Use of polymer derivatives and lipid nanoparticle modifiers

Abstract

The present invention relates to polymer derivatives comprising a hydrophilic segment obtained from the polymerization of N-methyl-N-vinylacetamide (PNMVA) coupled to at least one hydrophobic segment, wherein the hydrophobic segment comprises at least one aliphatic chain of 8 to 28 carbon atoms. The present invention also relates to methods for preparing such polymer derivatives. Lipid nanoparticles modified with PNMVA-based derivatives were prepared and advantages were demonstrated compared to PEG-modified lipid nanoparticles. The present invention also relates to the medical use of lipid nanoparticles containing PNMVA-based derivatives, in particular for nucleic acid delivery, e.g., for siRNA delivery to targeted tumor cells.

Description

[Technical Field]

[0001] The present invention relates to polymer derivatives and methods for preparing such polymer derivatives. The present invention also relates to lipid nanoparticles comprising such polymer derivatives and the use of such lipid nanoparticles in medical procedures, particularly for nucleic acid delivery. [Background technology]

[0002] Nanomedicines are nanotechnology-based products for drug delivery. They are considered a promising strategy for treating different diseases, such as cancer, or for vaccination. Nanomedicines use nanocarriers for the stable and efficient delivery of small active pharmaceuticals or activators such as nucleic acids. Lipid nanoparticles, such as liposomes, are the most common nanocarriers for activator delivery.

[0003] Liposomes are spherical, self-assembling structures formed by one or more concentric lipid bilayers composed of natural and / or synthetic lipids, with an encapsulated aqueous phase at their center. Liposomes offer several advantages in delivering therapeutic molecules, such as nucleic acids, to cells. For example, liposomes can encapsulate or complex charged molecules (both charged and uncharged), provide some protection to the encapsulated or complexed molecules from degradation processes, and target specific cells or tissues.

[0004] For example, as described in Lechanteur et al., Eur.J.Pharm.Sci.2016,93,493-503 and Berger et al., Int.J.Pharm.2021,605, liposomes are often modified with polyethylene glycol (PEG) to make them stealthy. Such PEGylation results in a reduced affinity of liposomes to phagocytic cells, allowing them to evade recognition by the reticuloendothelial system and extend their in vivo circulation time.

[0005] While PEGylated lipid nanoparticles offer many advantages, PEGylation presents several problems: (i) steric hindrance of the PEG chain inhibits liposome uptake by target cells; (ii) PEG prevents liposome "endosome escape," leading to degradation and impairment of encapsulated drugs within lysosomes (these first two problems are commonly known as the PEG dilemma); (iii) repeated injection of PEGylated liposomes in the same subject induces the phenomenon of "accelerated blood clearance," known as the ABC phenomenon; and (iv) hypersensitivity reactions can occur, potentially inducing serious allergic reactions.

[0006] Therefore, there is a great need for compounds that provide stealth properties to lipid nanoparticles while avoiding one or more of the above problems, in particular the PEG dilemma, and / or immunogenic reactions such as the ABC phenomenon and / or hypersensitivity. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] To our surprise, the inventors have found that polymer derivatives based on poly(N-methyl-N-vinylacetamide) (PNMVA) can solve one or more of the above-mentioned problems.

[0008] To our surprise, we have found that polymer derivatives based on N-methyl-N-vinylacetamide (NMVA), having at least one hydrophobic segment containing at least one aliphatic chain of 8 to 28 carbon atoms, can play an important role in modifying lipid nanoparticles, such as lipid nanoparticles specifically for siRNA delivery.

[0009] An advantage of the polymer derivative of the present invention is that it has amphiphilic properties. An advantage of the polymer derivative of the present invention is that it is not cytotoxic. An advantage of the polymer derivative of the present invention is that it is not toxic to living organisms. An advantage of the polymer derivative of the present invention is that it is blood-compatible. An advantage of the polymer derivative of the present invention is that it can be inserted into lipid nanoparticles.

[0010] An advantage of lipid nanoparticles containing the polymer derivative of the present invention is that they have good stealth properties. Lipid nanoparticles containing the polymer derivative of the present invention can effectively avoid protein corona formation. An advantage of lipid nanoparticles containing the polymer derivative of the present invention is that they have gene silencing efficiency. An advantage of lipid nanoparticles containing the polymer derivative of the present invention is that they have an extended in vivo circulation time. A further advantage of lipid nanoparticles containing the polymer derivative of the present invention is that they do not induce inflammatory responses in vivo. Compared to PEGylated nanoparticles, lipid nanoparticles containing the polymer derivative of the present invention are advantageous in that they exhibit a reduced ABC phenomenon. Lipid nanoparticles containing the polymer derivative of the present invention are also advantageous in that they exhibit less immunogenicity. An advantage of lipid nanoparticles containing the polymer derivative of the present invention is that they provide good efficacy in siRNA delivery. [Means for solving the problem]

[0011] A first object of the present invention is to provide a polymer derivative comprising a hydrophilic segment obtained from the polymerization of N-methyl-N-vinylacetamide, coupled to at least one hydrophobic segment, wherein the hydrophobic segment comprises at least one aliphatic chain of 8 to 28 carbon atoms.

[0012] The polymer derivative of the present invention is composed of a hydrophilic segment and a hydrophobic segment, which gives it unique amphiphilic properties. For example, thanks to the hydrophobic segment, the polymer derivative can interact with lipid-based substances. Thanks to the hydrophilic segment, the polymer derivative can be soluble in aqueous environments.

[0013] The hydrophilic segment in the polymer derivative of the present invention is a polymer segment. A polymer means that it is obtained from the polymerization of repeating units (also called monomers). It is obtained from the polymerization of a vinyl monomer, which is N-methyl-N-vinylacetamide. It may also be called poly(N-methyl-N-vinylacetamide) or PNMVA. Advantageously, PNMVA may be water-soluble.

[0014] The term "polymer segment" also refers to a copolymer. A copolymer means that it can be obtained from the polymerization of two or more repeating units (repeating units of different types). For example, a hydrophilic segment can be obtained from the copolymerization of N-methyl-N-vinylacetamide with one or more other monomers. For example, it can be obtained from the copolymerization of N-methyl-N-vinylacetamide with N-methylvinylpyrrolidone.

[0015] The hydrophobic segment in the polymer derivative of the present invention may consist, for example, of a single aliphatic chain of 8 to 28 carbon atoms, which may be saturated or unsaturated. Alternatively, the hydrophobic segment may consist of 2 to 4 aliphatic chains, which may be all saturated, all unsaturated, or a combination of saturated and unsaturated. The hydrophobic segment may consist of two aliphatic chains, both saturated, both unsaturated, or one saturated and one unsaturated. As long as one of the aliphatic chains has a length of 8 to 28 carbon atoms, the two or more aliphatic chains may have the same length or different lengths.

[0016] An aliphatic chain refers to a hydrocarbon chain, preferably containing only carbon and hydrogen atoms. Preferably, the aliphatic chain is an acyclic hydrocarbon chain. An aliphatic chain with 8 to 28 carbon atoms allows interaction with lipid-based substances. Interaction with lipid-based substances may, for example, involve the insertion of hydrophobic segments into lipid membranes.

[0017] Preferably, the aliphatic chain has a length of 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms.

[0018] Preferably, the polymer derivative includes at least one hydrophobic segment selected from the group of hydrophobic segments comprising: a single aliphatic chain of 8 to 28 carbon atoms; two aliphatic chains of 8 to 28 carbon atoms; one or two fatty acids having an aliphatic chain of 8 to 28 carbon atoms, preferably 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms; a diglyceride having two aliphatic chains of 8 to 28 carbon atoms, preferably 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms; a ceramide having an aliphatic chain of 8 to 28 carbon atoms, preferably 8 to 24 carbon atoms, more preferably 8 to 18 carbon atoms, and even more preferably 8 to 16 carbon atoms; or a phospholipid having an aliphatic chain of 8 to 28 carbon atoms, preferably 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms.

[0019] Preferably, the hydrophobic segment is composed of at least one saturated aliphatic chain having 8 to 28 carbon atoms, preferably 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and even more preferably 16 to 18 carbon atoms.

[0020] Preferably, the hydrophobic segment is a nonpolymer compound. Nonpolymer means it is not obtained from the polymerization of one or more repeating units. Preferably, the hydrophobic segment has up to four aliphatic chains, each having 8 to 28 carbon atoms. This is advantageous for efficient insertion into lipid membranes.

[0021] Preferably, at least one aliphatic chain of 8 to 28 carbon atoms is a terminal chain in the hydrophobic segment. A terminal chain refers to the position of a chain at the end of a hydrophobic segment that is not coupled to a hydrophilic segment. Here again, this can be advantageous for insertion into lipid-based materials.

[0022] A preferred phospholipid is phosphatidylethanolamine having two aliphatic chains of 8 to 28 carbon atoms. Other types of phospholipids may be used, such as phosphatidylcholine or phosphatidylserine, provided they contain at least one aliphatic chain of 8 to 28 carbon atoms.

[0023] Examples include dodecyl, hexadecyl, or octadecyl chains, which may be coupled to the hydrophilic segment, for example, by covalent bonding. The hydrophobic segment may also consist of fatty acids. Preferred examples are stearic acid and palmitic acid. In other examples, the hydrophobic segment may contain two aliphatic chains with 8 to 28 carbon atoms. In such cases, the lengths of the two aliphatic chains may be the same or different. The two aliphatic chains may be introduced, for example, in the form of glycerols acylated with fatty acids at positions 1 and 2. Such compounds may also be called diglycerides. Examples include 1,2-dimiristoyl-sn-glycerol (DMG), 1,2-dipalmitoyl-rac-glycerol (DPG), and 1,2-distearoyl-rac-glycerol (DSG). The two aliphatic chains may also be introduced in the form of phosphoethanolamines. Phosphoethanolamines possessing two aliphatic chains are generally referred to as phosphatidylethanolamines. They belong to the phospholipid family. Examples of phosphatidylethanolamines include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0024] Preferably, at least one hydrophobic segment is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). DSPE has two saturated aliphatic chains of 18 carbon atoms each.

[0025] PNMVA can advantageously have a controlled molecular weight. Such a controlled molecular weight can be obtained from any suitable controlled polymerization process. For example, it can be obtained by a controlled radical polymerization process. For example, it can be obtained by organometallic-mediated radical polymerization (OMRP), tellurium-mediated radical polymerization (TERP), or reversible addition-fraction chain transfer (RAFT) polymerization. A controlled polymerization process advantageously allows for the coupling of a desired segment to the polymer, or, in a further step, to a functional group that facilitates its coupling with the segment.

[0026] Coupling can be achieved, for example, through the formation of a covalent bond. This bond can be irreversible or cleavable. Cleavable bonds may be advantageous when it is desirable to release the PNMVA chain from the hydrophobic segment. Cleavage can be achieved, for example, by external stimuli such as light stimulation, or by internal stimuli such as pH changes. Examples of possible bonds include ether, ester, carbonate, amide, or urethane bonds. Examples of cleavable bonds include imine, oxime, or disulfur bonds.

[0027] In some embodiments, the functionalization or coupling rate is high, exceeding 50% of the coupling rate, preferably exceeding 60%, and more preferably exceeding 70%. The conversion rate can be determined, for example, by nuclear magnetic resonance.

[0028] In some embodiments, PNMVA has a molecular weight (M) of less than 50,000 g / mol or less than 20,000 g / mol, preferably 1,000 to 10,000 g / mol, more preferably 1,500 to 6,000 g / mol, and even more preferably 2,000 to 4,000 g / mol. n It has the following properties. The molecular weight can be measured, for example, by size exclusion chromatography, optionally combined with multi-angle light scattering (MALLS).

[0029] Preferably, the hydrophilic segment has a molecular weight of 1000 to 10000 g / mol, preferably 1500 to 6000 g / mol, and more preferably 2000 to 4000 g / mol.

[0030] In some embodiments, PNMVA has low polydispersity (D). Polydispersity represents the molecular weight distribution range. It is obtained by the ratio of molecular weight by mass to molecular weight by number. Favorably, the polydispersity is less than 2, preferably less than 1.5.

[0031] A second object of the present invention is a method for preparing the polymer derivatives listed above, (i) A step of coupling a hydrophobic segment comprising at least one aliphatic chain of 8 to 28 carbon atoms, as defined in the first object of the present invention, to a chain control agent. (ii) A step of polymerizing N-methyl-N-vinylacetamide in the presence of the coupled chain control agent and, optionally, a radical polymerization initiator. Or, (i) A step of polymerizing N-methyl-N-vinylacetamide in the presence of a chain control agent functionalized with a reactive group, and optionally, a radical polymerization initiator. (ii) To provide a method comprising the step of coupling the obtained polymer to a hydrophobic segment comprising at least one aliphatic chain of 8 to 28 carbon atoms as defined in the first object of the present invention, by reaction with the reactive group.

[0032] A chain control agent refers to a compound capable of adjusting the molar mass of a polymer chain and / or functionalizing the polymer chain. Functionalization is preferably performed at one end of the polymer chain.

[0033] Preferably, the chain control agent is a chain transfer agent.

[0034] A chain transfer agent refers to a compound capable of functionalizing the NMVA growth chain during polymerization and / or controlling the radical polymerization of NMVA. A suitable chain transfer agent is one known as a RAFT agent, also called a RAFT agent, which is used in RAFT polymerization. When using a RAFT agent, a radical polymerization initiator is added to polymerize N-methyl-N-vinylacetamide. An example of a RAFT agent is disclosed, for example, by Dupre-Demorsy et al. in Macromolecules, 2022, 55(4), 1127. Advantageously, RAFT agents can control the radical polymerization of NMVA.

[0035] The RAFT agent may also be represented by formula I: [ka] In the formula, Z is OR 1 It is either (referred to as dithiocarbonate or xanthate) or N-R2R3 (referred to as dithiocarbamate), and R 1 , R 2 , R 3 Each of these is individually either alkyl or phenyl, substituted or unsubstituted, preferably R 1 R2 and R3 are either methyl or ethyl, preferably both are unsubstituted phenyl groups or both are methyl. In the formula, R is any group that can initiate polymerization.

[0036] Examples of RAFT agents will be shown next. [ka]

[0037] The chain transfer agent can be functionalized with a reactive group, advantageously, to introduce a functional group into the growing chain during polymerization, and thus into the resulting polymer. This functional group is preferably introduced at the ends of the polymer chain. If the chain transfer agent is a xanthate species, such a xanthate species can be functionalized. Reactive groups may be, for example, acids, activated carbonates, esters, thiols, carboxylic acids, activated esters, N-hydroxysuccinimide esters (NHS esters), N-succinimidyl carbonates, amines, aldehydes, alkynes, and azides.

[0038] A radical polymerization initiator refers to any species capable of generating radicals under polymerization conditions. Preferred are azo initiators. An example is 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), also known as V70 or azobisisobutyronitrile (AIBN).

[0039] To obtain a polymer derivative, a hydrophobic segment may be coupled to a chain transfer agent, and then a PNMVA chain may be grown from the segment, or the PNMVA chain may be first polymerized, preferably by controlled polymerization, and then coupled to the hydrophobic segment in a second step. To enable such coupling, the chain transfer agent is preferably functionalized with a functional group that enables reaction with the desired hydrophobic segment. The reactive group may be as described above.

[0040] A third object of the present invention is to provide lipid nanoparticles containing the polymer derivatives listed above.

[0041] Thanks to its hydrophobic segment, the polymer derivative can be inserted into the lipid nanoparticle. The hydrophobic segment advantageously allows interaction with the lipid portion of the lipid nanoparticle through at least one aliphatic chain of 8 to 28 carbon atoms.

[0042] On the other hand, the hydrophilic segment extends, at least partially, from the lipid nanoparticles to interact with the aqueous environment of the lipid nanoparticles.

[0043] Lipid nanoparticles refer to spherical vesicles made from lipids. Lipids refer to any molecule that is hydrophobic and soluble in nonpolar solvents. Lipids may be of natural origin or synthetic origin. Examples of lipids include fatty acids, glycerides, and phospholipids.

[0044] Lipid nanoparticles can be made from neutral, ionic, ionizable, or exchangeable lipids. They are preferably positively charged and / or pH-sensitive, meaning they can be neutral at basic / neutral pH while retaining a positive charge at acidic pH. An example of an exchangeable lipid is CSL-3. An example of a pH-sensitive lipid is SM-102. Other examples of lipids include cholesterol (CHOL), DOTAP, DOPE, DSPC, SPC, EPC, HSPC, HEPC, and ceramide. The lipids used to form the nanoparticles may include, or differ from, one or more of the lipids used in the hydrophobic segment. Preferred compositions are different proportions of SPC / CHOL / polymer derivatives, or SM-102 / DSPC / CHOL / polymer derivatives, or DOTAP / CHOL / DOPE / polymer derivatives.

[0045] Preferably, the lipid nanoparticles have an average diameter in the nanometer range.

[0046] A particularly interesting example of suitable lipid nanoparticles is liposomes. Liposomes are constructed from a phospholipid bilayer and an aqueous core. Cationic liposomes are liposomes that contain at least one cationic lipid.

[0047] Preferably, the lipid nanoparticles may contain the polymer derivatives listed above in a molar percentage of 1 to 50%, preferably 40%, and more preferably 1 to 30%, relative to the total lipids.

[0048] Total lipids refer to the sum of the different lipids used in the preparation of lipid nanoparticles. For example, three different lipids may be used in the preparation of lipid nanoparticles. To arrive at total lipids, the molar percentages of each lipid are summed up. The molar percentage of polymer derivatives contained in lipid nanoparticles may depend on the use of the lipid nanoparticles. Specifically, it may depend on the need for stealth behavior of the lipid nanoparticles. For example, longer circulation times in the body, such as targeting tumor cells, require more stealthy nanoparticles and therefore require a higher input of polymer derivatives. When used in vaccine formulations, a lower input may be sufficient (e.g., as low as 1.5%).

[0049] The polymer derivative can advantageously provide good steric protection for lipid nanoparticles. Advantageously, it suppresses the adsorption of proteins or particles on the surface of lipid nanoparticles.

[0050] Preferably, lipid nanoparticles comprising a polymer derivative as defined in the first object of the present invention have a size of less than 500 nanometers, preferably less than 200 nanometers. The size is, for example, the hydrodynamic mean diameter of the lipid particles.

[0051] Advantageously, lipid nanoparticles may further contain surfactants.

[0052] An activator refers to any agent that has cosmetic or therapeutic activity. The activator may be a small pharmaceutical molecule, biomolecule, peptide-based agent, protein, or genetic material. Preferably, the activator is nucleic acid.

[0053] Nucleic acids refer to any of the following: plasmids, mRNA, miRNA, ASO, morpholino, siRNA, shRNA, small non-coding RNA, long non-coding RNA, rRNA, CRISPR / Cas9 guides, or catalytic oligonucleotides.

[0054] When lipid nanoparticles are cationic liposomes and the activator is an electrolytic nucleic acid, the resulting condensed particles can be called lipoplexes.

[0055] When lipid nanoparticles are composed of ionizable or exchangeable lipids, and the activator is an electrically charged nucleic acid, the resulting condensed particles may be referred to as lipid nanoparticles (LNPs).

[0056] Advantageously, the activator can be encapsulated and / or complexed and / or solubilized by lipid nanoparticles.

[0057] A fourth object of the present invention relates to lipid nanoparticles comprising the polymer derivatives of the present invention listed above for use in medical treatment or methods for preventing disease.

[0058] Lipid nanoparticles containing the polymer derivative of the present invention can deliver activators to subjects requiring medical treatment. They can also deliver activators to subjects requiring preventative measures, such as vaccines.

[0059] Lipid nanoparticles containing the polymer derivative of the present invention may be particularly suitable for use in drug delivery.

[0060] Lipid nanoparticles containing the polymer derivatives of the present invention can be loaded with, for example, substances suitable for targeting tumor cells. Before reaching the target, the activator is protected by the lipid nanoparticles. Thanks to its stealth properties provided by the hydrophilic PNMVA segment, protein corona formation is avoided. Furthermore, efficient delivery at the target becomes possible. In fact, lipid nanoparticles containing the polymer derivatives of the present invention enable higher cellular uptake compared to the same PEGylated lipid nanoparticles. For example, when tested in human lung adenocarcinoma cells A549, lipid nanoparticles containing the polymer derivatives of the present invention were internalized up to 6 times more than their PEGylated equivalents.

[0061] Repeated injections into mice also demonstrated that lipid nanoparticles containing the polymer derivative of the present invention were less immunogenic than their PEGylated equivalents. After the second injection, significantly lower production of IgG antibodies was observed, and no increase in IgM antibodies was seen. The ABC phenomenon, as observed with the PEGylated equivalents, was not observed.

[0062] Furthermore, lipid nanoparticles containing the polymer derivatives of the present invention are safe to use. No accumulation was observed in the liver and spleen. No inflammation (production of pro-inflammatory cytokines) was observed. The safe use of the derivatives and lipoplexes incorporating these derivatives has been demonstrated at the cellular level, in zebrafish and mouse animal models, and in human blood.

[0063] Due to all these properties, lipid nanoparticles containing the polymer derivatives of the present invention can be advantageously used in any application where PEGylated lipid nanoparticles are currently used. An example is Doxil®, the first liposomal drug approved by the U.S. FDA. It contains doxorubicin, a drug for chemotherapy, as its activator. A second example of PEGylated lipid nanoparticles currently in use is as an excipient in both the Comirnaty® and Spikevax® COVID-19 vaccines. The activator in both vaccines is mRNA. A third example is Onpattro, a lipid nanoparticle-based short-chain interfering RNA drug for the treatment of polyneuropathy with liver targeting.

[0064] Specifically, if lipid nanoparticles containing the polymer derivatives of the present invention, as listed above, contain nucleic acids, they can be used for nucleic acid delivery. For example, they can be used to deliver nucleic acids to targeted cells such as tumor cells. They can be used not only for medical procedures, such as vaccine administration, but also for the prevention of medical diseases.

[0065] The ratio of nucleic acid to polymer derivative may be expressed as an N / P ratio, where N and P correspond to the number of moles of positive charge in the lipid nanoparticles and the number of moles of negative charge in the nucleic acid, respectively. The number of moles of positive charge is based, for example, on the cationic lipid present, e.g., ammonium-based lipids, e.g., DOTAP. This enables the complexation of positively charged nucleic acids with positively charged lipid nanoparticles. The N / P ratio may vary depending on the genetic material. When using siRNA, an N / P ratio of 1 to 10, more preferably 2 to 5, for example, 2.5, is used.

[0066] Lipid nanoparticles having the polymer derivative of the present invention can advantageously enhance the cellular uptake of lipoplexes compared to PEGylated lipoplexes. Preferably, cellular uptake is favorable when the PNMVA chain is relatively short, for example, when the molecular weight of PNMVA is 2000-4000 g / mol. Advantageously, lipoplexes grafted with a polymer derivative containing siRNA and having 2500 g / mol of PNMVA can be internalized approximately six times more than PEGylated lipoplexes when tested in A549 cells.

[0067] Lipid nanoparticles containing the polymer derivative of the present invention can be administered by any preferred method of administration, such as oral administration, skin administration, injection, intravenous administration, subcutaneous administration, systemic administration, parenteral administration, digestive, vaginal, rectal, or transdermal administration, transmucosal administration, pulmonary, nasal, or sublingual administration, inhalation, spray, or aerosol administration. Preferably, the composition is administered by parenteral injection.

[0068] Thanks to the hydrophilization of lipid nanoparticles by the polymer derivative of the present invention, improved mucosal penetration of these nanoparticles is expected. This property allows for use, for example, in pulmonary or vaginal administration.

[0069] A further object of the present invention is the use of lipid nanoparticles containing the polymer derivatives of the present invention, as listed above, for cosmetic treatments.

[0070] Lipid nanoparticles containing the polymer derivative of the present invention may be combined with any activator that provides cosmetic treatments, such as cosmetic skin treatments. In this case, they are preferably administered as a cream or by any suitable topical or oral administration.

[0071] Another object of the present invention is to provide a pharmaceutical composition comprising lipid nanoparticles containing polymer derivatives of the present invention as listed above, and a pharmaceutically acceptable carrier, the pharmaceutical composition preferably selected from vaccine formulations.

[0072] A further object of the present invention is a method for preparing lipid nanoparticles comprising the polymer derivatives of the present invention as listed above, (i) step of providing lipid nanoparticles, (ii) Optionally, the step of adding an activator, preferably selected from nucleic acids, to form an electrolytic interaction between lipid nanoparticles and the activator, (iii) Adding the polymer derivative of the present invention to a solvent under stirring, (iv) To provide a method comprising the optional step of removing a solvent.

[0073] Preferably, the solvent is an aqueous solvent. Preferably, the solvent is water.

[0074] Preferably, the solvent is removed by filtration, evaporation, centrifugation, freeze-drying, and / or spray-drying.

[0075] Alternatively, all ingredients may be added at once.

[0076] Alternatively, this method, (i) If the activator is a hydrophobic compound in the first solvent, the step of solubilizing the lipid and optionally such activator so as to form the first solution. (ii) Optionally, if the activator is a hydrophilic compound in the second solvent, the step of solubilizing the activator to form a second solution. (iii) Optionally, a step of mixing the first solution and the second solution, (iv) The step of removing the first solvent and optionally the second solvent may be included, The polymer derivative of the present invention is added in step (i), in step (ii), or after step (iv).

[0077] These steps are presented in a suggestive order only. The order of the steps may be changed without departing from the scope of the invention. Some of the steps may also be repeated without departing from the scope of the invention.

[0078] If a polymer derivative is added after step (iv), it is preferably added under stirring in an aqueous solution.

[0079] Preferably, at least one activator is a nucleic acid. Preferably, the nucleic acid is considered a hydrophilic compound and is solubilized in step (ii).

[0080] Preferably, step (iii) is continuous mixing.

[0081] Preferably, step (iii) is carried out in a microfluidic apparatus (also known as a rapid mixer).

[0082] A microfluidic device refers to a device having channels with a diameter in the micrometer range of 1 mm or less.

[0083] Advantageously, the first solvent is a solvent capable of solubilizing all the substances provided in step (i) to form the first solution. Preferably, the first solvent is a water-miscible organic solvent. More preferably, the first solvent is ethanol. Alternatively, the first solvent is chloroform.

[0084] Advantageously, the second solvent is a solvent capable of solubilizing nucleic acids to form a second solution. Preferably, the second solvent is an aqueous solution. More preferably, the second solvent is water.

[0085] Advantageously, the first and second solutions are continuously mixed within a microfluidic device. Preferably, the flow rate of each solution is adjusted according to a desired N / P ratio.

[0086] Advantageously, the first solvent and / or the second solvent are supercritical fluids. Preferably, the supercritical fluid is carbon dioxide (CO2). When the supercritical fluid is a gas at ambient temperature, as in the case of CO2, the supercritical fluid can be advantageously removed by depressurization. [Brief explanation of the drawing]

[0087] [Figure 1] Naked lipoplex vs. grafted lipoplex with a mean Z diameter in nanometers (nm) of 2.5 (N / P ratio), 100 nM, and increased percentage of polymer derivative to total lipid molar ratio (10%, 15%, 30%, 40%), grafted with DSPE-PEG, DSPE-PNMVA24, and DPSE-PNMVA50. [Figure 2] The multivariance index (PdI) of naked lipoplex versus grafted lipoplex. [Figure 3] Zeta potential (Zp) (mV) of naked lipoplex versus grafted lipoplex. [Figure 4] siRNA encombination (%) between naked lipoplex and grafted lipoplex. [Figure 5] (a) Δf traces (7th harmonic) from QCM-D monitoring of deposits of Et-PNMVA27, (b) OD-PNMVA31, (c) DSPE-PNMVA24, and (d) DSPE-PNMVA50. [Figure 6] Average size increase of naked lipoplex and grafted lipoplex after 2 hours in fetal bovine serum (30%). [Figure 7] Cell viability of A549 cell lines treated for 24 hours with the polymer derivative of the present invention and comparative DPSE-PEG. [Figure 8] Cell viability of A549 cell lines treated with naked lipoplex and grafted lipoplex for 24 hours. [Figure 9] Fluorescence intensity of A549 / GFP cells in the presence of naked and grafted lipoplex. [Figure 10] Gene knockdown (MFI, %) in A549 / GFP cells transfected with anti-GFP siRNA (100 nM) lipoplex, compared to naked lipoplex, DSPE-PEG grafted lipoplex, and Lipofectamine® RNAiMAX (black line), 72 hours after transfection (n=3). [Figure 11] Fluorescence intensity of grafted lipoplex containing fluorescent Cy5.5 GL3 SiO2 after first (5-hour) and second (24-hour) injections into mice for in vivo imaging. [Figure 12] Fluorescence intensity of grafted lipoplexes containing fluorescent Cy5.5 GL3 SiO2, using ex vivo imaging in the kidneys, heart, liver, lungs, and spleen. [Figure 13] IgM and IgG levels in grafted lipoplex one week after the first and second injections, using comparative DSPE-PEG ELISA assays. [Figure 14] Levels of IgM and IgG in grafted lipoplex one week after first and second injections using an ELISA assay with the polymer derivative of the present invention. [Modes for carrying out the invention]

[0088] Herein, the present invention is described with reference to certain embodiments and certain drawings. It will be apparent that other embodiments of the present invention can be constructed in accordance with the knowledge of those skilled in the art without departing from the technical teachings of the present invention, and the present invention is limited only by the claims. [Examples]

[0089] 1. Synthesis of PNMVA derivatives To impart amphiphilicity to PNMVA and enable its possible immobilization to a lipoplex membrane, this water-soluble polymer sequence was terminally functionalized with two different hydrophobic groups, namely octadecyl (OD) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). These are further referred to as OD-PNMVA and DSPE-PNMVA, respectively (Scheme 1). [ka] Scheme 1: Examples of polymer derivatives having a PNMVA segment and a hydrophobic segment, which are octadecyl (OD) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0090] The synthesis strategies for these polymer derivatives are shown in Scheme 2 and Scheme 3, respectively. Both of these schemes involve reversible addition-fraction chain transfer (RAFT) polymerization.

[0091] The synthesis of OD-PNMVA was achieved by RAFT polymerization of NMVA using an OD-containing RAFT agent. The synthesis of DSPE-PNMVA derivatives was achieved by a two-step process based on (i) RAFT of NMVA initiated with a xanthate containing succinimidyl carbonate (SC-XA), and (ii) coupling of the resulting succinimidyl carbonate-PNMVA (SC-PNMVA-XA) with the amino group of DSPE. The DP and molar mass of the hydrophilic portion of PNMVA were adjusted by the monomer / RAFT agent ratio. [ka] Scheme 2: First example of synthesis of amphiphilic PNMVA derivatives [ka] Scheme 3: Second example of synthesis of amphiphilic PNMVA derivatives

[0092] The materials used were as follows: potassium ethylxanthogenic acid (96%, Aldrich), octadecylamine (97%, Aldrich), 2-bromopropionyl bromide (97%, Aldrich), N,N'-disuccinimidyl carbonate (DSC) (Aldrich), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) (Aldrich), ethylene glycol (Roth), magnesium sulfate (Abcr), ammonium chloride (Aldrich), sodium bicarbonate (Aldrich), lauroyl peroxide (Fluka), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V70, t1 / 2 = 30°C for 10 hours) (Wako), hydrochloric acid (Acros), monobasic potassium phosphate (Janssen) Chimica), potassium phosphate dibasic (VWR), sodium chloride (VWR), 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) (Avanti polar lipids), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti polar lipids), cholesterol (Aldrich), ammonium peroxide (Aldrich), 3-mercaptopropionic acid (MPA) (Aldrich), hydrogen peroxide (30%, Aldrich), sulfuric acid (95-97%, Merck), silica gel for column chromatography (60 Å, ROCC SA), triethylamine (99%, Acros) Organic ethanol, pyridine (Aldrich), diethyl ether (VWR), n-hexane (>99%, VWR), ethyl acetate (≧99.9%, VWR), acetonitrile (≧99.99%, Fisher), heptane (>97.5%, Fisher), anhydrous ethanol (Fisher), and isopropanol (Fisher) were used as received. N-methyl-N-vinylacetamide (Aldrich) was dried on calcium hydride, degassed, and distilled under reduced pressure before use. N,N-dimethylformamide (DMF, >99%, VWR) was dried on a molecular sieve and degassed before use. Dichloromethane (CH2Cl2) was degassed and dried on a 4 Å molecular sieve.For characterizing samples using quartz crystal microbalancing, the QCM-D sensor was a gold-coated AT-cut quartz crystal (Q-Sense, Sweden) with a fundamental frequency of approximately 5 MHz.

[0093] The following methods were used to characterize the samples. Polymer derivatives were analyzed by size exclusion chromatography (SEC) in DMF containing 0.025 M LiBr at 55°C using a Waters chromatograph equipped with three columns (Waters Styragel PSS GRAM 1000 Å (⊆ 2), 30 Å), a dual λ absorbance detector (Waters 2487), and a refractive index detector (Waters 2414). The system was operated at a flow rate of 1 mL / min. The molar mass of SC-PNMVA-XA was determined by SEC in DMF / LiBr (0.025 M) equipped with a multi-angle laser light scattering (MALL) detector. A Wyatt MALLs detector (120 mW solid-state laser, k 1 / 4 658 nm, DawnHeleos S / N342-H) measured the excess Rayleigh ratio Rh (related to scattering intensity) at different angles for each slice of the chromatogram. The specific refractive index increment (dn / dc) of the polymer was measured using a Wyatt Optilab refractive index detector (k 1 / 4 658nm). The data was processed using Astra V software (Wyatt Technology). 1 H and 13 Nuclear magnetic resonance (NMR) of 13C was recorded at 298K using a Bruker AVANCE III HD spectrometer (B0=9.04T) (400MHz) and processed with MestreNova software.

[0094] 2-Bromo-N-octadecylpropanamide (OD-Br) was obtained as follows. Octadecylamine (3.00 g, 11.1 mmol) and triethylamine (1.68 g, 16.6 mmol) were dissolved in 100 mL of THF dried and degassed under nitrogen, and placed in a 250 mL two-neck round-bottom flask equipped with a three-way stopcock and an addition funnel containing 2-bromopropionyl bromide (2.87 g, 13.3 mmol) in 10 mL of THF dried and degassed. After cooling the octadecylamine solution to 0 °C, the 2-bromopropionyl bromide solution was added dropwise. The reaction medium was stirred at 0 °C for 30 minutes and then stirred overnight at room temperature. The white precipitate was filtered off and the solvent was evaporated under vacuum. The crude product was solubilized in ethyl acetate / hexane 3 / 1 v / v (40 mL) at 50 °C, and the mixture was stored at 6 °C overnight. White crystals of 2-bromo-N-octadecylpropanamide were formed, filtered, and dried under vacuum (3.7 g, 83% yield). 1 H NMR (400 MHz, CDCl3): δ [ppm] = 6.37 (1H, s, C(O)NH), 4.41 (1H, q, J = 7.1 Hz, CH), 3.26 (2H, q, C(O)NHCH2), 1.88 (3H, d, J = 7.1 Hz, CHCH3), 1.52 (2H, m, C(O)NHCH2CH2), 1.27 (30H, m, (CH2) 15 ), 0.88 (3H, t, J = 7.0 Hz, CH3(CH2) 15 )。 13 C NMR (100.6 MHz, CDCl3): δ [ppm] = 169.12 (C=O), 45.67 (CH), 40.23 (C(O)NHCH2), 31.93, 29.71, 29.68, 29.67, 29.64, 29.57, 29.51, 29.37, 29.28, 29.25, 26.81, 23.34 (CHCH3), 22.70, 14.13 (C(S)OCH2CH3).

[0095] O-ethyl S-(1-(octadecylamino)-1-oxopropan-2-yl)carbonodithioate (OD-XA) was obtained as follows: OD-Br (5.00 g, 12.3 mmol) and potassium ethylxanthogenicate (7.90 g, 49.3 mmol) were placed in a 1 L flask under nitrogen, and a degassed solution of pyridine (65.2 g, 824 mmol) in dried and degassed dichloromethane (330 mL) was added. The reaction mixture was stirred at room temperature for 24 hours. After adding dichloromethane (100 mL) to the solution, it was washed sequentially with saturated NH4Cl (3 times with 50 mL), NaHCO3 (3 times with 50 mL), and water (3 times with 50 mL). The organic layer was dried on magnesium sulfate, filtered, and evaporated under vacuum. The crude product was solubilized in ethyl acetate / hexane 3 / 2v / v (40 mL) and stored overnight at 6°C. Pale yellow crystals of O-ethyl S-(1-(octadecylamino)-1-oxopropan-2-yl)carbonodithioate were formed, filtered, and dried under vacuum (4.37 g, 79% yield). 1 H NMR (400MHz, CDCl3): δ[ppm]=6.33(1H,s,C(O)NH),4.64(2H,q,J=7.2Hz,C(S)OCH2),4.27(1H,q,J=7.4Hz,CH),3.22(2H,q,J=7.0Hz,C(O)NHCH2),1. 54(3H,d,J=7.4Hz,CHCH3),1.46(2H,m,C(O)NHCH2CH2),1.41(3H,t,J=7.2Hz,C(S)OCH2CH3),1.24(30H,m,(CH2)15),0.87(3H,t,J=7.0Hz,CH3(CH2) 15 ). 13 C NMR (100.6MHz, CDCl3): δ[ppm]=213.83(C=S),170.83(C=O),70.85(C(S)OCH2),48.17(CH),39.97(C(O)NHCH2) ,32.07,29.85,29.82,29.80,29.72,29.69,29.53,29.51,29.40,26.99,22.84,16.63(CHCH3),14.26(CH3(CH2) 15 ), 13.88(C(S)OCH2CH3).

[0096] Synthesis of OD-PNMVA. OD-XA (703 mg, 1.58 mmol) as a chain transfer agent and V70 (487 mg, 1.58 mmol) as an initiator were placed in a Schenck tube under an inert atmosphere, distilled, dried, and degassed NMVA (7.8 g, 79 mmol) was added. The reaction mixture was then stirred at 35°C. After 6 hours, the reaction was stopped and the mixture was taken in CD2Cl2. 1 Monomer conversion, as measured by 1H NMR, reached 50%. The mixture was diluted in dichloromethane and subsequently precipitated in diethyl ether. The polymer was recovered by filtration and dried under vacuum at 40°C for 12 hours. The polymer was further purified by dialyzing in acetone through a 1 kDa porous membrane for 24 hours and in miliQ water through a 500 Da porous membrane for 12 hours, followed by freeze-drying. The desired OD-PNMVA 31 (0.98g) was recovered as a pale yellow powder and PS calibration (M n SEC (=1800g / mol, D=1.21) and in CD2Cl2 1 1H NMR (M n NMR PNMVA The characterization was performed by SEC in DMF using (=3100 g / mol, DP=31).

[0097] Synthesis of 2-hydroxyethyl 2-bromopropionate (HO-Br). Dried ethylene glycol (4.84 mol, 300 g, 271 mL) and pyridine (0.1 mol, 7.91 g, 8.1 mL) were diluted in 100 mL of dry THF in a 1 L two-necked round-bottom flask equipped with an addition funnel under an inert atmosphere. 2-bromopropionyl bromide (95 mmol, 20.51 g, 9.95 mL) was placed in an addition funnel containing 50 mL of dry THF. The flask was cooled in an ice bath, and 2-bromopropionyl bromide was added dropwise. The mixture was stirred at 0°C for 1 hour. The mixture was then stirred at room temperature for 16 hours. After this, the mixture was poured into 800 mL of acidic water (pH=2, hydrochloric acid), and the product was extracted with dichloromethane (6 × 100 mL). The organic fractions were combined, extracted with water, and dried on magnesium sulfate. The solvent was evaporated, and the product was obtained as a colorless liquid (MM = 197.03 g / mol, 14.92 g, yield = 80%).1 H NMR (400MHz, CDCl3): δ[ppm]=4.40(1H,s,J=7.0Hz,CH),4.28(2H,m,CH2OC(O)),3.84,(2H,m,HOCH2CH2OC(O)),2.27(1H,s,OH),1.82(3H,d,J=7Hz,CH3CH). 13 C NMR (100.6MHz, CDCl3): δ[ppm]=170.59(C=O), 67.45(CH2OC(O)), 61.00(HOCH2CH2OC(O)), 39.89(CH), 21.66(CH3CH).

[0098] S-(1-methyl-4-hydroxyethyl acetate)O-ethyldithiocarbonate (HO-XA). Potassium ethylxanthogenic acid (3.65 g, 22.7 mmol) was added to 15 mL of acetone in a round-bottom flask equipped with an addition funnel containing 2-hydroxyethyl 2-bromopropionate (4.00 g, 20.3 mmol) in 15 mL of acetone under an inert atmosphere. The latter solution was added dropwise over 30 minutes at room temperature, and the reaction mixture was stirred at room temperature for 24 hours. The white solid was filtered and washed with 50 mL of acetone, after which the solvent was evaporated. The residue was then dissolved in 50 mL of dichloromethane and extracted with distilled water (3 × 25 mL). The organic phase was dried over magnesium sulfate, filtered, and evaporated to dryness. The desired product was obtained as a viscous yellow liquid (2.95 g, yield 61%). 1 H NMR(400MHz,CDCl3):δ[ppm]=4.59(2H,q,J=7.1Hz,C(S)OCH2),4.37(1H,q,J=7.4Hz,CH),4.23(2H,m,CH2OC(O)),3.79 (2H,t,J=4.7Hz,HOCH2CH2OC(O)),2.32(1H,s,OH),1.55(3H,d,J=7.3Hz,CH3CH),1.37(3H,t,J=7.2Hz,C(S)OCH2CH3). 13C NMR (100.6MHz, CDCl3): δ[ppm]=212.38(C=S),171.85(C=O),70.55(C(S)OCH2),67.32( CH2OC(O)), 61.06(HOCH2CH2OC(O)), 47.24(CH), 16.80(CH3CH), 13.77(C(S)OCH2CH3).

[0099] 2-((((2,5-Dioxopyrrolidine-1-yl)oxy)carbonyl)oxy)ethyl 2-((ethoxycarbonothio)thio)propanoate (SC-XA). HO-XA (1.00 g, 4.19 mmol) and N,N'-Disuccinimidyl carbonate (2.13 g, 8.32 mmol) were placed in a 25 mL round-bottom flask containing triethylamine (2.13 g, 21.03 mmol) and 10 mL of acetonitrile under a nitrogen atmosphere. The mixture was stirred at room temperature for 24 hours. The reaction medium was then diluted with 20 mL of dichloromethane, and the solvent was evaporated to facilitate the removal of acetonitrile. The resulting viscous brown liquid was then diluted with 10 mL of dichloromethane and washed with aqueous NaHCO3 solution (3 × 25 mL) and distilled water (3 × 25 mL). The organic phase was dried on magnesium sulfate, filtered, and evaporated to dryness. SC-XA was obtained as a brown viscous liquid (1.04 g, 65% yield). 1 H NMR(400MHz,CDCl3):δ[ppm]=4.63(2H,q,J=7.1Hz,C(S)OCH2),4.53(2H,t,J=4.8Hz,CH2OC(O)),4.48-4.34(3H,m,CH+C( O)OCH2CH2OC(O)CH),2.84(4H,m,NC(O)(CH2)2C(O)N),1.58(3H,d,J=7.4Hz,CH3CH),1.41(3H,t,J=7.2Hz,C(S)OCH2CH3). 13C NMR (100.6MHz, CDCl3): δ[ppm]=211.75(C=S),171.29(C(O)CH),168.48(C(O)NC(O)),151.51((O)C(O)),70.40(C(S)OCH2),68 .35(CH2OC(O)),62.41(C(O)OCH2CH2OC(O)CH),46.85(CH),25.47(NC(O)(CH2)2C(O)N),16.56(CH3CH),13.67(C(S)OCH2CH3).

[0100] Synthesis of SC-PNMVA-XA. SC-XA (519 mg, 1.36 mmol) and V70 (420 mg, 1.36 mmol) were placed in a Schenck tube under an inert atmosphere, distilled, dried, and degassed NMVA (6.8 g, 68.1 mmol) was added. The reaction mixture was then stirred at 35°C. After 5 hours, the reaction was stopped and the solution was extracted in CDCl3. 1 Monomer conversion, as measured by 1H NMR, reached 70%. The mixture was diluted in dichloromethane, and the polymer was purified twice by precipitation in diethyl ether. After drying under vacuum at 40°C for 24 hours, SC-PNMVA 40 -XA (2.71g) was collected as a pale yellow powder and PS calibration (M n,SEC =3800g / mol, D=1.20) and MALLS(dn / dc=0.071)(M n,MALLS Characterization was performed using SEC in DMF (4300 g / mol, D=1.16). This procedure was performed using SC-PNMVA. 30 - Adapted for the generation of XA. [Table 1]

[0101] Synthesis of DSPE-PNMVA-XA. SC-PNMVA 40-XA (2g, 5.88 mmol), DSPE (440 mg, 5.88 mmol), and triethylamine (2.5g, 24.57 mmol) were placed in a 25 mL round-bottom flask under a nitrogen atmosphere, dried, and dissolved in 10 mL of degassed dichloromethane. The mixture was then stirred at 40°C for 1 hour. The solvent was then evaporated, and the residue was dissolved in acetonitrile (12 mL) and stored overnight at 6°C. The solution was then centrifuged twice (10000 rpm, 15 min, 6°C). The supernatant was collected and evaporated under vacuum to obtain a white powder. The white powder was then diluted in milli-Q water and sequentially dialyzed through a 1 kDa porous membrane in aqueous sodium chloride (0.3 M) for 12 hours and distilled water for 6 hours. After lyophilization, the desired DSPE-PNMVA was obtained. 53 -XA (1.8g) was obtained as a white powder, and PS calibration (M n,SEC (=5000g / mol, D=1.12) and in CDCl3 1 1H NMR (M n,NMR Characterization was performed by SEC in DMF using (=5300 g / mol, DP=53). This procedure was performed using DSPE-PNMVA 35 - Adapted for the generation of XA. [Table 2]

[0102] Removal of terminal xanthates. (See DSPE-PNMVA above.) 53 -XA (1.2 g, 0.2 mmol) and LPO (33.5 mg, 0.084 mmol) were placed in a 10 mL round-bottom flask under an inert atmosphere and dissolved in degassed isopropanol (5 mL). The mixture was then stirred at 80°C for 8 hours, during which time the LPO portion (16.8 mg, 0.042 mmol) was added every 2 hours. The mixture was stirred at 80°C for a further 16 hours. The polymer was recovered by precipitation in hexane, dried under vacuum at 40°C for 12 hours, and sequentially dialyzed through a 1 kDa porous membrane in aqueous sodium chloride (0.3 M) for 12 hours and then in water for 12 hours. After freeze-drying, the desired DSPE-PNMVA was obtained. 50-H was collected as a white powder (480 mg). The absence of xanthate was confirmed by SEC-UV by the disappearance of the characteristic absorption signal of xanthate at 290 nm. This procedure was performed using DSPE-PNMVA. 35 - Also applicable to XA, DSPE-PNMVA 24 -H was brought about. [Table 3]

[0103] Table 4 summarizes the PNMVA structures that were appropriately prepared and tested for decorating lipid nanoparticles (also known as lipoplexes) in the following section. Et-PNMVA 27 This is PNMVA modified with only an ethyl chain, and therefore only two carbon atoms. It was used as a comparative example. All polymers were purified by repeated precipitation, prolonged dialysis in water, and lyophilization. In the case of DSPE-PNMVA derivatives, removal of the terminal XA was achieved by treating with lauroyl peroxide (LPO) in isopropanol at 80°C. The successful exclusion of the XA terminal group of DSPE-PNMVA was confirmed by the disappearance of the absorption peak characteristic of xanthates at 290 mm in the size exclusion chromatography-UV curve. [Table 4]

[0104] 2. Preparation of lipid nanoparticles 2.a. By thin film hydration method Cationic liposomes were prepared by lipid membrane hydration from a mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) in a molar ratio of 1 / 0.75 / 0.5, cholesterol, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). DOTAP and DOPE were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol (Chol) was purchased from Sigma Aldrich (Belgium). The lipids were dissolved in chloroform or ethanol at a total concentration of 5.5 mM. The chloroform / ethanol was removed under reduced pressure using a rotary evaporator at 30°C for 1 hour. The resulting thin lipid membrane was hydrated with 2 mL of RNAse-free water (ThermoFisher Scientific, Walthman, MA, USA), vortexed, and directly extruded through a polycarbonate membrane (5 times with a pore size of 400 nm and 10 times with a pore size of 200 nm) (Lipex® Extruder, Tansferra Nanosciences Inc., Burnaby, Canada).

[0105] Next, cationic liposomes were complexed with anionic siRNAs via spontaneous charge interactions to form lipoplexes. The liposomes and siRNAs were mixed for 30 minutes at an N / P ratio of 2.5 (where N and P correspond to the moles of positive charge (DOTAP) and negative charge (siRNA), respectively). siRNAs targeting improved green fluorescent protein (EGFP) (siGFP), unrelated siRNAs directed against non-human luciferase genes (siGL3), and fluorescent-negative control siRNAs (siGL3 Cy5 and Cy5.5) were provided by Eurogentec® (Eurogentec SA, Liege, Belgium). The corresponding sequences are as follows: siGFP: sense strand: 5'-GCAAGCUGACCCUGAAGUUC55-3', antisense strand: 5'-GAACUUCAGGGUCAGCUUGC55-3', siGL3: sense strand: 5'-CUUACGCUGAGUACUUCGAUU55-3', antisense strand: 5'-AAUCGAAGUACUCAGCGUAAG55-3', siGL3 Cy5 and Cy5.5 are siGL3 chemically conjugated with the Cy5® or Cy5.5® dye at the 5' end of the sense strand.

[0106] To obtain so-called "grafted lipoplex" through post-insertion, DSPE-PNMVA 24 and DSPE-PNMVA 50 Polymers were added to lipoplexes obtained under stirring in RNAse-free water (1 mM) at different proportions (5, 10, 15, 30, and 40 mol%) relative to total lipids. The resulting mixtures were vortexed for 15 seconds and maintained at 37°C for 1 hour. DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), was used as a positive control.

[0107] 2.b. Rapid mixing / microfluidics pH-sensitive (SM-102) or exchangeable lipid (CSL3), DSPC, Chol, and DSPE-PNMVA 24 Or DSPE-PNMVA 50 The polymer was dissolved in ethanol (1 mM total lipid concentration) in a molar ratio of 50 / 10 / 37.5 / 2.5 (0.5 mL), and then mixed with an siRNA solution (1.5 mL) prepared in 25 mM acetate buffer (SM-102 formulation) at pH 4, or in PBS buffer pH 7.4 (CSL3 formulation) at an N / P ratio of 4. The mixing was performed according to a rapid mixing method using two syringe pumps (Chemyx Fusion 200-X, KR Analytical Ltd., Sandbach, UK) connected by a T-junction (PEEK® Tee, ThermoFisher Scientific [Walthman, MA, USA]) and Tube Peek (1 / 16 inch, 0.010 inch between the pump and the T-junction, and 1 / 16 inch, 0.020 inch after the T-junction). Mixing was performed at a total flow rate of 12 mL / min with a flow ratio of 3:1 (aqueous solution to ethanol). The lipid nanoparticles were then dialyzed overnight in PBS buffer at pH 7.4, 4°C, and under magnetic agitation. Pur-A-Lyzer® Maxi dialysis tubing MWCO 12-14 kDa (Sigma-Aldrich, St. Louis, MO, USA) was used.

[0108] 3. Physicochemical properties of Lipoplex The insertion of hydrophobic segments of amphiphilic polymers into lipoplexes was evaluated by measuring several physicochemical properties of the grafted lipoplexes. The Z-mean size (nm) and polydispersity index (PdI) of the lipoplexes were determined by dynamic light scattering (DLS) using a Malvern Zetasizer® (Nano ZS, Malvern Instrument, UK) in RNAse-free water with a fixed angle of 90°. Zeta potential (Zp) (mV) was determined using the same instrument. All experiments were performed three times (n=3) at 25°C. The values ​​in Figures 1-4 represent the mean + / - standard deviation of the three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett's test. Two polymer derivatives according to the present invention were tested: DSPE-PNMVA 24 and DSPE-PNMVA 50 Lipoplexes grafted with DSPE-PEG were also tested as a comparative example. Each was tested with different amounts of polymer derivative / PEG relative to total lipids (molar ratios of 10, 20, 30, and 40%).

[0109] The average Z size of the naked lipoplex was approximately 200 nm, and this did not increase significantly with increasing amounts of different polymers (Figure 1). However, a decreasing trend in size was observed with increasing polymer concentration. Therefore, except in the case of adding large amounts of DSPE-PNMVA, the size of the lipoplex was slightly affected by the addition of polymers, and this phenomenon was also observed for DSPE-PEG.

[0110] The polydispersity index (PdI) of lipoplexes (below 0.1 for naked lipoplexes) was significantly increased for DSPE-PNMVA lipoplexes and PEGylated lipoplexes (PdI > 0.2 for 10, 15, 30, and 40% DSPE-PNMVA and DSPE-PEG polymers), reflecting the higher heterogeneity of dispersion after insertion (here, post-insertion). The PdI of grafted lipoplexes was shown to be affected by the addition of polymers containing hydrophobic DSPE segments (DSPE-PEG and DSPE-PNMVA), which may be due to the effective insertion of the lipoplex membrane (Figure 2).

[0111] The surface charge (Zp) of all formulations was also evaluated (Figure 3). A significant decrease in Zp was observed for all formulations compared to the naked lipoplex (approximately +45mV). At 40%, the DSPE-PNMVA lipoplex showed a negative Zp (approximately -15mV), similar to that observed in the PEGylated lipoplex. These results confirm the efficient insertion of the hydrophobic segment of the polymer derivative in the lipoplex.

[0112] The effect of polymer grafting on siRNA complexation efficiency was evaluated. Uncomplexed siRNA was quantified using the Quant-it® RiboGreen® RNA assay (Invitrogen® (ThermoFisher Scientific, Walthman, MA, USA)) according to the manufacturer's instructions. Samples of 100 nM siRNA and siRNA calibration curves were prepared as follows. 100 μl of RiboGreen® reagent was added to each well, and fluorescence was measured using a FlexStation3 multimode microplate reader (Molecular Devices, CA, USA). The excitation and emission wavelengths were 485 and 530 nm, respectively. The detected fluorescence was calculated in relation to the concentration of free siRNA according to the blank and calibration curves. The level of complexed siRNA was then determined.

[0113] Naked lipoplexes showed a complexation rate higher than 90%, while the complexation rate observed with DSPE-PNMVA lipoplexes dropped to approximately 45% in the presence of 40% of these polymers, despite the hydrophilic chain length (Figure 4). Interestingly, similar results had been previously observed with PEGylated lipoplexes. No significant effects of hydrophilic chain length on size, PdI, surface charge, and encapsulation efficiency were observed.

[0114] 4. Membrane interaction To gain insight into the interactions of different PNMVA derivatives with lipoplexes, their binding to model lipid bilayers was monitored via a quartz crystal microbalance with dissipation analysis (QCM-D). The liposome membrane was mimicked by a supporting lipid bilayer (SLB) consisting of DOTAP / Chol / DOPE deposited on a gold-coated quartz crystal. QCM-D analysis of polymer / membrane interactions was performed as follows: A gold-coated sensor (Q-Sense AT-cut quartz crystal, 5 MHz fundamental frequency) was cleaned and functionalized with mercaptopropionic acid. DOTAP / Chol / DOPE (1 / 0.75 / 0.5 molar ratio) liposomes were prepared as described above, except that the lipid membrane was rehydrated with high-salt PBS buffer (20 mM PBS and 100 mM NaCl) instead of RNAse-free water (final liposome concentration 5.5 mM). Deposition of the DOTAP / Chol / DOPE membrane onto the sensor was achieved according to the membrane deposition method. The sensor was successively exposed to high-salt PBS buffer (20 mM PBS and 100 mM NaCl), liposome solution (0.1 mM in high-salt PBS), high-salt PBS buffer to remove excess liposomes, low-salt PBS buffer (20 mM PBS and 30 mM NaCl) to disrupt the liposome membrane by osmotic pressure, and high-salt PBS buffer to remove weakly bound lipids. Suitable deposition of the DOTAP / Chol / DOPE membrane (44.44% / 33.33% / 22.22%) was confirmed by Δf at approximately 14 Hz. Subsequently, the appropriately modified sensor surface was subjected to a successive flow of solutions of different PNMVA derivatives, i.e., (a) Et-PNMVA using the comparative example. 27(b) OD-PNMVA 31 (c)DSPE-PNMVA 24 , and (d) DSPE-PNMVA 50 The samples were exposed to (30 μM in high-salt PBS buffer). The change in sensor frequency due to polymer binding was converted to deposited mass according to Sauerbrey's formula (Δm = -C.Δf / n) (Figure 5). Arrows indicate the addition of polymer solution (upward arrow) and the final rinse with buffer (downward arrow). The gray area corresponds to the buffer elution period.

[0115] Lipid membrane Et-PNMVA 27 No change in frequency was observed even upon exposure, and therefore no adsorption was observed, confirming the lack of membrane interaction for PNMVA derivatives from which sufficiently long aliphatic chains had been removed.

[0116] OD-PNMVA showed transient interactions with lipid membranes through its hydrophobic segment, which is an aliphatic chain of 18 carbon atoms. DSPE showed high interaction with lipid membranes through its hydrophobic segment, which has two aliphatic chains at its tip. In fact, DSPE-PNMVA 24 Most of it remains on the surface of the film after rinsing, at 45 ng.cm -2 This corresponds to polymer deposition. The effect of the molar mass of the PNMVA segment was observed as a shorter PNMVA chain, allowing for better insertion of the hydrophobic segment of the polymer derivative into the lipid membrane.

[0117] These observations supported DLS, Zp, and NTA measurement results suggesting significant structural and charge changes in lipoplexes grafted with DSPE-PNMVA.

[0118] 5. Protein Corona Formation Protein corona formation was evaluated by nanoparticle tracking analysis (NTA). NTA measurements were performed using a NanoSight NS300 (Malvern Instruments Ltd., UK) equipped with a 642 nm red laser module. The protocol was inspired by Karim et al. Sixty lipoplexes were mixed with thermally inactivated fetal bovine serum (FBS) (Gibco (Invitrogen®, ThermoFisher Scientific, Walthman, MA, USA)) in a 2:1 ratio (v / v) and incubated at 37°C for 2 hours under a stirring process. The FBS concentration was selected to mimic physiological conditions. Protein corona formation around the lipoplexes was determined by measuring the evolution of the average particle size from 0 to 2 hours after FBS addition. Samples were measured three times (n=3) at 25°C with a red laser and 40 syringe pump speeds (arbitrary units).

[0119] For the naked lipoplex, protein corona formation resulted in a change in the distribution profile characterized by an increase in the average particle size (a shift in the curve towards larger sizes). Conversely, the DSPE-PNMVA and DSPE-PEG lipoplexes showed less interference and a more overlapping size distribution profile compared to the initial profile, indicating a strong reduction in FBS protein adsorption. These observations confirmed that DSPE-PEG and DSPE-PNMVA were effective in inhibiting protein corona formation around the lipoplex. Figure 6 shows the size increase of naked or PNMVA derivative-lipoplexes after 2 hours in FBS. Each value represents the mean + / - standard deviation (SD) of three independent experiments (n=3). For the naked lipoplex, a size increase (approximately 100 nm) was observed, while for the DSPE-PNMVA polymer, a 15% amount allowed for effective avoidance of the lipoplex size increase and thus avoided protein corona formation, as was also observed with 15% DSPE-PEG. These results confirm the importance of hydrophobic segments for efficient insertion into lipoplexes, thereby protecting particles from protein corona formation.

[0120] 6. Toxicity Assessment The toxicity of the polymer derivatives of the present invention and DSPE-PEG as a comparative example was evaluated in cellulose using an MTT assay (Figure 7). A549 cells were treated with different amounts of polymer in molar ratios up to 150. (p=0.6682 for Lipoplex, p<0.001 for DSPE-PEG, DSPE-PNMVA) 24 For this, p=0.9979, DSPE-PNMVA 50 For this case, p<0.001). Each value represents the mean + / - standard deviation (SD) of three independent experiments (n=3). For the MTT trial, each independent experiment represents the mean of four trials. Statistical analysis was performed using a one-way ANOVA trial, comparing each group to the untreated cell population.

[0121] All DSPE-PNMVA polymers did not show cell death (>80% of cells were viable 24 hours after treatment) and were comparable to DSPE-PEG for all tested doses. Next, cell viability was evaluated using grafted lipoplexes containing unrelated GL3 siRNA (Figure 8). Naked lipoplex and PNMVA 24 No significant decrease in cell viability was observed in cells treated with grafted lipoplex. DSPE-PEG and DSPE-PNMVA 50 A significant decrease in cell viability (approximately 40%) was observed in lipoplexes grafted with DSPE-PEG. However, viability was observed in cells treated with DSPE-PNMVA compared to cells treated with DSPE-PEG lipoplex. 50 It is important to note that the cells remained equivalent to those treated with lipoplex.

[0122] Human lung adenocarcinoma cells A549 were obtained from the American Type Culture Collection (ATCC, University Blvd, Manassas, VA, USA). A549 cells (A549 / GFP) that stably express green fluorescent protein (GFP) were maintained in Dulbecco's modified Eagle medium (DMEM) (Biowest, VWR, Leuven, Belgium) supplemented with 10% FBS and 1% PenStrep® (Gibco (Invitrogen®, ThermoFisher Scientific, Walthman, MA, USA)) at 37°C in a 5% CO2 humidified atmosphere. A549 cells were then plated in 96-well plates (5 × 10⁶). 3Cells were seeded in wells. After 24 hours, the cells were incubated for 4 hours with different concentrations of polymer (5–150 mol% different molar ratios compared to the total lipids used in the lipoplex formulation) or lipoplex (naked lipoplex and grafted lipoplex) in Opti-MEM (Gibco (Invitrogen (Trademark), ThermoFisher Scientific, Walthman, MA, USA)) prepared at a concentration of 100 nM GL3 siRNA. The formulations were then replaced with fresh culture medium containing 10% (v / v) MTT (3-(4,5-dimethylthiazole-2-yere)-2,5-diphenyltetrazolium bromide) reagent (Invitrogen (Trademark), ThermoFisher Scientific, Bleiswijk, Netherlands) at 37°C for 3 hours. Untreated cells were used as negative controls, and cells treated with 20 mM hydrogen peroxide (H2O2, Sigma-Aldrich Chimie GmbH) were used as positive controls. Absorbance was measured using MikroWin2010 software (Labsis Laborsysteme GmbH, Neunkirchen-Seelscheid, Germany) with TriStar. 2 Measurements were taken at 450 nm using an S LB 942 multimode reader (Berthold Technologies, Vilvoorde, Belgium). The number of viable cells is directly proportional to the absorbance value.

[0123] In vivo acute toxicity in a zebrafish model The toxicity of DSPE-PNMVA lipoplex was tested in zebrafish, a model widely used to elucidate the in vivo behavior (toxicity, biodistribution, pharmacokinetics, and therapeutic effects) of nanoparticles.

[0124] Adult zebrafish (Danio rerio) were maintained in accordance with the standards of the Ethics Committee for the Use of Laboratory Animals at the University of Liège (Protocol No. 21-2313). Fertilized eggs were collected, washed in E3 medium, and placed in petri dishes. Fertilized eggs were incubated at 28°C and maintained for 14 hours during the day and 10 hours at night throughout the experiment. Embryos were collected 24 hours post-fertilization (hpf), their chorionic membranes were removed, and the toxicity of the lipoplex was assessed. Ten embryos were used per 12-well plate condition. Embryos were treated once daily for 2 days and observed daily for a maximum of 72 hpf. Each well contained 1.5 ml of E3 medium with 100 μl of naked lipoplex or grafted lipoplex (15% polymer, 1 and 10 g of siRNA GL3 / kg). The E3 medium stock solution consists of 34.8 g of NaCl, 1.6 g of KCl, 5.8 g of CaCl2·2H2O, and 9.78 g of MgCl2·6H2O relative to a final volume of 2 L of H2O. The pH is adjusted to 7.2 with NaOH. The E3 stock solution is diluted 60-fold before use, and 100 μL of 1% methylene blue is added. The treatment dose was replaced once daily for 2 days, and the embryos were observed daily until 72 hpf.

[0125] After two doses, no specific toxicity was observed compared to the control. At all concentrations tested, embryonic morphology appeared normal: no tail curvature or axial deformation, no pericardium or yolk sac edema, and normal embryonic development compared to the control. Based on these results, it can be concluded that DSPE-PNMVA lipoplex is non-toxic to these living organisms.

[0126] To plan for parenteral administration, the biocompatibility of Lipoplex with blood was evaluated. Hemolysis was tested based on the Drabkin method. Washed red blood cells from three healthy donors were incubated with Lipoplex for 1 hour at 37°C or room temperature (RT) in the presence of 5% CO2. After centrifugation, the supernatant was collected, and hemoglobin concentration was determined by spectroscopic detection of cyanmethemoglobin at 540 nm, expressed as a percentage compared to Triton X-100 (which induces 100% hemolysis). Platelet aggregation was tested using the protocol described above, and with a final concentration of 200 nM siRNA in Lipoplex. Polymer derivatives were tested at a molar ratio of 15% of total lipids. The study protocol conformed to the Declaration of Helsinki and was approved by the Medical Ethics Committee of Centre Hospitalier Universitaire (CHU), UCL Namur (Yvoir, Belgium).

[0127] Compared to the control (Triton X-100), DSPE-PNMVA (15%) lipoplex did not show significant hemolysis or platelet aggregation. All of these results suggest the safe use of DSPE-PNMVA polymer as a PEG alternative. DSPE-PNMVA was found to be blood-compatible.

[0128] 7. Cell uptake and siRNA efficiency One crucial step for efficient gene silencing is the internalization and release of siRNA. This step is considered one of the most significant limitations of PEGylated lipoplexes, as polymer grafting is known to reduce interaction with the cell membrane, thereby decreasing cellular uptake and endosomal extrusion, which inhibits siRNA delivery and gene silencing.

[0129] To assess the ability of grafted lipoplexes to cross the cell membrane and access the cytoplasm (intracellular integration), the intracellular fluorescence intensity of labeled Cy5 GL3 siRNA-lipoplexes was measured. For this purpose, A549 cells were plated in 24-well plates (1 × 10⁶). 5Cells were seeded in wells. After 24 hours, a lipoplex prepared with 100 nM siRNA conjugated with Cy5 was added to the cells for 4 hours. Untreated cells were used as a negative control. After treatment, cells were washed with PBS, harvested with Trypsin-EDTA®, and resuspended in PBS. 1 × 10 4 Cells were analyzed using a BD FACS Canto® II flow cytometer (BD Biosciences, Franklin Lakes, USA) to measure intracellular fluorescence of Cy5.

[0130] As shown in Figure 9, compared to naked lipoplexes, intracellular integration of lipoplexes was reduced by approximately 80% after grafting DSPE-PEG onto their surface. This observation is consistent with the well-known PEG dilemma. Compared to naked lipoplexes, DSPE-PNMVA 24 The presence of the polymer did not reduce the amount of internalized fluorescent siRNA. DSPE-PNMVA 50 In the polymer, the cell permeability of lipoplex was reduced by approximately 50%. Therefore, for the DSPE-PNMVA polymer, cell uptake appears to depend on the length of the hydrophilic chain. Indeed, intracellular fluorescence intensity decreased with longer hydrophilic chains. DSPE-PNMVA 24 In comparison, DSPE-PNMVA 50 Lower uptake was observed when cells were treated with polymer-grafted lipoplexes. In all cases, internalization of lipoplexes grafted with DSPE-PNMVA was higher than that of lipoplexes grafted with DSPE-PEG. 24 and DSPE-PNMVA 50 Lipoplex exhibits approximately 6 times and 3 times higher cellular uptake than DSPE-PEG lipoplex, and therefore appears to be less susceptible to the dilemma than PEG.

[0131] The effect of polymers on lipoplex gene silencing efficiency was evaluated. To address this question, we used A549 cells that stably express GFP and determined the ability of naked lipoplexes and grafted lipoplexes to transfect GFP with siRNA. A549 / GFP cells were plated in 6-well plates (1.8 × 10⁶). 5 Cells were seeded in wells. After 24 hours, the cell culture medium was replaced with 1 mL of Opti-MEM® containing lipoplex prepared with 100 nM siRNA EGFP (siGFP) and grafted lipoplex. Lipofectamine® RNAiMAX (ThermoFisher Scientific, Walthman, MA, USA) associated with EGFP siRNA was used as a positive control. Cells were incubated at 37°C for 4 hours, then the preparation was replaced with fresh culture medium. After 72 hours, cells were washed with PBS at pH 7.4, harvested with Trypsin-EDTA®, and resuspended in 300 μL of PBS. 1 × 10⁶ cells were measured to determine the GFP fluorescence intensity of A549 cells. 4 Cells were analyzed using a CytoFLEX flow cytometer. The intracellular fluorescence intensity of siRNA GL3 Cy5 was detected by flow cytometry after a 4-hour incubation.

[0132] Suppression of GFP fluorescence intensity is an indicator of siRNA transfection efficiency, combining target mRNA entry into cells, delivery / release, and degradation. As shown in Figure 10, the naked lipoplex reduced fluorescence by approximately 60% after 72 hours. Due to the PEG dilemma, the PEGylated lipoplex did not reduce fluorescence as much as the naked lipoplex (approximately 40%). Interestingly, the DSPE-PNMVA lipoplex efficiently reduced fluorescence (fluorescence reduction accounted for 40%–65%). This transfection efficiency appears to correlate with cellular uptake. DSPE-PNMVA 24 Lipoplex (15%) reduced fluorescence by approximately 60%, while DSPE-PNMVA 50Lipoplex (15%) reduced fluorescence by approximately 40%, suggesting that even if the difference is not statistically significant, an increase in hydrophilic chain length tends to decrease efficiency. Furthermore, regarding the effect of polymer percentage, higher percentages appear to have a negative effect, suggesting that the best polymer concentration is 15%. 15% DSPE-PNMVA 50 Alternatively, lipoplexes grafted with DSPE-PEG give similar gene delivery efficiency, but DSPE-PNMVA 24 Lipoplex (15%) was able to reduce fluorescence by approximately 60% compared to DSPE-PEG. 24 15% avoids protein corona formation around the lipoplex while ensuring more efficient siRNA delivery.

[0133] 8. In vivo evaluation in mice Considering the physicochemical results, low toxicity, and good internalization and efficiency for silencing target mRNA, 15% DSPE-PNMVA 24 We selected grafted lipoplexes and compared them to DSPE-PEG in immune BALB / c mice.

[0134] Eight-week-old female BALB / c mice (20-25g) were purchased from Janvier Labs (Saint-Berthevin, France). All animal experiments were evaluated and approved by the Animal Ethics Review Board of the University of Liège (Protocol No. 21-2397). During the study, the animals were housed in a controlled climate and photoperiod (12-hour light-dark cycle) and had free access to water and food. Three groups of eight mice each were divided as follows: (i) a control group injected with PBS, (ii) a group injected with DSPE-PEG lipoplex, and (iii) a group injected with DSPE-PNMVA prepared at a concentration of 1 mg / kg siGL3 Cy5.5. 24 The group that received Lipoplex injections.

[0135] In vivo biodistribution and ABC phenomenon in mice were investigated using 100 μl of grafted lipoplex (15% DSPE-PEG and DSPE-PNMVA) containing 1 mg / kg of control siGL3 Cy5.5. 24 The polymer was intravenously injected at N / P2.5, isotonicized with mannitol, and then monitored weekly for two weeks. In vivo fluorescence imaging was performed on living animals 5 and 24 hours after each injection using the Living Image® software of the IVIS Spectrum In Vivo Imaging System (PerkinElmer). One week after the second injection, the mice were sacrificially killed, and the major organs (liver, kidney, heart, spleen, and lungs) were removed and ex vivo fluorescence imaging was performed.

[0136] Figure 11 shows DSPE-PEG and DSPE-PNMVA 5 hours after the first and second injections. 24 For p<0.0001, p=0.6929 for DSPE-PEG 24 hours after the first injection, and for DSPE-PNMVA 24 For the first injection, p=0.0852; for DSPE-PEG 24 hours after the second injection, p=0.0429; and for DSPE-PNMVA 24 For DSPE-PEG or DSPE-PNMVA, such in vivo distributions are observed with p=0.0435. 24 Significant fluorescence intensity was detected in mice injected with both formulations 5 hours after the first and second injections of Lipoplex. Fluorescence decreased 24 hours after each injection, but 24 hours after the second injection, DSPE-PEG and DSPE-PNMVA 24 Mice injected with lipoplex showed a weak but significant fluorescence signal. This result indicates that lipoplex circulates for a slightly longer period after the second injection, which tends to suggest less of the ABC phenomenon with these formulations.

[0137] Figure 12 shows DSPE-PEG and DSPE-PNMVA in the kidney and heart. 24For [specific item] p < 0.0001, for DSPE-PEG in the liver p = 0.1668, for DSPE-PNMVA in the liver 24 p = 0.1623, for DSPE-PEG in the lung p = 0.5622, for DSPE-PNMVA in the lung 24 p = 0.2249, for DSPE-PEG in the spleen p = 0.7348, and for DSPE-PNMVA in the spleen 24 Having p = 0.7319, for DSPE-PEG and DSPE-PNMVA against mouse organs including the liver, spleen, heart, lung, and kidney 24 Showing the postmortem investigation of the biodistribution of lipoplexes.

[0138] Quantification of fluorescence intensity was for DSPE-PEG and DSPE-PNMVA in the kidney 24 Showed a high signal of Cy5.5 of lipoplexes. Weak fluorescence was also detected in the heart, but the other organs (liver, lung, and spleen) were completely negative. The absence of liver accumulation for both formulations suggests good stealth properties for both the DSPE-PEG lipoplex formulation and the DSPE-PNMVA 24 Lipoplex formulations, and these do not seem to be cleared by the MPS (mononuclear phagocyte system). However, both lipoplex formulations were found to be mainly confined within the kidney, which suggests nanoparticle-kidney interactions and renal clearance. The ability of DSPE-PEG and DSPE-PNMVA to undergo complete renal clearance or accumulate in specific parts of the kidney 24 Of lipoplexes probably depends on a combination of various physicochemical properties such as their diameter, surface charge, structural characteristics, morphology, etc.

[0139] Detection of anti-PEG or anti-PNMVA antibodies of IgG and IgM and pro-inflammatory cytokines Blood was collected from the mouse tail 7 days after the first injection and by cardiac puncture on day 14. To obtain serum, the blood was left at room temperature for 30 minutes and then centrifuged at 1000 g for 15 minutes at 4 °C. Serum collected from mice injected with PBS was used as a negative control. The direct ELISA procedure was employed to detect PEG- or PNMVA-specific IgM and IgG antibodies in the serum. 2 μg of DSPE-PEG or DSPE-PNMVA in ethanol 24 was added to a 96-well plate. The coated plate was allowed to air dry completely. The plate was then washed with PBS and then blocked with PBS containing 2% BSA for 1 hour. Then, diluted serum samples (1:1000) were added to the wells, incubated for 1 hour, and washed 5 times with PBS. Horseradish peroxidase (HRP)-conjugated antibodies diluted 1 / 1000 in PBS (goat anti-mouse IgM-HRP (Invitrogen™, Camarillo, USA) or goat anti-mouse IgG-HRP (Cell Signaling, Leiden, The Netherlands)) were added to the wells. After 45 minutes, the wells were washed 5 times with PBS. Color development was initiated by adding TMB (Invitrogen™). After 30 minutes, the reaction was stopped by adding 2 M sulfuric acid. Absorbance was measured at 450 nm using the MikroWin2010 software (Labsis Laborsysteme GmbH, Neunkirchen-Seelscheid, Germany) of a TriStar 2 S LB 942 multimode reader (Berthold Technologies, Bad Wildbad, Germany). All incubations were performed at room temperature using a plate shaker (300 rpm).

[0140] IgG levels that rely on IgM stimulation are an important indicator of the immune response when foreign substances are injected into the body. DSPE-PEG or DSPE-PNMVA 24IgM and IgG levels were assessed in mouse serum one week after each injection using an ELISA assay, and are shown in Figures 13 and 14, respectively. The results were compared to a negative control group injected with PBS. For the ELISA assay, the results for each mouse represent the mean of two trials. Statistical analysis was performed using a two-way ANOVA assay. DSPE-PEG or DSPE-PNMVA 24 Production of IgG and IgM against DSPE-PNMVA was observed after the first injection. After the second injection, levels of anti-DSPE-PEG IgM and IgG increased further, with approximately a three-fold increase compared to the first injection. This higher presence of detectable anti-PEG IgG after the second injection suggests that immunological memory is likely present. Such a rapid increase in anti-PEG antibody levels upon repeated injection of liposomes could pose a real challenge to the individual by inducing serious adverse reactions. DSPE-PNMVA 24 After the second injection with Lipoplex, anti-DSPE-PNMVA 24 A slight but significant increase (approximately 1.5 times) in IgG was observed compared to the first injection, but anti-DSPE-PNMVA 24 No increase in IgM was detected. This result is related to DSPE-PNMVA 24 This demonstrates that lipoplex has lower immunogenicity than DSPE-PEG lipoplex.

[0141] With the aim of studying inflammation-related functions, the study ultimately aimed to investigate the systemic production of two pro-inflammatory cytokines (TNFα and IL-1 beta) after a second intravenous injection using commercially available ELISA kits (mouse IL-1 beta ELISA kit and mouse TNF-alpha ELISA kit; Invitrogen®, Vienna, Austria) with DSPE-PEG or DSPE-PNMVA. 24 The effects were investigated. Medication was administered according to the manufacturer's instructions. IL-1β and TNF-α levels were measured in vivo.

[0142] PBS control group (for DSPE-PEG, p=0.555, and for DSPE-PNMVA)24 (For DSPE-PEG, p=0.3847) and TNF-α (For DSPE-PEG, p=0.5865, and for DSPE-PNMVA) 24 For comparison (p=0.9226), DSPE-PEG lipoplex or DSPE-PNMVA 24 No increase in IL-1β and TNF-α levels was detected in the serum of mice injected with lipoplex, and each value represents the mean + / - standard deviation (SD) of at least 7 mice (n=7 or 8). This result indicates that intravenous administration of these grafted lipoplexes did not induce an inflammation-related response in vivo. Following the lack of toxicity to human blood in vitro and in zebrafish models in vivo, these results suggest that DSPE-PNMVA is suitable for in vivo use. 24 The safety of Lipoplex has been confirmed.

Claims

1. A polymer derivative comprising a hydrophilic segment obtained from the polymerization of N-methyl-N-vinylacetamide, coupled to at least one hydrophobic segment, wherein the hydrophobic segment comprises at least one aliphatic chain having 8 to 28 carbon atoms.

2. The polymer derivative according to claim 1, wherein the at least one hydrophobic segment is selected from the group of hydrophobic segments comprising: a single aliphatic chain having 8 to 28 carbon atoms; two aliphatic chains having 8 to 28 carbon atoms; one or two fatty acids having an aliphatic chain having 8 to 28 carbon atoms; a diglyceride having two aliphatic chains having 8 to 28 carbon atoms; a ceramide having an aliphatic chain having 8 to 28 carbon atoms; or a phospholipid having an aliphatic chain having 8 to 28 carbon atoms.

3. The polymer derivative according to claim 1 or 2, wherein the at least one aliphatic chain is a saturated aliphatic chain.

4. The polymer derivative according to any one of claims 1 to 3, wherein the at least one hydrophobic segment is a phosphatidylethanolamine having two aliphatic chains having 8 to 28 carbon atoms, preferably 12 to 24 carbon atoms, more preferably 14 to 20 carbon atoms, and most preferably 16 to 18 carbon atoms.

5. The polymer derivative according to any one of claims 1 to 4, wherein the at least one hydrophobic segment is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

6. The polymer derivative according to any one of claims 1 to 5, wherein the hydrophilic segment has a molecular weight of 1,000 to 10,000 g / mol, preferably 1,500 to 6,000 g / mol, and more preferably 2,000 to 4,000 g / mol.

7. A method for preparing a polymer derivative according to any one of claims 1 to 6, (i) A step of coupling a hydrophobic segment containing at least one aliphatic chain of 8 to 28 carbon atoms to a chain control agent. (ii) A step of polymerizing N-methyl-N-vinylacetamide in the presence of the coupled chain control agent and, optionally, a radical polymerization initiator. or (i) A step of polymerizing N-methyl-N-vinylacetamide in the presence of a chain control agent functionalized with a reactive group, and optionally a radical polymerization initiator. (ii) A method comprising the step of coupling the obtained polymer to a hydrophobic segment containing at least one aliphatic chain of 8 to 28 carbon atoms by reaction with the reactive group.

8. A polymer derivative according to any one of claims 1 to 6, for use in lipid nanoparticles.

9. Lipid nanoparticles containing the polymer derivative according to any one of claims 1 to 6 in a molar percentage of 1 to 50%, preferably 1 to 30%, relative to the total lipids.

10. Lipid nanoparticles according to claim 9, having a size of less than 500 nanometers, preferably less than 200 nanometers.

11. Lipid nanoparticles according to claim 9 or 10, further comprising an activator.

12. Lipid nanoparticles according to any one of claims 9 to 11, comprising nucleic acid.

13. Lipid nanoparticles according to any one of claims 9 to 12, for use in a medical procedure or a method of preventing disease.

14. Lipid nanoparticles according to claim 12 for use in nucleic acid delivery.

15. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 9 to 12 and one or more pharmaceutically acceptable carriers.

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