How to avoid pre-stored anti-PEG antibodies in humans

Abstract

The present invention discloses a method for avoiding anti-PEG antibodies pre-stored in humans, as well as the use of hydroxy-terminal PEGylated nanocarriers in the preparation of drugs that avoid anti-PEG antibodies pre-stored in humans. The present invention also discloses hydroxy-terminal PEGylated nanocarriers and nanoformulations. The hydroxy-terminal PEGylated nanocarriers and nanoformulations of the present invention exhibit low binding to anti-PEG antibodies pre-stored in humans, thereby avoiding rapid clearance in human blood and enabling better therapeutic effects. Furthermore, by avoiding binding to anti-PEG antibodies pre-stored in human blood, hydroxy-terminal PEGylated nanocarriers and nanoformulations can reduce complement activation and side effects such as clinical injection reactions.

Description

[Technical Field]

[0001] Technical Field The present invention belongs to the field of biomedicine technology and relates to a method for avoiding anti-PEG antibodies pre-stored in humans. Specifically, the present invention relates to hydroxyl-terminal PEGylated nanocarriers and nanoformulations, and the use of hydroxyl-terminal PEGylated nanocarriers in avoiding anti-PEG antibodies pre-stored in humans. [Background technology]

[0002] Background technology Pre-stored anti-polyethylene glycol (PEG) antibodies in humans refer to anti-PEG antibodies detected in the bodies of healthy individuals or clinical patients who have never used drugs or drug carriers containing PEG. According to research by Kozma et al., the mechanism of the generation of pre-stored anti-PEG antibodies is currently unknown and may be closely related to the widespread use of PEG as an additive in foods and cosmetics. Over time, the rate of anti-PEG antibody positivity has shown an increasing trend among people, increasing from 0.2% in 1984 to 40% in 2016 (Kozma, G.T., et al., Anti-PEG antibodies: Properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals. Advanced Drug Delivery Reviews, 2020. 154-155: pp. 163-175; Richter, A.W. and E. Akerblom, Polyethylene glycol reactive antibodies in man: titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors. Int Arch Allergy Appl Immunol, 1984. 74(1): pp. 36-9; Chen, B.-M., et al., Measurement of Pre-Existing IgG and IgM Antibodies against Polyethylene Glycol in Healthy Individuals. Analytical Chemistry, 2016. 88(21): p. 10661-10666).

[0003] PEG has been widely applied in the field of nanopharmaceuticals because it can increase the in vivo and in vivo stability and long-term circulation of modified nanocarriers or nanoformulations. In currently available formulations, the end group of PEG is generally a methoxy group. For example, the PEGylated lipid structure in the PEGylated lipid body (Doxil®) encapsulating doxorubicin is methoxypolyethylene glycol-distearoylphosphatidylethanolamine. The PEGylated lipid structure used in the already-released siRNA drug-lipid nanoparticles Onpattro and Moderna's mRNA COVID-19 vaccine is methoxypolyethylene glycol-dimyristoylglycerol, and the PEGylated lipid structure used in BioNTech's mRNA COVID-19 vaccine is methoxypolyethylene glycol-tetracosanylacetamide. One reason for using a methoxy group at one end of PEG is the simple structure of methoxyPEG (mPEG) and its inertness. This allows for derivatization of only one active end of PEG, minimizing crosslinking during the synthesis reaction and maximizing the homogeneity of the final product.

[0004] However, recent research has shown that while PEG improves the in vivo performance of nanocarriers or formulations, it also poses significant problems, such as the fact that anti-PEG antibodies present in the body can quickly identify and bind to PEGylated nanocarriers or nanoformulations, activating the complement system and causing complement C3 fragments to condition the particles, thereby accelerating the uptake of PEGylated nanocarriers or nanoformulations by the monocyte-macrophage system (MPS) and rapidly clearing them from the blood. Anti-PEG antibodies are the main cause of accelerated blood clearance, and clinical experience with PEGylated therapy has revealed that anti-PEG antibodies not only increase the clearance rate, resulting in a loss of therapeutic efficacy, but also causing serious side effects such as injection reactions.

[0005] In addition to the anti-PEG antibodies pre-stored in the body (the mechanism of origin is unknown, but anti-PEG antibodies are already present in the body before administration), anti-PEG antibodies may also be produced after repeated injections of PEGylated nanocarriers. For example, when PEGylated nanocarriers or nanoformulations were injected into animal models such as mice, rats, guinea pigs, rabbits, beagles, and Ganges monkeys, anti-PEG antibodies could be detected in the body one week later, and were mainly anti-PEG IgM antibodies, which showed a significant decrease after 2 to 3 weeks in the body. It has been reported that this anti-PEG IgM antibody is produced primarily by stimulating the proliferation and differentiation of splenic marginal zone B cells in a T cell-independent (TI) manner, and is generally produced by low doses of PEGylated nanocarriers, but high doses of PEGylated nanocarriers or loaded chemotherapeutic drugs (e.g., doxorubicin) have been reported to produce low or no PEG antibodies (Koide, H., et al., T cell-independent B cell response is responsible for ABC phenomenon induced by repeated injection of PEGylated liposomes. Int J Pharm, 2010. 392(1-2): p. 218-23).

[0006] There is considerable controversy regarding the effects of repeated administration on anti-PEG antibodies and the in vivo effects of PEGylated nanocarriers or nanoformulations. While many PEGylated nanocarriers that produce anti-PEG antibodies after repeated injections are administered at doses lower than clinically applicable doses, the predicted clinical correlation is relatively poor. On the other hand, PEGylated nanocarriers currently used clinically do not produce antibodies when used to deliver cytotoxic drugs. Finally, there are significant species differences in the effects of repeated injections on the in vivo performance of PEGylated nanocarriers or nanoformulations, affecting the effective evaluation of clinical translation (Suzuki, T., et al., Influence of dose and animal species on accelerated blood clearance of PEGylated liposomal doxorubicin. Int J Pharm, 2014. 476(1-2): pp. 205-12). To address the impact of repeated injections on the in vivo anti-PEG antibody production of PEGylated nanocarriers or nanoformulations, researchers have attempted to use materials such as PVP, PDMA, and HPMA instead of PEG, and many researchers have devoted considerable time to structural modifications of PEG. According to Chinese Patent Application CN11330512A, PEG is immunogenic, and a direct solution to this problem was proposed. Specifically, the initial injection of PEG2,n (branched PEG)-modified nanocarriers into rats significantly reduced the production of anti-PEG antibodies compared with PEG2k (linear PEG), suggesting that the use of PEG2,n-modified nanocarriers can reduce the production of anti-PEG antibodies. Furthermore, the initial injection of PEG2,n-modified emulsions, followed by repeated injections 7 days later, resulted in different levels of antibody binding. As the molecular weight of PEG2,n increased, the binding between the drug and antibody slowed, with PEG2,40k-modified antibody binding being the slowest. Nanocarriers modified with high molecular weight PEG2,n were more advantageous in extending the circulation time of nanocarriers, suggesting that nanocarriers modified with PEG2,n-lipid derivatives could address the issues of rapid blood clearance of PEGylated nanocarriers and significantly increased aggregation in the liver and spleen.

[0007] US Patent Application No. 20210046188A1 reported that branched polymers containing PEG side chains were used to reduce or eliminate the antigenicity of the molecule and eliminate reactivity against patient-derived PEG antibodies. This patent application disclosed that the terminal groups of the PEG side chains were methoxy groups, and pointed out that the key to eliminating pre-stored anti-PEG antibodies was the length and terminal group of the PEG side chains.

[0008] Shimizu et al. reported that the first PEGylated lipid bilayer (PL) (first agent) induces an anti-PEG IgM antibody response, which triggers rapid systemic clearance of the second PL (second agent) through a phenomenon known as "accelerated blood clearance (ABC)" (Shimizu, T., et al., "A hydroxyl PEG version of PEGylated liposomes and its impact on anti-PEG IgM induction and on the accelerated clearance of PEGylated liposomes." European Journal of Pharmaceutics and Biopharmaceutics, 2018. 127: pp. 142-149). In this paper, they confirmed that PL-OH essentially activates the complement system via the alternative pathway in mice without anti-PEG IgM. In the presence of anti-PEG IgM, PL-OH activates the complement system not only via the classical pathway mediated by anti-PEG IgM, but also via the alternative pathway. Therefore, due to such strong complement-activating properties, PL-OH may be rapidly cleared from the blood circulation even in the absence (first agent) or presence (second agent) of anti-PEG IgM. Summary of the Invention

[0009] Summary of the Invention Problems that the invention aims to solve During long-term research on PEGylated nanocarriers, the inventors discovered the following:

[0010] 1. The biological activity of anti-PEG antibodies stimulated by repeated administration of PEGylated nanocarriers in animal models was significantly different from that of pre-preserved anti-PEG antibodies in humans (the mechanism of origin is unknown, but anti-PEG antibodies are already present in the host before administration). The pre-preserved anti-PEG antibodies in humans have different binding avidity to different PEG end groups. The inventors examined the commonly used methoxy-terminated PEG (MeO-PEG), carboxyl-terminated PEG (HOOC-PEG), amino-terminated PEG (H2N-PEG), and hydroxyl-terminated PEG (HO-PEG) and found that the binding avidity of pre-preserved anti-PEG antibodies in humans was highest for MeO-PEG and lowest for HO-PEG. Because methoxy-PEG has a simple structure and the methoxy group is inert, only one active end of PEG can be derivatized, minimizing crosslinking during the synthesis and resulting in the highest homogeneity of the final product. Therefore, currently available PEGylated nanoparticle formulations are essentially MeO-PEG derivatives. The inventors discovered that the binding mechanisms of anti-PEG antibodies generated by injecting PEGylated nanocarriers in animals differ from those of anti-PEG antibodies pre-prepared in humans. The primary antigen-binding site of anti-PEG antibodies generated by injecting PEGylated nanocarriers in rats may be the ethylene glycol repeating units in the PEG chain. Because there is no preference for PEG end groups, the anti-PEG antibodies generated in rats have similar high binding activity to different PEG end groups. Because anti-PEG antibodies pre-prepared in humans primarily discriminate between PEG end groups, the anti-PEG antibodies pre-prepared in human serum have different binding activity to different PEG end groups, with HO-PEG being the lowest. Most drugs require animal testing before clinical trials. The inventors speculate that, due to a lack of understanding of different antibody binding mechanisms, the fact that anti-PEG antibodies generated in animals have the same binding activity to different PEG end groups has hindered researchers' research into the application of HO-PEGylated nanocarriers to humans and the further development of human drugs. Therefore, researchers are focusing more attention on inert MeO-PEGylated nanocarriers and nanoformulations.

[0011] 2. A study by Sherman et al. showed that proteins modified with hydroxyl-terminated PEG produced lower immunogenicity and better in vivo performance in rabbits compared with proteins modified with methoxy-terminated PEG (Sherman, MR, et al., Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates. Bioconjugate Chemistry, 2012. 23(3): pp. 485-499). Meanwhile, a study by Shimizu et al. (see above) found that lipid bodies modified with hydroxyl-terminated PEG enhanced complement activation and exhibited a stronger accelerated clearance phenomenon in the body after secondary injection than lipid bodies modified with methoxy-terminated PEG. Based on the contradictory research results of modification with hydroxyl-terminated PEG in animal bodies, the inventors examined the complement activation ability of various nanocarriers modified with hydroxyl-terminated PEG in different animal species and human blood (see Example 3). They found that liposomes modified with hydroxyl-terminated PEG showed some indication of enhancing complement activation in mice and rats, but no enhancement of complement activation was observed in human blood. Furthermore, when the nanocarriers were lipid nanoparticles, their complement activation ability in mouse and rat blood was equivalent to that of modification with methoxyl-terminated PEG. This indicates that the effect of modification with hydroxyl-terminated PEG on the complement activation ability of nanocarriers or nanoformulations is closely related to the species and type of nanocarrier. None of the nanocarriers detected in human blood showed significant enhancement of complement activation. This explains why the animal experimental results reported in the literature hinder the application of hydroxyl-terminated PEG-modified nanocarriers or nanoformulations in human research.

[0012] Based on the above findings, the objective of the present invention is to apply HO-PEGylated nanocarriers with low binding activity to anti-PEG antibodies in humans to nanopreparations for human use, thereby avoiding binding with anti-PEG antibodies pre-stored in humans and providing PEGylated nanopreparations that can be rapidly cleared in the body and avoid the occurrence of side effects such as injection reactions.

[0013] Means to solve the problem The present invention provides a use of PEGylated nanocarriers modified with terminal hydroxyl groups in the preparation of drugs that evade anti-PEG antibodies pre-existing in humans, wherein the PEGylated nanocarriers are one or more selected from liposomes, polymeric nanoparticles, micelles, and lipid nanoparticles.

[0014] In one embodiment, the molecular weight of the PEG is 500 to 10,000 Da. In one embodiment, the molecular weight of the PEG is 1,000 to 5,000 Da. In one embodiment, the molecular weight of the PEG is 2,000 Da.

[0015] In one embodiment, the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol % to 10 mol %, preferably 1 mol % to 5 mol %.

[0016] In one embodiment, the modification ratio of the PEG in the polymer nanoparticles and micelles is 0.5 mol % to 100 mol %, preferably 50 mol % to 100 mol %.

[0017] In one embodiment, the drug comprises one or more of a small molecule drug, a protein drug, and a nucleic acid drug.

[0018] In one embodiment, the nucleic acid drug is one or more selected from small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA).

[0019] In one embodiment, the terminal hydroxyl group modified PEG has a hydroxyl group at one end and a polymer such as an amphiphilic polymer or a lipid at the other end.

[0020] In one embodiment, the PEGylated nanocarriers comprise one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

[0021] The present invention also provides a nanoformulation for human use. In one embodiment, the nanoformulation comprises an active ingredient and a PEGylated nanocarrier modified with a terminal hydroxyl group. In one embodiment, the nanocarrier is one or more selected from liposomes, polymeric nanoparticles, micelles, and lipid nanoparticles (LNPs). In one embodiment, the active ingredient is one or more selected from small molecule drugs, protein drugs, and nucleic acid drugs.

[0022] In one embodiment, the molecular weight of PEG in the nanoformulation for humans of the present invention is 500 Da to 10,000 Da.

[0023] In one embodiment, in the nanopreparation for human use of the present invention, the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol% to 10 mol%, preferably 1 mol% to 5 mol%, and in one embodiment, the PEG modification ratio in the micelles and polymer nanoparticles is 0.5 mol% to 100 mol%, preferably 50 mol% to 100 mol%.

[0024] In one embodiment, in the nanoformulation for humans of the present invention, the nucleic acid drug is one or more selected from siRNA, DNA, and mRNA.

[0025] In one embodiment, in the nanoformulation for humans of the present invention, one end of the hydroxyl-terminal modified PEG is a hydroxyl group, and the other end is a polymer such as an amphiphilic polymer or a lipid.

[0026] In one embodiment, in the nanoformulations for humans of the present invention, the PEGylated nanocarriers comprise one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

[0027] The present invention also provides a method for treating a disease by circumventing pre-existing anti-PEG antibodies in humans, comprising administering to a human subject in need thereof a nanoformulation comprising an active ingredient and a PEGylated nanocarrier modified with a terminal hydroxyl group. In one embodiment, the nanocarrier is one or more selected from liposomes, polymeric nanoparticles, micelles, and lipid nanoparticles (LNPs). In one embodiment, the active ingredient is one or more selected from small molecule drugs, protein drugs, and nucleic acid drugs.

[0028] In one embodiment, the PEGylated nanocarriers containing terminal hydroxyl group modifications can reduce complement activation.

[0029] In one embodiment, the molecular weight of the PEG is 500 to 10,000 Da. In one embodiment, the molecular weight of the PEG is 1,000 to 5,000 Da. In one embodiment, the molecular weight of the PEG is 2,000 Da.

[0030] In one embodiment, the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol % to 10 mol %, preferably 1 mol % to 5 mol %.

[0031] In one embodiment, the modification ratio of the PEG in the polymer nanoparticles and micelles is 0.5 mol % to 100 mol %, preferably 50 mol % to 100 mol %.

[0032] In one embodiment, the nucleic acid drug is one or more selected from siRNA, DNA, and mRNA.

[0033] In one embodiment, the terminal hydroxyl group modified PEG has a hydroxyl group at one end and a polymer such as an amphiphilic polymer or a lipid at the other end.

[0034] In one embodiment, the PEGylated nanocarriers comprise one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

[0035] Effect of the invention The hydroxyl-terminated PEGylated nanocarriers and nanoformulations of the present invention exhibit low binding to anti-PEG antibodies pre-stored in humans, thereby avoiding rapid clearance in human blood and thereby enabling better therapeutic effects.Furthermore, by avoiding binding to anti-PEG antibodies pre-stored in human blood, the hydroxyl-terminated PEGylated nanoformulations of the present invention can reduce complement activation and avoid side effects such as injection reactions during clinical administration. [Brief explanation of the drawings]

[0036] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows a comparison of the binding of anti-PEG antibodies to MeO-PEG and HO-PEG as detection antigens in human blood samples.

[0037] [Figure 2] Figure 1 shows the anti-PEG-IgM content generated by repeated weekly injections of nanocarriers modified with HO-PEG or MeO-PEG into mice, where a, b, c, and d are the results of serum testing of mice after naive, 1 week, 3 weeks, and 6 weeks, respectively.

[0038] [Figure 3] FIG. 1 shows the complement activation results of nanocarriers modified with HO-PEG or MeO-PEG in different animal and human serum.

[0039] [Figure 4] Fig. 1 shows the binding activity of anti-PEG-IgM stimulated in rat serum and anti-PEG-IgM pre-stored in human serum to DSPE containing different PEG end group structures, where a shows the experimental results for rat serum and b shows the experimental results for human serum.

[0040] [Figure 5] Figure 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEGs with different hydrophobic groups or PEG chain lengths, where a, b, c, and d represent the experimental results for the hydrophobic groups and PEG chain lengths of PEG2000-DSPE, PEG2000-DMG, PEG10000-PLGA, and PEG500-DMG, respectively.

[0041] [Figure 6] Figure 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum with blank liposomes having different PEG modification ratios, where a, b, and c are the experimental results for PEG ratios of 1%, 3%, and 5%, respectively.

[0042] [Figure 7] Figure 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEG liposomes encapsulating different drugs, where a and b are experimental results for encapsulating ovalbumin and doxorubicin, respectively.

[0043] [Figure 8] FIG. 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEG micelles and MeO-PEG micelles.

[0044] [Figure 9] FIG. 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEG polymer nanoparticles and MeO-PEG polymer nanoparticles.

[0045] [Figure 10] This figure shows the binding activity of anti-PEG-IgM pre-stored in human serum with LNPs modified with different PEG ratios, where a, b, c, d, e, and f represent the experimental results for 5% modified DSPE end groups, 3% modified DSPE end groups, 1.5% modified DSPE end groups, 5% modified DMG end groups, 3% modified DMG end groups, and 1.5% modified DMG end groups, respectively.

[0046] [Figure 11] FIG. 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to LNPs encapsulating siRNA.

[0047] [Figure 12] FIG. 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to LNPs encapsulating mRNA.

[0048] [Figure 13] FIG. 1 shows the binding activity of anti-PEG-IgM pre-stored in human serum to DNA-encapsulated LNPs.

[0049] [Figure 14] FIG. 1 shows the complement activation results of PEGylated nanoformulations.

[0050] [Figure 15]FIG. 1 shows the complement activation results of PEGylated liposomes and lipid nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[0051] MODE FOR CARRYING OUT THE INVENTION The objective of the present invention is to provide a PEGylated nano-preparation that can avoid binding with PEG antibodies pre-stored in humans and rapid clearance in humans by applying HO-PEGylated nano-carriers with low binding activity to human drugs.

[0052] The inventors conducted experimental verification of various currently used end-group modified PEGylated nanocarriers and found that HO-PEGylated liposomes, polymer nanoparticles, micelles, and lipid nanoparticles (LNPs) exhibited low binding activity with anti-PEG antibodies pre-prepared in humans.

[0053] In the present invention, the molecular weight of PEG is preferably 500 to 10,000 Da, more preferably 1,000 to 5,000 Da, and most preferably 2,000 Da in terms of ease of availability.

[0054] In the liposomes and lipid nanoparticles, the PEG modification ratio is 0.5 mol % to 10 mol %, and in the polymer nanoparticles and micelles, the PEG modification ratio is 0.5 mol % to 100 mol %.

[0055] In the present invention, the hydroxyl-terminal modified PEG has one end hydroxyl and the other end is an arbitrary structure useful for embedding into nanocarriers. The structure useful for embedding into nanocarriers is not involved in antibody binding and is simply used to enable PEG modification of the nanocarriers, and is therefore not critical to the implementation of the present invention. Such a structure may be a polymer, such as an amphiphilic polymer, or a lipid. Examples of such structures include, but are not limited to, one or more of polylactic acid-hydroxyacetic acid copolymer, polylactic acid, distearoylphosphatidylethanolamine, dimyristoylglycerol, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, lecithin, cardiolipin, and sphingomyelin. In one embodiment, the fatty acid chain length of the phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine is between 4 and 28 carbon atoms and has a degree of unsaturation of 0 to 3.

[0056] In the present invention, the drug is not limited and may be one or more of small molecule drugs, protein drugs, and nucleic acid drugs, among which the nucleic acid drugs are also not particularly limited and may be one or more of siRNA, DNA, and mRNA.

[0057] In the present invention, the lipid is not particularly limited, and various commonly used lipids available on the market, such as dimyristoylglycerol, distearoylphosphatidylethanolamine, polylactic acid-hydroxyacetic acid, soybean lecithin, egg yolk lecithin, phosphatidylglycerol, EPG, phosphatidic acid, cardiolipin, sphingomyelin, serine phosphatidate, phosphatidylinositol, phosphatidylethanolamine, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, distearoylphosphatidylcholine, dipalmito ... phosphatidylcholine, dioleylphosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, didecanoylphosphatidylcholine, dioctanoylphosphatidylcholine, dihexanoylphosphatidylcholine, distearoylphosphatidylglycerol and its salts, dipalmitoylphosphatidylglycerol and its salts, L-α-dimyristoylphosphatidylglycerol and its salts, dilauroylphosphatidylglycerol, didecanoylphosphatidylglycerol , dioctanoylphosphatidylglycerol, dihexanoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine, dioleylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, bisdistearoylphosphatidylglycerol and its salts, bisdipalmitoylphosphatidylglycerol and its salts, bisdimyristoylphosphatidylglycerol and its salts, bisdilauroylphosphatidylglycerol One or more of the following may be used: distearoylphosphatidylinositol, dipalmitoylphosphatidylinositol, dioleylphosphatidylinositol, dimyristoylphosphatidylinositol, dilauroylphosphatidylinositol, palmitoyloleylphosphatidylcholine, palmitoyllinoleoylphosphatidylcholine, stearoyllinoleoylphosphatidylcholine, stearoyloleylphosphatidylcholine, and stearoylarachidonylphosphatidylcholine.

[0058] Example

[0059] The present invention will be described in detail below based on examples. The examples enumerate detailed embodiments of the present invention in order to fully disclose and demonstrate that the present invention can be carried out. However, the examples do not imply any limitations on the present invention, and the protection scope of the present invention is defined by the claims. Based on the specific examples of the present invention, those skilled in the art can obtain all aspects and scope of the present invention without any creative work.

[0060] In the examples of the present invention, unless otherwise specified, all raw materials are commercially available products well known to those skilled in the art. PEG derivatives used in the examples include MeO-PEG 2000 -DSPE, HO-PEG 2000 -DSPE, NH2-PEG 2000 -DSPE, COOH-PEG 2000 -DSPE, MeO-PEG 2000 -DMG, HO-PEG 2000 -DMG, etc. (all purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and MeO-PEG 10000 -PLGA (50:50) and HO-PEG 10000 Examples of suitable methods include, but are not limited to, PEG-100-PLGA (50:50) (purchased from Xi'an Ruixi Biological Technology Co., Ltd.). In the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art. The progress of the reaction of the present invention can be monitored using conventional monitoring methods in the art, such as TLC, HPLC, LCMS or NMR, and the disappearance of the reaction substrate is generally regarded as the end point of the reaction.

[0061] Experimental methods for which specific conditions are not specified in the examples of the specification generally follow the conditions common to the art or the conditions suggested by the manufacturer. In the present invention, unless otherwise specified, "parts" refers to parts by weight, and "weight" refers to mole percent. In the present invention, unless otherwise specified, "more than," "less than," and "within" refer to the inclusive numbers.

[0062] Abbreviations: [Table 1]

[0063] Unless otherwise specified, HO-PEG-DSPE is HO-PEG 2000 -DSPE, and MeO-PEG-DSPE refers to MeO-PEG 2000 -Refers to DSPE.

[0064] Terminology Small molecule drugs: Primarily refers to chemically synthesized drugs, which are organic compounds usually with a molecular weight of less than 1,000.

[0065] PEG modification ratio: The molar ratio of PEGylated material to all materials in the formulation or carrier. [Example]

[0066] Example 1

[0067] This example is used to demonstrate that pre-preserved anti-PEG antibodies in humans have high binding activity to MeO-PEG.

[0068] Sandwich ELISA was used to detect the proportion of the pre-stored anti-PEG antibody positive group.

[0069] The specific operations are as follows:

[0070] Step 1: 2 μg MeO-PEG per well in a medium binding ELISA plate 2000After adding -DSPE, the plate was left overnight at room temperature, washed three times with PBST, blocked with 5% BSA at 37°C for 1 hour, washed three times with PBST, and then gradient-diluted human serum (initial 4-fold dilution) in 1 wt% BSA-PBS was added. The plate was then incubated at 37°C for 1.5 hours. After washing three times with PBST, horseradish peroxidase-labeled anti-rat or human IgM antibody was added. After 1 hour, the plate was reacted with TMB coloring solution for 8 minutes, and the reaction was stopped with 0.18M H2SO4. The absorbance value was detected at 450 nm. Samples with an initial OD value of greater than 1.0 were considered MeO-PEG positive.

[0071] Step 2: Add 2 μg of HO-PEG per well to a medium-binding ELISA plate 2000 The cells were incubated overnight at room temperature, washed three times with PBST, blocked with 5 wt% BSA at 37°C for 1 hour, washed three times with PBST, and then gradient-diluted human serum (from the MeO-PEG-IgM-positive group selected in step 1 above) was added in 1% BSA-PBS. The cells were incubated at 37°C for 1.5 hours, washed three times with PBST, and then horseradish peroxidase-labeled anti-rat or human IgM antibody was added. After 1 hour, the cells were incubated with TMB color developer for 8 minutes. The reaction was stopped with 0.18M H2SO4, and the absorbance was measured at 450 nm. Samples with an initial OD value of greater than 1.0 were considered HO-PEG-positive.

[0072] We screened pre-stored antibodies against 854 clinical blood samples and found that approximately 25% of the blood samples had strong binding activity to MeO-PEG (detected by ELISA, OD > 1.0). When HO-PEG was substituted as the detection antigen, only approximately 10% of the samples were positive for MeO-PEG, indicating that the antibodies pre-stored in human blood samples have selectivity for MeO-PEG. Anti-PEG antibodies pre-stored in humans have high binding activity to MeO-PEG and low binding activity to HO-PEG.

[0073] The results are shown in Figure 1. [Example]

[0074] Example 2

[0075] This example is used to demonstrate that the immunogenicity of nanoformulations modified with HO-PEG-DSPE is lower than that of MeO-PEG-DSPE.

[0076] The specific operations are as follows:

[0077] LNPs modified with MeO-PEG-DSPE or HO-PEG-DSPE were repeatedly injected intravenously (IV) or intramuscularly (IM) into mice (10 mg LNP / kg, once a week). Serum samples were collected before the first injection and 1, 3, and 6 weeks after the first injection, respectively, and the anti-PEG-IgM content produced by the stimulation of the mice was detected using a sandwich ELISA. 2 μg of MeO-PEG per well was added to a medium-binding ELISA plate. 2000 -DSPE or HO-PEG 2000 -DSPE (corresponding to the injected formulation) was added, left at room temperature overnight, washed three times with PBST, blocked with 5% BSA at 37°C for 1 hour, washed three times with PBST, added mouse serum diluted with PBS gradient, incubated at 37°C for 1 hour, washed three times with PBST, added anti-mouse IgM antibody labeled with horseradish peroxidase, reacted with TMB coloring solution for 10 minutes after 1 hour, stopped with 0.18M H2SO4, and the absorbance value (OD value) was detected at a wavelength of 450 nm. The experimental results are shown in Figure 2.

[0078] By comparing the immunogenicity of LNPs modified with hydroxyl- or methoxyl-terminated PEG in mice, we found that the anti-PEG-IgM levels stimulated by LNPs modified with hydroxyl- or methoxyl-terminated PEG were significantly lower than those stimulated by methoxyl-terminated PEG, regardless of tail vein or intramuscular injection. The results were similar for single injections (1 time, Figure 2, b) and multiple injections (2 times, Figure 2, c; 5 times, Figure 2, d). This suggests that nanoparticles modified with hydroxyl-terminated PEG have the advantage of low immunogenicity. [Example]

[0079] Example 3

[0080] This example is used to demonstrate that the effect of modification with hydroxyl-terminated PEG on the complement activation ability of nanocarriers or nanoformulations is closely related to the species and type of nanocarrier.

[0081] Liposomes or LNPs modified with hydroxyl- or methoxy-terminated PEG were mixed with mouse, rat, or human serum and incubated at 37°C for 1.5 hours. The reaction was stopped with 10 mM EDTA-PBS, and the serum was diluted 500-fold. Loading buffer was added and the mixture was incubated at 95°C for 10 minutes to denature the proteins. Proteins were separated by molecular weight using 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was then used to detect the degree of C3 protein degradation in the serum, and the ratio of degradation products to C3 protein was statistically determined.

[0082] To compare the effect of modification with hydroxyl-terminated PEG on the complement activation activity of liposomes or LNPs, we incubated the formulations with serum and then detected C3 protein degradation using Western blot analysis. Because C3 protein degradation is an essential pathway for complement activation, it can be used to demonstrate complement activation activity. As shown in Figure 3, liposomes modified with hydroxyl-terminated PEG showed signs of enhanced complement activation in mice and rats compared with liposomes modified with methoxyl-terminated PEG. However, no enhanced complement activation was observed in human serum. Furthermore, when the nanocarriers were lipid nanoparticles, the complement activation activity in mouse and rat serum was comparable to that of liposomes modified with methoxyl-terminated PEG. None of the nanocarriers detected in human serum significantly enhanced complement activation. This suggests that the effect of modification with hydroxyl-terminated PEG on the complement activation activity of nanocarriers or nanoformulations is closely related to the species and nanocarrier type, and animal results may not accurately reflect the human condition. [Example]

[0083] Example 4

[0084] This example is used to explain that anti-PEG-IgM obtained by stimulation in rats has the same binding activity to different PEG end group structures, whereas anti-PEG-IgM pre-stored in human serum has different binding activities to different PEG end group structures (all end group-PEG-DSPE structures), with the binding activity when the end group is HO being the lowest.

[0085] A low dose of PEGylated lipid bodies was injected into rats (5 mg HSPC / kg) to stimulate the body's production of anti-PEG-IgM. On the sixth day, when the PEG antibody content reached its peak, rat serum was collected and compared with anti-PEG-IgM pre-stored in healthy human serum for differences in binding mode.

[0086] The binding activity of anti-PEG-IgM from rats and anti-PEG-IgM pre-stored in human serum with different PEG end group structures was detected using sandwich ELISA. The specific procedure is as follows: 2 μg of MeO-PEG per well was placed on an ELISA plate. 2000 -DSPE, HO-PEG 2000 -DSPE, H2N-PEG 2000 -DSPE and HOOC-PEG 2000 -DSPE was added, and the cells were incubated overnight at room temperature. After washing three times with PBST, they were blocked with 5% BSA at 37°C for 1 hour. After washing three times with PBST, rat or human serum diluted with PBS was added, incubated at 37°C for 1 hour, and washed three times with PBST. Horseradish peroxidase-labeled anti-rat or human IgM antibody was added, and after 1 hour, the cells were reacted with TMB color developer for 10 minutes. The reaction was stopped with 0.18M H2SO4, and the absorbance value (OD value) was detected at a wavelength of 450 nm. The experimental results are shown in Figure 4.

[0087] To compare the binding patterns of PEG to PEG-induced anti-PEG-IgM in vivo and to pre-preserved human anti-PEG-IgM, PEG liposomes were injected via the tail vein into rats. Six days later, blood was collected and serum was separated. A sandwich ELISA was used to detect the binding activity of anti-PEG-IgM to different PEG end-group structures in rat and healthy human serum. As shown in Figure 4a, rat serum showed similar binding activity to the four PEGylated materials. Analysis indicated this was due to the fact that the primary antigen-binding site of PEG-induced anti-PEG-IgM in rats is the PEG chain, with little selectivity for the PEG end-group. As shown in Figure 4b, human serum showed the strongest binding to MeO-modified PEG, followed by carboxyl- and amino-modified PEGs, and little binding to HO-modified PEG. Analysis indicated this was due to the fact that pre-preserved human anti-PEG-IgM primarily discriminates against PEG end-groups. These results demonstrated that the binding mechanisms of anti-PEG-IgM generated in animals by PEGylated drug injection differ from those of pre-preserved PEG antibodies in humans, and that pre-preserved anti-PEG-IgM in humans has different binding activities toward different PEG end-group structures, with the HO end-group structure being the lowest. [Example]

[0088] Example 5

[0089] This example is used to demonstrate that anti-PEG-IgM pre-stored in human serum has low binding activity to different HO-PEGs, regardless of the hydrophobic group or PEG chain length (comparing MeO-PEG and HO-PEG, grafted with DSPE, DMG, and PLGA, with PEG molecular weights of 500, 2000, and 10000).

[0090] The binding activity of anti-PEG-IgM pre-stored in human serum to PEGs with different hydrophobic groups was detected by sandwich ELISA. 2 μg of MeO-PEG was added per well to an ELISA plate. 2000 -DSPE, HO-PEG 2000 -DSPE, MeO-PEG 2000 -DMG, HO-PEG 2000 -DMG, MeO-PEG 10000 -PLGA, HO-PEG 10000 -PLGA, MeO-PEG 500 -DMG and HO-PEG 500 -DMG was added, and the binding status of different materials with anti-PEG-IgM was detected using the method of Example 4. The experimental results are shown in Figure 5.

[0091] From the results of Example 4, it is clear that HO-terminal group-modified PEG 2000 Since DSPE materials can avoid binding with anti-PEG-IgM, we further examined the binding activity of PEG-IgM with other commonly used PEG materials to investigate whether the low binding advantage of HO-PEG is related to the hydrophobic group or PEG molecular weight. PEG-DMG is commonly used to manufacture lipid nanoparticles encapsulating nucleic acid drugs, such as the commercially available novel coronavirus mRNA-LNP vaccine, while PEG-PLGA is commonly used to manufacture nanoparticles encapsulating chemotherapy drugs. After changing the MeO end group of both materials to HO, we examined their binding ability with anti-PEG-IgM by ELISA. As shown in Figure 5, in addition to DSPE, the binding ability of the HO end group with anti-PEG-IgM was also significantly weaker than that of the MeO end group for PEG-modified DMG and PLGA materials. At PEG chain lengths of 500, 2000, and 10,000, the binding ability of hydroxyl-end-group-modified PEG with IgM was weaker than that of methoxy end-groups. Therefore, the low binding activity of anti-PEG-IgM pre-stored in human serum to different HO-PEGs is independent of the hydrophobic group or the molecular weight of the PEG chain and has universal applicability. [Example]

[0092] Example 6

[0093] This example is used to demonstrate that the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEG liposomes is independent of the PEG modification ratio.

[0094] Liposomes with three different PEG modification ratios were prepared, and their formulations are shown in Table 1.

[0095] [Table 2]

[0096] The above membrane material was dissolved in 5 mL of trichloromethane and rotary evaporated at 60°C to form a membrane, which was then vacuum dried to remove the organic solvent. 1 mL of double-distilled water was added and the membrane was uniformly hydrated in a water bath at 60°C. Liposomes with different MeO-PEG or HO-PEG modification ratios were obtained by extrusion through filter membranes with pore sizes of 200 nm and 100 nm in a liposome extruder.

[0097] A PBS solution containing 20 μg of liposomes per well was added to an ELISA plate and coated overnight at 4°C. Then, the binding activity of liposomes with different PEG-modification ratios to anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA. The results are shown in Figure 6.

[0098] Blank liposomes with different PEG modification ratios were prepared and their binding activity with anti-PEG-IgM was examined. As shown in Figure 6, the binding activity of HO-PEG liposomes with anti-PEG-IgM pre-stored in human serum was significantly weaker than that of MeO-PEG liposomes, regardless of the PEG modification ratio (1%, 3%, or 5%). This indicates that HO-PEG liposomes have low binding activity with PEG-IgM, and this is unrelated to the PEG modification ratio. [Example]

[0099] Example 7

[0100] This example is used to demonstrate that the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PEG liposomes is independent of the type of encapsulated drug.

[0101] 5% PEG-modified liposomes encapsulating doxorubicin (small molecule drug, sLip / DOX) and ovalbumin (protein antigen, sLip / OVA) were prepared, respectively, by the following method.

[0102] sLip / DOX: A uniform lipid membrane containing 5% MeO-PEG or HO-PEG was prepared according to the method of Example 6. 1 mL of 0.25 M ammonium sulfate solution was added, and the membrane was uniformly hydrated in a 60°C water bath. Liposomes with pore sizes of 200 nm and 100 nm were extruded using a liposome extruder to obtain liposomes hydrated with ammonium sulfate solution. The outer aqueous phase of the liposomes was replaced with saline using a dextran gel G-50 column. An aqueous doxorubicin solution (drug-lipid ratio 1:10) was then added, and unencapsulated doxorubicin was removed through a G50 chromatography column to obtain sLip / DOX.

[0103] sLip / OVA: A uniform lipid membrane containing 5% MeO-PEG or HO-PEG was prepared according to the method described in Example 6. Liposomes hydrated with 1 mL of 1 mg / mL OVA aqueous solution were added, and liposomes hydrated with the OVA aqueous solution were obtained by extrusion into pores with 200 nm and 100 nm diameters using a liposome extruder. Unencapsulated OVA was removed by passing through a dextran gel G-75 column, and the external aqueous phase was replaced with saline.

[0104] A PBS solution containing 20 μg of liposomes per well was added to an ELISA plate and coated overnight at 4°C. Then, the binding activity of liposomes encapsulating different drugs with anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA. The results are shown in Figure 7.

[0105] Liposomes modified with MeO-PEG or HO-PEG were prepared and encapsulated with two different drugs: the small molecule chemotherapy drug doxorubicin and the protein antigen drug ovalbumin. These were coated on ELISA plates (20 μg of formulation / well) as antigens, and binding of native anti-PEG-IgM in human serum was detected using a sandwich ELISA. As shown in Figure 7, the binding activity of HO-PEG liposomes encapsulating different drugs to anti-PEG-IgM stored in human serum was lower than that of MeO-PEG liposomes. This indicates that the lower binding activity of HO-PEG liposomes and stored anti-PEG-IgM is unrelated to the encapsulated drug. [Example]

[0106] Example 8

[0107] This example is used to illustrate that anti-PEG-IgM pre-stored in human serum has low binding activity to HO-PEG micelles.

[0108] Paclitaxel (PTX) encapsulated MeO-PEG 2000 -DSPE or HO-PEG 2000 Micelle (PM) consisting of MeO-PEG and 10 mg of MeO-DSPE was prepared as follows. 2000 -DSPE or HO-PEG 2000 MeO-DSPE and 1 mg of paclitaxel were weighed and dissolved in 5 mL of trichloromethane. The film was formed by rotary evaporation at 60 °C and dried under vacuum to remove the organic solvent. 1 mL of saline solution was added and the film was uniformly hydrated in a water bath at 60 °C. Filtering through a 0.22 μm filter membrane yielded MeO-paclitaxel micelles (MeO-PM / PTX) and HO-paclitaxel micelles (HO-PM / PTX). MeO-PM / PTX and HO-PM / PTX were each added to wells of an ELISA plate at 20 μg lipid / well to detect binding activity with anti-PEG-IgM pre-stored in human serum. The results are shown in Figure 8.

[0109] To demonstrate that HO-PEG-containing micelles have low binding activity to anti-PEG-IgM pre-stored in human serum, paclitaxel was encapsulated in MeO-PEG. 2000 -DSPE or HO-PEG 2000 Micelles composed of HO-DSPE were prepared and added to wells of an ELISA plate at 20 μg lipid / well to detect binding activity with anti-PEG-IgM pre-stored in human serum. As shown in Figure 8, the binding activity of anti-PEG-IgM pre-stored in human serum to HO-PM / PTX was lower, meaning that the binding-preventing effect of the HO-PEG material on PEG-IgM still exists in the micelles. [Example]

[0110] Example 9

[0111] This example is used to illustrate that anti-PEG-IgM pre-stored in human serum has low binding activity to HO-PEG polymer nanoparticles.

[0112] Paclitaxel-encapsulated MeO-PEG 10000 -PLGA or HO-PEG 10000 Polymer nanoparticles composed of PEG-PLGA were prepared as follows: 10 mg of PLGA, 10 mg of MeO-PEG10000-PLGA or HO-PEG10000-PLGA, and 1 mg of paclitaxel were weighed and dissolved in 1 mL of dichloromethane. 4 mL of 0.5% aqueous sodium cholate solution was added and ultrasonically emulsified at 34% (650 W) power for 8 min. The organic solvent was removed by rotary evaporation, followed by centrifugation at 3000 rpm to remove large particles and 12000 rpm to remove small particles. Uniform paclitaxel nanoparticles were obtained and dispersed in saline. MEO-NPs / PTX and HO-NPs / PTX were each added at 20 μg / well to wells of an ELISA plate to detect binding activity with anti-PEG-IgM pre-stored in human serum. The results are shown in Figure 9.

[0113] To demonstrate that HO-PEG-containing polymer nanoparticles have low binding activity to anti-PEG-IgM pre-stored in human serum, paclitaxel was encapsulated in MeO-PEG. 10000 -PLGA or HO-PEG 10000 Nanoparticles composed of HO-PLGA were prepared and added to the wells of an ELISA plate at 20 μg / well to detect the binding activity of anti-PEG-IgM pre-stored in human serum. As shown in Figure 9, the binding activity of anti-PEG-IgM pre-stored in human serum to HO-NPs / PTX was lower, meaning that the binding-preventing effect of the HO-PEG material on PEG-IgM still exists in the polymer nanoparticles. [Example]

[0114] Example 10

[0115] This example is used to demonstrate that anti-PEG-IgM pre-stored in human serum has low binding activity to HO-PEG / LNP (lipid nanoparticles), and also to compare MeO-PEG and HO-PEG with different modification ratios.

[0116] Solid lipid nanoparticles (LNPs) with different PEG modification ratios were prepared, and their formulations are shown in Table 2.

[0117] [Table 3]

[0118] The membrane material was dissolved in 2 mL of ethanol and mixed with 6.25 mM sodium acetate buffer at a volume and flow rate ratio of 1:3 in a microfluidic device (PDMS chip). Six volumes of pre-chilled PBS were added to dilute and stabilize the system. After concentration by ultrafiltration, the membrane material was purified by HO / MeO-PEG with different PEG modification ratios. 2000 -DSPE and HO / MeO-PEG 2000 -DMG LNP was obtained.

[0119] A PBS solution containing 200 μg of LNP per well was added to an ELISA plate and coated overnight at 4°C. Then, the binding activity of LNPs with different PEG modification ratios to anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA, as shown in Figure 10 .

[0120] Solid lipid nanoparticles with different PEG-DSPE or PEG-DMG modification ratios were prepared and their binding activity with anti-PEG-IgM was examined. As shown in Figure 10, despite the different hydrophobic groups (DSPE / DMG) of the modified PEG, all HO-LNPs exhibited low PEG-IgM binding, and the change in PEG modification ratio did not affect the low binding activity of anti-PEG-IgM pre-stored in human serum to HO-LNP. In other words, HO-LNPs exhibited low binding activity with PEG-IgM, regardless of the PEG modification ratio or the hydrophobic groups of the modified PEG. [Example]

[0121] Example 11

[0122] This example is used to illustrate that anti-PEG-IgM pre-stored in human serum has low binding activity to HO-PEG / LNP encapsulating siRNA.

[0123] Example 10 demonstrated that the binding activity of HO-LNP with PEG-IgM was low regardless of the PEG modification ratio or the hydrophobic group of the modified PEG. Therefore, siRNA was encapsulated in LNP modified with 3% PEG-DMG, and the binding activity with anti-PEG-IgM was detected. The method was as follows: 6.1 mg of SM-102, 0.136 mg of DSPC, 2.56 mg of CHO, and 1.31 mg of MeO-PEG. 2000 -DMG or HO-PEG 2000-DMG was weighed, dissolved in 2 mL of ethanol, and mixed with 6.25 mM sodium acetate buffer (containing 10 μg / mL ApoB siRNA) in a microfluidic device (PDMS chip) at a volume and flow rate ratio of 1:3. The system was then diluted and stabilized by adding 6 volumes of pre-chilled PBS, and concentrated by ultrafiltration to obtain siRNA-encapsulated LNPs (HO / MeO-LNP / siRNA).

[0124] PBS solution containing 200 μg of LNP per well was added to the ELISA plate and coated overnight at 4 °C, after which the binding activity of siRNA-encapsulated LNPs with anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA, as shown in Figure 11 .

[0125] We prepared solid lipid nanoparticles encapsulating siRNA and examined their binding activity with anti-PEG-IgM. As shown in Figure 11, HO-NP / siRNA exhibited weak PEG-IgM binding. That is, even after encapsulating siRNA, HO-LNP still maintained low binding activity with PEG-IgM. [Example]

[0126] Example 12

[0127] This example is used to illustrate that anti-PEG-IgM pre-stored in human serum has low binding activity to mRNA-encapsulated HO-PEG / LNP.

[0128] LNPs modified with 3% PEG-DMG were selected to encapsulate mRNA and detect its binding activity with anti-PEG-IgM. The method was as follows: 6.1 mg of SM-102, 0.136 mg of DSPC, 2.56 mg of CHO, and 1.31 mg of MeO-PEG-DMG or HO-PEG-DMG were weighed and dissolved in 2 mL of ethanol. The solution was mixed with 6.25 mM sodium acetate buffer (containing 10 μg / mL luciferase mRNA) at a volume-to-flow ratio of 1:3 in a microfluidic device (PDMS chip). Six volumes of pre-chilled PBS were added to dilute and stabilize the mixture. The resulting mRNA-encapsulated LNPs (HO / MeO-LNP / mRNA) were then concentrated by ultrafiltration.

[0129] A PBS solution containing 200 μg of LNP per well was added to the ELISA plate and coated overnight at 4°C, after which the binding activity of the LNPs encapsulating firefly luciferase mRNA with anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA, as shown in Figure 12 .

[0130] Solid lipid nanoparticles encapsulating firefly luciferase mRNA were prepared and their binding activity with anti-PEG-IgM was examined. As shown in Figure 12, HO-NP / mRNA exhibited weak PEG-IgM binding. That is, even after encapsulating mRNA, HO-LNP still maintained low binding activity with PEG-IgM. [Example]

[0131] Example 13

[0132] This example is used to illustrate that anti-PEG-IgM pre-stored in human serum has low binding activity to DNA-encapsulated HO-PEG / LNPs.

[0133] LNPs modified with 3% PEG-DMG were selected to encapsulate DNA and assay its binding activity with anti-PEG-IgM. The method was as follows: 6.1 mg of SM-102, 0.136 mg of DSPC, 2.56 mg of CHO, and 1.31 mg of MeO-PEG-DMG or HO-PEG-DMG were weighed and dissolved in 2 mL of ethanol. The solution was mixed with 6.25 mM sodium acetate buffer (containing 10 μg / mL PTEN DNA) at a volume-to-flow ratio of 1:3 in a microfluidic device (PDMS chip). Six volumes of pre-chilled PBS were added to dilute and stabilize the system, which was then concentrated by ultrafiltration to obtain DNA-encapsulated LNPs (HO / MeO-LNP / DNA).

[0134] A PBS solution containing 200 μg of LNP per well was added to the ELISA plate and coated overnight at 4°C. After that, the binding activity of LNPs encapsulating tumor suppressor gene PTEN DNA and anti-PEG-IgM pre-stored in human serum was detected using sandwich ELISA, as shown in Figure 13 .

[0135] Solid lipid nanoparticles encapsulating the tumor suppressor gene PTEN DNA were prepared and their binding activity with anti-PEG-IgM was examined. As shown in Figure 13, HO-NP / DNA exhibited weak PEG-IgM binding. That is, even after encapsulating DNA, HO-LNP still maintained low binding activity with PEG-IgM. [Example]

[0136] Example 14

[0137] This example is used to illustrate that hydroxyPEG can reduce complement activation by avoiding binding to natural anti-PEG-IgM in human serum.

[0138] The complement activation ability of methoxy or hydroxy PEG nanoparticles was determined using a sheep red blood cell hemolysis experiment. The method was as follows: Sheep red blood cells were washed twice with PBS and adjusted to a 4% concentration. Hemolysin was diluted 1:1000 and mixed with 4% sheep red blood cells. The hemolysin was then incubated at 37°C for 30 minutes to sensitize the red blood cells. Human serum and the PEG nanoparticles were then incubated at 37°C for 30 minutes to consume complement. Sensitized sheep red blood cells were then added and incubated at 37°C for 30 minutes. The mixture was then centrifuged to remove undigested sheep red blood cells. The supernatant was analyzed for absorbance at OD 542nm, as shown in Figure 14.

[0139] Antigen binding with antibodies activates and depletes complement proteins in the blood via the classical pathway, resulting in hypersensitivity reactions and shortening blood circulation time by accelerating phagocytosis by the monocyte-macrophage system. Clinically occurring infusion reactions to liposomal doxorubicin and allergic reactions to novel coronavirus mRNA vaccines (LNPs encapsulating SARS-CoV-2 mRNA) are both caused by complement activation. Therefore, evaluating the complement activation ability of hydroxyl-modified PEGylated nanoformulations is an important indicator for predicting clinical allergic reactions. The complement activation ability of formulations was detected using a sheep red blood cell hemolysis experiment. Sensitized sheep red blood cells were obtained by incubating them with hemolysin. Upon encountering complement proteins, complement activation was induced, generating the membrane attack complex (C5b-9), leading to red blood cell rupture and the release of hemoglobin. The remaining red blood cells were centrifuged, and the absorbance value of the supernatant was measured, allowing for comparison of differences in complement content between samples. Healthy human serum was incubated with different PEGylated preparations at 37°C for 30 minutes to activate complement, and sensitized sheep red blood cells were used to detect the residual complement content in the serum. As shown in Figure 14, the complement activation ability of the nanoparticles modified with hydroxyPEG was weaker than that modified with methoxyPEG, and the residual complement caused strong hemolysis of sheep red blood cells.

[0140] Furthermore, using the method of Example 3, the complement activation status of the nanoparticles modified with hydroxyPEG in human serum with PEG antibodies was detected. As shown in Figure 15, the complement activation level of liposomes or LNPs modified with hydroxyPEG in PEG antibody-positive human serum was weaker than that of methoxyPEG. Combined with the results of the sheep red blood cell hemolysis experiment, this revealed that the nanoparticles modified with hydroxyPEG can reduce complement activation by avoiding binding to natural anti-PEG-IgM in human blood, thereby alleviating clinical allergic reactions.

[0141] The protection content of the present invention is not limited to the above embodiments. All modifications and substitutions that can be thought of by those skilled in the art without departing from the spirit and scope of the invention are included in the present invention and are defined in the scope of the accompanying claims.

Claims

1. The PEGylated nanocarrier is one or more selected from liposomes, polymer nanoparticles, micelles and lipid nanoparticles. The PEGylated nanocarrier is modified with a terminal hydroxyl group and is used in the preparation of a drug to circumvent anti-PEG antibodies pre-stored in humans.

2. The use of claim 1, wherein the drug comprises one or more of a small molecule drug, a protein drug, and a nucleic acid drug.

3. The use according to claim 2, wherein the nucleic acid drug is one or more selected from small interfering ribonucleic acid (siRNA), deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA).

4. The molecular weight of the PEG containing the terminal hydroxyl group modification is 500 to 10,000 Da, and / or The use according to any one of claims 1 to 3, wherein the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol% to 10 mol%, and the PEG modification ratio in the micelles and polymer nanoparticles is 0.5 mol% to 100 mol%.

5. The use according to any one of claims 1 to 3, wherein one end of the hydroxyl-terminal modified PEG is a hydroxyl group and the other end is a polymer or lipid.

6. The use according to any one of claims 1 to 3, wherein the PEGylated nanocarriers comprise one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

7. A nano-preparation for human use, comprising an active ingredient and a PEGylated nano-carrier modified with a terminal hydroxyl group, wherein the nano-carrier is one or more selected from liposomes, polymer nanoparticles, lipid nanoparticles and micelles, and the active ingredient is one or more selected from small molecule drugs, protein drugs and nucleic acid drugs.

8. The molecular weight of the PEG containing the terminal hydroxyl group modification is 500 to 10,000 Da, and / or The nano-preparation for human use according to claim 7, wherein the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol% to 10 mol%, and the PEG modification ratio in the micelles and polymer nanoparticles is 0.5 mol% to 100 mol%.

9. The nanoformulation for human use according to claim 7 or 8, wherein the nucleic acid drug is one or more selected from siRNA, DNA and mRNA.

10. A nanoformulation for human use according to claim 7 or 8, wherein one end of the PEG modified with a terminal hydroxyl group is a hydroxyl group and the other end is a polymer or lipid.

11. The nanoformulation for human use according to claim 7 or 8, wherein the PEGylated nanocarrier contains one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

12. A method for treating a disease by circumventing anti-PEG antibodies pre-stored in humans, comprising administering a nanoformulation to a human subject in need thereof, the nanoformulation comprising an active ingredient and a PEGylated nanocarrier modified with a terminal hydroxyl group, the nanocarrier being one or more selected from liposomes, polymeric nanoparticles, lipid nanoparticles and micelles, and the active ingredient being one or more selected from small molecule drugs, protein drugs and nucleic acid drugs.

13. The molecular weight of the PEG containing the terminal hydroxyl group modification is 500 to 10,000 Da, and / or The method according to claim 12, wherein the PEG modification ratio in the liposomes and lipid nanoparticles is 0.5 mol% to 10 mol%, and the PEG modification ratio in the micelles and polymer nanoparticles is 0.5 mol% to 100 mol%.

14. The method of claim 12 or 13, wherein the nucleic acid drug is one or more selected from siRNA, DNA, and mRNA.

15. The method of claim 12 or 13, wherein one end of the hydroxyl-terminal modified PEG is a hydroxyl group and the other end is a polymer or lipid.

16. The method of claim 12 or 13, wherein the PEGylated nanocarriers comprise one or more of hydroxypolyethylene glycol polylactic acid-hydroxyacetic acid copolymer (HO-PEG-PLGA), hydroxypolyethylene glycol polylactic acid copolymer (HO-PEG-PLA), hydroxypolyethylene glycol-distearoylphosphatidylethanolamine (HO-PEG-DSPE), and hydroxypolyethylene glycol-dimyristoylglycerol (HO-PEG-DMG).

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