Multivalent cell-penetrating peptide-based biological macromolecule delivery carriers and uses thereof

Abstract

The present application belongs to the field of pharmaceutical preparations, and relates to a series of multivalent TAT delivery carriers and complexes thereof constructed by using multi-arm polyethylene glycol and TAT or TAT derivatives. The multivalent TAT delivery carrier has an octopus-like flexible structure, can self-assemble with biological macromolecules, especially genes, to form a non-covalent complex, has a strong ability to carry, deliver and penetrate ocular tissues, does not produce ocular tissue toxicity, can realize effective delivery of biological macromolecule drugs into the eye or the fundus through a non-invasive route, and increases the uptake of ocular tissues to the biological macromolecule drugs. The complex formed by self-assembly of the multivalent TAT delivery carrier and biological macromolecules is used for eye drop administration, can replace the poor patient compliance of intraocular injection administration, and enhances the convenience and safety of treating intraocular and fundus diseases.

Description

Technical Field

[0001] This invention pertains to the field of pharmaceutical formulations and relates to multivalent membrane-penetrating peptide delivery carriers, specifically to a series of multivalent membrane-penetrating peptide delivery carriers and their complexes constructed using multi-arm polyethylene glycol and membrane-penetrating peptides or their derivatives. These multivalent membrane-penetrating peptide delivery carriers possess an octopus-like flexible structure, enabling them to self-assemble with biomolecules, especially genes, to form non-covalent complexes. They exhibit strong capabilities in carrying and delivering biomolecules, as well as penetrating ocular tissues, without producing ocular toxicity. This allows for the effective delivery of biomolecule drugs into the eye or fundus via a non-invasive route, increasing the uptake of biomolecule drugs by ocular tissues. Technical Background

[0002] According to data, the eye, as the most important sensory organ in the human body, has a unique physiological structure that protects it from the invasion of exogenous substances, but it is also not conducive to intraocular drug delivery. The physiological barriers of the eye include static barriers (such as the corneal epithelial barrier, blood-eye barrier, etc.) and dynamic barriers (such as tear flushing, etc.), which are the main reasons for hindering drug absorption (DrugDiscovery Today, 2008, 13(3-4):135-143; Adv.Drug Delivery Rev., 2006, 58(11):1131-1135). Studies have shown that the eye is an ideal site for the treatment of biomolecules such as peptides, proteins, and genes. This is because the eyeball is relatively small, requiring small doses of biomolecules for administration; the eye has immune privilege, which can reduce inflammation and immune responses caused by exogenous substances to a certain extent; local administration is convenient and can effectively avoid the complex physiological environment in the body (Br.J.Ophthalmol., 2011, 95(5):604-612.).

[0003] Currently, commercially available ophthalmic preparations are mainly in the form of eye drops, ophthalmic gels, and ointments. In clinical practice, after eye drops are instilled into the conjunctival sac, the drug is mainly transported into the eye through the cornea or conjunctiva. Due to the limited volume of the human conjunctival sac, coupled with tear dilution and nasolacrimal duct loss, the bioavailability of eye drops is usually less than 5%. Moreover, due to the long diffusion distance from the ocular surface to the fundus and the convection of aqueous humor within the eye, very little drug (<0.001%) can reach the posterior segment of the eye (J. Controlled Release, 2014, 193:100-112).

[0004] Systemic administration is another clinical approach to treating eye diseases. However, practice has shown that due to the obstruction of the blood-eye barrier (such as the blood-retinal barrier), it is difficult for drugs to reach the retinal tissue and vitreous cavity after systemic administration. In addition, due to the large amount of drugs entering the systemic circulation, frequent administration of high doses also carries the risk of causing systemic side effects (Invest. Ophthalmol. Visual Sci., 2000, 41(5): 961-964). Using biomolecular drugs for eye treatment via this route not only exacerbates the risk of side effects at non-target sites but also leads to unnecessary drug waste.

[0005] Intravitreal injection (such as intravitreal injection) and periocular injection (such as subscleral injection) are among the most effective routes of administration for the treatment of intraocular and fundus diseases using biological macromolecular drugs in clinical practice (Eur.J.Pharm.Biopharm.,2015,95:331-342). For example, in the treatment of age-related macular degeneration, vascular endothelial growth factor inhibitors, such as monoclonal antibody drugs ranibizumab, bevacizumab, and conbercept, as well as gene therapy drugs such as pilgatani sodium, are mainly administered via intravitreal injection. With the help of these invasive administration methods, the drugs can directly reach the eye, have a rapid onset of action, and have high bioavailability. However, repeated injections may cause a variety of complications (such as retinal detachment, endophthalmitis, etc.), which are difficult for patients to accept and have poor compliance (EYE,2013, 27(7):787-794).

[0006] Considering various intraocular and fundus drug delivery methods, eye drops are the most ideal ophthalmic dosage form in clinical practice due to their non-invasiveness, low manufacturing cost, ease of use, and good patient compliance. However, their main problem lies in the difficulty of drug absorption into the eye, and even more so in reaching the fundus. By employing pharmaceutical methods to add absorption enhancers to the eye drop formulation, the intraocular delivery efficiency of drugs can be effectively improved.

[0007] Cell-penetrating peptides (CPPs) are short peptides that are positively charged under physiological conditions and can mediate the entry of covalently or non-covalently linked molecules (such as genes, peptides, and proteins) or drug delivery systems (such as liposomes and nanoparticles) into cells (J. Controlled Release, 2011, 155(1SI):26-33; Biomaterials, 2013, 34(32):7980-7993). Penetratin, a cell-penetrating peptide derived from the DNA-binding domain of Drosophila antennae, has strong ocular tissue penetration ability and does not produce ocular cytotoxicity (Mol. Pharm. 2014, 11(4):1218-1227). In several non-invasive ocular drug delivery systems, penetratin has been reported as an absorption enhancer that can mediate the delivery of reporter genes to the posterior segment of the eye and their efficient expression in the retina (ACS Appl. Mater. Interfaces, 2016, 8(30): 19256-19267; Nanomedicine: NBM, 2017, 13: 2091-2100; Int. J Pharmaceut., 2017, 529: 347-356; Chinese invention patent application: CN201610560173.8).

[0008] Based on the publicly available literature reporting that penetratin, a naturally derived polypeptide with strong penetrability to ocular tissues, a series of penetratin derivatives were obtained through artificial design and modification (Chinese invention patent application: CN2017104143347). These polypeptide derivatives, as ocular absorption enhancers, can more effectively deliver covalently or non-covalently linked drug molecules to the posterior segment of the eye after being instilled into the conjunctival sac, while retaining the good ocular safety of wild-type penetratin.

[0009] Membrane-penetrating peptides can self-assemble with biomacromolecules such as genes, peptides, and proteins to form complexes, delivering these macromolecules into cells or carrying them across biological membrane barriers. However, the capacity of a single membrane-penetrating peptide to carry biomacromolecules is very limited, resulting in complexes with large particle sizes, poor stability, and a weak ability to promote cellular uptake or penetration of biomembrane barriers. Therefore, membrane-penetrating peptides need to be used in conjunction with polymer carriers to carry biomacromolecules more effectively, facilitating their escape from endosomes. However, commonly used polymer carriers, such as polyethyleneimine (PEI) and polyamide-amine (PAMAM), are non-degradable in vivo, especially at larger molecular weights or high concentrations in formulations, which can lead to cellular or tissue toxicity, thus limiting their clinical application.

[0010] Enhancing the ability of transmembrane peptides to carry biomolecules, enabling them to better perform their delivery function, while ensuring that the constructed delivery system has good biosafety and can be degraded and eliminated in vivo, is a problem that has not yet been solved by existing technologies.

[0011] Based on the current state of existing technologies, this invention aims to provide a series of multivalent membrane-penetrating peptide-based biomolecule delivery carriers. These carriers are prepared by modifying the ends of multi-arm polyethylene glycol with membrane-penetrating peptides or their derivatives, exhibiting an octopus-like flexible structure. Each molecule contains multiple membrane-penetrating peptides or their derivatives, which can self-assemble with biomolecules to form non-covalent complexes, thereby improving the carrying efficiency of biomolecules such as genes, peptides, and proteins. This allows for more effective delivery of biomolecules into cells or through biological membrane barriers, and utilizes the "proton sponge" effect to help biomolecules escape from endosomes. Biomolecule complexes prepared using these carriers, administered via intraconjunctival eye drops, can overcome the ocular absorption barrier, efficiently delivering biomolecules into the eye or fundus. Subsequently, these carriers are degraded by enzymes and ultimately eliminated from the eye, exhibiting good biocompatibility. This non-invasive delivery method improves the local absorption of biomolecules, avoids their accumulation in non-target tissues, and significantly improves compliance and safety when using biomolecules for treatment. Summary of the Invention

[0012] The purpose of this invention is to overcome the problems of low delivery efficiency and easy ocular tissue toxicity of existing polymer-based biomolecule delivery carriers in ocular applications, and to provide a class of artificially synthesized multivalent transmembrane peptide biomolecule delivery carriers and their preparation method. The multivalent transmembrane peptide delivery carrier has an octopus-like flexible structure, is biodegradable, and can self-assemble with biomolecules such as genes, peptides, and proteins to form complexes. Through non-invasive ocular instillation, biomolecules can be delivered to the intraocular or fundus region, thereby promoting the therapeutic effect of these drugs.

[0013] This invention addresses problems existing in current clinical practice. For example, when using biomolecular drugs to treat intraocular or fundus diseases, the main route of administration is intravitreal injection, but patient compliance is poor and it may lead to serious complications. Transmembrane peptides or their derivatives, as ocular absorption enhancers, can self-assemble with biomolecular macromolecules such as genes, peptides, and proteins to form complexes and mediate the penetration of non-covalently bound biomolecular macromolecules through the ocular absorption barrier. However, the ability of transmembrane peptides to carry biomolecular macromolecules is very limited, resulting in large particle size and poor stability of the formed complexes. Moreover, their ability to promote the uptake of biomolecular macromolecules by cells or their penetration of biological membrane barriers is also relatively weak, etc.

[0014] This invention provides a multivalent membrane-penetrating peptide-based biomolecule delivery carrier. The membrane-penetrating peptide or its derivative is covalently modified to the ends of multi-armed polyethylene glycol, resulting in a delivery carrier containing multiple membrane-penetrating peptides and possessing an octopus-like, flexible multi-arm structure, as shown in the diagram. Figure 1 As shown, compared with free membrane-penetrating peptides or their derivatives, the multivalent membrane-penetrating peptide delivery carrier prepared in this invention has a stronger ability to carry biomolecules while retaining the inherent absorption-promoting function of membrane-penetrating peptides or their derivatives. It can form complexes with smaller particle sizes and more compact structures with biomolecules such as genes, peptides, and proteins, significantly improving the delivery effect of biomolecules. This not only promotes the uptake of biomolecules by cells but also helps biomolecules escape into the cytoplasm to exert their biological functions. In particular, the multivalent membrane-penetrating peptide delivery carrier can be degraded by enzymes in vivo, exhibiting better biocompatibility compared to existing polymer carriers. The complexes formed by the multivalent membrane-penetrating peptide delivery carrier and biomolecules have strong penetration ability and high biocompatibility in ocular tissues. When used for ocular drug delivery, it can mediate the efficient penetration of biomolecule drugs, especially gene drugs, through the ocular absorption barrier. It can deliver biomolecule drugs such as genes, peptides, and proteins to the intraocular or fundus sites via a non-invasive route, thereby improving the ocular bioavailability and therapeutic effect of biomolecule drugs.

[0015] More specifically, the multivalent transmembrane peptide biomolecule delivery carrier provided by the present invention comprises covalently modifying the transmembrane peptide or its derivative at the ends of multi-armed polyethylene glycol side arms to form a multivalent transmembrane peptide delivery carrier with an octopus-like multi-armed, flexible structure; wherein the multi-armed polyethylene glycol has a core of polyols such as glycerol, pentaerythritol, dipentaerythritol, tripentaerythritol, or hexaglycerol, with polyethylene glycol side arms extending from the hydroxyl groups of the core; the multi-armed polyethylene glycol mainly includes 3-armed, 4-armed, 6-armed, and 8-armed polyethylene glycols, and each polyethylene glycol side arm contains an ethoxylated [(CH2CH2O)] group. n The degree of polymerization n is between 1 and 225, preferably between 2 and 100, and more preferably between 5 and 50;

[0016] In this invention, the number of side arms and the ethoxy groups [(CH2CH2O) contained in the side arms are used to determine the composition of the components. n The degree of polymerization varies, and the molecular weight of multi-arm polyethylene glycol ranges from 200 to 80,000.

[0017] In this invention, the free end of each polyethylene glycol side arm can be modified with specific chemical groups to facilitate interconnection with membrane-penetrating peptides or membrane-penetrating peptide derivatives. Common modification groups are maleimide, isothiocyanate, or carboxyl groups.

[0018] In this invention, the membrane-penetrating peptide or its derivative is selected from, but not limited to, the transactivator protein TAT (Science 1999, 285:1569–1572) found in HIV, penetratin derived from the DNA-binding domain of Drosophila antennae (Nat. Cell Biol. 2004, 6:189–196), and artificially synthesized oligoarginine (poly(arginine)). n (where n = 4-20)(Proc.Natl.Acad.Sci.USA, 2000, 97:13003–13008), protamine, a nucleoprotein bound to DNA in the nucleus of fish sperm cells, and low molecular weight protamine extracted from a portion of its functional sequence; the membrane-penetrating peptides of this invention include not only the wild-type polypeptide penetratin (amino acid sequence: RQIKIWFQNRRMKWKK), but also a series of its lipophilic derivatives;

[0019] The amino acid sequences of the lipophilic derivatives of Penetratin are shown in Table 1 (see Chinese invention patent application CN2017104143347), which are quoted here:

[0020] Table 1 Amino acid sequences of penetratin derivatives

[0021]

[0022]

[0023]

[0024] The multivalent membrane-penetrating peptide biomolecule delivery carrier described in this invention retains the characteristics of membrane-penetrating peptides and can self-assemble with biomolecules such as genes, peptides, and proteins to form complexes. Because multiple membrane-penetrating peptide molecules are linked together by multi-arm polyethylene glycol, compared with a single free membrane-penetrating peptide, the multivalent membrane-penetrating peptides bind more tightly to biomolecules and have better stability, which is more conducive to the in vivo delivery of biomolecules.

[0025] In this invention, the aforementioned biopharmaceutical is selected from, but is not limited to, one of the following drugs or a combination thereof:

[0026] 1) Gene therapy: Selected from plasmid DNA (pDNA), pegaptanib, bevasiranib, antisense oligonucleotide, small interfering RNA, etc.;

[0027] 2) Monoclonal antibody drugs: selected from bevacizumab, ranibizumab, ramucirumab, etc.;

[0028] 3) Other polypeptide and protein drugs: selected from anti-vascular endothelial growth factor (VEGF) fusion protein Conbercept, epidermal growth factor (EGF), interferon-α, etc.

[0029] In this invention, the multivalent transmembrane peptide delivery carrier self-assembles with biomolecules such as genes, peptides, and proteins to form a non-covalent complex. After intraconjunctival instillation, the complex facilitates the drug's passage through numerous ocular absorption barriers (cornea, conjunctiva, sclera, etc.) into the eye, and may even deliver the biomolecules such as genes, peptides, and proteins it carries to the posterior segment of the retina.

[0030] In this invention, the concentration of the membrane-penetrating peptide contained in the non-covalent complex constructed by the multivalent membrane-penetrating peptide delivery carrier and the biomacromolecule, after being converted to a single-molecule membrane-penetrating peptide, is between 0.1 μM and 1200 μM, preferably between 1 μM and 600 μM, and more preferably between 1 μM and 100 μM.

[0031] In this invention, the non-covalent complex constructed using a multivalent membrane-penetrating peptide delivery carrier and a biomacromolecule contains a multivalent membrane-penetrating peptide to biomacromolecule molar ratio between 1:2 and 300:1 (multivalent membrane-penetrating peptide: biomacromolecule, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide); preferably 1:1 to 200:1 (multivalent membrane-penetrating peptide: biomacromolecule, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide); more preferably 1:1 to 100:1 (multivalent membrane-penetrating peptide: biomacromolecule, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide).

[0032] In this invention, the non-covalent complex constructed by the multivalent transmembrane peptide delivery carrier and the biomacromolecule has a particle size between 1 nm and 1000 nm, preferably 10 nm to 800 nm, and more preferably 50 nm to 500 nm. This intraocular drug delivery system, which delivers drugs via a non-invasive route, helps promote the absorption of biomacromolecule drugs in the eye and improves the ocular bioavailability of biomacromolecule drugs. Clinically, it can replace drug delivery methods with low patient compliance, such as intraocular injection.

[0033] To visually demonstrate the ocular absorption-enhancing effect of the multivalent transmembrane peptide delivery carrier described in this invention on biopharmaceutical drugs, this invention uses multivalent penetratin as an example and antisense oligonucleotides and plasmid DNA (pDNA) as biopharmaceutical drug models to construct non-covalent complexes carrying antisense oligonucleotides and plasmid DNA, respectively. Through a series of in vitro and in vivo experiments, the ocular cellular uptake capacity of the non-covalent complexes constructed based on multivalent penetratin and their absorption and distribution in the eyes of live animals after intraconjunctival sac eye drops were investigated. The results showed that, compared with free single-molecule penetratin, multivalent penetratin significantly enhances the intraocular gene delivery effect.

[0034] The advantages of the multivalent permeable membrane peptide delivery carrier described in this invention are that, compared with existing biomolecule delivery carriers, the multivalent permeable membrane peptide is non-pathogenic and easily degraded in vivo, thus exhibiting better biosafety. On the other hand, the multivalent permeable membrane peptide retains the inherent good intraocular drug delivery capability of free permeable membrane peptides, enabling intraocular delivery of biomolecule drugs through a non-invasive route of administration, which is beneficial for improving patient compliance. Moreover, the multivalent permeable membrane peptide has a stronger ability to carry and deliver biomolecules into the eye compared to free permeable membrane peptides. Attached Figure Description

[0035] Figure 1 Schematic diagram of the structure of a multivalent transmembrane peptide biomolecule delivery carrier.

[0036] Figure 2 Synthesis of polyvalent penetratin.

[0037] Figure 3 Structural characterization of multivalent penetratin

[0038] The sample concentration was 5 mg / mL for HPLC detection and 3 mg / mL for 1H NMR characterization.

[0039] Figure 4 Evaluation of the cytotoxicity of multivalent penetratin.

[0040] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with multivalent penetratin for 12 hours, and the cell viability of the experimental group relative to the negative control group was detected by the MTT assay.

[0041] Figure 5 Qualitative evaluation of cellular uptake of polyvalent penetratin covalently linked to small molecules.

[0042] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with the multivalent penetratin-FAM covalent complex for 4 hours before measurement, and the sample concentration was 3 μM (calculated as a single molecule of penetratin).

[0043] Figure 6 Evaluation of the intracellular distribution of polyvalent penetratin covalently linked complexes with small molecules.

[0044] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with the multivalent penetratin-FAM covalent complex for 4 hours before measurement, and the sample concentration was 3 μM (calculated as a single molecule of penetratin).

[0045] Figure 7 Quantitative evaluation of cellular uptake of polyvalent penetratin covalently linked to small molecules.

[0046] Among them, human corneal epithelial cells and human conjunctival epithelial cells were incubated with the multivalent penetratin derivative-FAM covalent complex for 4 hours and then measured. Fm is the average fluorescence intensity value of the cells.

[0047] Figure 8 Formulation screening of non-covalently linked complexes constructed from multivalent penetratin and antisense oligonucleotides.

[0048] Figure A shows the polyacrylamide gel electrophoresis results of the polyvalent penetratin and antisense oligonucleotide non-covalent complex under different molar ratios (where the molar amount of multivalent penetratin is converted to the molar amount of single-molecule penetratin); Figure B shows the uptake of 8-valent penetratin and antisense oligonucleotide non-covalent complex with corneal epithelial cells after 1 h of incubation under different molar ratios (where the molar amount of multivalent penetratin is converted to the molar amount of single-molecule penetratin); Figure C shows the uptake of 8-valent penetratin and antisense oligonucleotide non-covalent complex with conjunctival epithelial cells after 1 h of incubation under different molar ratios (where the molar amount of multivalent penetratin is converted to the molar amount of single-molecule penetratin).

[0049] Figure 9Evaluation of particle size, potential, and stability of non-covalently linked complexes of multivalent penetratin and antisense oligonucleotides.

[0050] Figure A shows the particle size of each group of non-covalent complexes; Figure B shows the surface potential of each group of non-covalent complexes; Figure C shows the particle size changes of each group of non-covalent complexes at different time points (0, 0.5h, 1h, 2h, 4h, 6h, 12h, 24h, 48h).

[0051] Figure 10 Morphological evaluation of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides.

[0052] The scale in column A is 200nm, the scale in column B is 100nm, and the scale in column C is 50nm.

[0053] Figure 11 Evaluation of intracellular distribution of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides.

[0054] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with a non-covalent penetratin / antisense oligonucleotide complex for 4 hours before measurement. Each complex contained 1.32 μg of antisense oligonucleotide. LIPO / ASO is a non-covalent complex of the commercially available gene transfection reagent cationic liposome Lipofectamine 2000 and the antisense oligonucleotide.

[0055] Figure 12 Quantitative evaluation of cellular uptake of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides.

[0056] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with a non-covalent penetratin / antisense oligonucleotide complex for 4 hours before measurement. Each complex contained 1.32 μg of antisense oligonucleotide. LIPO / ASO is a non-covalent complex of the commercially available gene transfection reagent cationic liposome Lipofectamine 2000 and the antisense oligonucleotide.

[0057] Figure 13 Cytotoxicity evaluation of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides.

[0058] Human corneal epithelial cells and human conjunctival epithelial cells were incubated with complexes Pe / ASO, 4VP / ASO, and 8VP / ASO for 12 hours, respectively. The cell viability of the experimental group relative to the negative control group was detected by the MTT assay.

[0059] Figure 14Distribution of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides in the eyes of live mice.

[0060] Figure A shows the distribution of non-covalent complexes in the whole eye of mice 10 minutes after administration to the conjunctival sac; Figure B shows the distribution of non-covalent complexes in the anterior segment (cornea) and posterior segment (retina) of mice 10 minutes after administration to the conjunctival sac.

[0061] Figure 15 Evaluation of ocular elimination of the multivalent penetratin-antisense oligonucleotide non-covalently linked complex.

[0062] The distribution of free antisense oligonucleotides and non-covalent complexes (Pe / ASO, 4VP / ASO, 8VP / ASO) in the mouse retina at different time points (10 min, 30 min, 1 h, 2 h, 4 h, 6 h) after administration to the conjunctival sac was analyzed.

[0063] Figure 16 Evaluation of cell transfection efficacy of the non-covalently linked complex of multivalent penetratin and plasmid DNA (pDNA).

[0064] In this study, 293T cells were incubated with multivalent penetratin / plasmid DNA non-covalent complex and Lipofectamine 2000 / plasmid DNA non-covalent complex for 4 h, and then cultured for another 48 h after changing the medium. The plasmid concentration was measured to be 1 μg / mL. Detailed Implementation

[0065] The present invention will be further illustrated below with reference to specific embodiments thereof, but this does not limit its scope of protection.

[0066] Example 1

[0067] Preparation of multivalent penetratin: The multivalent penetratin was prepared by a one-step reaction of a 4-arm polyethylene glycol maleimide derivative (4-arm PEG, molecular weight 5000±500 Da) or an 8-arm polyethylene glycol maleimide derivative (8-arm PEG, molecular weight 10000±1000 Da) with N-terminal cysteine-modified penetratin (Cys-penetratin). Specifically, 1.0 mg of 4-arm PEG was dissolved in phosphate buffer (10 mM, pH 7.2), and 2.0 mg of Cys-penetratin dissolved in the same medium was added under stirring. The mixture was stirred overnight to allow the reaction to complete. After the reaction, the resulting mixture was dialyzed under ice bath conditions for 2 days and freeze-dried to obtain a white flocculent tetravalent penetratin (4VP). 8-valent penetratin (8VP) was prepared using the same synthetic method.

[0068] Other multivalent membrane-penetrating peptides are prepared using the same method, obtained by a one-step reaction between a multi-arm polyethylene glycol derivative (PEG) and the membrane-penetrating peptide. This includes a one-step reaction between the terminal maleimide group of the PEG and the thiol group of cysteine ​​in the membrane-penetrating peptide molecule, the terminal isothiocyanate group of the PEG and the free amino group in the membrane-penetrating peptide molecule, and the terminal carboxyl group of the PEG and the free amino group in the membrane-penetrating peptide molecule. The reaction route is as follows:

[0069] multi-arm PEG+peptide→multi-valent peptide

[0070] The selection of multi-arm polyethylene glycol (PEG) derivatives includes 3-arm, 4-arm, 6-arm, and 8-arm PEG derivatives, with terminal modification groups including maleimide, isothiocyanate, and carboxyl groups. The PEG side chain contains an ethoxy group [(CH2CH2O]. n The degree of polymerization n of the [] is between 1 and 225, preferably 2 to 100, more preferably 5 to 50, or the molecular weight of the multi-arm polyethylene glycol is between 200 and 80,000;

[0071] The choice of peptides includes TAT, penetratin, and poly(arginine). n(where n = 4-20), penetrating peptides such as protamine or low molecular weight protamine, and derivatives of the above penetrating peptides. The amino acid sequence of the penetrating peptide penetrating peptide penetrating tin is RQIKIWFQNRRMKWKK, and the amino acid sequences of its derivatives are shown in Table 1. To facilitate reaction with the terminal groups of multi-arm polyethylene glycol, an additional cysteine ​​(C) or lysine (K) residue may be added to the N-terminus or C-terminus of the above penetrating peptides and their derivatives.

[0072] Example 2

[0073] Characterization of polyvalent penetratin: Characterization was performed using high-performance liquid chromatography (HPLC). A Sepax Bio-C4 column (4.6 × 150 mm, 5 μm) was selected; column temperature 25 °C; mobile phase acetonitrile (0.1% trifluoroacetic acid): water (0.1% trifluoroacetic acid), 5–65% acetonitrile gradient, 30 min; flow rate 0.8 mL / min; detection wavelength 214 nm; injection volume 20 μL; sample concentration 5 mg / mL.

[0074] The results showed that, compared with multi-arm polyethylene glycol maleimide derivatives, the polyvalent penetratin elutes approximately 2 minutes earlier, indicating increased product polarity. Furthermore, the liquid chromatography spectrum also indicated that the purity of the obtained product was above 95%.

[0075] In the characterization by proton NMR, multi-arm polyethylene glycol maleimide derivative and polyvalent penetratin were dissolved in heavy water at a concentration of 3 mg / mL and scanned 120 times at room temperature.

[0076] The results showed that the characteristic peak of maleimide (δ6.8) disappeared in the polyvalent penetratin spectrum compared with the multi-arm polyethylene glycol maleimide derivative, proving that the peptide penetratin had been successfully linked to the end of polyethylene glycol.

[0077] Example 3

[0078] Evaluation of the cytotoxicity of multivalent penetratin: HCEC and NHC cells in good growth status during the logarithmic growth phase were collected at a concentration of 5 × 10⁻⁶ cells / cells. 3 cells / cm 2Different concentrations of penetratin were seeded into the middle 60 wells of a 96-well plate, and the edges were filled with sterile PBS buffer. The plates were cultured at 37°C and 5% CO2 until a monolayer of cells covered the bottom of the plate. The culture medium was discarded, and the cells were washed three times with sterile PBS buffer. Then, 200 μL of culture medium containing different concentrations of penetratin was added, and the plates were incubated for 12 h. After discarding the drug solution, the cells were washed three times with sterile PBS buffer, and then complete culture medium was added, followed by another 12 h of incubation. Next, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for another 4 h. The liquid was carefully discarded, and the cells were washed three times with PBS buffer. Then, 150 μL of DMSO was added to each well, and the plates were shaken at low speed for 20 min. The absorbance of each well was measured at OD490 nm using a microplate reader. Blank control wells (culture medium, MTT, DMSO) and negative control wells (cells, culture medium, MTT, DMSO) were also included.

[0079] The results showed that when the concentrations of penetratin, 4VP, and 8VP (based on penetratin) were less than 100 μM, HCEC and NHC cells exhibited good growth, and the materials used were non-toxic to the cells.

[0080] Example 4

[0081] Qualitative evaluation of cellular uptake of the multivalent penetratin covalently linked complex with small molecules: HCEC and NHC cells in good growth condition were analyzed at a concentration of 5 × 10⁻⁶ cells / year. 3 cells / cm 2 Cells were seeded into 24-well plates. After culturing for 24 hours at 37°C and 5% CO2 in 500 μL of DMEM medium containing 10% FBS, experiments were performed. The culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free DMEM solutions containing 3 μM (calculated as penetratin) fluorescein 5-FAM-labeled penetratin and multivalent penetratin were added, respectively, and incubated at 37°C and 5% CO2 for a specified time. After incubation, the solutions were discarded, and positively charged adsorbed substances were washed away with PBS buffer containing 0.02 mg / mL heparin sodium. Cells were then covered with PBS / glycerol and observed under a laser confocal microscope.

[0082] The results showed that even after incubation for up to 4 hours, HCEC and NHC cells showed no significant fluorescence signal in the penetratin group. However, in the multivalent penetratin group, cells showed a strong green fluorescence signal after only 1 hour of incubation. Even after 4 hours of incubation, the green fluorescence signal produced by 4VP and 8VP in HCEC cells was almost diffuse throughout the entire cell, indicating the good cellular penetration ability of multivalent penetratin.

[0083] Example 5

[0084] Evaluation of intracellular distribution of the polyvalent penetratin covalently linked complex with small molecules: HCEC and NHC cells in good growth condition were collected at a concentration of 5 × 10⁻⁶ cells / cells. 3 cells / cm 2 Cells were seeded into 35 mm four-cell confocal microscopy dishes. After culturing at 37°C and 5% CO2 in 500 μL of DMEM medium containing 10% FBS for 24 h, experiments were performed. The culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free DMEM solutions containing 3 μM (calculated as penetratin) fluorescein 5-FAM-labeled penetratin and multivalent penetratin were added, respectively, and incubated at 37°C and 5% CO2 for a specified time. After incubation, the solutions were discarded, and positively charged adsorbed substances were washed away with PBS buffer containing 0.02 mg / mL heparin sodium. Cells were then covered with PBS / glycerol and observed under a laser confocal microscope.

[0085] The results showed that HCEC and NHC cells had low penetratin uptake 4 hours after administration, while 4VP and 8VP were uptaken in large quantities and distributed in the cytoplasm. Furthermore, some green fluorescent signals did not co-localize with red fluorescently labeled lysosomes, indicating that the multivalent penetratin delivery vector possesses endosome escape properties.

[0086] Example 6

[0087] Quantitative evaluation of cellular uptake of the multivalent penetratin covalently linked complex with small molecules: HCEC and NHC cells in good growth condition were analyzed at a concentration of 5 × 10⁻⁶ cells / year. 3 cells / cm 2 Cells were seeded into 12-well plates, with the medium changed daily after seeding. Experiments were performed after 2–3 days of culture. After discarding the culture medium, the cells were washed three times with sterile PBS, and serum-free DMEM solution containing 3 μM (based on penetratin) fluorescently labeled penetratin and multivalent penetratin were added, respectively. The cells were incubated at 37°C and 5% CO2 for 4 hours. After incubation, the solution was discarded, and positively charged adsorbed substances were washed away with PBS buffer containing 0.02 mg / mL heparin sodium. The cells were then digested, resuspended in 200 μL of sterile PBS buffer, and thoroughly mixed before flow cytometry analysis. Cell counts were performed at approximately 10⁻⁶ cells per sample. 4 Untreated cells served as a negative control group;

[0088] The results showed that cellular uptake of 4VP and 8VP was significantly higher than that of penetratin (p<0.001). In HCEC cells, the mean fluorescence intensity of the 4VP group was 18 times that of the penetratin group, and the mean fluorescence intensity of the 8VP group was 8 times that of the penetratin group. In NHC cells, the mean fluorescence intensity of the 4VP group was 24 times that of the penetratin group, and the mean fluorescence intensity of the 8VP group was 14 times that of the penetratin group.

[0089] Example 7

[0090] Formulation screening of multivalent penetratin-antisense oligonucleotide (ASO) non-covalent linkage complexes: Preliminary formulation screening of gene delivery systems was performed using polyacrylamide gel electrophoresis (PAGE). Free penetratin and multivalent penetratin (4VP, 8VP) were mixed with 1.32 μg ASO at different molar ratios (the molar ratios of multivalent penetratin converted to monomolecule penetratin to ASO, from left to right, were 3:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1 and 60:1; where the concentrations of multivalent penetratin converted to monomolecule penetratin were 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 800 μM, 1000 μM and 1200 μM, respectively) in 20 μL of DEPC water, vortexed for 30 s, and incubated at 37 °C for 30 min to obtain complexes Pe / ASO, 4VP / ASO and 8VP / ASO. The complexes were carefully added to the wells of the PAGE gel, with a free ASO group included. After electrophoresis, the gels were soaked in GelRed working solution in the dark for 30 min, and then imaged under UV 302 nm conditions.

[0091] The results showed that for the Pe / ASO complex, there were still noticeable free ASO spots at a molar ratio of 20:1, indicating that free penetratin did not completely shield the negative charge of ASO or fully compress it under this ratio. However, for the 4VP / ASO and 8VP / ASO complexes constructed with multivalent penetratin, almost no free ASO spots were observed at a molar ratio of 20:1, indicating that both complexes could effectively shield the negative charge of ASO and achieve better ASO compression under this ratio.

[0092] Flow cytometry was used for further formulation screening. HCEC and NHC cells in good growth condition were collected at a concentration of 5 × 10⁶ cells / mL.3 cells / cm 2 Cells were seeded into 12-well plates, with the medium changed daily after seeding. After 2–3 days of culture, experiments were performed. The culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free DMEM solutions containing complexes with different charge ratios were added, and the cells were incubated at 37°C and 5% CO2 for 1 hour. After incubation, the DMEM solution was discarded, and the cells were washed three times with PBS buffer. The cells were then digested, resuspended in 200 μL of sterile PBS buffer, and thoroughly mixed before flow cytometry analysis. Cell counts were performed at approximately 10⁻⁶ cells per sample. 4 Untreated cells served as a negative control group;

[0093] The results showed that as the molar ratio of multivalent penetratin to ASO increased, the uptake of ASO in corneal and conjunctival cells also increased, indicating that the gene delivery efficiency of multivalent penetratin increased with the increase of their molar ratio.

[0094] Example 8

[0095] Evaluation of particle size, potential and stability of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotide: The non-covalently linked complex of multivalent penetratin and antisense oligonucleotide was prepared according to the method in Example 7, and the particle size and potential were measured.

[0096] The results (Table 2) show that, compared with the Pe / ASO complex formed by free penetratin, the composites obtained by compressing ASO with multivalent penetratin have relatively smaller particle sizes, especially the 8VP / ASO composite, whose particle size is reduced by about 60 nm compared with Pe / ASO. The polydispersity index of all three composites is less than 0.3, indicating that the particle size distribution is relatively uniform. The surface potential of the Pe / ASO and 8VP / ASO composites is around +20 mV, and the particle count of 8VP / ASO is significantly higher than that of Pe / ASO, indicating that its structure is more compact.

[0097] Table 2. Particle size, polydispersity index, and potential characterization of antisense oligonucleotide non-covalent complexes.

[0098]

[0099] The complex was incubated at 34℃, and the particle size was measured at different time points (0, 0.5h, 1h, 2h, 4h, 6h, 12h, 24h, 48h) to investigate the particle size stability of the complex.

[0100] The results showed that, under incubation conditions at 34℃, the composite formed by polyvalent penetratin compressed ASO had better stability than the composite Pe / ASO formed by free penetratin. The former maintained a stable particle size within 48 hours, while the composite Pe / ASO increased in particle size to about 800 nm after 48 hours, indicating that the composite had aggregated.

[0101] Example 9

[0102] Morphological evaluation of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotide: The non-covalently linked complex of multivalent penetratin and antisense oligonucleotide was prepared according to the method in Example 7, and the morphology of the complex was observed under an electron microscope.

[0103] The results showed that all four complexes were generally spherical, and their particle size distributions were consistent with those in Example 8. For the Pe / ASO complex, the particle size distribution was between 100 and 200 nm; for the 4VP / ASO complex, it was between 100 and 150 nm; and for the 8VP / ASO complex, it was around 100 nm. The complex constructed based on Lipofectamine 2000 had a larger particle size and was particularly prone to aggregation.

[0104] Example 10

[0105] Cytotoxicity evaluation of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides: HCEC and NHC cells in good growth status during the logarithmic growth phase were collected at a concentration of 5 × 10⁻⁶ cells / year. 3 cells / cm 2 Cells of different concentrations were seeded into the middle 60 wells of a 96-well plate, with the edges filled with sterile PBS buffer. The plates were cultured at 37°C and 5% CO2 until a monolayer of cells covered the bottom of the plate. The culture medium was discarded, and the cells were washed three times with sterile PBS buffer. Then, 200 μL of culture medium containing different concentrations of the complex was added, and the plates were incubated for 12 h. After discarding the solution, the cells were washed three times with sterile PBS buffer, and then complete culture medium was added, followed by another 12 h of incubation. Next, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated for another 4 h. The liquid was carefully discarded, and the cells were washed three times with PBS buffer. Then, 150 μL of LDMSO was added to each well, and the plates were shaken at low speed for 20 min. The absorbance of each well was measured at OD490 nm using a microplate reader. Blank control wells (culture medium, MTT, DMSO) and negative control wells (cells, culture medium, MTT, DMSO) were also included.

[0106] The results showed that HCEC and NHC cells grew well at 16 times the experimental concentration (3.2 μM ASO), and the complex did not show any toxic effects on ocular cells.

[0107] Example 11

[0108] Evaluation of intracellular distribution of the non-covalently linked complex of multivalent penetratin and antisense oligonucleotides: HCEC and NHC cells in good growth condition were divided into groups of 5 × 10⁻⁶ cells. 3 cells / cm 2 Cells were seeded at a density of 35 mm in four-cell confocal microscopy dishes. The medium was changed daily after seeding, and experiments were performed after 2–3 days of incubation. After discarding the culture medium, the cells were washed three times with sterile PBS, and serum-free DMEM solution containing different complexes (each containing 1.32 μg of green fluorescently labeled ASO) was added. The cells were incubated at 37 °C and 5% CO2 for 4 h. After incubation, the solution was discarded, and the cells were washed three times with PBS buffer. Lysosomes were stained with LysoTracker staining, and cell nuclei were stained with DAPI staining. The cells were then observed under a laser confocal microscope.

[0109] The results showed that almost no fluorescently labeled ASO was distributed in HCEC and NHC cells in the free ASO group; in the Pe / ASO complex formed by free penetratin, a small amount of ASO entered the cells, but most of it co-localized with red fluorescently labeled lysosomes; while in the 4VP / ASO and 8VP / ASO complexes formed by multivalent penetratin, more ASO entered the cells, and a considerable portion of the ASO did not co-localize with lysosomes. The commercially available gene transfection reagent, cationic liposome Lipofectamine 2000, and the non-covalent complex LIPO / ASO with antisense oligonucleotides, resulted in a relatively small amount of ASO entering the cells under the same conditions.

[0110] Example 12

[0111] Quantitative evaluation of cellular uptake of the multivalent penetratin-antisense oligonucleotide non-covalently linked complex: HCEC and NHC cells in good growth condition were divided into groups of 5 × 10⁻⁶ cells. 3 cells / cm 2 Cells were seeded at a density of [insert density here] in 12-well plates, with the medium changed daily after seeding. After 2–3 days of culture, experiments were performed. The culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free DMEM solution containing different complexes (each containing 1.32 μg of green fluorescently labeled ASO) was added, and the cells were incubated at 37°C and 5% CO2 for 4 h. After incubation, the solution was discarded, and the cells were washed three times with PBS buffer. The cells were then digested, resuspended in 200 μL of sterile PBS buffer, and thoroughly mixed before flow cytometry analysis. Cell counts were performed at approximately 10-1 per sample. 4 Untreated cells served as a negative control group;

[0112] The results showed that in both cell types, the uptake of ASO was minimal in both the free ASO group and the Pe / ASO complex formed by free penetratin. However, both multivalent penetratin and the commercially available gene transfection reagent, cationic liposome Lipofectamine 2000, significantly increased ASO uptake. In HCEC cells, 4VP-mediated ASO uptake was 3.7 times that of free ASO (p<0.001), while 8VP was 45 times (p<0.001). In NHC cells, 4VP-mediated ASO uptake was 6.6 times that of free ASO (p<0.01), while 8VP was 90 times (p<0.001). Notably, compared to the commercially available gene transfection reagent, cationic liposome Lipofectamine 2000, 8VP-mediated ASO uptake was 8.6 and 12.4 times (p<0.001), respectively, indicating a significant improvement in gene delivery.

[0113] Example 13

[0114] Distribution of multivalent penetratin-antisense oligonucleotide non-covalently linked complexes in the eyes of live mice: The distribution and pharmacokinetic characteristics of green fluorescently labeled ASO in each complex were investigated using an intraconjunctival sac ocular instillation experiment in mice. Three complexes, Pe / ASO, 4VP / ASO, and 8VP / ASO, were prepared, each containing 2.64 μg of fluorescently labeled ASO dissolved in 5 μL of simulated tear fluid. Free ASO, Pe / ASO, 4VP / ASO, and 8VP / ASO groups, as well as a blank control group (no treatment), were set up. Free ASO or the complexes were instilled into the conjunctival sac of mice, and the eyelids were gently closed repeatedly to ensure even distribution on the ocular surface. The end of administration was recorded as time zero. Mice were sacrificed 10 minutes later, and the eyeballs were removed, fixed in Davidson's solution for 30 minutes, and then dehydrated overnight in 30% sucrose solution. DAPI-stained frozen sections were prepared and observed.

[0115] The results showed that no green fluorescence signal was observed in the anterior and posterior segments of the eyeballs in the blank group, indicating the absence of fluorescent background interference. No obvious green fluorescence signal was observed in the free ASO group; weak green fluorescence signals were observed in both the anterior and posterior segments of the eye in the Pe / ASO group; strong fluorescence signals were observed in both the anterior and posterior segments of the eye in the 4VP / ASO and 8VP / ASO groups, with the fluorescence signal mainly distributed in the posterior segment. Furthermore, magnified local results showed ( Figure 14B) Free ASO or penetratin-mediated ASO has difficulty entering the cornea, while 4VP and 8VP can mediate ASO into the corneal stroma. In the posterior segment of the eye, no green fluorescence signal was observed in the free ASO group. Penetratin can mediate some ASO reaching the choroid. 4VP and 8VP can mediate ASO distribution throughout the posterior segment of the eye, including the choroid and retina, and 8VP has a better delivery effect than 4VP.

[0116] Example 14

[0117] Evaluation of ocular elimination of multivalent penetratin and antisense oligonucleotide non-covalently linked complexes: The ocular distribution and pharmacokinetic characteristics of green fluorescently labeled ASO in each complex were investigated by intraconjunctival sac dosing in mice. Three complexes, Pe / ASO, 4VP / ASO, and 8VP / ASO, were prepared, each containing 2.64 μg of fluorescently labeled ASO dissolved in 5 μL of simulated tear fluid. Free ASO group, Pe / ASO group, 4VP / ASO group, 8VP / ASO group, and blank control group (no treatment) were set up. Free ASO or complexes were instilled into the conjunctival sac of mice, and the eyelids were gently closed repeatedly to ensure even distribution on the ocular surface. The end of administration was recorded as time zero. Mice were sacrificed and their eyeballs were removed at 10 min, 30 min, 1 h, 2 h, 4 h, and 6 h. The eyeballs were fixed in Davidson's solution for 30 min and then dehydrated overnight in 30% sucrose solution. DAPI-stained frozen sections were prepared and observed.

[0118] The results showed that after intraconjunctival sac eye drops, no green fluorescence signal was observed in the cornea and retina of the free ASO group and the Pe / ASO group. For the 4VP / ASO and 8VP / ASO groups, ASO could enter the eye 10 minutes after administration and was mainly distributed in the retinal tissue. The fluorescence signal reached its peak within 1 hour and then gradually weakened over time, with a residence time of up to 6 hours.

[0119] Example 15

[0120] Preparation of non-covalent penetratin / plasmid complexes (8VP / plasmid) with plasmid DNA (Plasmid-Cas1, Plasmid-Cas2, Plasmid-Cas3): 8-valent penetratin (8VP) was mixed with plasmid DNA at different molar ratios (1:1, 5:1, 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 150:1, 200:1, 250:1, and 300:1; 8-valent penetratin:plasmid DNA, where the molar amount of 8-valent penetratin was converted to the molar amount of a single penetratin molecule) in 100 μL of ultrapure water. The mixture was vortexed for 30 s and incubated at 37 °C for 30 min to obtain the 8VP / plasmid complex.

[0121] Example 16

[0122] Evaluation of cell transfection efficacy of multivalent penetratin and plasmid DNA (Plasmid-Cas1, Plasmid-Cas2, Plasmid-Cas3) non-covalent linkage complex (8VP / plasmid): 293T cells in good growth condition were transfected at a rate of 5 × 10⁻⁶ cells / year. 3 cells / cm 2 Cells were seeded into 24-well plates. After culturing for 24 hours at 37°C and 5% CO2 in 500 μL of DMEM medium containing 10% FBS, experiments were performed. The culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free DMEM solutions containing complexes 8VP / Plasmid-Cas1, 8VP / Plasmid-Cas2, 8VP / Plasmid-Cas3, Lipofectamine 2000 / Plasmid-Cas1, Lipofectamine 2000 / Plasmid-Cas2, and Lipofectamine 2000 / Plasmid-Cas3 were added, and the cells were incubated at 37°C and 5% CO2 for 4 hours. After incubation, the solutions were discarded, and the cells were washed with PBS buffer containing 0.02 mg / mL heparin sodium to remove adsorbed substances from the cell surface. Cells were cultured under normal conditions for another 48 hours. The culture medium was then discarded, and the cells were washed three times with PBS buffer. After covering with PBS / glycerol, the cells were observed under a laser confocal microscope. Cells in the untreated group served as a blank control.

[0123] The results showed that all experimental groups expressed some green fluorescent protein. Compared with the complex constructed with Lipofectamine 2000, 8VP was more effective in mediating plasmid DNA entry into 293T cells and expression.

[0124] Example 17

[0125] Preparation of non-covalent complexes of polyvalent TAT and small interfering RNA (siRNA): Trivalent TAT and hexavalent TAT were synthesized by reacting TAT with 3-arm polyethylene glycol and 6-arm polyethylene glycol, respectively. These were then mixed with 0.5 nM siRNA at molar ratios (1:2, 1:1, 5:1, 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1, 160:1, 180:1, and 200:1; polyvalent TAT:siRNA, where the molar amount of polyvalent TAT was converted to the molar amount of a single TAT molecule) in 50 μL of DEPC water. The mixture was vortexed for 30 s and incubated at 37 °C for 30 min to obtain the non-covalent complexes of polyvalent TAT and siRNA.

[0126] Example 17

[0127] Poly(arginine) n Preparation of non-covalent complexes of pegaptanib (where n = 6, 8, and 12): using poly(arginine) n Synthesize 8-valent poly(arginine)6, 8-valent poly(arginine)8, and 8-valent poly(arginine) with 8-arm polyethylene glycol. 12 Poly(arginine) was mixed with 100 nM piperatanib at molar ratios (1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 120:1, 140:1, and 160:1; poly(arginine):piperazine, where the molar amount of poly(arginine) was converted to the molar amount of monomeric oligo(arginine)) in 30 μL LEPC water, vortexed for 30 s, and incubated at 37 °C for 30 min to obtain poly(arginine). n (where n = 6, 8 and 12) non-covalent complexes with pilgatanib.

[0128] Example 18

[0129] Preparation of non-covalent complexes of protamine and bevasiranib: Hexavalent protamine was synthesized by reacting protamine with 6-arm polyethylene glycol. This hexavalent protamine was then mixed with 20 nM bevasiranib at molar ratios (1:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, and 100:1; polyvalent protamine:bevasiranib, where the molar amount of polyvalent protamine was converted to the molar amount of monomolecule protamine) in 20 μL of DEPC water. The mixture was vortexed for 30 s and incubated at 37 °C for 30 min to obtain the non-covalent complexes of protamine and bevasiranib.

[0130] Example 19

[0131] Preparation of non-covalent complexes of multivalent penetratin derivatives (28-W) and monoclonal antibody drugs: A tetravalent penetratin derivative was synthesized using penetratin derivatives (28-W) and four-arm polyethylene glycol. This tetravalent derivative was then mixed with 1 nM monoclonal antibody drugs bevacizumab, ranibizumab, or ramucirumab at molar ratios (1:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, and 100:1; multivalent penetratin derivative: monoclonal antibody, where the molar amount of the multivalent penetratin derivative is converted to the molar amount of a single molecule of penetratin derivative) in 200 μL. In ultrapure water, gently shake for 30 seconds and incubate at 37°C for 30 minutes to obtain a non-covalent complex of a multivalent penetratin derivative and a monoclonal antibody drug.

[0132] Example 20

[0133] Preparation of non-covalent complexes of polyvalent penetratin derivative (289-F) and protein drugs: A hexavalent penetratin derivative was synthesized by combining penetratin derivative (289-F) with 6-arm polyethylene glycol. This hexavalent penetratin derivative was then mixed with 5 nM protein drugs Conbercept, Epidermal Growth Factor, or Interferon-α at molar ratios (1:1, 5:1, 10:1, 20:1, 40:1, 60:1, 80:1, and 100:1; polyvalent penetratin derivative: protein drug, where the molar amount of the polyvalent penetratin derivative was converted to the molar amount of a single molecule of penetratin derivative) in 300 μL of ultrapure water. The mixture was gently shaken for 30 s and incubated at 37 °C for 30 min to obtain non-covalent complexes of polyvalent penetratin derivative and protein drugs.

Claims

1. A multivalent transmembrane peptide-based biologic macromolecule delivery carrier, characterized by, It includes, multi-price The membrane-penetrating peptide has a multi-armed polyethylene glycol core, with each polyethylene glycol side arm covalently linked to the membrane-penetrating peptide at its terminal, forming an octopus-like multi-armed, flexible, multivalent structure. Its characteristic is that... The multi-arm polyethylene glycol (PEG) serving as the core is selected from 3-arm PEG, 4-arm PEG, 6-arm PEG, or 8-arm PEG, wherein each PEG side arm contains an ethoxylated (CH2CH2O)n with a degree of polymerization n between 5 and 50, and the molecular weight of the multi-arm PEG is between 200 and 80,000. The membrane-penetrating peptide covalently linked to the end of the polyethylene glycol side arm is penetratin; and The multivalent membrane-penetrating peptide and the biomolecular drug self-assemble to form a non-covalent complex. In the non-covalent complex formed by the multivalent membrane-penetrating peptide and the biomolecular drug, the biomolecular drug is selected from gene drug plasmid DNA (pDNA), pegaptanib, bevasiranib, antisense oligonucleotide, small interfering RNA, or from monoclonal antibody drugs bevacizumab, ranibizumab, ramucirumab, or from polypeptide protein drugs conbercept, epidermal growth factor, and interferon-α, as well as combinations thereof.

2. The multivalent TFP-based biological macromolecule delivery carrier of claim 1, wherein, The membrane-penetrating peptide covalently linked to the end of the polyethylene glycol side arm is wild-type penetratin, and the amino acid sequence of wild-type penetratin is RQIKIWFQNRRMKWKK.

3. The multivalent TFP-based biological macromolecule delivery carrier of claim 1, wherein, In the non-covalent complex formed by the self-assembly of the multivalent membrane-penetrating peptide and the biomacromolecule, the molar ratio of the multivalent membrane-penetrating peptide to the biomacromolecule is between 1:2 and 300:1, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide.

4. The multivalent TFP-based biological macromolecule delivery carrier of claim 1, wherein, In the non-covalent complex formed by the self-assembly of the multivalent membrane-penetrating peptide and the biomolecular drug, the molar ratio of the multivalent membrane-penetrating peptide to the biomolecular drug is 1:1 to 200:1, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide.

5. The multivalent TFP-based biologic macromolecule delivery carrier of claim 1, wherein, In the non-covalent complex formed by the self-assembly of the multivalent membrane-penetrating peptide and the biomacromolecule, the molar ratio of the multivalent membrane-penetrating peptide to the biomacromolecule is 1:1 to 100:1, wherein the molar amount of the multivalent membrane-penetrating peptide is converted to the molar amount of a single membrane-penetrating peptide.

6. The multivalent TFP-based biologic macromolecule delivery carrier of claim 1, wherein, The particle size of the non-covalent complex formed by the self-assembly of the multivalent membrane-penetrating peptide and the biomacromolecule drug is between 1 nm and 1000 nm.

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