorporated herein by reference in their entirety), providing a final structure with improved mechanical properties and colloidal stability. Furthermore, the flexible nature of copolymer synthesis allows for the application of different compositions and functions across a wide range of molecular weights, and consequently, membrane thicknesses. Therefore, the use of these block copolymers as delivery vehicles offers significant advantages.

[0018] Polymersomes are often substantially spherical. Polymersomes typically comprise an amphiphilic membrane. The membrane is generally formed from two monolayers of amphiphilic molecules that align and entangle to form a core surrounded by hydrophilic head groups that face the exterior of the core and vesicle, and hydrophilic tail groups that form the interior of the membrane.

[0019] The thickness of the bilayer is generally between 2 and 100 nm, more typically between 2 and 50 nm (e.g., between 5 and 20 nm). These dimensions can be routinely measured, for example, by using transmission electron microscopy (TEM) and / or small-angle X-ray scattering (SAXS) (see, e.g., J. Am. Chem. Soc. 2005, 127, 8757, the contents of which are incorporated herein by reference in their entirety).

[0020] When a polymersome is formed from more than one type of copolymer, different regions of the polymersome typically have different bilayer thicknesses. For example, when a polymersome is formed from two different types of copolymer, the polymersome bilayer thickness in the first region is preferably 1-10 nm, more preferably 2-5 nm. The polymersome bilayer thickness in the second region is preferably 5-50 nm, e.g., 10-40 nm. The polymersome bilayer thickness in the second region is more preferably 5-20 nm. Preferably, the polymersome bilayer thickness in the first region is thinner than the polymersome bilayer thickness in the second region. Alternatively, the copolymers can be the same thickness but have different chemical compositions, creating two different permeabilities, with one copolymer forming a bilayer that is less permeable than the other.

[0021] In aqueous solutions, there is usually an equilibrium between different types of structures, for example, between polymersomes and micelles. Preferably, at least 80% by weight, more preferably at least 90% or 95% by weight, and most preferably, all of the structures in the solution are present as polymersomes. This can be achieved using the methods outlined herein.

[0022] It is known that when two different polymersome-forming copolymers are mixed to form hybrid vesicles, they undergo phase separation, resulting in polymersomes containing distinct regions corresponding to the individual copolymers. For example, this phenomenon is described in detail in ACS NANO, 5(3), 1775-1784 2011 (the contents of which are incorporated herein by reference in their entirety). Polymersomes can be easily produced by applying these known synthetic principles.

[0023] The polymersomes are preferably capable of dissociating and releasing the encapsulated drug once it reaches the target tissue (i.e., the target tissue). Non-limiting exemplary target tissues are discussed in more detail below and include cells that cross the blood-brain barrier (e.g., CNS cells), immune cells, and cancer cells. Preferably, the polymersomes are capable of dissociating and releasing the encapsulated drug after being taken up into the target cells (e.g., CNS cells, immune cells, or cancer cells) via endocytosis.

[0024] Dissociation can be driven by a variety of mechanisms, such as pH sensitivity of the block copolymer, heat sensitivity of the block copolymer, hydrolysis (ie, water sensitivity of the block copolymer), and / or redox sensitivity of the block copolymer.

[0025] The hydrophobic block of the copolymer contained in the polymersome may also contain pendant cationizable moieties as pendant groups. Cationizable moieties are, for example, primary, secondary, or tertiary amine and imidazole groups that can be protonated at a pH below 3 to 6.9. Alternatively, the groups may be phosphines.

[0026] The hydrophobic block of the polymersome preferably has a degree of polymerization of at least 50, more preferably at least 70. The degree of polymerization of the hydrophobic block is preferably 250 or less, even more preferably 200 or less. The degree of polymerization of the hydrophilic block is typically at least 10, preferably at least 15, more preferably at least 20. The ratio of the degree of polymerization of the (hydrophilic) to (hydrophobic) block is preferably in the range of 1:2.5 to 1:8. All of these limitations promote polymersomes, not micelle formation.

[0027] The hydrophilic block can be based on condensation polymers such as polyesters, polyamides, polyanhydrides, polyurethanes, polyethers (including polyalkylene glycols, particularly polyethylene glycol (PEG)), polyimines, polypeptides, polypeptoids, polyureas, polyacetals, and polysaccharides. Preferably, the hydrophilic block is based on a polymer selected from poly(alkylene glycol), poly(vinylpyrrolidone) (PVP), poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), poly(glycerol), poly(amino acids), polysarcosine, poly(2-oxazoline), poly[oligo(ethylene glycol)methyl methacrylate], and poly(N-(2-hydroxypropyl)methacrylamide). Most preferably, the hydrophilic block is based on PEG, poly(propylene glycol), or poly[oligo(ethylene glycol)methyl methacrylate]. The hydrophilic block can have zwitterionic pendant groups, in which case the zwitterionic pendant groups can be present in the monomer and can remain unchanged during the polymerization process. Alternatively, the pendant functional groups of the monomer can be derivatized to become zwitterionic after polymerization.

[0028] The polymersomes of the present invention can include any of the structural and / or functional features of polymersomes described in any of WO 2017 / 144849, WO 2017 / 158382, WO 2017 / 199023, and WO 2017 / 191444 (the contents of each of which are incorporated herein by reference in their entirety).

[0029] In one embodiment of the present invention, the monomer that forms the hydrophobic block is 2-(diisopropylamino)ethyl methacrylate (DPA) or 2-(diethylamino)ethyl methacrylate (DEA).

[0030] In another embodiment, the hydrophobic block is formed from 2-(diisopropylamino)ethyl methacrylate (DPA) or 2-(diethylamino)ethyl methacrylate (DEA), and the hydrophilic block is based on polyesters, polyamides, polyanhydrides, polyurethanes, polyethers, polyimines, polypeptides, polypeptoids, polyureas, polyacetals, or polysaccharides. Preferably, the hydrophobic block is formed from 2-(diisopropylamino)ethyl methacrylate (DPA) or 2-(diethylamino)ethyl methacrylate (DEA), and the hydrophilic block is based on PEG, poly(propylene glycol), or polyoligo(ethylene glycol)methyl methacrylate. More preferably, the polymersomes of the present invention comprise a diblock PEG-PDPA (where PEG is poly(ethylene glycol) and PDPA is poly(2-(diisopropylamino)ethyl methacrylate)). Alternatively, the polymersomes of the present invention comprise a diblock POEGMA-PDPA, where POEGMA is poly[oligo(ethylene glycol)methyl methacrylate] and PDPA is poly(2-(diisopropylamino)ethyl methacrylate). A particularly preferred diblock copolymer is (P[(OEG) 10 MA 20 ]-PDPA 100 ) These copolymers have the ability to self-assemble in water or PBS to create vesicles with aqueous lumens that can be loaded with drugs. PEG functionality provides pendant hydroxyl groups that serve as handles for easy and reliable functionalization of the polymer with ligands (as discussed below) while avoiding protein opsonization (giving polymersomes long circulation time and low nonspecific binding). On the other hand, PDPA is a pH-sensitive blocker that causes polymersome degradation at pH values ​​below 6.4, a typical pH during early-stage endocytosis. Due to the pH sensitivity, internalization of polymersomes into cells releases the drug payload into the cytosol.

[0031] The block copolymer may be a simple AB block copolymer, or an ABA or BAB block linear triblock copolymer, or an (A)2B or A(B)2 star copolymer (where A is a hydrophilic block and B is a hydrophobic block). It may also be an ABC, ACB, or BAC block linear triblock copolymer, or an ABC star copolymer (blocks linked together by the same end) (where C is a different type of block). The C block may contain, for example, functional groups, such as crosslinkable or ionic groups, to enable copolymer reaction in novel compositions. Crosslinking reactions, particularly of ACB-type copolymers, can impart useful stability to polymersomes. Essentially, crosslinking may be covalent or sometimes electrostatic. Crosslinking may involve the addition of a separate reagent to link functional groups, such as using a bifunctional alkylating agent to link two amino groups. Block copolymers may alternatively be star-shaped molecules with a hydrophilic or hydrophobic core, or comb-shaped polymers with a hydrophilic backbone (block) and hydrophobic pendant blocks, or vice versa. Such polymers can be formed, for example, by random copolymerization of monounsaturated macromers and monomers.

[0032] Further details of suitable processes for polymerizing the monomers can be found in WO 03 / 074090, the contents of which are incorporated herein by reference in their entirety.

[0033] Exemplary methods that can be used to polymerize the monomers include atom transfer radical polymerization (ATRP) (see, for example, the exemplary method described in Journal of the American Chemical Society 127, 17982-17983), living radical polymerization, efficient post-polymerization modification, and functional NCA (N-carboxyanhydride) polymerization with ring-opening polymerization (ROP). Living radical polymerization has been found to provide polymers of the monomers with a polydispersity (in terms of molecular weight) of less than 1.5, as determined by gel permeation chromatography. A polydispersity in the range of 1.2 to 1.4 for the or each block is preferred. Polymersomes can be loaded using a pH shift system, electroporation, or film hydration. In the pH shift system process, the polymer is dispersed in an aqueous liquid in an ionized form and solubilized at relatively high concentrations without forming polymersomes. The pH is then shifted so that some or all of the ionized groups are deprotonated, converting them to a non-ionic form. At a second pH, the hydrophobicity of the blocks increases, and polymersomes form spontaneously.

[0034] A method for forming polymersomes having an encapsulated material (e.g., an encapsulated drug) in the core can include the following steps: (i) dispersing an amphiphilic copolymer in an aqueous medium; (ii) acidifying the composition formed in step (i); (iii) adding the material to be encapsulated to the acidified composition; and (iv) raising the pH to near neutral to encapsulate the material.

[0035] The method preferably includes a preliminary step of dispersing the amphiphilic copolymer in an organic solvent in a reaction vessel and then evaporating the solvent to form a film on the inside of the reaction vessel.

[0036] Step (ii) of acidifying the composition typically lowers the pH to a value below the pKa of the pendant group.

[0037] Another method for forming polymersomes with encapsulated material in the core can include the following steps: (i) dispersing the amphiphilic copolymer, and optionally the material to be encapsulated, in an organic solvent (e.g., a 2:1 chloroform:methanol mixture) in a reaction vessel; (ii) evaporating the solvent to form a film on the inside of the reaction vessel; and (iii) rehydrating the film with an aqueous solution that may contain the solubilized material to be encapsulated.

[0038] Another method for forming polymersomes with encapsulated material in their cores can include the following steps: (i) dispersing an amphiphilic copolymer, and optionally an encapsulated material, in an organic solvent in a reaction vessel; (ii) adding an aqueous solvent to allow for solvent exchange and formation of polymersomes inside the reaction vessel; and (iii) optionally electroporating the resulting polymersomes to allow for encapsulation of water-soluble bioactive molecules.

[0039] UV spectroscopy and HPLC chromatography can be used to calculate encapsulation efficiency using techniques well known in the art.An alternative method for forming polymersomes with encapsulated materials can involve simple electroporation of the material and polymer vesicles in water.For example, a drug can be contacted in solid form with an aqueous dispersion of polymer vesicles and an electric field to form pores on the polymersome membrane.The solubilized material molecules can then enter the polymersome vesicles through the pores.This is followed by a self-repairing process of the membrane, which continuously traps the material molecules within the polymersome.

[0040] Alternatively, materials dissolved in organic solvents can be emulsified into an aqueous dispersion of polymer vesicles, thereby incorporating the solvent and materials into the core of the vesicles, followed by evaporation of the solvent from the system.

[0041] The polymersomes used in the present invention may be formed from two or more different block copolymers. In this embodiment, a mixture of two or more block copolymers is used in the method of forming the polymersomes.

[0042] For example, 0.01% to 10% (w / w) of the material to be encapsulated is mixed with the copolymer in the manner described above.

[0043] Alternatively, the nanoparticles or microparticles of the present invention can be liposomes. Liposomes are spherical vesicles with at least one lipid bilayer. Typically, liposomes contain phospholipids, such as phosphatidylcholine, but can also contain other lipids, such as egg phosphatidylethanolamine, as long as they are compatible with the lipid bilayer structure. The main types of liposomes include multilamellar vesicles (MLVs, having multiple lamellar phase lipid bilayers), small unilamellar vesicles (SUVs, having one lipid bilayer), large unilamellar vesicles (LUVs), and cochleate vesicles.

[0044] Typically, liposomes are fusogenic. This means that they can fuse with membranes, such as the cell surface membrane of a target cell or the membrane of an endosome within the cell. Fusion of the fusogenic liposome bilayer with the cell surface membrane results in the incorporation of the liposome bilayer into the cell surface membrane and the release of the drug cargo contained within the lysosome into the cell cytosol. Alternatively, liposomes are taken up into target cells via endocytosis, and the drug cargo carried within the liposome is released after the liposome bilayer fuses with the endosomal membrane. The pH within endosomes is slightly acidic, and therefore it is advantageous for liposomes to be pH-sensitive; for example, the stability of the liposome structure decreases at lower pH, promoting fusion with the endosomal membrane. Other environments with low pH (e.g., the low pH found in tumors or inflammatory sites) can also cause such liposomes to fuse.

[0045] Liposomes can be zwitterionic structures. Alternatively, liposomes can be amphoteric liposomes. This means that liposomes have an isoelectric point, becoming negatively charged at higher pH values ​​and positively charged at lower pH values. Typical pH-responsive components of pH-sensitive liposomes include cholesterol hemisuccinate (CHEMS), palmitoyl homocysteine, dioleoylglycerol hemisuccinate (DOG-Succ), etc.

[0046] Alternatively, the nanoparticles or microparticles of the invention can be synthosomes, which are a particular type of polymersome engineered to contain channels (transmembrane proteins) that selectively allow certain chemicals to pass through the membrane into or out of the vesicle.

[0047] Alternatively, the nanoparticles or microparticles of the present invention may be micelles. Micelles are aggregates (or supramolecular assemblies) of molecules having both hydrophilic and hydrophobic regions, dispersed in a liquid. Typically, in aqueous solutions, aggregated micelles are arranged such that the hydrophobic regions of the molecules are isolated in the center of the micelle, while the hydrophilic regions of the molecules are present on the outer surface of the micelle and in contact with the aqueous medium. Typically, micelles are substantially spherical, although other shapes, such as ellipsoids, cylinders, tori, and discs, are also possible.

[0048] Alternatively, the nanoparticles or microparticles of the present invention can be anything that can encapsulate and / or conjugate any kind of bioactive molecule, such as anti-cancer drugs, proteins, peptides (natural or non-natural), antibodies, antibody fragments, dyes, etc.

[0049] Encapsulated drugs The nanoparticles or microparticles of the present invention may include a drug encapsulated within the nanoparticle or microparticle. For the avoidance of doubt, it is also possible to encapsulate multiple different drugs within a single nanoparticle or microparticle, or to provide multiple nanoparticles or microparticles, each containing a particular encapsulated drug.

[0050] As will be readily appreciated, the encapsulated drug will be selected according to the disorder to be treated, non-limiting examples of which are described elsewhere in this disclosure.

[0051] Non-limiting examples of drugs include drugs effective for treating or preventing brain disorders, drugs effective for treating or preventing immune and / or inflammatory disorders, and drugs effective for treating or preventing cancer. There is no particular limit to the identity of the drug, and drugs can be selected from those known in the art for treating or preventing the disorder of interest in any given embodiment.

[0052] Non-limiting examples of drugs include neuroprotective agents, immunomodulatory agents ("immunomodulators"), nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, TNF-alpha inhibitors, and anti-cancer agents.

[0053] Illustrative and non-limiting examples of specific drugs that may be encapsulated include fumarates and fumarate esters, glutamate antagonists (e.g., estrogen, ginsenoside Rd, progesterone, simvastatin, memantine), antioxidants (e.g., acetylcysteine, crocin, fish oil, minocycline, pyrroloquinoline quinone (PQQ), resveratrol, vinpocetine, vitamin E), stimulants (e.g., selegiline, nicotine, caffeine), caspase inhibitors, trophic factors (e.g., CNTF, IGF-1, VEGF, and BDNF), protein aggregation inhibitors, and the like. agents) (e.g., sodium 4-phenylbutyrate, trehalose, and polyQ-binding peptide), erythropoietin, lithium, carnosine, asiatic acid, flavonoids (e.g., xanthohumol, naringenin, galangin, fisetin, and baicalin), cannabinoids (e.g., WIN55,212-2, JWH-133, and TAK-937), citicoline, minocycline, cerebrolysin, ginsenosoid-Rd, granulocyte-colony stimulating factor, Tat-NR2B9c, magnesium, albumin, paracetamol, aspirin, choline, and magnesium salicylate, celecoxib, diclofenac (e.g., diclofenac potassium, diclofenac sodium), diflunisal, etodolac, fenoprofen, flurbiprofen , ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen (including naproxen sodium), oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin, valdecoxib, corticosteroids, alemtuzumab, interferon beta-1b, fingolimod, glatiramer acetate, natalizumab, plegridy, peginterferon beta-1a, teriflunomide, methotrexate, sulfasalazine, leflunomide, adalimumab, etanercept, golimumab, ustekinumab, azathioprine, cyclosporine, infliximab, golimumab, certolizumab, hydroxychloroquine, methotrexate, azathioprine,Mycophenolate, acitretin, hydrair, isotretinoin, mycophenolate mofetil, sulfasalazine, 6-thioguanine, calcipotriol, calcitriol, tacalcitol, tacrolimus, pimecrolimus, dithranol, endamustine, bendamustine, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, melphalan, procarbazine, streptozocin, temozolomide, capecitabine, 5-fluorouracil, fludarabine, gemcitabine, methotrexate, pemetrexed, raltitrexed, actinomycin D, bleomycin, doxorubicin, epirubicin Benzene, mitomycin, mitoxantrone, etopoxel, docetaxel, irinotecan, paclitaxel, topotecan, vinblastine, vincristine, vinorelbine, eribulin, carboplatin, cisplatin, oxaliplatin, afatinib, aflibercept, BCG, bevacizumab, brentuximab, cetuximab, crizotinib, denosumab, erlotinib, gefitinib, imatinib, interferon, ipilimumab, lapatinib, panitumumab, pertuzumab, rituximab, sunitinib, sorafenib, trastuzumab emtansine, temsirolimus, trastuzumab, vemurafenib, clodronate, ibandronate, pamidronate, zolendronic acid acid), anastrozole, abiraterone, bexarotene, bicalutamide, buserelin, cyproterone, degarelix, exemestane, flutamide, folinic acid, fulvestrant, goserelin, lanreotide, lenalidomide, letrozole, leuprolide, medroxyprogesterone, megestrol, mesna, octreotide, stilbestrol, tamoxifen, and thalidomide.

[0054] Targeting Ligands The nanoparticle or microparticle comprises at least two different ligand species on its exterior surface. The "ligands" are sometimes referred to herein as "targeting moieties." By "on its exterior surface," we mean that each ligand is positioned so that it can interact with its target (as opposed to being encapsulated within the nanoparticle or microparticle, for example, and thereby positioned in an inaccessible location that would prevent interaction with the target).

[0055] Typically, the nanoparticles or microparticles of the present invention are intended to bind to a cell surface and comprise at least a first ligand species on their outer surface and at least a second ligand species on their outer surface, wherein the first ligand species is capable of binding to a first receptor species on the cell surface and the second ligand species is capable of binding to a second receptor species on the cell surface. Typically, the nanoparticles or microparticles of the present invention comprise two to seven different ligand species on their outer surface, each of which is capable of binding to a complementary receptor species on the cell surface. Preferably, the nanoparticles or microparticles of the present invention comprise two to six different ligand species on their outer surface, more preferably three to five different ligand species, and most preferably four different ligand species.

[0056] Without wishing to be bound by any particular theory, it is believed that multiplexing ligands on the surface of nanoparticles or microparticles in this manner confers the property of "superselectivity" for target cells.

[0057] As shown in Figure 1b, for a monovalent ligand, the probability of saturating the receptor (i.e., the bound particle fraction, θ = 1, right side of Figure 1b) increases linearly with the number of receptors expressed on the cell membrane (ρ). This means that the stronger the affinity of the ligand-receptor binding, the stronger the ligand association. However, this also means that if a ligand has low affinity for its target receptor, a very high dose of the ligand may be required to produce a response in the target (i.e., diseased) cell. Conversely, if a ligand has high affinity for its target receptor, the majority of the ligand may bind to any cell expressing a lower number of target receptors (i.e., there may be undesirable off-target effects).

[0058] However, when multiple ligands of the same type are confined in nanoscopic scaffolds such as polymersomes, binding increases nonlinearly with receptor density according to a sigmoidal function (Figure 1b, green line). This corresponds to an "on-off" association, where binding occurs only above a certain onset receptor density. Such ultraselective association enables the creation of nanoscopic or microscopic multipolymersomes that more specifically target desired cell populations. However, even these scaffolds suffer from several off-target effects and require moderate to high levels of receptor expression on the target cell surface. Furthermore, relying on binding to only a single target receptor leaves open the possibility that receptors on the target cell surface (e.g., those on tumor cells) may mutate over time, avoiding binding to nanoparticles / microparticles. The nanoparticles and microparticles of the present invention overcome these challenges through the multiplexing of ligands on the surface of the scaffold (i.e., the presence of multiple different ligand species on the surface of the scaffold). This strategy is believed to further enhance the selectivity of nanoparticles / microparticles to target cells, targeting only very specific cell populations (i.e., diseased cells) and leaving other (healthy) cells alone.

[0059] For the superselective interactions observed in the context of nanoparticles and microparticles, it is also advantageous for each ligand to individually have a very low binding affinity for its target receptor. In practice, selective ligands with such low binding energies to target receptors are not readily available. The inventors have discovered that this challenge can also be overcome by providing the surface of the nanoparticle or microparticle with moieties, such as polymer brushes, that create an interference steric potential with the surface of the target cell. Typically, polymer brushes can comprise naturally occurring polymers, such as polypeptides or polysaccharides, or synthetic polymers, such as any of the amphiphilic block copolymers described above. Components on the outer surface of target cells, such as glycans, glycoproteins, and glycolipids (collectively referred to as the "glycocalyx"), are also thought to contribute to this repulsive steric potential. Preferred polymer components of the polymer brushes include poly(ethylene glycol) (PEG), poly(vinylpyrrolidone) (PVP), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), poly(glycerol), poly(sulfobetaine), poly(carboxybetaine), poly(amino acids), polysarcosine, poly(2-oxazoline), poly(N-(2-hydroxypropyl)methacrylamide), polyglycols, heparin, dextran, poly(ethylene glycol)-poly(2-(diisopropylamino))ethyl methacrylate), and / or poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA). Figure 2 shows some examples of polymer brushes on a cell surface (a) and on the outer surface of a nanoparticle (b).

[0060] The polymer brushes preferably have a degree of polymerization of at least 5, more preferably at least 10. The degree of polymerization of the polymer brushes is preferably 500 or less, for example, 300 or less, or 200 or less. The polymer brushes preferably have a length of 1.5 to 350 nm, more preferably 3 to 210 nm.

[0061] Thus, polymersomes functionalized with two or more ligand species, each with a relatively low affinity for the target receptor, may avoid targeting unwanted cells while still effectively binding to target cells for drug delivery. Furthermore, mutations in cell surface receptors may be less likely to lead to evasion of detection by nanoparticles or microparticles. A schematic diagram of the ligand binding strategy used in the present invention is shown in Figure 1a.

[0062] The number of each type of ligand on the outer surface of a nanoparticle or microparticle is an important parameter in the design of nanoparticles / microparticles. The inventors have discovered that, once certain physical parameters of the ligand-receptor system are known, the optimal number of ligands of each ligand type can be calculated in a very simple manner. These relevant parameters can be easily determined by a person skilled in the art. Specifically, the number of ligands of each type on the outer surface of a nanoparticle or microparticle is determined by the number of ligands of each type on the outer surface of the nanoparticle or microparticle. th ) Ligand species (l i The optimal number of ligands in the .sup.-dimer is determined according to the following formula (1):

[0063]

number

[0064] (In the formula: a is the activity of the nanoparticles or microparticles, a = [P]V P N A where [P] is the molar concentration of nanoparticles or microparticles in the bulk solution, and N A is Avogadro's constant, and V P is the geometric volume); k B is the Boltzmann constant; T is the absolute temperature; E B (i) is the total energy of binding of the ith ligand-receptor pair, and (a) the ligand / receptor binding affinity, k B TlnK D (i) (where KD (i) is the dissociation constant of the ith receptor / ligand couple) and (b) the steric hindrance E between the nanoparticle or microparticle and the cell surface. s is given by the sum of; and r i is the density of receptors for species i on the cell surface.)

[0065] This formula therefore provides a useful and novel empirical tool for determining the optimal number of each ligand species on the exterior surface of the nanoparticles or microparticles of the invention.

[0066] For spherical (or substantially spherical) particles, the parameter a is a = [P]N A ( π / 3)[3(R+d) 3 -2R 3 )] where R is the radius of the nanoparticle or microparticle and d is the ligand tether length.

[0067] The number (and density) of each type of ligand on the outer surface of a polymersome can typically be controlled by varying the ratio of ligand-conjugated copolymer and "pristine" copolymer (i.e., unliganded diblock copolymer) during polymersome synthesis. For any given system, the number of ligands per polymersome is given by the copolymer's self-assembly parameters (related to the polymer's molecular weight and packing ratio) and the size of the polymersome. The number of each type of ligand (and therefore receptor density) on the outer surface of a polymersome can typically be confirmed using mass spectrometry.

[0068] Typically, the ligand is attached to a polymer component on the outer surface of the nanoparticle or microparticle. In the case of polymersomes, the ligand is typically attached to the hydrophilic block of an amphiphilic diblock copolymer. Thus, the tether length d of the ligand is given by the molecular weight of the hydrophilic block; typically, d = 0.3N nm, where N is the degree of polymerization of the hydrophilic block.

[0069] Total steric potential E s is the sum of the steric potential arising from the glycocalyx brush on the cell surface and the steric potential arising from the polymer brush coating the nanoparticle. The magnitude of both depends on the accessibility of the ligand and receptor. This, in turn, depends on: (i) the relative height (δ) of the receptor to the glycan / glycoprotein / glycolipid / etc. chains on the cell surface; G h G , where h G is the length of the glycan / glycoprotein / glycolipid, and δ G is between 0 and 1 and is a measure of how embedded the receptor is in the glycocalyx), and (ii) the tether length of the ligand relative to the length of the polymer chains of the brush on the outer surface of the nanoparticle (δ P h P , where h P is the chain length of the polymer, and δ P (where σ is between 0 and 1 and is a measure of how buried the ligand is in the polymer brush). These parameters can be readily obtained for any given system from structural biology databases known in the art.

[0070] Typically, the nanoparticles or microparticles comprise 2 to 1000 ligands of the first ligand species. Preferably, the nanoparticles or microparticles comprise 5 to 1000 ligands of the first ligand species, more preferably 10 to 500 ligands of the first ligand species, even more preferably 20 to 200 ligands of the first ligand species, and most preferably 50 to 100 ligands of the first ligand species.

[0071] Typically, the nanoparticles or microparticles comprise 2 to 1000 ligands of the second ligand species. Preferably, the nanoparticles or microparticles comprise 5 to 1000 ligands of the second ligand species, more preferably 10 to 500 ligands of the second ligand species, even more preferably 20 to 200 ligands of the second ligand species, and most preferably 50 to 100 ligands of the second ligand species.

[0072] Typically, the nanoparticles or microparticles comprise 2 to 1000 ligands of the subsequent (i.e., third or more) ligand species. Preferably, the nanoparticles or microparticles comprise 5 to 1000 ligands of the subsequent ligand species, more preferably 10 to 500 ligands of the subsequent ligand species, even more preferably 20 to 200 ligands of the subsequent ligand species, and most preferably 50 to 100 ligands of the subsequent ligand species.

[0073] Typically, the combination of ligands on the surface of the nanoparticles or microparticles is 8kJ / 2000 kJ / 2000 kcal / 3000 kJ / 4000 kcal / 5000 kcal / 6000 kcal / 7000 kcal / 8000 kcal / 9000 kcal / 9000 kcal / 1 ... B T~30k B T (where k B is the Boltzmann constant and T is temperature), which results in an on-off association profile for the nanoparticle or microparticle, in which the receptors are saturated only above a given starting receptor density, while the nanoparticle or microparticle does not bind at all at lower receptor densities.

[0074] Each ligand species is adapted to allow the nanoparticle or microparticle to bind to a target. Typically, the ligand selectively binds to the target. The target is a chemical substance on or near the tissue of interest (thus allowing the nanoparticle or microparticle to accumulate specifically in the tissue of interest rather than other sites). The target is preferably a receptor, for example, a receptor that is particularly abundant in the target tissue of interest. Most preferably, the target is a receptor on or in a cell surface membrane.

[0075] Each ligand species can be any ligand that specifically binds to a target. As is well known in the art, a wide range of substances can be used as ligands, for example, to target receptors, from the well-developed field of bioconjugates.

[0076] In one embodiment, each ligand is a moiety attached to the outer surface of the nanoparticle or microparticle. Examples of suitable ligands include antibodies, antibody fragments, aptamers, oligonucleotides, small molecules, peptides, and carbohydrates. Peptide, protein, antibody, and antibody fragment ligands are particularly preferred. However, any such moiety can be used as a ligand in the present invention. The suitability of any given moiety for targeting any given receptor can be determined using routine assays, including testing the moiety's ability to specifically bind to the receptor.

[0077] An example of a ligand is one that is adapted to enable the nanoparticle or microparticle to cross the blood-brain barrier (BBB). This property of the ligand arises through the ligand's ability to bind to a target (e.g., receptor) at the blood-brain barrier, where the target (e.g., receptor) mediates transcytosis across the blood-brain barrier.

[0078] Examples of receptors for receptor-mediated transcytosis that are highly expressed in endothelial cells that form the blood-brain barrier include low-density lipoprotein receptor-related protein 1 (LRP-1), scavenger receptor class B, member 1 (SCARB1), insulin receptor (IR), and transferrin receptor 1 (TFRC), all of which are suitable targets for targeting moieties.

[0079] In one embodiment, at least one ligand species, preferably one ligand species, targets the LRP-1 receptor. LRP-1 is a member of the LDL receptor family and plays diverse roles in various biological processes, including lipoprotein metabolism, protease degradation, lysosomal enzyme activation, and cellular entry of bacterial toxins and viruses. Deletion of the LRP-1 gene leads to lethality in mice, revealing an important, yet still unclear, role in development. Tissue-specific gene deletion studies reveal important contributions of LRP-1 in the vasculature, central nervous system, macrophages, and adipocytes. Three key properties of LRP-1 determine its diverse physiological roles: first, its ability to recognize more than 30 different ligands; second, its ability to bind to multiple cytoplasmic adaptor proteins in a phosphorylation-specific manner via determinants located in its cytoplasmic domain; and third, its ability to associate with and regulate the activity of other transmembrane receptors, such as integrins and receptor tyrosine kinases.

[0080] We have found that providing nanoparticles or microparticles featuring ligands targeting the LRP-1 receptor enables them to cross the BBB and effectively deliver encapsulated drugs to both CNS parenchyma and CNS cells. In particular, we have found that the endothelial transcytosis mechanism does not involve acidification of nanoparticles or microparticles in membrane transport organelles, which is important for avoiding premature degradation of polymersomes and the concomitant release of encapsulated drugs. Furthermore, the LRP-1 receptor is involved in conventional endocytosis in CNS cells, aiding in the delivery of drugs (via disintegration of nanoparticles or microparticles) within the cytosol after crossing the BBB. In particular, we have found that providing ligands targeting the LRP-1 receptor enables nanoparticles or microparticles to achieve effective neuroprotection in the treatment of stroke.

[0081] Peptides that bind to receptor LRP-1 are known in the art.For example, Angiochem (Montreal, Canada) has developed a peptide that uses the LRP-1 pathway to cross the blood-brain barrier when conjugated to a drug cargo.One example of a peptide suitable for use in the present invention is Angiopep-2, a peptide with the sequence TFFYGGSRGKRNNFKTEEY.Further examples of suitable targeting moieties are disclosed in WO2013 / 078562, the contents of which are incorporated herein by reference in their entirety (specifically, the disclosed ligand peptide is incorporated herein by reference).

[0082] In one embodiment, at least one ligand species, preferably one ligand species, targets the SCARB1 receptor. The protein encoded by this gene is a plasma membrane receptor for high-density lipoprotein cholesterol (HDL). The encoded protein mediates cholesterol transfer from HDL to HDL. Furthermore, this protein is a receptor for hepatitis C virus glycoprotein E2.

[0083] Malignant tumors exhibit remarkable heterogeneity, to the extent that different cell types with distinct genetic backgrounds can be found even within the same tissue site. In contrast, scavenger receptor type B1 (SR-B1), a relatively consistent marker, has been found to be consistently overexpressed by most tumor cells. Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that promotes the uptake of cholesterol esters from circulating lipoproteins. Additional findings suggest an important role for SR-BI in cholesterol metabolism, signaling, motility, and cancer cell proliferation, potentially leading to key influences in carcinogenesis and metastasis. Recent findings indicate that the level of SR-BI expression correlates with aggressiveness and poor survival in breast and prostate cancer. Furthermore, genomic data indicate that high or low expression of SR-BI can promote poor survival, depending on the cancer type. SR-BI is considered a diagnostic and prognostic indicator of cancer, helping to elucidate the contribution of this protein to cancer initiation, progression, and survival.

[0084] Ligands that bind to SCARB1 are known in the art. One such ligand is poly(2-(methacryloyloxy)ethylphosphorylcholine) (PMPC).

[0085] Preferably, one ligand species on the nanoparticle or microparticle scaffold targets LRP-1 and another ligand species on the scaffold targets SCARB1.

[0086] In another embodiment, at least one ligand species, preferably one ligand species, is a ligand adapted to allow nanoparticles or microparticles to bind to cancer cells.Cancer cells typically have a high density of membrane receptors.Illustrative and non-limiting examples of such targeting moieties include proteins (mainly antibodies and their fragments), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates.

[0087] Examples of receptors for receptor-mediated transcytosis that are highly expressed in tumor cells include LRP-1, SCARB1, TFRC, folate receptor 1 (FOLR1), and epidermal growth factor receptor (EGFR). For example, SCARB1 is highly expressed in HeLa cells (cervical carcinoma) and FaDu cells (squamous cell carcinoma of the hypopharynx).

[0088] In one embodiment, at least one ligand species, preferably one ligand species, targets LRP-1. In another embodiment, at least one ligand species, preferably one ligand species, targets SCARB1. In another embodiment, at least one ligand species, preferably one ligand species, targets TFRC. In another embodiment, at least one ligand species, preferably one ligand species, targets FOLR1. In another embodiment, at least one ligand species, preferably one ligand species, targets EGFR.

[0089] In one embodiment, at least one ligand species, preferably one ligand species, targets the TFRC receptor, which encodes a cell surface receptor required for cellular iron uptake by the process of receptor-mediated endocytosis, which is required for erythropoiesis and neurological development.

[0090] Iron, as an essential element, plays a crucial role in various physiological and pathological processes. Iron metabolism operates at two levels: systemic and cellular, which are normally in balance. Disturbances in iron metabolism balance are associated with many diseases, including Alzheimer's disease, osteoporosis, and various cancers. In systemic iron metabolism, regulated by the hepcidin-ferroportin axis, plasma iron binds to transferrin (TF), which has two high-affinity binding sites for ferric iron. General cellular iron metabolism consists of iron uptake, utilization, and efflux. During the general cellular iron uptake process, transferrin receptors (TFRs) function as the most important receptor-mediated controls. TFR1 and TFR2 are two subtypes of TFRs that bind to the iron-transferrin complex and promote iron translocation into the cell. TFR1 is ubiquitously expressed on the surface of general cells, while TFR2 is specifically expressed in liver cells. TFR1 has received more attention than TFR2 due to its diverse functions in both invertebrates and vertebrates. Recent reports have shown that TFR1 is involved in many types of diseases, including anemia, neurodegenerative diseases, and cancer. Most importantly, TFR1 has been found to be abnormally expressed in various cancers. Therefore, TFR1 has been hypothesized as a potential molecular target for the diagnosis and treatment of cancer therapy.

[0091] In one embodiment, at least one ligand species, preferably one ligand species, targets folate receptor 1 (FOLR1). The protein encoded by this gene is a member of the folate receptor family. Members of this gene family bind folic acid and its reduced derivatives and transport 5-methyltetrahydrofolate into cells. The gene product is a secreted protein that is membrane-anchored via a glycosyl-phosphatidylinositol bond or exists in a soluble form. Mutations in this gene are associated with neurodegeneration due to impaired folate transport in the brain.

[0092] The folate cycle maintains important metabolic reactions and is essential for rapidly growing cells. Under physiological conditions, exogenous reduced folate (a water-soluble B vitamin) is primarily transported into cells via the low-affinity, high-capacity, ubiquitously expressed reduced folate carrier (RFC; a bidirectional anion exchange mechanism). Once inside the cell, folate plays an essential role in the biosynthesis of purines and thymidine, which are then required for DNA synthesis, methylation, and repair. Folate is also transported by high-affinity FRs. In humans, there are four FR isoforms (FRα, FRβ, FRγ, and FRδ). FRα, FRβ, and FRδ are attached to the cell surface by glycosylphosphatidylinositol anchors, while FRγ is a secreted protein. FRα is expressed on the cell surface in a tumor-specific manner, potentially enabling tumor localization as well as selective delivery of therapeutics to malignant tissues, minimizing secondary toxic side effects.

[0093] There are many unique advantages to utilizing FRs as diagnostic and therapeutic targets. First, FRα is located on the luminal surface of epithelial cells in most proliferating, non-tumor tissues and is inaccessible to the circulation. In contrast, FRα is expressed throughout cells in malignant tissues and is accessible via the circulation. Second, FRs have the ability to bind folate, a relatively harmless small molecule that can rapidly penetrate solid tumors and is amenable to chemical conjugation with other molecules. Once the folate conjugate binds to the FR, it is internalized, and FRα is rapidly recycled to the cell surface via the FR-mediated endocytic pathway. All of these factors highlight the potential role of FRα in the diagnosis and treatment of specific tumor types.

[0094] In one embodiment, at least one ligand species, preferably one ligand species, targets epidermal growth factor receptor (EGFR). The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to the ligand induces receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation.

[0095] Epidermal growth factor receptors (EGFRs) are a large family of receptor tyrosine kinases (TKs) expressed in several types of cancer, including breast, lung, esophageal, and head and neck cancers. EGFR and its family members are key contributors to a complex signaling cascade that regulates cancer cell proliferation, signaling, differentiation, adhesion, migration, and survival. EGFR binds to its cognate ligand, EGF, which further induces tyrosine phosphorylation and receptor dimerization with other family members, leading to enhanced and uncontrolled proliferation. Due to their multifaceted roles in cancer progression, EGFR and its family members have emerged as attractive candidates for anticancer therapy. Specifically, aberrant EGFR activity has been shown to play a critical role in tumor cell development and proliferation, among which it is involved in numerous cellular responses, including proliferation and apoptosis. The epidermal growth factor receptor (EGFR) signaling pathway is also a strong candidate for both initiating and determining the clinical outcome of many respiratory diseases. Deregulation of the EGFR pathway, leading to aberrant EGFR signaling, is associated with the early pathogenesis of pulmonary fibrosis, cancer, and numerous airway hypersecretory diseases, including COPD, asthma, and cystic fibrosis.

[0096] Ligands for binding to each of these receptors are well known in the art. Exemplary ligands for LRP-1 and SCARB1 are described above. Exemplary ligands for TFRCs, such as TFR1, are transferrin and transferrin mimic peptides. Exemplary ligands for FOLR1 are folic acid. Exemplary ligands for EGFR are the peptide YHWYGYTPQNVI.

[0097] Another example of the ligand is a ligand that allows nanoparticles or microparticles to bind with immune cells.Illustrative and non-limiting examples of such ligands include phosphorylcholine (discussed in more detail below), peptidoglycan, lipoprotein, glycolipid, lipopolysaccharide, lipopeptide, synthetic compounds such as loxoribine and bropirimine, peptidoglycan, acetylated / maleylated protein, modified low-density lipoprotein, polyanionic ligand, sulfated sugar, mannose-modified polysaccharide, fucose-modified polysaccharide, galactose-modified polysaccharide, protein and β-glucan.In targeting immune system cells, specific and precise targeting requires a particularly high level of discrimination / precision, which can be provided by the nanoparticles and microparticles of the present invention.

[0098] Targeting immune cells is thought to be important for the treatment of immune-related diseases such as autoimmune diseases and graft rejection, as well as for the improvement of prophylactic / therapeutic vaccines. Cell membranes provide a remarkable example of the spatiotemporal control of complex biological interactions, thanks to hundreds of different ligand-receptor interactions selected through evolution with the right amount of affinity and multicombinatorial binding.

[0099] Packaging antigens and adjuvants into vehicles such as polymersomes may offer significant advantages. This approach allows the contents of the vaccine carrier to be protected from potential degradation and shielded from premature interactions with undesired receptors, such as nucleic acids and scavenger receptors. Polymersomes are considered a good choice for future vaccine formulations. Polymer particles overcome the general stability challenges of liposomes and, in contrast to virus-like particles, can constitute a non-immunogenic vehicle, which may enable potential prime-boost regimens. Improved antigen processing and presentation as a result of co-localization of antigen and stimulus within the same phagosome may explain why co-delivery of antigen and adjuvant improves T cell responses. At the same time, this approach ensures activation of antigen-encountered cells, which is essential for effective CD8+ T cell priming. The main arguments in favor of co-targeting of antigen and adjuvant are a more controlled vaccine application and a reduced risk of side effects such as autoimmune reactions, induction of tolerance, or unwanted systemic cytokine release.

[0100] This concept can be applied to several different pathologies, such as incorporating targeting ligands to target APC cells, with applications in anti-cancer vaccines. A further example of a ligand is a ligand adapted to enable nanoparticles or microparticles to bind to neutrophils. Neutrophils are key effector cells in inflammation and play a key role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before being removed by macrophages, but delayed apoptosis can cause extensive tissue damage and chronic disease. Promoting neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, but neutrophils have proven experimentally to be difficult cells to manipulate.

[0101] Therapies targeting components of the defense system, such as neutrophils and neutrophil-associated effectors, hold promise for adjunctive host-directed therapy to improve antibiotic efficacy, i.e., in tuberculosis treatment, as well as to shorten both treatment time and long-term pathological sequelae.

[0102] However, neutrophils have proven to be very difficult cells to manipulate, and to our knowledge, there are no commercially available vectors that allow for efficient intracellular delivery of cargo within their short lifespan without compromising their viability and activation state.

[0103] High levels of immune cells, including neutrophils, are associated with adverse outcomes in some solid tumors, and new strategies to reduce their presence and activity are currently in clinical development. Thus, neutrophils are desirable targets for the nanoparticles and microparticles of the present invention.

[0104] Ligands can be attached to the outer surface of nanoparticles or microparticles using routine techniques, for example, by adapting well-known methods for attaching ligands to polymers, drugs, nucleic acids, antibodies, and other substances. Attachment can be non-covalent (e.g., electrostatic) or covalent, but is preferably covalent. For example, if the nanoparticle or microparticle is a polymersome, the targeting moiety can be attached by reacting a suitable functional group (including, but not limited to, amine, carboxyl, and thiol groups) on the targeting moiety with a corresponding functional group on at least one copolymer that forms or will form the polymersome. Attachment can be performed before the polymersome structure is formed from the copolymer, or after the polymersome is formed.

[0105] In a particularly preferred embodiment, the nanoparticles or microparticles are polymersomes comprising, on their outer surface, polymer brushes containing poly(ethylene glycol)-poly(2-(diisopropylamino)ethyl methacrylate) and the respective ligand species. Thus, the ligands are inserted into the polymer brushes of polymersomes made from poly(ethylene glycol)-poly(2-(diisopropylamino)ethyl methacrylate), typically by using a solvent-switching method. Typically, the density of the ligand within the brush can also be varied.

[0106] It is also possible to attach the ligand to the copolymer by first chemically activating either or both of the ligand and the copolymer.For example, a peptide ligand can be activated by adding a reactive species to one of its termini, such as a cysteine ​​moiety (its thiol group is well known to easily react with functional groups such as the widely used maleimide moiety).Similarly, a copolymer can be activated by functionalizing it with a reactive species (for example, a maleimide moiety when the targeting moiety has a thiol group).This reactive species can be provided to the copolymer by functionalizing the copolymer itself, or by providing a suitable monomer before polymerization to form the copolymer, or by providing a suitable initiator for polymerization.

[0107] In a particularly preferred embodiment, the nanoparticles or microparticles are polymersomes in which one or more ligands on the outer surface of the polymersome are covalently attached to poly(ethylene glycol) molecules. Tethering the ligands to PEG molecules of different chain lengths in this manner allows for control of the depth of ligand insertion into the polymer brushes. This, in turn, affects the steric repulsion potential, Es, between the ligand and the target cell surface receptor. As discussed above, this steric potential is an important factor in determining the optimal number of ligands on the surface of a nanoparticle or microparticle for binding to a particular cell type.

[0108] The ligand can be attached directly onto the exterior surface of the nanoparticle or microparticle, or alternatively, can be attached via a chemical spacer.

[0109] When the nanoparticle or microparticle is a polymersome, the ligand may be a pendant group of the polymer contained in the polymersome (i.e., at least one of the copolymers forming the polymersome itself). Obviously, in this embodiment, there is no need to perform a separate synthetic step to attach the ligand to the copolymer or the resulting polymersome.

[0110] Suitable pendant groups generally include any group that corresponds to a ligand as defined elsewhere herein. In one illustrative embodiment, the targeting moiety is a phosphorylcholine moiety, i.e., a group of the formula:

[0111] [ka]

[0112] is a group having the formula: The phosphorylcholine moiety is a zwitterionic moiety that can constitute a pendant group in one or more of the monomers that form the copolymer contained in the polymersome.

[0113] The phosphorylcholine moiety selectively targets scavenger receptor class B, member 1 (SCARB1), which is overexpressed by macrophages and other immune cells; in particular, it allows polymersomes featuring phosphorylcholine moieties to enter such cells. Thus, polymersomes featuring phosphorylcholine targeting moieties are particularly suitable for use in the treatment of inflammatory and / or immune disorders.

[0114] Pharmaceutical Composition The nanoparticles or microparticles of the invention can be formulated into pharmaceutical compositions using routine techniques known in the art, such as those already utilized to formulate nanoparticles or microparticles such as polymersomes or drug-containing liposomes.

[0115] The pharmaceutical composition comprises a plurality of nanoparticles or microparticles of the present invention. It also comprises one or more pharmaceutically acceptable excipients or diluents. The one or more pharmaceutically acceptable excipients or diluents can be any suitable excipients or diluents. The pharmaceutical composition is typically aqueous, i.e., it comprises water (especially sterile water).

[0116] The typical pH of the aqueous pharmaceutical composition is 7.0 to 7.6, preferably 7.2 to 7.4. Pharmaceutically acceptable buffers may be used to achieve the required pH. The pharmaceutical composition may be in the form of a sterile, isotonic saline solution.

[0117] Typically, the pharmaceutical composition is an injectable composition, for example, it is suitable for intravenous delivery, for example, it is suitable for infusion.

[0118] Medical Uses of Nanoparticles or Microparticles The nanoparticles or microparticles of the present invention can target tissues, including, but not limited to, cells across the blood-brain barrier (e.g., CNS cells), immune cells, and cancer cells, and release drugs upon localization at the target. As described above, the high efficiency of targeting is at least partially due to the presence of multiple different ligand species (i.e., targeting moieties) on the outer surface of the nanoparticles or microparticles (e.g., as part of the polymer itself or as separate moieties attached thereto).

[0119] Thus, the nanoparticles or microparticles of the present invention can be used in methods for improved targeted treatment of diseases and other pathological conditions.

[0120] As can be easily understood, the ligand and the encapsulated drug are selected according to the disease to be treated. For example, if the disorder is a brain disorder, the ligand is a ligand adapted to allow the nanoparticles or microparticles to pass through the BBB and typically cross the BBB to enter cells such as CNS cells, while the drug is a drug effective for treating or preventing brain disorders. If the disorder is an immune and / or inflammatory disorder, the ligand can be a ligand adapted to allow the nanoparticles or microparticles to bind to (and typically enter) immune cells, while the drug is a drug effective for treating or preventing immune and / or inflammatory disorders. If the disorder is cancer, the ligand can be a ligand adapted to allow the nanoparticles or microparticles to bind to (and typically enter) cancer cells, while the drug is a drug effective for treating or preventing cancer.

[0121] Examples of brain disorders include stroke, neurodegenerative diseases, traumatic brain injury (TBS), spinal cord injury, and neurotoxin consumption (e.g., methamphetamine overdose). Neurodegenerative diseases include conditions such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Stroke can be ischemic or hemorrhagic.

[0122] Examples of immune and / or inflammatory disorders include multiple sclerosis, psoriatic arthritis, rheumatoid arthritis, lupus erythematosus, and psoriasis.

[0123] Examples of cancers include: cancers of the skin, such as melanoma; lymph nodes; breast; cervix; uterus; gastrointestinal tract; lung; ovaries; prostate; colon; rectum; mouth; brain; head and neck; throat; testes; thyroid; kidney; pancreas; bone; spleen; liver; bladder; larynx; nasal cavity; AIDS-related cancers; cancers of the blood and bone marrow, such as multiple myeloma and acute and chronic leukemias, e.g., lymphoblastic, myeloid, lymphocytic, and myeloid leukemia; advanced malignancies, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforme multiformes), glioblastoma, brainstem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, malignant astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C&D colorectal cancer, unresectable colorectal cancer, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma, low-grade follicular lymphoma, metastatic melanoma (localized melanoma, including but not limited to intraocular melanoma), malignant mesothelioma, malignant pleural effusion syndrome mesothelioma syndrome, peritoneal carcinoma, serous papillary carcinoma, gynecological sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, eiomyosarcoma, fibrodysplasia ossificans progressive, hormone-refractory prostate cancer, resected high-risk soft tissue sarcoma, unresectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube carcinoma, androgen-independent prostate cancer, androgen-dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary, follicular, and medullary thyroid carcinoma, and leiomyoma.

[0124] Additional disorders that may be amenable to treatment or prevention with the nanoparticles or microparticles of the present invention include HIV, atherosclerosis, ischemic heart disease, and obstructive sleep apnea.

[0125] Medical uses and methods of treatment naturally involve the administration of a therapeutically effective amount of nanoparticles or microparticles. A therapeutically effective amount of nanoparticles or microparticles is administered to a patient. Typical doses are 0.001 to 1000 mg measured as the weight of the drug, depending on the activity of the particular drug, the age, weight, and condition of the subject being treated, the type and severity of the disease, and the frequency and route of administration. Preferably, the daily dosage level is 0.001 mg to 4000 mg.

[0126] The present invention further provides a method for treating or preventing a disorder, comprising administering a therapeutically effective amount of the nanoparticles or microparticles of the present invention to a patient in need thereof. For example, the present invention provides a method for treating or preventing a disorder selected from any of the disorders identified in this disclosure, wherein the drug is a drug capable of treating or preventing said disorder, such as a brain disorder, an immune and / or inflammatory disorder, or cancer. The present invention further provides the use of the nanoparticles or microparticles of the present invention in the manufacture of a medicament for use in the method for treating or preventing the above-identified disorders.

[0127] The present invention further provides a vaccine comprising the nanoparticles or microparticles of the present invention and an antigen. The antigen is any agent that can elicit an immune response in an animal's immune system, such as a bacterium, virus, or pollen. Typically, the vaccine is administered to a human or animal recipient to induce memory function in the adaptive immune system against the specific antigen contained in the vaccine. Preferably, the nanoparticles or microparticles in the vaccine selectively bind to dendritic cells. More preferably, the vaccine is a cancer vaccine. [Example]

[0128] Example The present invention will now be illustrated by the following examples, which, however, do not limit the scope of the present invention.

[0129] Example 1: Preparation of polymersomes Synthetic vesicles were fabricated using amphiphilic copolymers made with poly(ethylene glycol) (PEG) as the hydrophobic block and poly(2-(diisopropylamino)ethyl methacrylate) (PDPA) as the hydrophilic block.

[0130] P[(OEG) 10 MA] 20 -PDPA 100 , Cy5-P[(OEG) 10 MA] 20 -PDPA 100 , Angiopep-P [(OEG) 10 MA] 20 -PDPA 100 and PMPC 25 -PDPA 70 The copolymer was synthesized as reported in Tian et al., Sci Rep, 2015, 5, 11990, the contents of which are incorporated herein by reference in their entirety.

[0131] The Angiopep-2 peptide on the surface of the polymersome targets the LRP1 receptor, while the PMPC ligand targets the SCARB1 receptor. Approximately 5% of the POEGMA-PDPA chains were labeled with Cy5 dye to allow for fluorescence quantification. The Angiopep peptide was conjugated to POEGMA-PDPA copolymers, which were then mixed with the original POEGMA-PDPA at different concentrations. The resulting peptide sequences were expressed on the surface and expressed as δ p Oligoethylene oxide chains (N p PMPC chains were copolymerized with DPA to form PMPC 24 -PDPA 70 These were mixed with the original POEGMA-PDPA chains at different concentrations.

[0132] To prepare 10 mg / mL polymersomes, the copolymer was weighed and dissolved in PBS at pH 2. Once the film dissolved, the pH was raised to 5.0. To avoid acidic degradation, the peptide-functionalized copolymer was then added. The pH was gradually increased from 6.8 to 7.0 and finally stopped at 7.4 to 7.5. Polymersomes formed during prolonged stirring at pH 6.8 to 7.0. The polymersomes were then sonicated for 15 to 30 minutes at 4 °C. Finally, polymersome purification was performed by passing them through a gel permeation chromatography column packed with Sepharose 4B (Sigma-Aldrich). For long-term storage, polymersomes can be kept at 4 °C and protected from light once conjugated with the dye. Peptide-functionalized polymersomes were prepared fresh immediately before use. In this regard, it is important to note that although POEGMA-PDPA and PMPC-PDPA chains can undergo phase separation to form patchy polymersomes (see LoPresti et al., ACS Nano, 2011, 5(3), 1775-1784), the cell experiments were performed immediately after preparation and therefore did not allow sufficient time (3–5 days) for separation.

[0133] The size distribution of the polymersomes was measured by dynamic light scattering (DLS) (see Figures 3a-3c). The average radius of all formulations was 50 nm (+ / - 10 nm), and the addition of ligands did not alter the final structure, as confirmed by both TEM and DLS. The polymersomes were further characterized by transmission electron microscopy (FEI Tecnai G2) and dynamic light scattering (Malvern Nanosizer) using phosphotungstenic acid as a stain (see Figures 3d and 3e).

[0134] Example 2: Binding of polymersomes to brain endothelial cells Brain endothelial bEND.3 cells (ATCC CRL-2299) were seeded onto rat tail collagen type I (Sigma-Aldrich, C3867)-precoated T-75 flasks maintained in DMEM (Dulbecco's Modified Eagle's Medium - High Glucose, D5671-Sigma) supplemented with 2 mM glutamine, 100 IU / mL penicillin, 100 mg / mL streptomycin, and 10% fetal calf serum (FCS). Cultures were maintained at 37°C in an atmosphere of 5% CO2 and 95% air. Upon reaching 100% confluence, they were routinely subcultured using 0.02% (w / v) EDTA trypsin (5 mL, 5 min, 37°C, 5% CO2 incubation). LADMAC macrophages were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% (v / v) FCS and 2 mM glutamine.

[0135] Cells were placed in 96-well plates with a plastic image-reading bottom and treated with different polymersome formulations for 1 hour. Their medium was then replenished. Although nanoparticle / cell interactions are kinetically controlled by binding and endocytosis, a short incubation time was chosen to ensure that this accounted for a negligible portion of the overall process. Cells were imaged using confocal laser scanning microscopy with an incubation chamber connected to a ZEISS temperature control unit 37-2 and CO2 controller (temperature and CO2 concentration stabilization was performed 1–2 hours before the experiment). Confocal laser scanning microscopy was performed on a ZEISS LSM 510 microscope equipped with the following lasers: Ar laser, 30 mW; HeNe laser, 1 mW; and HeNe laser, 5 mW. The laser excitation wavelengths used were 405 nm (DAPI) and 548 nm (Cy5-polymersome). Fluorescence intensity was quantified in triplicate on 30 micrographs per formulation. Images were analyzed by ImageJ by creating ad hoc regions of interest around nuclei (pre-stained with DAPI) and measuring the intensity of the Cy5 channel.

[0136] The results are shown in Figure 4. In Figure 4a, an example of a micrograph used for quantification is shown to illustrate the effective binding of ligand-modified polymersomes to brain endothelial cells, while Figures 4b and 4c show the mean fluorescence per cell measured after 1 hour of incubation with brain endothelial cells, macrophages, and lymphocytes. To assess the ability of polymersomes to selectively target a given cell phenotype, a parameter known as the selectivity index, s, was calculated:

[0137]

number

[0138] (In the formula, F BE is the mean fluorescence per cell in the brain endothelial cells (i.e., target cells), and F Swhere s is the mean fluorescence per cell in lymphocytes or macrophages (here considered sentinel cells). Formulations with s > 1 interact preferentially with target cells over sentinel cells, while formulations with s < 0 interact preferentially with sentinel cells, and formulations with 1 ≥ s ≥ 0 are promiscuous and do not bind with high selectivity to either target or sentinel cells.

[0139] As shown in Figure 4b, Angiopep-decorated polymersomes preferentially interact with brain endothelial cells over the two sentinel cell types, with selectivity peaking at 2.5 when approximately 30 ligands are present. As expected, with higher ligand numbers, selectivity is lost and polymersomes interact equally with all cell populations.

[0140] Very different results were observed with PMPC-decorated chains, where (although the fluorescence output of brain endothelial cells was similar in magnitude to that observed with Angiopep polymersomes) macrophages showed the highest uptake, followed by lymphocytes and finally brain endothelial cells, where selectivity was negative for brain endothelial cells.

[0141] Three PEG-PDPA polymersomes expressing both Angiopep and PMPC ligands were also synthesized and incubated with brain endothelial cells. The results are plotted in Figure 5. Using Equation (1) for this particular system, the optimal number of Angiopep ligands is 20-30, and the optimal number of PMPC ligands is 400-600 (room temperature, 25°C). As suggested, these show good correlation between the predictive empirical equation (1) and the experimental data. For this particular system, the key parameters for applying equation (1) are:

[0142] PMPC parameters: [P]=2nM R=50nm d=10nm N(PEO)=10 Density PEO = 0.5 nm 2 Density sugar coating = 5nm 2 δ P =0.6 δ G =0.2 K D (PMPC) = 5 × 10 -8 M <srb1>Density = 34um for brain endothelial cells -2 <srb1>Density=センチネルcell (white blood cell)について24um -2

[0143] Angiopep parameters: [P]=2nM R=50nm d=10nm N(PEO) = 10 Density PEO = 0.5 nm 2 Density sugar coating = 5nm 2 δ P =0.65 δ G =0.95 K D (Angiopep) = 3.13 × 10 -7 M <lrp1>Density = 14um for brain endothelial cells -2 <lrp1>Density = 28um for sentinel cells (white blood cells) -2

[0144] Polymersome radius R, and insertion parameter δ G and δ P Steric potential E as a function of s The calculation of Θ ... steric , and the steric hindrance exerted by polymersomes inserting into the glycocalyx, U steric are R and δ in Figures 6f and 6g, respectively. P or δ G It is mathematically modeled as a function of E s U demonstrated by inserting LRP1 into a PEO brush steric and U exerted by polymersomes that insert into the glycocalyx. steric is the sum of

[0145] Interestingly, experimental results show that the two ligands act synergistically, enabling targeting using a number of ligands that would not be compatible with either interaction alone. These lower limits on the effective number of ligands are also accurately predicted by empirical equation (1). This validates the ligand multiplexing strategy as a highly effective method for designing nanoparticles or microparticles for targeted drug delivery.

[0146] Example 3: Binding of polymersomes to antigen-presenting cells Here, we illustrate the incorporation of targeting ligands to target APC cells with application to anti-cancer vaccines.

[0147] Using data from the human protein atlas, a bioinformatics search was performed for cross-matching receptors expressed on antigen-presenting cells. The results are shown in the spider plot in Figure 7, which shows the expression of scavenger receptor B1 (SRB1), transferrin (TFRC), lipoprotein receptor-related protein 1 (LRP1), and mannose receptor in healthy and tumor tissues. The data indicate that only tumor tissue expresses high levels of all three receptors, while healthy tissue expresses only one or two receptors at high levels.

[0148] These receptors will be targeted using clinically approved ligands attached to the surface of polymersomes to create the desired superselective effect.

[0149] Example 4: Binding of polymersomes to neutrophils Using data from the human protein atlas, a bioinformatics search was performed for cross-matching receptors expressed on antigen-presenting cells. Figure 8 shows a spider plot illustrating the expression of SRB1, FCAR, and SLFN12L receptors in healthy and inflamed tissues. The data indicate particularly high levels of expression of SRB1 and SLFN12L in inflamed tissues.

[0150] These receptors will be targeted using clinically approved ligands attached to the surface of polymersomes to create the desired superselective effect.

[0151] Example 5: Binding of polymersomes to tumor cells Peritoneal carcinomatosis (PC) is the metastatic invasion of tumor cells into the lining of the peritoneal cavity and intraperitoneal organs. PC is a rare primary tumor, occurring in 60% of gastric, 40% of ovarian, and 35% of colon malignancies. Median survival is 1-3 months. After treating the tumor with a balanced combination of chemotherapy and immunomodulation, it has been proposed to stimulate the immune system with "find and kill-me" signals to attack tumor metastases.

[0152] In combination, we identified three distinct receptors overexpressed by tumor tissue: scavenger receptor B1 (SRB1), transferrin (TFRC), and epidermal growth factor receptor (EGFR). The spider plot in Figure 9 shows the expression of these receptors in healthy peritoneal tissues (liver, colon, female reproductive tract) as well as in the three most common PC-associated tumors: stomach, colorectum, and ovary. The data show that only tumor tissue expresses all three receptors at high levels, while healthy tissue expresses only one or two receptors at high levels.

[0153] These receptors can be targeted using clinically approved ligands attached to the surface of the particles of the present invention (polyNauts particles), creating the desired ultraselectivity. These are poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) for SRB1, folic acid for FOLR1, and cetuximab for EGFR. PMPC has been approved for coating stents, contact lenses, and catheters and has been in clinical use for 10 years. Folic acid, also known as vitamin B9, is an FDA-approved common dietary supplement and a ligand used in several anticancer therapies. Finally, cetuximab is a monoclonal antibody approved for the treatment of colon cancer driven by wild-type KRAS and is under clinical evaluation for several other malignancies.

[0154] Example 6: Binding of polymersomes to glioma cells Insufficient and inefficient drug delivery is a major reason for the failure of pediatric brain tumor treatment. The integrity of the blood-brain barrier (BBB) ​​can confer complete resistance to drug therapy to some pediatric tumors (e.g., diffuse midline gliomas), as can partial disruption of the BBB (e.g., medulloblastomas and ependymomas). High doses of systemic chemotherapy are also required, resulting in significant and dose-limiting side effects. Therefore, improving drug transport across the BBB in a tumor-specific manner could result in both improved survival and reduced systemic toxicity.

[0155] The challenge of creating a targeted formulation that crosses the BBB and enters brain tumors limits the selection of potential receptors. Using data from the human protein atlas, we performed a bioinformatics search for cross-matching receptors that are expressed at high levels in both brain endothelial cells and glioma cells, but not together in other tissues. The data shown in Figures 10a and 10b confirm that the BBB markers low-density lipoprotein receptor-related protein 1 (LRP1) and glucose transporter (GLUT1) are also expressed in glioma cells. LRP1 coincides with the expression level of the established adult cancer-associated marker, epidermal growth factor receptor (EGFR). Similarly, the pediatric tumor marker platelet-derived growth factor receptor alpha (PDGFR-α) is expressed at high levels in BECs. These data suggest that it is possible to create the binding profile required to target only both classes of cells. As shown in the spider plot in Figure 10c, this data can be used to tailor the correct ligand composition to the polymersome surface (using the empirical model described in Equation (1)) through superselective design to achieve selective targeting of these cells.

[0156] Example 7: Determining the optimal number of ligands on the polymersome surface Experiments were performed to determine the effect of Angiopep-2 ligand density on the polymersome surface on the extent of adsorption to target cells. The results of this experiment were compared to the trends predicted by the theoretical model and are shown in Figure 11.

[0157] A. Theoretical Model As previously shown (see, for example, Martinez-Veracoeachea and Frenkel, PNAS, 2011, 108(27), 10963-10968 and Angioletti-Urberti, Phys Rev Lett, 2017, 118(6):068001), the adsorption probability θ for nanoparticle binding to a cell surface can be described by a Langmuir-type equation:

[0158]

number

[0159] where N x=L,R are the numbers of ligand and receptor, respectively, and ΔG is the binding free energy (throughout this example, energy is always expressed in terms of thermal energy k B It is considered to be scaled by T, where k B is the Boltzmann constant and T is the temperature). < > denotes the mean of the Poisson distribution. This mean takes into account the heterogeneity of the spatial distribution of receptors and / or ligands, which can be related either to the grafting procedure or to the binder mobility on the surface. z is the activity of the nanoparticles in bulk solution, which can be equal to their number density for uniformly coated nanoparticles and dilute solutions (see Martinez-Veracoeachea and Frenkel, PNAS, 2011, 108(27), 10963-10968).

[0160] The central parameter in equation (2) is q, the partition function of the bound nanoparticle, which depends on the number of ligands and receptors available for binding and the strength of their binding. This partition function is given by q=v bind exp(-F tot ), where v bind =π(R np ) 2 L is the binding volume of the absorption site, and R np is the size of the nanoparticle, and L is the distance range over which the particles can bind, which can be approximated as the radius of gyration of the ligand tether. tot is the free energy of adsorption, which is F att (attractive contribution resulting from the formation of the ligand-receptor bond) and F rep (equal to the sum of contributions of repulsions due to steric repulsion between different components of the nanoparticle and the target cell within the crowded environment of the binding region, e.g., receptor interactions with polymer brushes on the nanoparticle surface, and ligand interactions with the cell glycocalyx).

[0161] Attractive contribution F to the free energy of adsorption of a polymersome to its target receptor att Assuming that is governed by the bond formation between the ligand and receptor, we have:

[0162]

number

[0163] where the sum is N φ over all configurations φ with a given number of bonds. ΔG is the energy of a single bond (which depends on the particular ligand-receptor pair chosen), and Ω(φ) is the number of configurations with a particular number of bonds. To calculate this quantity, a particular binding scenario must be chosen. The different binding scenarios are determined by the number of allowed configurations Ω(φ), which is given by S=k B The contribution of the weakest possible bond is to the "indifferent binding" (as described in Kitov and Bundle, J Am Chem Soc, 2003, 125(52), 16271-16284). In this case, only a single ligand can bind to the receptor at a time, and we have Ω(φ)=N R N L where N R is the number of receptors on the cell surface, and N L is the number of ligands on the polymersome surface, Nφ=1, and F att =-ln(N R )-ln(N L Alternatively, in the case of radial binding, all ligands can bind to all receptors (but in each configuration, only a single ligand can bind to a particular receptor, and vice versa):

[0164]

number

[0165] The sum in (3) ranges from 0 to min(N L ,N R ) This partition function cannot be written in an exact form, and calculating it by force is computationally inefficient. However, as shown by Angioletti-Uberti et al. (J Chem Phys, 2013, 138, 21102-21106), the ligand-receptor mediated energy in all possible binding scenarios (except when both the number of ligands and the number of receptors are simultaneously 1; see Tito and Angioletti-Uberti, J Chem Phys, 2016, 144(16), 161101), including the radial case, can be calculated by a set of binding equations k B It can be approximated to within some accuracy of T:

[0166]

number

[0167]

number

[0168] Here, χ = exp(-ΔG) can be interpreted as the strength of a single bond (see Angioletti-Uberti, Phys Rev Lett, 2017, 118(6):068001), which increases for lower values ​​of ΔG. In (6), the index i refers to any ligand or receptor in the system, and the sum extends to all binding partners j of i. Thus, the N L +N R In a radial binding scenario, each ligand or receptor has the same number of neighbors (N for ligands). R or N for receptors L ), so the previous expression reduces to just two combined expressions:

[0169]

number

[0170] The simultaneous solution is

[0171]

number

[0172]

number

[0173] (and p R We derive a symmetric equation for varying the pedices L, R for , which is used to plot the curves in Figure 11 for the radial coupling scenario.

[0174] F rep Regarding , by combining previous results by Halperin (Langmuir, 1999, 15(7), 2525-2533) and Zhulina (Eur Phys JE: Soft Matter Biol Phys, 2006, 20(3), 243-256 and Macromolecules, 2012, 45(11), 4429-4440), we can build a model to calculate the repulsive free energy for inserting an object into a polymer brush on a curved surface. Within this model, we can derive:

[0175]

number

[0176]

number

[0177] where V R is the volume of the receptor, σ is the average area per polymer chain, δ = (z / h0) ∈ [0,1] is the average brush height h0 = N(ν a 2 / 3σ) 1 / 3 The distance between the surface and the object measured by, N is the degree of polymerization, ν=a 3 is the volume of a monomer of size a, and finally γ is a parameter that depends on the radius R of the nanoparticle core relative to the brush height, and γ = 3 if h0 / R > (√3-1), otherwise γ = (1 + h0 / R). 2 is.

[0178] B. Fitting of experimental data The theoretical curves in Figure 4 were obtained by fitting the experimental data using equations (2)–(10) above, where double Poisson averaging was taken for both the number of receptors per site and the number of interacting ligands to account for heterogeneity in the polymersome functionalization. To calculate the attractive force contribution, a radial binding scenario was assumed. The binding distance L was approximated to be the most likely end-to-end distance of the tether used to graft the ligand to the nanoparticle, which, when treated as a Gaussian chain, was L = 2R in the test experimental system. G = 6 nm (see below). Furthermore, R = R np +h, h = 8 nm is the brush height estimated from the degree of polymerization of the protective PEO coating and its grafting density using the Zhulina model (see Eur Phys JE: Soft Matter Biol Phys, 2006, 20(3), 243-256), and R np = 50 nm is the size of the nanoparticles experimentally determined by dynamic light scattering and cryoTEM experiments.

[0179] These values ​​also give δ = 0.75 in (10) and γ = (1 + h0 / R) for the estimation of the repulsive contribution by the receptor through (10), which allows for V for the Angiopep receptor as predicted from known structural data. P =188nm 3 (See Xiaohe Tian et al., 2019, Sci Adv.) This leaves three fitting parameters: the reference grafting density of ligands on the surface, σ L (Only the ratio of the number of ligands between different polymersomes is known, not the absolute values, see below); the average grafting density of the receptors and the repulsive contribution per ligand, thus similar to (9), can be calculated by the simple formula F rep =AN L applies, where A includes the combined effects of all unknown parameters describing the cellular glycocalyx, as well as all excluded volume interactions between the ligand and the cell surface. Taking these equations into account, experimental data can be fitted using Monte Carlo annealing to minimize the amount

[0180]

number

[0181] where w i is the experimental data θ i,exp , can be considered as the reciprocal of the msrd, the experimentally measured adsorption normalized by the maximum value among all polymersomes of different grafting densities. The procedure starts with an effective temperature of 1 and continues until the temperature is increased to 10 -7 The temperature is scaled by a factor of 0.95 every 100 MC sweeps until a value of σ is reached, at which point the system stops evolving. L =1.88 10 -3 / nm 2 fitting parameters of , or ∼7 interacting ligands within the polymersome region (relative to the reference polymersome) that are in contact with the binding site, ρ R =4.3×10 -5 / nm 2 , or an average of 0.32 receptors per adsorption site, and A = 0.62k B The repulsion parameters of T were generated.

[0182] C. Preparation of Polymersomes PEO-b-PDPA and N3-PEO-b-PDPA copolymers were synthesized by atom transfer radical polymerization as previously reported (see Blanazs et al., Adv Functional Mat, 2009, 19(18), 2906-2914 and Gaitzsch et al., Polymer Chem, 2016, 7(17), 3046-3055). 113 -PDPA 100 ) and ligand conjugation (Angiopep2-PEG 68 -PDPA 90 For the PEG-conjugated (AP-alkyne) conjugation, 1 equivalent of N3-PEO-b-PDPA was first assembled in PBS by a pH-switch procedure (as described in Contini et al., IScience, 2018, 7, 132-144). The self-assembled polymer solution was then degassed by sonication and inert gas flow under stirring. The degassed solution was mixed with 1.2 equivalents of the corresponding ligand. For the peptide conjugation (AP-alkyne), the peptide was dissolved in degassed PBS pH 7.4, and a water-insoluble ligand, such as Cy5-alkyne, was added to degassed dimethyl sulfoxide (DMSO) to a final DMSO:PBS ratio of 10:1. Sodium ascorbate (5 equivalents) was then added, and the mixture was further degassed for at least 30 minutes. Finally, 1 equivalent of CuSO4 was added under an inert atmosphere and the reaction was allowed to proceed at 40 °C for 72 hours, protected from light. The labeled polymers were purified by dialysis against DMSO and then against water (MWCO of at least 5 KDa for peptide purification and 3.5 KD for dye purification). The labeled polymers were recovered after lyophilization. To prepare polymersomes with increasing amounts of ligand, copolymer PEG was used. 113 -PDPA 80 Angiopep2-PEG 68 -PDPA 90 (0-10 mol%) and Cy5-PEG 113 -PDPA 100 The polymersome solution was mixed with 10 mol% PBS (10 mol%) and dissolved in tetrahydrofuran:dimethyl sulfoxide (90:10) to a final total polymer concentration of 20 mg / mL. 2.3 mL of PBS pH 7.4 (aqueous phase) was injected into the organic solution at 2 L / min using an automated syringe pump. The addition of the aqueous phase was performed under continuous stirring at 40 °C. After injection, an additional volume of PBS (pH 7.4) (3.7 mL) was added manually. To remove residual organic solvent, the polymersome solution was transferred to a semipermeable cellulose membrane (3.5 kDa cutoff) and dialyzed against PBS (pH 7.4) at room temperature for over 24 h. The sample was centrifuged at 1000 r.cf for 10 min, sonicated at 4 °C for 20 min, and purified on a size-exclusion chromatography (SEC) column packed with Sepharose 4B. All samples were stored at 4 °C and protected from light until further use. The average size and morphology of polymersomes were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS measurements were performed using a Malvern Zeta sizer equipped with a He-Ne 4 mW 633 nm laser, diluting the polymersome solution with PBS (pH 7.4) in a disposable polystyrene cuvette. For TEM analysis, polymersomes were deposited on glow-discharged carbon-coated copper grids for 1 min and then stained with 0.5% (w / v) PTA solution for 2 s.

[0183] D. Measurement of Adsorption Probability FaDu cells (ATCC HTB-43) were seeded at a density of 20,000 cells per well in 8-well chamber slides (iBidi) and maintained in MEME (Minimum Essential Medium Eagle M5650 - Sigma) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in 5% CO2. After 24 hours, the medium was removed, the cells were washed three times with Dulbecco's phosphate-buffered saline (DPBS), and fixed with 3.7% paraformaldehyde (v / v in DPBS) for 10 minutes at room temperature. The fixed cells were incubated with Cy5-labeled and Angiopep2-decorated polymersomes (0.15 mg / mL) in PBS (pH 7.4) for 1 hour at 37°C. After 1 hour, the polymersome solution was removed, the cells were washed three times with DPBS, and treated with CellMask Green (1:1000 in DPBS) for 5 minutes at room temperature. Cells were left in Live Imaging Solution, and polymersome adsorption to the cell membrane was analyzed using a Leica SP8 confocal laser scanning microscope equipped with a 63X oil immersion lens. Total emission fluorescence of Cy5-labeled polymersomes was measured in the range of 650–700 nm using an excitation wavelength of 633 nm and collected using a z-stack of 30 images. Image data were acquired on 50 cells for each Angiopep2 fraction, which were examined and processed using ImageJ software. All experiments were performed in triplicate.

Claims

1. 1. A polymersome for selectively binding to the surface of a brain endothelial cell, comprising: the polymersome comprises at least a first ligand species on its outer surface and at least a second ligand species on its outer surface, the first ligand species being Angiopep-2 and capable of binding to low density lipoprotein receptor-related protein 1 (LRP-1) on the surface of the cell, and the second ligand species being poly(2-(methacryloyloxy)ethyl phosphorylcholine) and capable of binding to scavenger receptor class B, member 1 (SCARB1) on the surface of the cell, and the polymersome further comprises 10 to 30 Angiopep-2 ligands and 200 to 800 poly(2-(methacryloyloxy)ethyl phosphorylcholine) ligands, or 20 to 30 Angiopep-2 ligands and 50 to 800 poly(2-(methacryloyloxy)ethyl phosphorylcholine) ligands; Additionally, the polymersome comprises polymer brushes on its outer surface, the polymer brushes comprising poly(ethylene glycol) (PEG) or poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), and The polymersomes have a diameter of 50-150 nm.

2. 10. The polymersome of claim 1, further comprising a drug encapsulated within the polymersome.

3. 3. The polymersome of claim 2, wherein the drug is selected from neuroprotective agents, immunomodulatory agents, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying antirheumatic drugs (DMARDs), immunosuppressants, TNF-alpha inhibitors, and anticancer agents.

4. The polymersome of any one of claims 1 to 3, wherein the polymersome comprises 20 to 30 Angiopep-2 ligands.

5. The polymersome of any one of claims 1 to 4, wherein the polymersome comprises 400 to 600 poly(2-(methacryloyloxy)ethylphosphorylcholine) ligands.

6. 6. The polymersome of any one of claims 1 to 5, wherein the polymersome comprises 20 to 30 Angiopep-2 ligands and 400 to 600 poly(2-(methacryloyloxy)ethylphosphorylcholine) ligands, and the polymersome comprises, on its outer surface, a polymer brush comprising poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA).

7. A pharmaceutical composition comprising a plurality of polymersomes according to any one of claims 1 to 6, and one or more pharmaceutically acceptable excipients or diluents.

8. A polymersome according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use in the treatment of a disease.

9. 9. The polymersome or pharmaceutical composition for use according to claim 8, wherein the disease is cancer.

10. Use of a polymersome according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for treating cancer in a patient.

11. A vaccine comprising the polymersome of any one of claims 1 to 6 and an antigen.

Images