Amphiphilic drug carrier and use thereof in ocular drug delivery

Abstract

An amphiphilic drug carrier and the use thereof in the ocular delivery of a biomacromolecular drug. The amphiphilic drug carrier is capable of penetrating the ocular mucus barrier, loading a biomacromolecular drug by means of non-covalent interaction, and delivering the biomacromolecular drug for the treatment of fundus diseases.

Description

An amphiphilic drug carrier and its application in ocular drug delivery Technical Field

[0001] This invention relates to the field of biopharmaceutical drug delivery, and more particularly to an amphiphilic drug carrier and its application in ocular drug delivery. Background Technology

[0002] Therapeutic biological macromolecules such as proteins, peptides, and nucleic acids have been widely used to treat eye diseases. However, non-invasive methods of drug delivery, such as eye drops or ointments, are not the primary form of treatment for fundus diseases. The main treatments for chronic fundus diseases such as diabetic retinopathy and age-related macular degeneration (AMD) involve high-dose oral antioxidants or intravitreal injections of therapeutic protein drugs. This is because the complex structure of the eye, with its barrier systems including tears, cornea, conjunctiva, blood aqueous humor, and retina, hinders the absorption of drugs after eye drops; intravenous infusion requires excessive dosage to achieve effective local drug concentrations, thus potentially causing serious side effects.

[0003] Currently, intraocular injection remains the most common method of drug delivery, but it still carries risks of intraocular infection, ocular hemorrhage, and retinal detachment. For lesions requiring frequent administration, frequent intraocular injections can lead to crusting and scarring, further reducing patient compliance. Therefore, there is an urgent need to develop non-invasive ocular drug delivery methods that combine safety and efficacy to deliver large molecule drugs (such as therapeutic protein drugs) to the posterior segment of the eye.

[0004] Mucus, a complex viscoelastic hydrogel, covers the surface of human mucous membranes. The cornea and conjunctiva of the eye are covered with a layer of mucus, which serves functions such as lubrication, cell signal transduction, and protection of the epithelium from harmful external substances. The mucus layer is a dynamic system with a natural self-renewal capacity; for example, the eye completes a mucus renewal cycle approximately every 5-7 minutes. If the active ingredients delivered via mucosal administration cannot penetrate the mucus in time, they will be cleared away during this renewal process, reducing their bioavailability. This mucus barrier is a challenge that mucosal drug delivery systems must address, and it is a problem that non-invasive ocular drug delivery must solve. Summary of the Invention

[0005] To address the aforementioned issues, this application provides an amphiphilic drug carrier for ocular drug delivery, specifically for delivering macromolecular biological drugs via eye drops. Compared to existing ocular drug carriers, the amphiphilic drug carrier described in this application exhibits excellent hydrophilicity and resistance to nonspecific adsorption, enabling it to effectively penetrate the ocular mucus barrier and deliver macromolecular biological drugs for the treatment of fundus diseases. This amphiphilic drug carrier can load macromolecular biological drugs through non-covalent interactions, thereby effectively protecting drug activity. Furthermore, it has a small molecular weight, a relatively simple and well-defined structure, and can be synthesized and scaled up for production in a controlled manner, demonstrating promising industrialization prospects.

[0006] The first objective of this invention is to provide an amphiphilic drug carrier for ocular drug delivery, comprising a hydrophobic region and an amphoteric region, wherein the hydrophobic region is a hydrophobic peptide segment; and the amphoteric region is an amphoteric peptide segment formed by alternating basic amino acids and acidic amino acids.

[0007] The amphiphilic drug carrier further includes a positively charged region, which is a basic polypeptide chain. Specifically, the hydrophobic region and the amphoteric region are connected by an amide bond. When the hydrophobic region is a hydrophobic peptide segment, the carboxyl terminus of the hydrophobic peptide segment undergoes a dehydration condensation reaction with the amino terminus of the amphoteric peptide segment to form an amide bond connecting the hydrophobic region and the amphoteric region.

[0008] Furthermore, the hydrophobic peptide segment comprises at least a hydrophobic amino acid, wherein the hydrophobic amino acid is selected from any one or more combinations of phenylalanine, valine, leucine, and isoleucine.

[0009] Furthermore, the hydrophobic amino acid can also be selected from non-natural hydrophobic amino acids. Specifically, the non-natural hydrophobic amino acid is a derivative of the hydrophobic amino acid.

[0010] Furthermore, the hydrophobic peptide contains 6-20 amino acids.

[0011] Further, the hydrophobic peptide contains 2-20 amino acids. Specifically, the hydrophobic peptide contains 2-6 phenylalanines, or 6-20 leucines or isoleucines, or 10-20 valine. Even further, the hydrophobic peptide contains 6-15 leucines or isoleucines.

[0012] Specifically, the hydrophobic peptide contains 2, 3, 4, 5 or 6 phenylalanines.

[0013] Specifically, the hydrophobic peptide contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 leucine or isoleucine.

[0014] Specifically, the hydrophobic peptide contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 valine residues.

[0015] Furthermore, the basic amino acid is selected from one or both of lysine and arginine; and / or the acidic amino acid is selected from one or both of glutamic acid and aspartic acid.

[0016] Further, the amphoteric peptide contains 8-32 amino acids. More preferably, the amphoteric peptide contains 8-30 amino acids.

[0017] More preferably, the amphoteric peptide contains 9-33 amino acids.

[0018] Furthermore, the number of amino acids in the amphoteric peptide is odd, and it contains a basic amino acid at the end.

[0019] Furthermore, the amphoteric peptide contains equal numbers of acidic amino acids and basic amino acids, which are arranged and linked together in any order.

[0020] Furthermore, the amphoteric peptide contains an equal number of free amino and free carboxyl groups. When the amphoteric peptide contains 2 to 10 amino acids, the carboxyl terminus is not an acidic amino acid.

[0021] Preferably, the interval between each basic amino acid or acidic amino acid is one or two amino acids of the same type. For example, one or two basic amino acids are connected to one or two acidic amino acids, and then connected to one or two more basic amino acids, and so on.

[0022] Further, the amphoteric peptide contains 2 to 16 minimal repeating units, wherein the minimal repeating unit is a combination of any basic amino acid and any acidic amino acid in the form of AB or AABB. More preferably, the minimal repeating unit is a combination of any basic amino acid and any acidic amino acid in the form of A1B1 or A1A2B1B2, wherein A1 and A2 are the same basic amino acid or different basic amino acids, and B1 and B2 are the same acidic amino acid or different acidic amino acids. Even more preferably, the amphoteric peptide contains 8 to 16 minimal repeating units.

[0023] Furthermore, the amphoteric peptide contains 2, 4, 6, 8, 10, 12, 14 or 16 minimal repeating units.

[0024] Specifically, the smallest repeating unit is selected from one or more of the following: lysine + glutamic acid, lysine + aspartic acid, arginine + glutamic acid, arginine + aspartic acid, two lysine + two glutamic acid, two lysine + two aspartic acid, two arginine + two glutamic acid, and two arginine + two aspartic acid.

[0025] Furthermore, the positively charged region and the hydrophobic region are connected by amide bonds.

[0026] Furthermore, the positively charged region is a basic polypeptide chain, which contains any one or more basic amino acids selected from lysine, arginine, and histidine.

[0027] Specifically, the basic polypeptide chain is connected to the amino terminus of the hydrophobic region via an amide bond, and the carboxyl terminus of the hydrophobic region is connected to the amino terminus of the amphoteric peptide. When the hydrophobic region is selected from the hydrophobic peptide, the amino terminus of the hydrophobic peptide is connected to the carboxyl terminus of the basic polypeptide chain, and the carboxyl terminus of the hydrophobic peptide is connected to the amino terminus of the amphoteric peptide.

[0028] Further, the basic polypeptide chain contains 0-8 basic amino acids. More preferably, the basic polypeptide chain contains 1-8 basic amino acids. Specifically, the basic polypeptide chain contains 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

[0029] Furthermore, the hermaphroditic region includes a linear structure or a branched structure.

[0030] Specifically, the linear structure is formed by the dehydration condensation reaction between amino acids to form a single chain.

[0031] Specifically, the branched structure is obtained by the reaction of amino or carboxyl groups on the side chain of an amino acid molecule.

[0032] Furthermore, the terminus of the linear amphipathic region is either lysine K or arginine R.

[0033] Furthermore, the number of terminal branches of the branch structure is 2, 4, 8, 16 or 32.

[0034] Furthermore, the amphiphilic drug carrier contains hydrophilic and hydrophobic amino acids, with a ratio of 1.0 to 4.0. Even further, the ratio of hydrophilic to hydrophobic amino acids is between 1.3 and 3.7.

[0035] Furthermore, the amphiphilic drug carrier possesses suitable hydrophilicity and hydrophobicity.

[0036] The appropriate hydrophilicity / hydrophobicity refers to a reasonable ratio between the length of the hydrophilic region and the length of the hydrophobic region in the amphiphilic drug carrier, which is between 1.0 and 4.0. That is, the ratio of the number of hydrophilic amino acids in the hydrophilic region to the number of hydrophobic amino acids in the hydrophobic region is between 1.0 and 4.0.

[0037] Furthermore, the ratio of the number of amino acids in the hydrophobic peptide to the number of amino acids in the amphipathic peptide is 5:(4-20).

[0038] By adjusting the ratio of the number of amino acids in hydrophobic peptides to the number of amino acids in amphiphilic peptides, the hydrophilicity and hydrophobicity of amphiphilic drug carriers can be modulated, thereby enabling them to assemble with biopharmaceutical drugs through non-covalent bonds to form macromolecular complexes.

[0039] A second objective of this invention is to provide a macromolecular complex comprising a biological macromolecular drug and an amphiphilic drug carrier as described in the above technical solution, wherein the biological macromolecular drug and the amphiphilic drug carrier are non-covalently assembled.

[0040] Furthermore, the macromolecular complex is used in formulations for the treatment of eye diseases.

[0041] The eye diseases mentioned include, but are not limited to, retinal-related diseases, retinopathy caused by various factors, retinal vasculitis, proliferative eye diseases, polypoid choroidal angiopathy, idiopathic choroidal neovascularization, retinopathy of prematurity, outer exudative retinopathy, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, cataracts, uveitis, keratitis, retinoblastoma, central retinal vein occlusion, retinal vein occlusion, primary retinitis pigmentosa, central serous chorioretinopathy, retinal periphlebitis, retinal artery occlusion, and glaucoma.

[0042] Specifically, the non-covalent bond assembly method can be electrostatic interaction, hydrophobic interaction, etc.

[0043] Compared to covalent assembly, non-covalent assembly has less impact on drug activity, thus drugs assembled with amphiphilic drug carriers have better efficacy at the same dosage.

[0044] Furthermore, the molecular weight of the said biopharmaceutical is 3-300 kDa.

[0045] Furthermore, the molecular weight of the said biopharmaceutical is 3–10 kD, 10–20 kD, 20–50 kD, 50–60 kD, 60–80 kD, 80–100 kD, 100–120 kD, 120–150 kD, 150–180 kD, 180–200 kD, 200–250 kD, or 250–300 kD.

[0046] Furthermore, the biological macromolecular drug includes, but is not limited to, any one of biological macromolecular therapeutic agents such as proteins, peptides, nucleic acids, and polysaccharides.

[0047] Furthermore, the aforementioned biopharmaceutical drugs include, but are not limited to, anti-vascular endothelial growth factor (VEGF) drugs, anti-angiogenic factor (Ang) drugs, anti-tumor necrosis factor a (TNF-α) drugs, programmed cell death-ligand 1 antibody drugs, cytotoxic T lymphocyte-associated protein 4 antibody drugs, programmed death receptor 1 drugs, anti-lymphocyte activation gene 3 (LAG3) antibody drugs, T cell immunoglobulin domain and mucin domain-3 (Tim-3) antibody drugs, and T cell immunoglobulin and ITIM domain protein (TIGIT) antibody drugs.

[0048] Furthermore, the aforementioned biological macromolecular drugs include, but are not limited to, one or more of the following: adalimumab, infliximab, etanercept, golimumab, ranibizumab, aflibercept, conbercept, bromizumab, bevacizumab, falimab, rituximab, trastuzumab, cetuximab, metuximab, nimotuzumab, faraximab, ipilimumab, nivolumab, pembrolizumab, pecelizumab, omalizumab, aliximab, evolocumab, and emecizumab.

[0049] Furthermore, the biopharmaceutical includes, but is not limited to, one or more of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and peroxide reductase (PRX).

[0050] Furthermore, the nucleic acid is selected from siRNA, mRNA, shRNA, lncRNA, pDNA, polyIC, CpG, or cyclic dinucleotides.

[0051] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is (1-16):1.

[0052] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is (1-8):1.

[0053] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1.

[0054] The third objective of this invention is to provide a method for preparing the macromolecular complex described in the above technical solution, comprising the following steps: mixing an amphiphilic drug carrier with a biological macromolecular drug, adjusting the pH to a value greater than the isoelectric point of the biological macromolecular drug, and obtaining the macromolecular complex.

[0055] Furthermore, the pH value is approximately 5 to 7.

[0056] Furthermore, the pH value is approximately 5.5 (±0.3) to 6.5 (±0.3).

[0057] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is (1-16):1.

[0058] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is (1-8):1.

[0059] Furthermore, the mass ratio of the amphiphilic drug carrier to the biomolecular drug is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1.

[0060] A fourth objective of this invention is to provide the application of the amphiphilic drug carrier or the macromolecular complex described above in the delivery of drugs across biological barriers.

[0061] Furthermore, biological barriers include, but are not limited to, the eye barrier.

[0062] Furthermore, the ocular barriers include, but are not limited to, the tear barrier, the corneal / conjunctival barrier, the blood-aqueous humor barrier, the blood-retinal barrier, and the blood-eye barrier.

[0063] The fifth objective of this invention is to provide the use of the amphiphilic drug carrier or the macromolecular complex described in the above-described technical solutions in eye drop formulations.

[0064] A sixth objective of this invention is to provide a medicament for treating fundus diseases, comprising an amphiphilic drug carrier as described in the above technical solutions or a macromolecular complex as described in the above technical solutions.

[0065] Furthermore, the fundus diseases mentioned include retinal-related diseases, retinopathy caused by various factors, retinal vasculitis, proliferative ocular diseases, polypoid choroidal vascular disease, idiopathic choroidal neovascularization, retinopathy of prematurity, outer exudative retinopathy, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, cataracts, uveitis, keratitis, retinoblastoma, central retinal vein occlusion, retinal vein occlusion, primary retinitis pigmentosa, central serous chorioretinopathy, retinal periphlebitis, retinal artery occlusion, and glaucoma, etc.

[0066] Furthermore, the medicine for treating fundus diseases also includes pharmaceutically acceptable excipients.

[0067] Furthermore, the pharmaceutically acceptable excipients include thickeners.

[0068] Furthermore, the thickener is hydroxypropyl methylcellulose.

[0069] Furthermore, the hydroxypropyl methylcellulose in the drug has a mass fraction of 0.1% to 5%.

[0070] Further, the hydroxypropyl methylcellulose in the drug has a mass fraction of 0.1%–0.5%, 0.5%–1%, 1%–2%, 2%–3%, 3%–4%, or 4%–5%.

[0071] Furthermore, the pH value of the drug used to treat fundus diseases is 5.0 to 7.0.

[0072] Furthermore, the pH value of the drug used to treat fundus diseases is 5.5 to 6.5.

[0073] Furthermore, the pH value of the drug used to treat eye diseases is 5.5 (±0.3) to 6.5 (±0.3).

[0074] Furthermore, the drug is a lyophilized preparation containing a lyophilization protectant.

[0075] Furthermore, the freeze-drying protectant is mannitol.

[0076] Furthermore, the content of the freeze-drying protectant is 1%wt to 10%wt.

[0077] Furthermore, the osmotic pressure of the drug for treating fundus diseases is in the range of 260 mOsmol / kg to 320 mOsmol / kg.

[0078] A seventh objective of this invention is to provide a method of use for treating fundus diseases, comprising administering to a patient an eye drop containing an amphiphilic drug carrier as described in the above-described technical solutions or a macromolecular complex as described in the above-described technical solutions.

[0079] The present invention also provides an amphiphilic drug carrier having the following general structural formula: X1-X2-X3;

[0080] X2 is a hydrophobic peptide composed of hydrophobic amino acids;

[0081] Furthermore, the hydrophobic amino acids comprise 6 to 20 types;

[0082] Furthermore, the hydrophobic peptide contains 2 to 20 amino acids.

[0083] Specifically, the hydrophobic peptide contains 2-6 phenylalanines, or 6-20 leucines or isoleucines, or 10-20 valine. Further, the hydrophobic peptide contains 6-15 leucines or isoleucines.

[0084] Specifically, the hydrophobic peptide contains 2, 3, 4, 5 or 6 phenylalanines.

[0085] Specifically, the hydrophobic peptide contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 leucine or isoleucine.

[0086] Specifically, the hydrophobic peptide contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 valine residues.

[0087] X3 is a basic polypeptide chain composed of basic amino acids, and the number of basic amino acids is 0 to 8.

[0088] Furthermore, X1 can be a dendritic structure containing 2, 4, 8, 16 or 32 terminal branches.

[0089] Furthermore, when X1 has a dendritic structure, the X1-X2-X3 structure is illustrated in the following example (of course, it is not limited to the following structure):

[0090] X2 is a hydrophobic peptide composed of hydrophobic amino acids, and further, there are 6 to 20 hydrophobic amino acids; X3 is a basic polypeptide chain composed of basic amino acids, and the number of basic amino acids is 1 to 8.

[0091] Alternatively, X2 may be a basic polypeptide chain composed of basic amino acids, wherein the number of basic amino acids is 1 to 8; X3 may be a hydrophobic peptide segment composed of hydrophobic amino acids, wherein the number of hydrophobic amino acids is 6 to 20.

[0092] Compared with the prior art, the amphiphilic drug carrier provided by the present invention has at least the following advantages:

[0093] 1) Amphiphilic drug carriers have amphiphilic peptide segments with alternating arrangement of amphiphilic amino acids (basic and acidic amino acids). Their positive and negative charges are evenly distributed at the molecular level, which endows them with superhydrophilicity and excellent anti-bioadhesion. This further endows amphiphilic drug carriers with the ability to diffuse rapidly in the mucus layer. At the same time, they can effectively deliver biomolecule drugs to ocular structures such as the retina and choroid without damaging the tight connection of the corneal and conjunctival tissue barriers, so as to achieve the purpose of treating eye diseases.

[0094] 2) In addition to the amphiphilic region, the amphiphilic drug carrier also contains a hydrophobic region and a positively charged region. The hydrophobic region can provide the assembly driving force for the amphiphilic drug carrier as a whole, and can load biological macromolecular drugs in polar solvents and assemble them into stable macromolecular complexes, thereby achieving good drug-like properties.

[0095] 3) The further introduction of positively charged regions enables this amphiphilic drug carrier to load macromolecular drugs via electrostatic adsorption, improving loading efficiency and thus increasing drug loading capacity. This results in better versatility and expanded application scenarios. Furthermore, the presence of positively charged regions further increases the stability of the complex as it crosses the eye barrier, preventing dissociation between the carrier and the protein.

[0096] 4) This amphiphilic drug carrier loads biopharmaceuticals via non-covalent bonding methods such as hydrophilic / hydrophobic / electrostatic adsorption. This avoids the impact and damage to drug activity that occurs when using covalent bonding or covalent modification in existing technologies for delivering biopharmaceuticals, thus better protecting the activity of the active ingredient and ensuring efficacy. Furthermore, this amphiphilic drug carrier has a wide adaptability range for biopharmaceuticals of various molecular weights, achieving good assembly and transocular barrier delivery even for drugs with larger molecular weights.

[0097] 5) This amphiphilic drug carrier is covalently linked by peptide bonds, giving it the advantage of biodegradability and rapid metabolism in vivo, achieving good biocompatibility and avoiding cumulative toxicity. The preparation method of this amphiphilic drug carrier-biomolecule complex is simple, requiring fewer formulation components, which helps improve production efficiency and safety. It also offers good ease of use and has broad market prospects.

[0098] 6) This amphiphilic drug carrier can safely and effectively deliver biological macromolecular drugs into the eye via eye drops. This non-invasive drug delivery method reduces patient discomfort, improves patient compliance, and has good prospects for clinical translation.

[0099] In summary, the amphiphilic drug carriers described in this invention can all self-assemble with biopharmaceutical drugs at room temperature, and the resulting macromolecular complexes have uniform and stable particle sizes, showing no significant change in particle size after standing overnight (at least 12 hours) at room temperature. Furthermore, under different assembly ratios and pH levels, the amphiphilic drug carriers can form macromolecular complexes with drugs of different molecular weights, exhibiting stable assembly performance, minimal particle size variation, and independence from drug molecular weight. This demonstrates that the amphiphilic drug carriers described in this invention have universal applicability for loading biopharmaceutical drugs, and the preparation conditions for the macromolecular complexes are simple, requiring only simple mixing at room temperature, thus showing promise for mass production and commercialization.

[0100] A certain amount of thickener can effectively prolong the penetration efficiency of the complex in the eye, increase the probability of the complex crossing the biological barrier, and also help the stability of the formulation. In addition, the researchers unexpectedly discovered that preparing traditional eye drop formulations into lyophilized powder form, or reconstituted after lyophilization, results in a complex with better dispersibility and stability, and significantly increases the concentration of the active ingredient in the final formulation. This breaks through the concentration limitations of directly formulating and producing complex solution formulations, increases the range of specifications available for the final product, and facilitates practical applications. Attached Figure Description

[0101] Figure 1 shows the relative catalase activity test results of the macromolecular complex in Example 2.6. Figure 1a shows the relative catalase activity test results of the macromolecular complex after being stored at 37°C for 7 days, and Figure 1b shows the relative catalase activity test results of the macromolecular complex after being stored at 4°C for 7 days.

[0102] Figure 2 shows the fluorescence staining results of the drug distribution in the eyeball after eye drop administration in Example 3.1. Figure 2a shows the immunofluorescence staining images of longitudinal sections of the mouse eyeballs in each group of Examples 3.1.1-3.1.3, and Figure 2b shows the quantitative analysis results of the fluorescence intensity in Figure 2a.

[0103] Figure 3 shows the ocular angiography images of mice in Examples 4.1.1 to 4.1.4;

[0104] Figure 4 is a statistical chart of the relative lesion area of ​​the mouse choroid in Examples 4.1.1 to 4.1.4;

[0105] Figure 5 shows color fundus photographs of mice in each group of Examples 4.2.1-4.2.5 after 7 and 14 days of continuous eye drop treatment;

[0106] Figure 6 shows fundus fluorescein angiography (FFA) images of mice in each group of Examples 4.2.1-4.2.5 after 7 and 14 days of continuous eye drop treatment;

[0107] Figure 7 shows the optical coherence tomography (OCT) images of the retina of mice in each group of Examples 4.2.1-4.2.5 after 7 and 14 days of continuous eye drop treatment.

[0108] Figure 8 shows the maximum amplitudes of A and B waves in mice from Examples 4.2.1 to 4.2.5 after 7 and 14 days of continuous eye drop treatment, as assessed by electroretinography (ERG). Figure 8a shows the maximum amplitude of B waves in mice from each group after 7 days of eye drop treatment, Figure 8b shows the maximum amplitude of B waves in mice from each group after 14 days of eye drop treatment, Figure 8c shows the maximum amplitude of A waves in mice from each group after 7 days of eye drop treatment, and Figure 8d shows the maximum amplitude of A waves in mice from each group after 14 days of eye drop treatment.

[0109] Figure 9 shows the TUNEL staining results of longitudinal sections of mouse eyeballs in each group of Examples 4.2.1-4.2.5 after 14 days of continuous eye drop treatment;

[0110] Figure 10 shows the ROS staining results of longitudinal sections of mouse eyeballs in each group of Examples 4.2.1-4.2.5 after 14 days of continuous eye drop treatment;

[0111] Figure 11 is a statistical chart of IgG content in retinal and choroidal tissue samples from New Zealand rabbits in each group in Example 5.2;

[0112] Figure 12 is a statistical chart of aflibercept content in the tissue supernatant of retinal and choroidal samples from different groups of New Zealand rabbits in Example 5.3;

[0113] Figure 13 is a statistical chart of aflibercept content in retinal and choroidal tissue samples from different groups of New Zealand rabbits in Example 5.4;

[0114] Figure 14 is a statistical chart of protein content in different tissue samples in Example 5.5;

[0115] Figure 15 is a statistical chart of aflibercept content in the retina and choroid of two groups of New Zealand rabbits in Example 6.

[0116] Figure 16 is a statistical graph showing the absorbance of samples with different concentrations of the complex bound to VEGF156 before and after lyophilization and reconstitution in Example 7.1.4.

[0117] Figure 17 is a statistical graph of aflibercept concentration in the retina and choroid of New Zealand rabbits in Example 7.2. Detailed Implementation

[0118] To further illustrate the technical means and effects of the present invention in order to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and preferred embodiments, describes in detail the specific implementation methods, steps, structures, features and effects of the amphiphilic drug carrier proposed according to the present invention.

[0119] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of preferred embodiments with reference to the accompanying drawings. Through the description of the specific embodiments, a more in-depth and specific understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the present invention.

[0120] Description of the detection instruments, detection methods, detection parameters, and reagent kits used in this example:

[0121] Particle size measurement instrument: Nano particle size analyzer and ZETA potentiometer, model Nano ZS90.

[0122] Average particle size: Samples were tested using a nanoparticle size analyzer and a ZETA potentiometer, model Nano ZS90, and the Z-average value was recorded based on the instrument readings.

[0123] PDI: PDI is a dimensionless value reflecting the width of particle size distribution, ranging from 0 to 1. The smaller the value, the more uniform the particle size, the more concentrated the particle size distribution, and the higher the particle uniformity. The macromolecular complex formed by the amphiphilic drug carrier and the biopharmaceutical described in this invention is mainly used for ophthalmic drop delivery. The PDI value is a reference data used to characterize the uniformity of the particle size distribution of the complex. A PDI less than 0.5 allows for further research, while a PDI less than 0.3 is the preferred option. When the PDI is greater than 0.5, it can be used as long as there is no obvious aggregation, visible particles, precipitation, or suspended matter. Adjusting the feed ratio, pH, and other conditions can significantly improve the uniformity of the particle size distribution of the complex.

[0124] Unless otherwise specified, the letters in the amphiphilic drug carrier structure described in this disclosure have the following meanings:

[0125] R: Arginine;

[0126] L: Leucine;

[0127] K: Lysine;

[0128] E: Glutamic acid;

[0129] D: Aspartic acid;

[0130] V: Valine;

[0131] F: Phenylalanine.

[0132] Example 1: Synthesis of Amphiphilic Drug Carrier

[0133] All amphiphilic drug carriers involved in this invention were synthesized stepwise using conventional solid-phase peptide synthesis (SPPS) to obtain the required amino acid sequences and the amphiphilic drug carrier. The specific synthesized sequences are as follows:

[0134] When the amphiphilic drug carrier has a linear structure, to balance the overall charge, the amino acid at the end of the amphiphilic region is arginine or lysine, and the number of amino acids in the amphiphilic region is odd. When the amphiphilic drug carrier has a branched structure, the above restrictions on the amphiphilic region are not required.

[0135] Example 2: Preparation and Assembly Performance Testing of Macromolecular Complexes

[0136] Example 2.1: Preparation of macromolecular complexes

[0137] A macromolecular complex includes an amphiphilic drug carrier and a biomolecular drug, wherein the biomolecular drug is non-covalently assembled on the amphiphilic drug carrier. The macromolecular complex is prepared as follows:

[0138] An amphiphilic drug carrier is dissolved in ultrapure water, and a biomolecular drug is dissolved in PBS (phosphate-buffered saline). Equal volumes of the two are then mixed, and the pH of the mixture is adjusted with sodium bicarbonate or hydrochloric acid. The mixture is then allowed to stand at room temperature for 2 hours to stabilize, yielding a macromolecular complex formed by loading the biomolecular drug onto the amphiphilic drug carrier. The concentrations of the amphiphilic drug carrier and the biomolecular drug in the mixed solution can be adjusted as needed to obtain macromolecular complexes with different mass ratios. Alternatively, the amphiphilic drug carrier can be dissolved in other solvents, such as ultrapure water, physiological saline, or phosphate-buffered saline. The resulting liquid has an osmotic pressure range of 260 to 320 mOsmol / kg and is non-irritating to the eyes and surrounding area.

[0139] Experiments have shown that adjusting the pH of the mixed solution to between the isoelectric point of the peptide or protein drug and the isoelectric point of the basic peptide chain is more conducive to complex formation. This is because for amino acids and proteins, when the pH of the environmental solution is higher than their isoelectric point, they carry a negative charge; when the pH is lower, they carry a positive charge. Since the isoelectric point of the basic peptide chain is relatively high (9-11), adjusting the pH of the mixed solution to between the isoelectric point of the peptide or protein drug and the basic peptide chain allows the amphiphilic drug carrier, with one end carrying the basic peptide chain, to carry a positive charge, while the protein drug carries a negative charge. This enables the amphiphilic drug carrier to assemble with the protein drug through electrostatic interactions, thereby improving protein loading efficiency.

[0140] Example 2.2: Effect of assembly pH on particle size of macromolecular complexes

[0141] A mixed solution of amphiphilic drug carrier and H-IgG (human immunoglobulin G, 150kD) was prepared at an assembly mass ratio of 2:1 (amphiphilic drug carrier: biological macromolecular drug). After thorough mixing, the pH of the mixed solution was adjusted to 5-7 with sodium bicarbonate or hydrochloric acid. The particle size distribution of the macromolecular complex was detected using a nanoparticle size analyzer and a ZETA potentiometer. The amphiphilic drug carrier sequence, pH value of the mixed solution, and corresponding particle size test results involved in this embodiment are shown in Table 1.

[0142] Table 1: Average particle size test results of macromolecular complexes prepared at different pH values

[0143] Particle size analysis results showed that the macromolecular complexes prepared within the above pH range had uniform particle sizes, around 90 nm, and did not exhibit significant fluctuations with pH changes. This indicates that the amphiphilic drug carrier exhibits high solubility within a pH range of 5-7 and can assemble with biopharmaceuticals to form uniform and stable macromolecular complexes. Furthermore, this pH range meets the requirements of commercial eye drop formulations (5-8), minimizing irritation during ocular application and effectively preventing tear secretion caused by eye irritation, which could accelerate drug loss.

[0144] Example 2.3: Effect of assembly ratio on particle size of macromolecular complex

[0145] Mixed solutions of amphiphilic drug carriers and aflibercept (115kD) were prepared according to different assembly mass ratios (amphiphilic drug carrier: biomacromolecule drug). After thorough mixing, the pH of the mixed solution was adjusted to 6±0.3 with sodium bicarbonate or hydrochloric acid. The particle size distribution of the macromolecular complex was detected using a nanoparticle size analyzer and a ZETA potentiometer. The amphiphilic drug carrier sequences, assembly mass ratios, and corresponding particle size test results involved in this embodiment are shown in Table 2.

[0146] Table 2: Average particle size test results of macromolecular complexes prepared with different assembly mass ratios

[0147] Particle size analysis results showed that as the assembly ratio of amphiphilic drug carriers increased, the particle size of the macromolecular complex gradually increased, the polymer dispersibility index (PDI) gradually decreased, and the particle size of the product macromolecular complex became more uniform. This indicates that increasing the relative content of amphiphilic drug carriers can improve the assembly effect of biological macromolecular drugs, thereby protecting the activity of biological macromolecular drugs and improving delivery efficiency and efficacy.

[0148] Example 2.4: Particle size test of macromolecular complexes formed by assembling amphiphilic drug carriers with different biomolecular drugs

[0149] A mixed solution of amphiphilic drug carrier and different biomolecular drugs was prepared at an assembly mass ratio of 8:1 (amphiphilic drug carrier: biomolecular drug). After thorough mixing, the pH of the mixed solution was adjusted to be greater than the isoelectric point of the biomolecular drugs using sodium bicarbonate or hydrochloric acid. The particle size distribution of the macromolecular complexes prepared by loading different biomolecular drugs onto the amphiphilic drug carrier was detected using a nanoparticle size analyzer and a ZETA potentiometer. The amphiphilic drug carrier sequence, biomolecular drug type and molecular weight, and particle size test results involved in this embodiment are shown in Table 3.

[0150] Table 3: Average particle size test results of macromolecular complexes formed by different drug assemblies

[0151] Particle size analysis results showed that amphiphilic drug carriers were able to successfully load and assemble into macromolecular complexes for biopharmaceuticals with molecular weights ranging from 3 to 300 kDa. Furthermore, the assembly efficiency of the amphiphilic drug carriers for biopharmaceuticals was not affected by the molecular weight of the biopharmaceuticals, and the particle size of the macromolecular complexes was uniform and stable. These results indicate that amphiphilic drug carriers have excellent loading performance for biopharmaceuticals and possess the potential for further in vivo drug delivery.

[0152] In summary, the amphiphilic drug carriers described in Examples 2.1-2.4 can all self-assemble with biopharmaceuticals at room temperature, and the resulting macromolecular complexes exhibit uniform and stable particle sizes, showing no significant change in particle size after standing overnight (12 hours) at room temperature. Furthermore, under different assembly ratios and pH levels, the amphiphilic drug carriers can form macromolecular complexes with biopharmaceuticals of varying molecular weights, demonstrating stable assembly performance and minimal particle size variation unaffected by the molecular weight of the biopharmaceuticals. This indicates that the amphiphilic drug carriers described in this invention have universal applicability for loading biopharmaceuticals, and the preparation conditions for the macromolecular complexes are simple, requiring only simple mixing at room temperature, thus showing promise for mass production and commercialization.

[0153] The protein samples used in this embodiment are only representative of biological macromolecules with different molecular weights. In practice, they can be replaced with other biological macromolecules with similar molecular weights. The assembly pH can be adjusted according to the actual situation without affecting the formation of the final macromolecular complex. Amphiphilic drug carriers have the potential to load biological macromolecules with a molecular weight not exceeding 300 kDa. Generally, the smaller the molecular weight, the easier it is to load; the higher the molecular weight, the more difficult it is to load. However, amphiphilic drug carriers have good adaptability, and by adjusting the ratio, pH, etc., a wide range of biological macromolecules can be loaded.

[0154] Example 2.5: Particle size test of macromolecular complexes assembled from amphiphilic drug carriers with different sequences

[0155] Unless otherwise specified, the macromolecular complexes in Example 2.5 were all prepared using the following method:

[0156] Mixed solutions of amphiphilic drug carriers with the biomacromolecule aflibercept (150kD) with different sequences were prepared according to an assembly ratio of amphiphilic drug carrier: aflibercept = 8:1. After uniform mixing, the pH of the mixed solution was adjusted to 6±0.3 with sodium bicarbonate or hydrochloric acid. The particle size distribution of the macromolecular complex was detected by nanoparticle size analyzer and ZETA potentiometer to verify its assembly behavior.

[0157] The effect of positively charged regions (basic polypeptide chains) on assembly behavior:

[0158] The number of amino acids contained in the basic polypeptide chain in the amphiphilic drug carrier was adjusted, and the particle size of the macromolecular complex formed after its assembly with the biomolecular drug aflibercept was evaluated. The results are shown in Table 4.

[0159] Table 4: Particle size testing of macromolecular complexes containing basic polypeptide chains of different lengths

[0160] In Examples 2.5.1-2.5.5, different amphiphilic drug carriers were able to form stable complexes with proteins, and the particle sizes were all relatively high. Comparing the particle size detection results of Examples 2.5.1 and 2.5.2, the particle size of the macromolecular complex formed by the amphiphilic drug carrier in Example 2.5.2 was slightly larger than that in Example 2.5.1, indicating that the loading efficiency of the two biomolecules for aflibercept was similar. That is, the assembly efficiency of the amphiphilic drug carrier in Example 2.5.2 loading the biomolecule aflibercept under the hydrophobic interaction provided by the hydrophobic region (hydrophobic peptides formed by hydrophobic amino acids) was similar to that of the amphiphilic drug carrier in Example 2.5.1 loading the biomolecule aflibercept through electrostatic interaction, and both were able to assemble into stable complexes.

[0161] Further comparison of the test results of Examples 2.5.1 and 2.5.3-2.5.5 shows that the particle size test results are similar, indicating that within a certain range, appropriately lengthening (i.e. increasing the number of amino acids contained in the basic polypeptide chain) or shortening (i.e. reducing the number of amino acids contained in the basic polypeptide chain) the basic polypeptide chain has little effect on the assembly behavior of the amphiphilic drug carrier.

[0162] Without altering the length of the basic polypeptide chain, changing the types of amino acids contained in the basic polypeptide chain did not significantly affect the particle size of the macromolecular complex, suggesting that all basic amino acids are suitable for amphiphilic drug carriers. Verification showed that within a certain length range (0-8 amino acids), the assembly of the carrier and protein drug was relatively stable, and the particle size of the macromolecular complex did not change significantly with the extension or shortening of the positively charged region.

[0163] The influence of hydrophobic regions (hydrophobic peptides formed by hydrophobic amino acids) on the properties of macromolecular complexes and their barrier permeability:

[0164] Increasing the length of the hydrophobic peptide (i.e., increasing the number of amino acids contained in the hydrophobic peptide) enhances the hydrophobicity of the amphiphilic drug carrier, which may lead to decreased solubility and hinder its practical application. Conversely, increasing the length of the amphoteric peptide (i.e., increasing the number of amino acids contained in the amphoteric peptide) enhances the hydrophilicity and permeability of the amphiphilic drug carrier, but this also results in reduced assembly efficiency and stability of biopharmaceuticals. Therefore, it is necessary to regulate the lengths of both the hydrophobic and amphoteric peptides to adjust the hydrophilicity and hydrophobicity of the amphiphilic drug carrier, enabling it to successfully carry biopharmaceuticals.

[0165] Based on this, the types and numbers of amino acids contained in the amphoteric peptides are fixed (i.e., (KE)). 10 The outermost layer also contains K to balance the charge, adjust the types and amounts of amino acids in the hydrophobic peptide, and explore the effect of the above adjustments on the particle size of the macromolecular complex. The relevant test results are shown in Table 5.

[0166] Table 5: Effect of hydrophobic peptides on particle size of macromolecular complexes

[0167] The particle size test results of Examples 2.5.1 and 2.5.6-2.5.9 are similar, indicating that the type of hydrophobic amino acid, the lengthening (i.e., increasing the number of hydrophobic amino acids) or shortening (i.e., decreasing the number of hydrophobic amino acids) of the hydrophobic peptide segment has little effect on the assembly behavior of amphiphilic drug carriers assembled via electrostatic interactions (i.e., amphiphilic drug carriers with basic polypeptide chains). Comparing the particle size test results of Examples 2.5.8 and 2.5.9, the particle size of the macromolecular complex formed by the carrier sequence described in Example 2.5.9 is slightly smaller than that in Example 2.5.8. This indicates that for amphiphilic drug carriers assembled via hydrophobic interactions (i.e., amphiphilic drug carriers without basic polypeptide chains), shortening the hydrophobic segment increases the hydrophilicity of the amphiphilic drug carrier, thereby reducing the product particle size.

[0168] In Examples 2.5.12 and 2.5.13, the complexes formed by the carrier and aflibercept had slightly larger particle sizes, but were still within the usable particle size range. No obvious agglomeration or precipitation was observed, and the dispersibility was good.

[0169] Overall, the specific composition of the hydrophobic peptides has little impact on the assembly particle size of the macromolecular complex. Furthermore, hydrophobic peptides with 6-20 amino acids can all function as carriers of biological macromolecular drugs. The number and types of amino acids in the hydrophobic peptides can be reasonably adjusted according to actual needs to achieve similar assembly effects, further verifying the universality of the amphiphilic drug carrier described in this invention.

[0170] The influence of the sex region on assembly behavior:

[0171] First, the effect of the length of the amphiphilic region (i.e., the length of the amphiphilic peptide) on the assembly behavior of the amphiphilic drug carrier was investigated: the minimum repeating unit (the combination of lysine and glutamic acid, KE), the hydrophobic region, and the positively charged region of the amphiphilic peptide were kept constant. The amphiphilic peptide was lengthened or shortened (the number of amino acids contained in the amphiphilic peptide was increased or decreased) and its assembly behavior with aflibercept was tested. The amphiphilic drug carrier sequence and the corresponding test results are shown in Table 6.

[0172] Table 6: Effects of amphoteric peptides on the properties of macromolecular complexes

[0173] The particle size analysis results in Examples 2.5.14-2.5.17 show that, with the inclusion of positively charged regions, amphiphilic drug carriers containing 8-32 amino acids in the amphoteric peptides can be assembled with aflibercept. Furthermore, with the elongation of the amphoteric peptides, the hydrophilicity of the carrier structure increases, and the particle size and PDI of the macromolecular complex gradually decrease. This verifies the stability and high efficiency of assembling amphiphilic drug carriers containing 8-32 amino acids with biomolecular drugs.

[0174] The particle size test results of Examples 2.5.10 and 2.5.18-2.5.19 show that, in the absence of positively charged regions and with hydrophobic peptides in the hydrophobic regions, amphiphilic drug carriers containing 16-36 amino acids in the amphoteric peptides can be assembled with aflibercept. Furthermore, with the elongation of the amphoteric peptides, the particle size and PDI of the macromolecular complex gradually decrease, showing a trend consistent with that of Examples 2.5.14-2.5.17.

[0175] The results of Examples 2.5.14-2.5.19 show that by selecting an appropriate length range of amphiphilic peptides, the hydrophilicity and hydrophobicity of the carrier can be regulated, and then biopharmaceutical drugs can be successfully loaded through non-covalent mechanisms such as electrostatic adsorption and hydrophilic-hydrophobic interactions. This demonstrates the stability and high efficiency of amphiphilic drug carriers for loading macromolecular drugs.

[0176] By fixing the chain length of the amphiphilic peptides (i.e., ensuring the number of amino acids contained in the amphiphilic peptides remained unchanged), the smallest repeating unit of the amphiphilic peptides was changed, and the assembly effect of the corresponding amphiphilic drug carriers on biopharmaceuticals was tested. The smallest repeating units involved in this embodiment were: arginine + aspartic acid (RD), lysine + aspartic acid (KD), arginine + glutamic acid (RE), and two lysines + two glutamic acids (KKEE). The corresponding test results are shown in Table 7.

[0177] Table 7: Effect of the minimum repeating unit of amphoteric peptides on the performance of macromolecular complexes

[0178] The test results of Examples 2.5.20-2.5.23 show that any combination of basic and acidic amino acids exhibits similar macromolecular complex assembly effects, and the form of the minimum repeating unit is variable. It can be a combination of a single basic amino acid and a single acidic amino acid (AB type) or a combination of two basic amino acids and two acidic amino acids (AABB). Peptides with a total length of 8-36 amino acids are applicable, further verifying the universality of amphiphilic drug carriers, which can assemble amphiphilic peptides and biological macromolecular drugs based on a variety of minimum repeating units.

[0179] Since different combinations of repeating units have different isoelectric points, the pH values ​​at which they achieve the most significant mucus permeation effect are not the same. In practical applications, the ideal smallest repeating unit can be selected to synthesize amphiphilic drug carriers and assemble them with biological macromolecular drugs based on factors such as the molecular weight, isoelectric point, and pH of the delivery site of the biological macromolecular drug.

[0180] Based on the test results of Examples 2.5.6-2.5.23, it can be concluded that in amphiphilic drug carriers that do not contain positively charged regions, the ratio of the number of amino acids in the amphoteric peptide to the number of amino acids in the hydrophobic peptide is in the range of (1.6-3.6):1, and the amphiphilic drug carriers that do not contain positively charged regions have ideal hydrophilicity and hydrophobicity, and can effectively load biological macromolecular drugs.

[0181] For amphiphilic drug carriers containing positively charged regions, the introduction of positively charged regions is more conducive to self-assembly. It has been verified that amphiphilic drug carriers containing positively charged regions with a ratio of amino acids in the amphoteric peptide to the hydrophobic peptide in the range of (0.8-3.3):1 can stably load macromolecular drugs and achieve barrier-free biological delivery of macromolecular drugs.

[0182] Assembly effect of branched-chain amphiphilic drug carriers with proteins:

[0183] A branched amphiphilic drug carrier was constructed by grafting different numbers of amphiphilic branches onto the carboxyl-containing end of a hydrophobic peptide segment through an amide reaction provided by a basic amino acid. The carrier was then further combined with a biomolecular drug, and its assembly efficiency was investigated. The sequence of the amphiphilic drug carrier and the corresponding particle size test results are shown in Table 8.

[0184] Table 8: Particle size test results of macromolecular complexes formed by assembling branched amphiphilic drug carriers and biomolecular drugs

[0185] In Example 2.5.24, the sequence is (EKEKE)8-K4-K2-KKL 15 The specific sequence of the -R4 amphiphilic drug carrier is as follows:

[0186] The sequence described in Example 2.5.25 is (EKEKE)4-K2-KL 10 The specific sequence of the -R4 amphiphilic drug carrier is as follows:

[0187] The particle size test results in Table 8 show that the assembly effect of the branched amphiphilic drug carrier is slightly worse than that of the straight-chain amphiphilic drug carrier. The macromolecular complex has a larger particle size, but it can still load biological macromolecular drugs to form a relatively stable macromolecular complex. This further verifies the universality of the amphiphilic drug carrier described in this invention in terms of amphiphilic region structure and number of amino acids. Amphiphilic drug carriers with branched structures commonly found in traditional polymer materials can also load drugs and complete delivery, showing good prospects for transformation and application.

[0188] Verification of the necessity of gender regions:

[0189] To verify the necessity of alternating amphiphilic regions formed by amphiphilic amino acids in the assembly process with biomolecular drugs, the original amphiphilic peptide segments were replaced with straight chains of polypeptides composed of a single basic amino acid (lysine) or an acidic amino acid (glutamic acid), and their assembly efficiency with biomolecular drugs was further investigated. The corresponding test results are shown in Table 9.

[0190] Table 9: Assembly behavior test of peptide carriers formed by replacing amphipathic peptide segments with peptide chains possessing a single charge.

[0191] When using basic polypeptide chains (K) 10 When the amphoteric peptide was replaced with an acidic polypeptide chain (E) (Example 2.5.26), the resulting macromolecular complex, after being mixed and assembled with the biopharmaceutical solution, exhibited a large particle size and significant aggregation, which was unfavorable for practical use; while when an acidic polypeptide chain (E) was used... 10 When the amphiphilic peptide was replaced with a homozygous amino acid (Example 2.5.27), the water solubility of the drug carrier with the sequence shown in Example 2.5.27 was significantly reduced, and it could not be dissolved in ultrapure water, thus its assembly behavior could not be investigated. The results show that the uniform arrangement of positive and negative charges can endow the carrier structure with superhydrophilicity, while a single charge cannot achieve the above effect. Therefore, only amphiphilic drug carriers formed by alternating arrangements of amphiphilic amino acids can successfully carry protein drugs, thus verifying the necessity of the amphiphilic region in the amphiphilic drug carrier described in this example.

[0192] In summary, as verified by Example 2.5, the number of amino acids contained in the hydrophobic peptides is between 6 and 20, the number of amino acids contained in the amphoteric peptides is between 8 and 36, and the number of amino acids contained in the basic polypeptide chains is between 0 and 8. The hydrophobicity provided by the hydrophobic peptides and the hydrophilicity provided by the amphoteric peptides and the basic polypeptide chains can achieve a relative balance. They can assemble with biopharmaceutical drugs to form macromolecular complexes, and the macromolecular complexes have relatively uniform and stable particle sizes, showing good application prospects and demonstrating the potential for cross-barrier delivery of biopharmaceutical drugs.

[0193] Example 2.6: Enzyme activity assay of macromolecular complexes formed by the assembly of amphiphilic drug carriers and enzyme drugs

[0194] Amphiphilic drug carriers (specific sequence R4L) were prepared at assembly mass ratios of 4:1 and 8:1 (amphiphilic drug carrier: biomacromolecule drug). 10 (KE) 10 A mixed solution of K and CAT (catalase, 240kD) was prepared and the pH of the mixed solution was adjusted to 5-7 with sodium bicarbonate or hydrochloric acid. The particle size distribution of the macromolecular complex was detected using a nanoparticle size analyzer and a ZETA potentiometer. The test results of the concentration and particle size of each component in the mixed solution are shown in Table 10.

[0195] Table 10: Particle size test results of macromolecular complexes assembled from amphiphilic drug carriers and CAT at different assembly mass ratios.

[0196] The particle size and polymer dispersibility index (PDI) test results of the macromolecular complexes in Examples 2.6.1 and 2.6.2 are close, and the particle size of the product macromolecular complexes is ideal and the uniformity is good. The above conclusions are similar to those in Example 2.3.

[0197] The above-mentioned macromolecular complexes were stored at 37°C or 4°C, respectively, and the relative catalase activity of the macromolecular complexes was measured daily by UV absorption. The specific test method is as follows: 10 μL of the macromolecular complex solution prepared as described in Examples 2.6.1-2.6.2 or 1 mg / mL catalase solution (as a control) was mixed with 190 μL of CAT detection working solution (Solepro, BC0205) preheated in a 25°C water bath. Immediately after mixing, the absorbance of the mixed solution at 240 nm was measured using a UV spectrophotometer (A1), and the absorbance at 240 nm was measured again after 1 min (A2). Since H2O2 has a strong absorption effect on UV light at a wavelength of 240 nm, the consumption rate of H2O2 can be calculated by the change in absorbance of the mixed solution at this wavelength (A1-A2). The formula for calculating the relative enzyme activity of each group is as follows:

[0198] Relative enzyme activity = (Change in absorbance of each mixed solution at 240 nm / Change in absorbance of the control catalase solution at 240 nm) * 100%

[0199] Figure 1 shows the relative catalase activity test results of the macromolecular complexes described in Examples 2.6.1 and 2.6.2 after being stored at 37°C (Figure 1a) or 4°C (Figure 1b) for different numbers of days. The results indicate that storing the macromolecular complexes at 37°C or 4°C for 7 days has little effect on the enzyme activity of the drugs contained therein. This suggests that the macromolecular complexes prepared in this example can be stored for a long time and can continue to catalyze reactions after delivery into the body, thus improving the therapeutic effect.

[0200] Example 3: Application Study of Amphiphilic Drug Carriers in Drug Delivery Across the Ocular Barrier

[0201] Example 3.1: Immunofluorescence staining to assess the penetrating power of macromolecular complexes into the eyeball

[0202] This embodiment investigates the ability of amphiphilic drug carriers to deliver macromolecular drugs across the eye barrier. CAT was labeled with Cy5.5, and macromolecular complexes of the amphiphilic drug carrier and fluorescently labeled CAT were prepared at different assembly mass ratios according to the method described in Example 2.6. The specific sequence of the amphiphilic drug carrier is R4L. 10 (KE) 10 K.

[0203] Mice were anesthetized and administered a macromolecular complex of CAT and an amphiphilic drug carrier via eye drops. 50 μg of CAT was administered to each eye, with free CAT serving as a control. Mice were sacrificed 12 hours after administration. The eye surface was rinsed with neutral PBS, and the eyeballs were enucleated, frozen sections were prepared, and the distribution of CAT fluorescence signal on the central longitudinal section of the eyeball was observed using a confocal microscope.

[0204] The specific groupings for the experiment are as follows:

[0205] Example 3.1.1: Adding free CAT to mouse eyeball tissue;

[0206] Example 3.1.2: Mouse ocular tissue containing a macromolecular complex formed by an amphiphilic drug carrier and CAT was added by drop, with the assembly mass ratio of the amphiphilic drug carrier to CAT being 4:1;

[0207] Example 3.1.3: Mouse eye tissue containing a macromolecular complex formed by an amphiphilic drug carrier and CAT was added, with an assembly mass ratio of 8:1 between the amphiphilic drug carrier and CAT.

[0208] The results of fluorescence staining of the central longitudinal section of the mouse eyeball (Figure 2a) and the quantitative analysis of fluorescence intensity (Figure 2b) are shown in Figure 2. Red fluorescence indicates the distribution of Cy5.5-labeled CAT. The fluorescence staining in Figure 2a shows that the red fluorescence in Example 3.1.1 is weak and remains on the surface of the eyeball, not penetrating into the eye. In Example 3.1.2, the red fluorescence is uniformly distributed in the cell nucleus region indicated by blue fluorescence. The quantitative analysis in Figure 2b shows that the red fluorescence intensity in the mouse eye in Example 3.1.2 is significantly higher than that in Example 3.1.1, indicating that the macromolecular complex in Example 3.1.2 can penetrate the ocular barrier and diffuse into the eyeball. Example 3.1.3 also shows a certain increase in red fluorescence intensity and is also distributed within the ocular tissue. These results demonstrate that amphiphilic drug carriers can help biopharmaceutical drugs penetrate the ocular barrier and reach deep into the eyeball to exert their therapeutic effects.

[0209] Because the positive and negative charges from the amphiphilic regions are evenly distributed at the molecular level, they can endow the entire carrier structure with hydrophilicity and excellent anti-bioadhesion. The amphiphilic drug carrier described in this application has the ability to diffuse rapidly in the mucus layer, avoiding the removal of the drug as the mucus is renewed, and improving the bioavailability of eye drops.

[0210] Example 4: Macromolecular complexes for the treatment of fundus diseases

[0211] Example 4.1: Macromolecular complex for the treatment of wet age-related macular degeneration

[0212] Aflibercept is a vascular endothelial growth factor inhibitor used clinically to treat wet age-related macular degeneration. However, due to the presence of the ocular barrier, it is difficult to administer via eye drops; currently, it is still administered via periocular or intraocular injection, resulting in poor patient compliance. This embodiment describes the use of an amphiphilic drug carrier to deliver a large molecular complex of aflibercept via eye drops for the treatment of wet age-related macular degeneration.

[0213] Choroidal neovascularization (CNV) is a prominent feature of wet age-related macular degeneration (AMD). In this study, 8-week-old male C57 mice were used to establish a CNV model and verify the therapeutic effect of the ophthalmic macromolecular complex. Laser photocoagulation was used to induce choroidal neovascularization to construct a mouse CNV model to simulate the main symptoms of wet AMD. Seven days after laser photocoagulation induction, high fluorescence signals were observed in the fundus via fluorescein angiography (FFA), indicating successful establishment of the CNV model. This was recorded as day 0, and mice were randomly assigned to groups and treated with the drug. The relative area of ​​CNV lesions was assessed every 7 days using FFA.

[0214] The specific groupings for the experiment are as follows:

[0215] Example 4.1.1: CNV mice, without treatment;

[0216] Example 4.1.2: CNV mice were given aflibercept via eye drops at a dose of 10 μg / eye / day for 28 consecutive days;

[0217] Example 4.1.3: CNV mice were administered aflibercept via the vitreous humor at a dose of 80 μg / eye / dose, once on day 0;

[0218] Example 4.1.4: CNV mice, intraocular administration of a macromolecular complex of an amphiphilic drug carrier and aflibercept, the specific sequence of which is R4L. 10 (KE) 10 K, with an assembly mass ratio of 8:1, and a dosage of 10 μg / eye / day based on the mass of aflibercept, administered continuously for 28 days;

[0219] Figure 3 shows the ocular angiography images of mice in Examples 4.1.1 to 4.1.4. The light spot leakage area in the images was measured and statistically analyzed using ImageJ, and the specific formula is as follows:

[0220] Relative lesion area = light spot leakage area on day X / light spot leakage area on day 0.

[0221] Figure 4 is a statistical chart of the relative lesion area of ​​the mouse choroid in Examples 4.1.1–4.1.4. The results show that the mice in Example 4.1.2 had a relatively large lesion area and weak therapeutic effect, indicating that direct eye drops of macromolecular drugs cannot penetrate the ocular barrier to reach the lesion site and therefore cannot exert their therapeutic effect. However, after 14 days of continuous eye drop administration, the CNV lesion area in the mice in Example 4.1.4 was significantly reduced, and choroidal neovascularization was significantly inhibited. After 28 days of continuous eye drop administration, the CNV lesion area in the mice in Example 4.1.4 was further reduced, achieving an efficacy similar to that of a single injection.

[0222] Quantitative analysis of the thickness of CNV lesions in representative OCT images of mice from each group showed that the thickness of CNV lesions in mice in Examples 4.1.3-4.1.4 was significantly reduced. Combining the evaluation results of FFA and OCT, mice treated with the macromolecular complex via eye drops showed a significant reduction in both the area and thickness of CNV lesions, achieving efficacy similar to that of retinal injection.

[0223] Full-field electroretinography (ERG) was used to evaluate changes in photoreceptor function in mice after different treatments. Compared with other groups, the mice in the Example 4.1.3-4.1.4 groups showed significantly improved dark response, indicating that the above treatment significantly protected the visual function of CNV mice.

[0224] Further immunofluorescence staining was used to assess the expression of VEGF, which is directly related to CNV progression and deterioration, and RPE65, which is related to retinal function. Immunofluorescence staining results of mouse retina showed that, in Examples 4.1.3-4.1.4, VEGF expression levels were significantly decreased, while RPE65 expression levels were significantly increased. This indicates that the amphiphilic drug carrier successfully achieved intraocular delivery of aflibercept, effectively reducing VEGF expression in the mouse retina while avoiding damage to retinal pigment cells, thus enhancing the efficacy of ophthalmic administration in treating wet AMD.

[0225] Wet macular degeneration (AMD) has been widely reported to be associated with inflammation; therefore, we further evaluated cytokine levels in mouse ocular tissue. qPCR results showed that both ocular administration of the amphiphilic drug carrier complex with aflibercept and intravitreal injection of aflibercept significantly reduced IL-1β and TNF-α levels, indicating that the inflammatory response induced by wet AMD was significantly suppressed.

[0226] The above results collectively demonstrate that amphiphilic drug carriers can effectively load aflibercept and cross the ocular barrier to treat fundus diseases with efficacy similar to intravitreal injection. It is estimated that the dosage of eye drops is about 3.5 times that of injection. Considering the significant reduction in the industrial manufacturing cost of recombinant antibodies in recent years, as well as the convenience of non-invasive administration and patient compliance, the increase in antibody usage is very reasonable and has extremely high clinical translation potential.

[0227] Example 4.2: Macromolecular complex for the treatment of dry age-related macular degeneration

[0228] Age-related macular degeneration (AMD) is clinically classified into wet AMD (wAMD) and dry AMD (dAMD). dAMD is more common, and current clinical treatment usually relies on the use of antioxidants, multivitamins, and zinc supplements. However, there is still a need to explore and develop effective treatments for dAMD.

[0229] This embodiment describes the use of an eye drop to administer a macromolecular complex formed by an amphiphilic drug carrier and catalase for the treatment of dAMD.

[0230] Sodium iodate (NaIO3) has been shown to induce the production of reactive oxygen species, leading to RPE cell damage, and has therefore been widely used as a preclinical model for dAMD. Following a standard protocol, we established a dAMD model in mice by intravenous injection of NaIO3 via the tail vein. The control group received PBS. Six hours later, the modeled mice were randomly divided into four groups and treated accordingly. The specific groupings are as follows:

[0231] Example 4.2.1: Unmodeled mice, without any treatment;

[0232] Example 4.2.2: dAMD modeling mice, without any treatment;

[0233] Example 4.2.3: dAMD modeling mice were given catalase via eye drops at a dose of 50 μg / eye / day for 14 consecutive days;

[0234] Example 4.2.4: dAMD modeling mice were injected with catalase via intravitreal injection at a dose of 200 μg / eye / time, once on day 0;

[0235] Example 4.2.5: dAMD modeling mice, intraocular administration of a macromolecular complex of an amphiphilic drug carrier and catalase, the specific sequence of which is R4L. 10 (KE) 10 K, with an assembly mass ratio of 4:1, was administered at a dose of 50 μg / eye / day based on the mass of catalase, for 14 consecutive days.

[0236] After 7 and 14 days of continuous eye drop treatment, the condition of the fundus and retina was observed by fundus photography, fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) of animal retinal imaging microscopy to evaluate the treatment effect.

[0237] One of the most common early signs of dAMD is the presence of drusen, tiny yellow deposits under the retina, or pigment clumps, which can be observed through fundus photography. Figure 5 shows fundus photography images of mice in each group of Examples 4.2.1-4.2.5 after 7 and 14 days of continuous ophthalmic instillation treatment. The results show that the tiny yellow deposits under the retina of mice in Examples 4.2.4-4.2.5 were significantly reduced, indicating that after administration of the macromolecular complex via ophthalmic instillation, drusen in the macular region of the eyes of dAMD-modeling mice were significantly reduced, and symptoms were alleviated.

[0238] Further observation of choroidal leakage in mice was conducted using fundus fluorescein angiography (FFA). Figure 6 shows fundus fluorescein angiography (FFA) images of mice in each group from Examples 4.2.1 to 4.2.5 after 7 and 14 days of continuous ophthalmic instillation treatment. The results showed that, compared with the normal mice in Example 4.2.1, the untreated dAMD modeling mice in Example 4.2.2 exhibited significant choroidal vascular leakage and edema, indicating that the retina of the dAMD modeling mice was significantly damaged. In Examples 4.2.4 to 4.2.5, the blood leakage in the fundus of the mice was significantly reduced, indicating that intraocular injection of catalase and ophthalmic administration of the amphiphilic drug carrier and the macromolecular complex of catalase can both enable catalase to reach the fundus and exert its effect, alleviating the choroidal lesions caused by dry age-related macular degeneration.

[0239] Figure 7 shows the optical coherence tomography (OCT) images of the animal retina of mice in Examples 4.2.1-4.2.5 after 7 and 14 days of continuous ophthalmic instillation treatment. It can be observed that the retinal thickness of the dAMD-modeled mice was significantly reduced, indicating that dAMD causes retinal thinning in mice, thus posing a risk of decreased vision. After treatment, the thickness of the outer nuclear layer (ONL) of the retina in mice in Examples 4.2.4-4.2.5 significantly increased, while the change in ONL thickness in mice treated with ophthalmic instillation of free catalase in Example 4.2.3 was smaller, indicating that the biomolecules administered via ophthalmic instillation alone cannot reach the fundus to exert their effects.

[0240] The test results in Figures 5-7 indicate that single-injection administration of catalase and ophthalmic administration of the amphiphilic drug carrier-catalase macromolecular complex achieved similar therapeutic effects in dAMD-modeled mice, effectively reducing the number of tiny yellow deposits under the retina, alleviating choroidal vascular leakage, and restoring retinal thickness. This demonstrates both the effectiveness of catalase in treating oxidative stress-related fundus diseases and the ability of the ophthalmic administration macromolecular complex to effectively cross the ocular barrier and deliver the carried biomolecular drug (catalase) to the fundus to exert its effect.

[0241] The functional changes of mouse photoreceptors were assessed using electroretinography (ERG). Figure 8 shows the maximum amplitude of A-wave and B-wave in each group of mice measured under dim light conditions 7 or 14 days after ophthalmic instillation treatment. The results indicate that in Examples 4.2.2-4.2.3, the amplitudes of both A-wave and B-wave in mice were significantly reduced, indicating severe damage to their retinal photoreceptors. After treatment, in Examples 4.2.4-4.2.5, the amplitudes of A-wave and B-wave in mice significantly increased, indicating that the functional impairment of their retinal photoreceptors due to oxidative damage was alleviated, further demonstrating the effectiveness of the ophthalmic administration of the macromolecular complex.

[0242] Fourteen days later, the mice were sacrificed, and ocular tissue was collected. Immunofluorescence staining was performed on the central longitudinal section of the mouse eyeball to observe RPE cell apoptosis and oxidative stress. Figure 9 shows the in situ terminal labeling (TUNEL) images of mice in each group of Examples 4.2.1-4.2.5. The TUNEL staining results showed that the ocular cells of untreated mice exhibited obvious apoptosis and necrosis, with high red fluorescence intensity. In contrast, the apoptosis of cells in the ocular cells of mice treated in Examples 4.2.4-4.2.5 was significantly improved, with almost no red fluorescence observed, indicating that the above treatment significantly inhibited ocular cell apoptosis. Figure 10 shows the reactive oxygen species (ROS) staining images of mice in each group of Examples 4.2.1-4.2.5. The ROS staining results were similar to the above conclusions. The ROS level in the ocular cells of mice in Examples 4.2.2-4.2.3 was high and evenly distributed throughout the retinal region. The ROS level in the ocular cells of mice treated in Examples 4.2.4-4.2.5 was significantly downregulated. The combined results of TUNEL and ROS staining indicate that the ophthalmic administration of the macromolecular complex effectively scavenged reactive oxygen species in the fundus, improved oxidative stress levels, thereby inhibiting ocular cell apoptosis and alleviating dAMD-induced lesions.

[0243] The above results indicate that the amphiphilic drug carrier described in this application has excellent ability to deliver drugs through the mucus layer, thereby changing the delivery mode of various drugs, such as antibody drugs and enzyme drugs, from injection formulations to eye drops, alleviating the negative emotions caused by intraocular injections to patients, while maintaining similar efficacy. It has good clinical application prospects and high patient acceptance and recognition.

[0244] Example 5: Optimization of Formulation

[0245] Example 5.1: Preparation of a drug mimicry for treating fundus diseases, comprising the sequence R4L 10 (KE) 10K's amphiphilic drug carrier and human immunoglobulin (IgG) form a large molecular complex. The mass ratio of the amphiphilic drug carrier to IgG is 4:1, the pH is adjusted to approximately 5.5–6.0, and the protein concentration is approximately 8 mg / mL.

[0246] In addition, hydroxypropyl methylcellulose with mass fractions of 2%, 4%, and 10% was prepared. The different mass fractions of hydroxypropyl methylcellulose were mixed with the macromolecular complex at a volume ratio of 1:1 to prepare three different drug mimicry samples. The final composition and concentration of the three drug mimicry samples are shown in the table below:

[0247] Example 5.2: New Zealand rabbit eye drop test

[0248] New Zealand rabbits were immobilized, and 25 μL of each sample prepared in Example 5.1 was applied to the rabbit's eye, specifically near the conjunctival sac at the outer corner of the eye. The rabbit's eye was gently closed for 10 seconds. After 6 hours, the New Zealand rabbits were sacrificed, and biological samples of the retina and choroid were obtained. The biological samples were ground and decomposed using a lysis buffer containing protease inhibitors and a disperser. The supernatant of the ground biological samples was separated by high-speed centrifugation. The content of IgG in each group of biological samples was detected using a human IgG ELISA kit, and the efficiency of different samples in promoting the penetration of the biomolecular drug mimicry into the ocular barrier was calculated.

[0249] The specific test groups and sample information used are shown in the table below:

[0250] Figure 11 is a statistical chart of IgG content in each group of biological samples in Example 5.2. The results show that the IgG content in the retina and choroid of New Zealand rabbits in Example 5.2.4 is significantly higher than that in Control Example 5.1.1, indicating that adding 2% hydroxypropyl methylcellulose to the drug mimic sample has a significant effect on improving the entry of macromolecular drugs into ocular tissues. Furthermore, adding 1% or 5% hydroxypropyl methylcellulose also helps to improve the penetration of biological macromolecular drugs. Improvements in drug formulation can help the amphiphilic drug carrier and macromolecular complex described in this invention cross the ocular barrier, improving the bioavailability of biological macromolecular drugs during ocular administration.

[0251] Example 5.3:

[0252] The aflibercept-loaded complex was prepared using a method similar to that in Example 5.1. Specifically, a 40 mg / mL R4L10(KE)10K peptide solution and a 40 mg / mL aflibercept solution were prepared. The peptide solution and the aflibercept solution were mixed at a volume ratio of 4:1, and the pH was adjusted to 5.5–6.0 to obtain the complex solution. Then, 2%, 4%, 6%, and 10% hydroxypropyl methylcellulose were mixed with an equal volume of the complex solution to obtain the final formulation of the eye drop complex shown in the table below.

[0253] The sample numbers of the prepared eye drop complex formulations and the actual content of each component in the samples are shown in the table below:

[0254] Using healthy New Zealand rabbits as experimental animals, the above-mentioned samples were dripped into the conjunctival sac region of different New Zealand rabbits, with a sample volume of 25 μL added to each eye. After dripping, the rabbit's eye was closed for 10 seconds. After 6 hours, tissue samples were taken from the New Zealand rabbit eyes, and the retinal and choroidal samples were ground and lysed to obtain tissue supernatants. The content of aflibercept in the tissue supernatants was detected using an ELISA kit. Figure 12 is a statistical graph of the aflibercept content in the tissue supernatants of retinal and choroidal samples from different groups of New Zealand rabbits in Example 5.3. The results show that the addition of hydroxypropyl methylcellulose helps to significantly increase the content of the active ingredient in the retina and choroid, and has the potential to help the polypeptide carrier described in this invention deliver aflibercept in the form of eye drops for the treatment of ocular diseases.

[0255] Example 5.4:

[0256] The eye drop complex formulation samples were prepared according to the method in Example 5.3. The sample numbers and the actual contents of each component in the samples are shown in the table below:

[0257] Using the same animal experiment method as in Example 5.3, the efficiency of the samples from Examples 5.4.1 to 5.4.3 in penetrating the ocular barrier was tested. Figure 13 is a statistical chart of aflibercept content in retinal and choroidal tissue samples from different groups of New Zealand rabbits in Example 5.4. The results show that the higher the content of the active ingredient in the formulation, the higher the content of the active ingredient that can penetrate, i.e., the higher the utilization rate. Calculating the penetration amount at different active ingredient concentrations, the penetration amount of aflibercept at a concentration of 4 mg / mL is 2.7 times that of the formulation at a concentration of 2 mg / mL, indicating that the concentration of the polypeptide carrier described in this invention is related to the penetration-enhancing effect and has a high upper limit of use, demonstrating the great potential of the polypeptide delivery carrier described in this invention in promoting the penetration of active ingredients.

[0258] Example 5.5:

[0259] The samples from Example 5.4.1 were used to administer eye drops to the eyes of New Zealand rabbits, with 25 μL per eye. Tissue samples were collected from the New Zealand rabbits at 1, 3, 6, and 12 hours after the eye drops. Aqueous humor, vitreous humor, retina, and choroid were separated. The vitreous humor, retina, and choroid were lysed to obtain tissue supernatants. The protein content in the aqueous humor, vitreous humor, retina, and choroid obtained at different time points was detected using an ELISA kit.

[0260] Figure 14 is a statistical chart of protein content in different tissue samples. The results show that the active ingredients can penetrate the retina and enter the vitreous body and aqueous humor. As time goes on, the amount of active ingredients entering the aqueous humor gradually increases, while the amount of active ingredients on the retina gradually decreases. This indicates that the polypeptide carrier described in this invention can promote the penetration of proteins through the ocular barrier into the vitreous body and aqueous humor, and has a good permeation-promoting effect. It can be further applied to the treatment of ocular diseases.

[0261] Example 6:

[0262] Healthy New Zealand rabbits were randomly divided into two groups. The control group (6.1) received a single intravitreal injection of commercially available aflibercept (25 μL) to simulate a clinical procedure. The experimental group (6.2) received 25 μL of the sample from Example 5.4.1 via eye drops twice daily for 28 days. The aflibercept levels in the retina and choroid of the rabbits' eyes were measured at days 7, 14, 21, and 28 using ELISA.

[0263] Figure 15 is a statistical chart of aflibercept content in the retina and choroid of two groups of New Zealand rabbits in Example 6. The results show that the aflibercept content in the retina and choroid of rabbits in the control group 6.1 gradually decreased, while the aflibercept content in the retina and choroid of rabbits in the experimental group 6.2 gradually increased. At day 14, the aflibercept content in the retina and choroid of the two groups was almost equal. Subsequently, the aflibercept content in the retina and choroid of rabbits in the control group 6.1 continued to decrease, while the aflibercept content in the retina and choroid of rabbits in the experimental group 6.2 continued to increase. The traditional intravitreal injection method used in the control group 6.1 is an invasive therapy with poor patient compliance. The eye drops preparation described in this invention can achieve similar effects to intravitreal injection through a non-invasive method, even though it requires some time. It has significant advantages in terms of ease of operation and patient acceptance. In particular, the amphiphilic carrier described in this invention enables the ocular delivery of bioactive macromolecular ingredients, representing a major breakthrough in the treatment of eye diseases.

[0264] Example 7.1:

[0265] The formulation was prepared according to the formula described in Table 11 and lyophilized to obtain lyophilized powder. The morphology of the lyophilized powder was observed, and the powder was reconstituted. The reconstitution process was observed and recorded. For samples that were successfully reconstituted, the particle size of the complex was detected.

[0266] Table 11: Evaluation and Recording of Sample Lyophilization and Reconstitution States after Adding Different Lyophilization Protectants

[0267] The results showed that antibody affinity was tested on the samples from Example 7.1.4 before lyophilization and after reconstitution. Specifically, 0.1 μg / well of VEGF156 was coated onto ELISA plates, and different samples were added to the plates and incubated for 3 hours. The plates were then washed to remove unbound aflibercept or the complex, and the absorbance at 450 nm was measured. Higher absorbance indicated more bound antibody. The results are shown in Figure 16, which is a statistical graph of absorbance after the complex bound to VEGF156. The horizontal axis represents the concentration of aflibercept when the sample was added, and the vertical axis represents the absorbance after binding. The control example can be understood as the standard curve in ELISA. The results showed that the samples from Example 7.1.4, in the presence of the lyophilization protectant, all exhibited similar antibody affinity to the control group (aflibercept solution). This indicates that the addition of the lyophilization protectant, lyophilization, and reconstitution after lyophilization do not affect antibody activity and have potential for further application in formulation development.

[0268] Furthermore, the experiment observed that the ratio of protein to carrier and the concentration of the complex were somewhat limited during the preparation process. When the concentration reached 9–10 mg / mL, dissolution became difficult, resulting in agglomeration, precipitation, and uneven dispersion. However, the researchers unexpectedly discovered that when the lyophilized complex was reconstituted with solvents such as water or physiological solutions, the concentration of protein in the reconstituted solution could be significantly increased. The protein concentration in the reconstituted solution could reach 20 mg / mL, and the complex formed by the carrier and protein exhibited good stability, was not prone to agglomeration or aggregation, and had uniform particle size. This indicates that adding a lyophilization protectant during lyophilization can effectively help the complex form a morphologically stable lyophilized formulation with good reconstitution properties, which helps to increase the final usage concentration and dosage, thereby improving drug delivery efficiency.

[0269] Example 7.2:

[0270] The sample from Example 7.1.4 was used to administer aflibercept eye drops to New Zealand rabbits. The aflibercept content in the retina and choroid of the rabbit eyes was measured and compared with intravitreal injection of aflibercept. Higher aflibercept content in the retina and choroid is more beneficial for the treatment of retinal diseases such as wet macular degeneration. The results are shown in Figure 17, which is a statistical graph of aflibercept concentration in the retina and choroid of New Zealand rabbit eyes. Grouping and dosing frequency were as follows:

[0271] Control Example 7.2.0: On day 1, a single injection of aflibercept solution, 25 uL * 40 mg / mL, for a total of 1 mg / eye, with a total monthly dose of 1 mg / eye.

[0272] Example 7.2.1: A lyophilized and reconstituted solution of the aflibercept and carrier complex was instilled into the eye. From day 1 to day 7, 25 μL * 20 mg / mL was administered 4 times a day, with a dose of 0.5 mg per dose. From day 8 to day 28, 25 μL * 20 mg / mL was administered 2 times a day, with a dose of 0.5 mg per dose. The total monthly dose was 35 mg per eye. All doses were calculated based on the aflibercept content.

[0273] The results showed that in the New Zealand rabbits of Control Example 7.2.0, the aflibercept concentration in the retina and choroid quickly reached a high level and then gradually decreased. In Example 7.2.1, by administering eye drops, after 7 consecutive days of instillation, the same aflibercept concentration in the retina and choroid as that obtained by intravitreal injection was achieved. Even with continued eye drops and a reduction in the frequency of administration, the aflibercept concentration in the retina and choroid remained stable, consistently exceeding the effect of intravitreal injection, and is expected to achieve therapeutic efficacy superior to intravitreal injection. Furthermore, since a higher concentration of eye drops was obtained through lyophilization and reconstitution, it has better universality and flexibility in actual clinical use, facilitating adjustments to the dosage concentration and frequency according to patient needs and the development of personalized treatment plans.

[0274] In summary, the complex formed by aflibercept and an amphiphilic drug carrier enables the delivery of macromolecular drugs, such as antibodies, via eye drops for the treatment of eye diseases. The lyophilized formulation further enhances the flexibility of drug use and increases its clinical feasibility. Compared to intravitreal injection, eye drops offer better patient compliance, reduce patient discomfort, and, because they maintain a high concentration of the drug in the eye for an extended period, are expected to provide better efficacy and thus shorten the treatment cycle.

[0275] Obviously, those skilled in the art can make various modifications and variations to the invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations fall within the scope of the claims of the invention and their equivalents, the invention is also intended to include these modifications and variations.

Claims

1. An amphiphilic drug carrier for ocular drug delivery, characterized in that, The amphiphilic drug carrier includes a hydrophobic region and an amphoteric region, wherein the hydrophobic region is a hydrophobic peptide segment; the amphoteric region is an amphoteric peptide segment formed by alternating basic amino acids and acidic amino acids; the amphiphilic drug carrier also includes a positively charged region, wherein the positively charged region is a basic polypeptide chain.

2. The amphiphilic drug carrier as described in claim 1, characterized in that, The hydrophobic peptide segment comprises at least a hydrophobic amino acid, wherein the hydrophobic amino acid is selected from any one or more of phenylalanine, valine, leucine, and isoleucine; or The hydrophobic amino acids are selected from non-natural hydrophobic amino acids.

3. The amphiphilic drug carrier as described in claim 1, characterized in that, The hydrophobic peptide contains 6-20 amino acids.

4. The amphiphilic drug carrier as described in claim 1, characterized in that, The hydrophobic peptide contains 2-20 amino acids.

5. The amphiphilic drug carrier as described in claim 1, characterized in that, The basic amino acid is selected from one or both of lysine and arginine; and / or the acidic amino acid is selected from one or both of glutamic acid and aspartic acid.

6. The amphiphilic drug carrier as described in claim 1, characterized in that, The amphipathic peptide contains 8-32 amino acids, or the amphipathic peptide contains 9-33 amino acids.

7. The amphiphilic drug carrier according to claims 1 to 5, characterized in that, The number of amino acids in the amphoteric peptide is odd, and the terminal amino acid is a basic amino acid.

8. The amphiphilic drug carrier as described in claim 1, characterized in that, Each segment of basic or acidic amino acids is spaced one or two amino acids of the same type.

9. The amphiphilic drug carrier as described in claim 1 or 8, characterized in that, The amphoteric peptide contains 2 to 16 minimal repeating units, wherein each minimal repeating unit is a combination of any basic amino acid and any acidic amino acid in the form of A1B1 or A1A2B1B2, wherein A1 and A2 are the same basic amino acid or different basic amino acids, and B1 and B2 are the same acidic amino acid or different acidic amino acids.

10. The amphiphilic drug carrier as described in claim 9, characterized in that, The smallest repeating unit is selected from one or more of the following: lysine + glutamic acid, lysine + aspartic acid, arginine + glutamic acid, arginine + aspartic acid, two lysine + two glutamic acid, two lysine + two aspartic acid, two arginine + two glutamic acid, and two arginine + two aspartic acid.

11. The amphiphilic drug carrier as described in claim 1, characterized in that, The positively charged region is a basic polypeptide chain, which contains any one or more basic amino acids selected from lysine, arginine, and histidine.

12. The amphiphilic drug carrier as described in claim 11, characterized in that, The basic polypeptide chain contains 0-8 basic amino acids.

13. The amphiphilic drug carrier as described in claim 11, characterized in that, The basic polypeptide chain contains 1-8 basic amino acids.

14. The amphiphilic drug carrier as described in claim 1, characterized in that, The hermaphroditic regions have a linear or branched structure.

15. The amphiphilic drug carrier as described in claim 14, characterized in that, The number of terminal branches in the branched structure is 2, 4, 8, 16 or 32.

16. The amphiphilic drug carrier as described in claim 1, characterized in that, The amphiphilic drug carrier contains hydrophilic and hydrophobic amino acids, and the ratio of the hydrophilic to hydrophobic amino acids is 1.0-4.

0.

17. The amphiphilic drug carrier as described in claim 1, characterized in that, The ratio of the number of amino acids in the hydrophobic peptide to the number of amino acids in the amphipathic peptide is 5:(4-20).

18. A macromolecular complex, characterized in that, The invention includes a biological macromolecular drug and an amphiphilic drug carrier as described in any one of claims 1-18, wherein the biological macromolecular drug and the amphiphilic drug carrier are non-covalently assembled, and the macromolecular complex is used to prepare a therapeutic agent for ocular diseases.

19. The macromolecular complex according to claim 18, characterized in that, The molecular weight of the biological macromolecular drug is 3-300 kDa.

20. The macromolecular complex according to claim 18, characterized in that, The biological macromolecular drug is selected from any one of biological macromolecular therapeutic agents such as proteins, peptides, nucleic acids, and polysaccharides.

21. The macromolecular complex according to claim 18, characterized in that, The biological macromolecular drug is selected from any one of the following: anti-vascular endothelial growth factor drugs, anti-angiogenic drugs, anti-tumor necrosis factor a drugs, programmed cell death-ligand 1 antibody drugs, cytotoxic T lymphocyte-associated protein 4 antibody drugs, programmed death receptor 1 drugs, anti-lymphocyte activation gene 3 antibody drugs, T cell immunoglobulin domain and mucin domain-3 antibody drugs, and T cell immunoglobulin and ITIM domain protein antibody drugs.

22. The macromolecular complex according to claim 18, characterized in that, The biological macromolecular drug is selected from one or more of the following: adalimumab, infliximab, etanercept, golimumab, ranibizumab, aflibercept, conbercept, bromizumab, bevacizumab, falimab, rituximab, trastuzumab, cetuximab, metuximab, nimotuzumab, faliximab, ipilimumab, nivolumab, pembrolizumab, peceliximab, omaliximab, aliximumab, evolox, and emecizumab.

23. The macromolecular complex according to claim 18, characterized in that, The biomolecular drug is selected from one or more of catalase, superoxide dismutase, glutathione peroxidase, and peroxide reductase.

24. The macromolecular complex according to claim 20, characterized in that, The nucleic acid is selected from siRNA, mRNA, shRNA, lncRNA, pDNA, polyIC, CpG, or cyclic dinucleotides.

25. The macromolecular complex according to any one of claims 18-24, characterized in that, The mass ratio of the amphiphilic drug carrier to the biological macromolecular drug is (1-16):

1.

26. A method for preparing the macromolecular complex according to any one of claims 18-25, comprising the following steps: mixing an amphiphilic drug carrier with a biological macromolecular drug, adjusting the pH to a value greater than the isoelectric point of the biological macromolecular drug, and obtaining the macromolecular complex.

27. The preparation method according to claim 26, characterized in that, The mass ratio of the amphiphilic drug carrier to the biological macromolecular drug is (1-16):

1.

28. The use of the amphiphilic drug carrier as described in any one of claims 1-17 or the macromolecular complex as described in any one of claims 18-25 in drug delivery across biological barriers.

29. The application as described in claim 28, characterized in that, Biological barriers include the eye barrier.

30. The application as described in claim 29, characterized in that, The ocular barriers include the tear barrier, corneal / conjunctival barrier, blood-aqueous humor barrier, blood-retinal barrier, and blood-eye barrier.

31. The use of the amphiphilic drug carrier as described in any one of claims 1-17 or the macromolecular complex as described in any one of claims 18-25 in an eye drop formulation.

32. A medicament for treating fundus diseases, comprising an amphiphilic drug carrier as described in any one of claims 1-17 or a macromolecular complex as described in any one of claims 18-25.

33. The medicament as described in claim 32, characterized in that, The fundus diseases mentioned include retinal-related diseases, retinopathy caused by various factors, retinal vasculitis, proliferative ocular diseases, polypoid choroidal vascular disease, idiopathic choroidal neovascularization, retinopathy of prematurity, outer exudative retinopathy, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, cataracts, uveitis, keratitis, retinoblastoma, central retinal vein occlusion, retinal vein occlusion, primary retinitis pigmentosa, central serous chorioretinopathy, retinal periphlebitis, retinal artery occlusion, or glaucoma.

34. The medicament as described in claim 32, characterized in that, It also includes pharmaceutical excipients.

35. The medicament as described in claim 34, characterized in that, The pharmaceutical excipients include thickeners.

36. The medicament as described in claim 33, characterized in that, The drug is a lyophilized preparation containing a lyophilization protectant.

37. The medicament as claimed in claim 36, characterized in that, The freeze-drying protectant is mannitol.

38. The medicament as described in claim 36 or 37, characterized in that, The content of the freeze-drying protectant is 1%wt to 10%wt.

39. A method of use for treating fundus diseases, comprising administering to a patient an eye drop containing an amphiphilic drug carrier as described in any one of claims 1-17 or a macromolecular complex as described in any one of claims 18-25.

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