WO2024067874A1 - 一种改性壳聚糖及其在大分子活性成分递送中的应用 - Google Patents

一种改性壳聚糖及其在大分子活性成分递送中的应用 Download PDF

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WO2024067874A1
WO2024067874A1 PCT/CN2023/123053 CN2023123053W WO2024067874A1 WO 2024067874 A1 WO2024067874 A1 WO 2024067874A1 CN 2023123053 W CN2023123053 W CN 2023123053W WO 2024067874 A1 WO2024067874 A1 WO 2024067874A1
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chitosan
active ingredients
modified chitosan
modified
barrier
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PCT/CN2023/123053
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French (fr)
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韦婷
柴钰
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苏州百迈生物医药有限公司
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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08BPOLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/00272-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0048Eye, e.g. artificial tears
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00Drugs for disorders of the senses
    • A61P27/02Ophthalmic agents
    • A61P27/06Antiglaucoma agents or miotics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to the field of macromolecular active component delivery, in particular to a modified chitosan and application thereof in macromolecular active component delivery.
  • Macromolecular active ingredients mainly include peptides, proteins, antibodies, nucleic acid active ingredients, etc.
  • the market has expanded rapidly in recent years. With the continuous deepening of research and the continuous development of technology, the clinical application of macromolecular active ingredients has expanded to multiple therapeutic fields.
  • macromolecular active ingredients themselves are easily metabolized and decomposed by gastric acid and enzymes in the digestive tract, resulting in the ineffectiveness of the active ingredients.
  • macromolecular active ingredients such as proteins are often difficult to pass through various mucosal barriers and be absorbed (such as intestinal mucosa, etc.) due to their large molecular weight. At this stage, most macromolecular active ingredients approved for clinical use are for injection.
  • Non-invasive delivery methods include transdermal, eye drops, transmucosal, oral, etc.
  • the key to this type of delivery route is to protect the activity of the delivered ingredients and allow the active ingredients to pass through the human biological barrier.
  • biomacromolecules such as proteins have poor permeability due to their larger molecular weight and size, making it difficult to penetrate biological barriers, and are easily affected by the environment to aggregate or degrade.
  • How to protect the activity of biomacromolecules while promoting their penetration through biological barriers is the focus of current research.
  • biomacromolecules In view of the many unfavorable factors of biomacromolecules in the delivery through biological barriers, suitable excipients are needed to protect their structure and activity from being destroyed. At present, different excipients are developed to help biomacromolecules (most of the existing product R&D pipelines are polypeptide active ingredients, such as semaglutide with a molecular weight of about 4.1kD) to be administered through biological barriers for specific delivery methods.
  • the Technosphere technology developed by MannKind can assemble excipients to obtain insulin-loaded microspheres, which are freeze-dried and prepared as pulmonary inhalation powders;
  • Emisphere's Eligen technology uses the non-covalent binding of SNAC and polypeptides to protect insulin from gastric acid damage, and release active ingredients in the blood after entering the intestine and being absorbed by the gastrointestinal mucosa; or add protease inhibitors to oral preparations to reduce the degradation of biomacromolecule active ingredients in the gastrointestinal tract.
  • these excipients have a single function, mainly to protect activity, and still need to add a variety of traditional excipients to the preparations to help the active ingredients pass through the biological barrier.
  • the preparation of such preparations is complicated, and can only deliver one or several specific active ingredients, and cannot achieve the delivery of protein active ingredients (such as antibodies with a molecular weight of tens of KD or even higher), and is not universal.
  • biomacromolecule active ingredient preparations such as therapeutic protein active ingredients, antibodies, etc.
  • biomacromolecule active ingredient preparations such as therapeutic protein active ingredients, antibodies, etc.
  • the present application provides a modified chitosan that promotes macromolecular active ingredients to penetrate biological barriers and a preparation method thereof.
  • the modified chitosan described in the present application has good controllability of the active ingredient loading amount, high penetration efficiency, and can produce a binding effect with a variety of biological macromolecular active ingredients, which is more universal.
  • the complex formed by the modified chitosan and the active ingredient can penetrate the biological barrier in a non-invasive manner, can temporarily open the cell gap, will not damage the cell barrier and mucosal system, has higher safety, and the preparation method is simple, which is convenient for transformation and application.
  • the application of physical barriers provides a new carrier.
  • the first object of the present invention is to provide a modified chitosan, which is a product generated by the reaction of chitosan or a derivative of chitosan with a modified molecule.
  • the modified chitosan has a general formula I: CS- X1 - R1 , wherein CS is chitosan or a derivative of chitosan, and X1 is selected from any one of an amide bond, a sulfonamide bond, a Schiff base, a secondary amine, an alkylamine, a carbamate, an ester bond, an ether, a phosphate ester, a sulfonate ester, a urea, a phosphoramide, and an amidine bond, and R1 comes from the modified molecule.
  • the molecular weight of the chitosan or chitosan derivative is in the range of 1000-5000000, and the degree of deacetylation is not less than 85%.
  • the chitosan or chitosan derivative is selected from one or more of chitosan, chitosan oligosaccharides, chitin, hydroxyethyl chitosan, hydroxypropyl chitosan, carboxymethyl chitosan, ethylene glycol chitosan, N-trimethyl chitosan, trimethylammonium glycol chitosan iodide, chitosan quaternary ammonium salt, chitosan hydrochloride, chitosan lactate, chitosan nitrate, chitosan glutamate, chitosan azelaic acid, etc.
  • the modifying molecule is selected from aliphatic compounds.
  • the aliphatic compound is selected from fatty acids, fatty aldehydes, fatty acyl halides, fatty acid anhydrides, and aliphatic polyesters.
  • the aliphatic compound contains an aliphatic chain of n carbon atoms, wherein 3 ⁇ n ⁇ 21.
  • X1 is an amide bond
  • R1 is any one of a fatty chain containing 6 carbon atoms, a fatty chain containing 7 carbon atoms, a fatty chain containing 11 carbon atoms, and a fatty chain containing 15 carbon atoms.
  • the degree of substitution of the modified molecules on the modified chitosan does not exceed 40%.
  • the second object of the present invention is to provide a modified chitosan, wherein the modified chitosan has the general formula II:
  • the CS is chitosan or a derivative of chitosan
  • the X1 is selected from any one of an amide bond, a sulfonamide bond, a Schiff base, a secondary amine, an alkylamine, a carbamate, an ester bond, an ether, a phosphate ester, a sulfonate ester, a urea, a phosphoramide, and an amidine bond.
  • R 2 is an aliphatic chain containing n carbon atoms, wherein 2 ⁇ n ⁇ 20;
  • the R 3 contains one or more of a phenolic hydroxyl group or a derivative thereof, a catechol group or a derivative thereof, and a pyrogallol group or a derivative thereof;
  • the R 3 is connected to R 2 via a connecting group.
  • the linking group is selected from any one of an amide bond, a sulfonamide bond, a Schiff base, a secondary amine, an alkylamine, a carbamate, an ester bond, an ether, a phosphate ester, a sulfonate ester, a urea, a phosphoramide, and an amidine bond.
  • the molecular weight of the chitosan is in the range of 1000-5000000, and the degree of deacetylation is not less than 55%.
  • the molar ratio of the phenolic hydroxyl group to chitosan is no more than 20%.
  • the molar ratio of the phenolic hydroxyl group to chitosan is 8% to 18%.
  • the degree of substitution of the modified molecules on the modified chitosan does not exceed 20%.
  • the CS-X1-R2-R3 can be obtained by reacting an intermediate with chitosan or a derivative thereof, and the intermediate is selected from the following structures:
  • the molar ratio of the intermediate to chitosan is not greater than 20%.
  • the feed ratio of the intermediate to chitosan is 1% to 18%.
  • the feed ratio of the intermediate to chitosan is not less than 10% and not more than 20%.
  • the molecular weight of the chitosan or chitosan derivative is in the range of 1000-5000000, and the deacetylation degree is not less than 55%.
  • the molecular weight of the chitosan or chitosan derivative is in the range of 1000-5000000, and the degree of deacetylation is not less than 85%.
  • the third object of the present invention is to provide a method for preparing modified chitosan CS-X 1 -R 1 , characterized in that the modified molecule reacts with chitosan or a chitosan derivative.
  • the modified molecule contains an active group capable of reacting with the primary amino group of chitosan.
  • the modified molecules contain the following active groups: carboxylic acid group, sulfonic acid group, acid anhydride, carbonate, acyl halide group, sulfonyl chloride, acyl chloride, NHS ester, imidate, pentafluorophenyl ester, aldehyde group, isocyanate, isocyanate, epoxy group, double bond, alkynyl group, hydroxymethylphosphine, carboxylate.
  • the molar ratio of the modified molecule to chitosan is not greater than 40%.
  • the feed ratio of the modified molecule to chitosan is 1% to 40%.
  • the feed ratio of the modified molecule to chitosan is not less than 10% and not more than 40%.
  • the fourth object of the present invention is to provide a biological barrier-penetrating composite comprising modified chitosan and an active ingredient.
  • modified chitosan and the active ingredient are combined physically or chemically to form the biobarrier-penetrating complex.
  • the active ingredient is selected from small molecule active ingredients and/or macromolecular active ingredients.
  • the mass ratio of the modified chitosan to the active ingredient is 1:0.1-10.
  • the mass ratio of the modified chitosan to the active ingredient is 1:0.2-5.
  • the biological barrier includes any one of the skin barrier, oral mucosa, vaginal mucosa, bladder mucosa, eye barrier, tear barrier, cornea/conjunctiva barrier, blood-aqueous humor barrier, and blood-retina barrier.
  • the molecular weight of the active ingredient is 5 to 260 kD.
  • the molecular weight of the active ingredient is approximately 5kD, 10kD, 20kD, 40kD, 60kD, 140kD, 180kD, 200kD, 240kD, or 260kD.
  • the active ingredient is selected from anti-tumor active ingredients, antibiotics, immunomodulators, analgesics, eye disease treatment active ingredients, cardiovascular system drugs, anti-infective drugs, endocrine system metabolism drugs, digestive system drugs, immune system drugs, nervous system drugs, mental disorder drugs, blood system drugs, skin disease active ingredients, and other active ingredients except sex hormones and insulin.
  • the macromolecular active ingredient is selected from one or more of therapeutic vaccines, preventive vaccines, mRNA vaccines, monospecific antibodies, bispecific antibodies, double antibody conjugates, antibody fusion proteins, multispecific antibodies, other antibodies, albumins, immunoglobulins, coagulation factors, enzymes, cytokines, non-antibody fusion proteins, radionuclide-coupled active ingredients, siRNA, mRNA, ASO, other nucleic acids, polypeptides, oncolytic viruses, protein degradation targeted chimeras, diagnostic reagents, companion diagnostic reagents, and exosomes.
  • the macromolecular active ingredients are selected from one or more of protein active ingredients, active ingredients for treating diabetes, polypeptide-like active ingredients for treating obesity, other polypeptide active ingredients, hormone active ingredients, antibody active ingredients, growth factors, and nucleic acid substances.
  • the active ingredient for treating eye diseases is selected from ranibizumab, bevacizumab, VEGF antibody, MP0112, ARC1905, FCFD4514S, One or more of Conbercept, Aflibercept, complement pathway inhibitors, visual cycle inhibitors, rapamycin target inhibitors, serine/threonine protein kinase inhibitors, LIM kinase inhibitors, and non-steroidal EP2 receptor agonists.
  • the growth factor is selected from one or more of platelet growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor, nerve growth factor, interleukin growth factor, erythrocyte growth factor, colony stimulating factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, and growth hormone release inhibitory factor.
  • the nucleic acid substance is selected from one or more of siRNA, mRNA, shRNA, lnc RNA, pDNA, poly IC, CpG or cyclic dinucleotide.
  • the protein active ingredient is selected from one or more of antibodies, monospecific antibodies, bispecific antibodies, vascular endothelial growth factor antibodies, catalase, superoxide dismutase, and glutathione peroxidase.
  • the macromolecular active ingredients are selected from one or more of collagen, ceramide, high molecular weight sodium hyaluronate, tremella polysaccharide or other mucopolysaccharides, polyglutamic acid (PGA), chondroitin sulfate, cellulose, natural colloid, blue copper peptide, cono peptide, acetyl hexapeptide, nonapeptide-1, palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, palmitoyl pentapeptide-4, palmitoyl tripeptide-5, and catalase.
  • collagen collagen
  • ceramide high molecular weight sodium hyaluronate
  • tremella polysaccharide or other mucopolysaccharides polyglutamic acid (PGA)
  • PGA polyglutamic acid
  • chondroitin sulfate chondroitin sulfate
  • cellulose natural colloid
  • blue copper peptide con
  • the small molecule active ingredient is selected from one or more of glycyrrhizin, astaxanthin, salicylic acid, ferulic acid, phenethylresorcinol, resveratrol; tranexamic acid, nicotinamide, vitamin C, ergothioneine, small molecule peptides, aminobutyric acid, deoxyribonucleic acid, bosine or ectoine, arginine/lysine polypeptide, ubiquinone, dipeptide diaminobutyrylbenzylamide diacetate, and ivermectin.
  • the fifth object of the present invention is to provide a modified chitosan or a bio-barrier-penetrating composite for use in the preparation of disease treatment drugs or medical cosmetic products.
  • the disease is selected from any one of tumors, eye diseases, metabolic diseases, pain, inflammation, immune diseases, nervous system diseases, mental diseases, reproductive system diseases, bone diseases, oral diseases, hormone disorders, and respiratory system diseases.
  • a sixth object of the present invention is to provide a transdermal preparation comprising a complex of modified chitosan and an active ingredient.
  • the transdermal preparation also includes a thickener, an excipient, an anti-allergic component, a moisturizing component, an antibacterial agent, a crystal One or more of inhibitors and solubilizers.
  • a seventh object of the present invention is to provide an eye drop preparation comprising a complex of modified chitosan and an active ingredient.
  • the eye drop preparation also includes one or more of a dispersant, an excipient, a thickener, a moisturizing component, an antibacterial agent, a crystallization inhibitor, and a preservative, and provides an application in the preparation of a disease treatment preparation.
  • the modified chitosan provided by the present invention has the following beneficial effects:
  • Modified chitosan has the advantages of easy raw material availability and simple preparation, and the product with appropriate degree of substitution can be obtained according to the needs and further used for transdermal delivery of macromolecular active ingredients.
  • the modified chitosan can efficiently load macromolecular active ingredients and enable macromolecular active ingredients to penetrate biological barriers, thereby realizing non-invasive administration of some traditional active ingredients, greatly increasing the convenience of clinical use. At the same time, since the modified chitosan has a high penetration efficiency, it helps to improve the utilization rate of active ingredients.
  • the modified chitosan has the characteristics of non-destructiveness in opening the biological barrier, which improves the application safety and comfort compared with the existing technology.
  • the modified chitosan described in this application can help active ingredients stay more in the subcutaneous tissue, thereby better inducing immune responses, or help active ingredients (such as collagen) to exert their functions in the subcutaneous tissue.
  • the present application provides a platform technology.
  • the modified chitosan of the present invention has good water solubility, can help active ingredients of various molecular weights to penetrate biological barriers, and provides a new carrier for transdermal delivery and eye drop administration, which has great application potential.
  • FIG1 is a graph showing the CD values of the composites in Embodiment B1;
  • FIG2 is the calculated permeability and intradermal retention rate of BSA-FITC after 12 h in Example B2;
  • FIG3 is a statistical diagram of the relative content of IgG protein retention in mouse skin for about 12 hours in different groups of in vitro transdermal experiments in Example B3;
  • Fig. 4 is a statistical diagram of the relative wound areas of mice after treatment in Example B4;
  • Fig. 5 is an electron microscopic image of H&E stained sections of the back skin of mice in Example B5;
  • FIG. 6 is an electron microscopic image of a Masson-stained section of the mouse back skin in Example B5.
  • Fig. 7 is a photo of the affected area of mouse skin in Example B6;
  • FIG8 is a microscopic image of H&E sections of mouse skin tissue obtained on day 5 in Example B6;
  • FIG9 is the ImageJ quantitative analysis result of the mouse skin tissue H&E section data (i.e., FIG3 ) in Example B6;
  • FIG10 is a statistical graph of OVA antibody titers in mouse serum after administration on different days in Example B7;
  • FIG11 is a statistical diagram of the relative contents of IFN- ⁇ + CD4 + T cells and CD8 + T cells in the spleen of mice in Example B7;
  • FIG12 is an immunofluorescence staining signal diagram of mouse skin tissue sections in Example B8;
  • FIG13 is the distribution of fluorescence signals of the protein penetrating into the retina of the mouse eyeball after different eye drop treatments in Example C1;
  • FIG14 is an image of choroidal neovascularization in mice in Example C2, showing the location of blood vessels through the fluorescent signal of sodium fluorescein entering the blood, and showing the growth of new blood vessels in the mouse choroidal neovascularization model after treatment with different means;
  • FIG15 is an angiographic image of the mouse eye in Example D1;
  • FIG. 16 is a statistical diagram of the relative lesion area of the mouse choroid in Example D1.
  • Example A1 A modified chitosan having a structure of CS-X1-R1, wherein CS is chitosan, X1 is an amide bond obtained by the reaction of the carboxyl group of heptanoic acid and the free amino group on chitosan, and R1 is a fatty chain containing 6 carbon atoms.
  • the chitosan with this modified structure is named CCS-7.
  • Chitosan with a molecular weight of 50KDa and a deacetylation degree of >90% was used as the raw material, and heptanoic acid was selected as the fatty acid modification molecule to synthesize a series of heptanoic acid-modified chitosans (CCS-7) through chemical coupling reactions.
  • CCS-7 heptanoic acid-modified chitosans
  • the specific synthesis method is as follows: accurately weigh 1g chitosan, dissolve it in 100mL 0.1M hydrochloric acid solution, adjust the pH to between 5-7 with 1M sodium hydroxide aqueous solution, add 0.005-0.0125mol N-hydroxysuccinimide (NHS) to dissolve it, and react at room temperature with magnetic stirring for 1 hour; separately weigh 0.001-0.0025mol of heptanoic acid and dissolve it in 20mL of anhydrous dimethyl sulfoxide, add 0.005-0.0125mol of carbodiimide (EDC) (the molar ratio of carboxyl group to EDC and NHS is 1:5:5), and react at room temperature with magnetic stirring for 1 hour.
  • NHS N-hydroxysuccinimide
  • the chitosan reaction solution and the heptanoic acid reaction solution were mixed and continued to react for 48 hours at room temperature under magnetic stirring.
  • a large amount of anhydrous ethanol was added to the final reaction solution, and low-temperature precipitation was performed to obtain a flocculent product, which was filtered and vacuum dried to obtain a series of heptanoic acid-modified chitosan derivatives (CCS-7).
  • CCS-7 amino substitution degree of CCS-7 (i.e. the grafting rate of hydrocarbon chains) increased from the lowest 9.1% to the highest 36.4%.
  • the proportion of hydrophobic segments of the amphiphilic polymer carrier has a causal relationship with its assembly efficiency and assembly stability with natural macromolecular active ingredients. Therefore, CCS-7 has a wide adjustment space for the grafting efficiency of hydrocarbon chains, which can provide more options for subsequent applications.
  • the feed ratio refers to the molar percentage of the carboxyl group of the fatty acid added during preparation to the amino group of chitosan
  • the modified chitosan prepared with the same feed ratio has a similar degree of substitution.
  • the degree of substitution is about 36-38%.
  • the preparation method is repeatable, and on the other hand, it has the characteristics of high reaction efficiency and high yield.
  • Example A2 A complex (CCS-7/BSA) comprising modified chitosan and protein, wherein the modified chitosan is CCS-7 obtained in Example A1, and the protein is bovine serum albumin (BSA for short), to illustrate that modified chitosan and protein can form a complex and have a significant penetration-enhancing effect.
  • CCS-7/BSA modified chitosan and protein
  • Example A1.1 to Example A1.4 The water-soluble product of CCS-7 obtained in Example A1.1 to Example A1.4 was selected as a carrier, and a complex of modified chitosan and protein was prepared with bovine serum albumin (BSA), and then the penetration enhancement effect was verified through in vitro mucosal penetration enhancement experiments and in vitro transdermal penetration enhancement experiments.
  • BSA bovine serum albumin
  • PB phosphate buffer
  • BSA and CCS-7 are combined with each other through electrostatic and hydrophobic interactions to obtain a CCS-7/BSA complex.
  • Example A3 A modified chitosan having a structure of CS-X1-R1, wherein CS is chitosan and X1 is an amide bond, is obtained by reacting modified molecules of different carbon chain lengths with free amino groups on chitosan.
  • the modified chitosan obtained by covalently modifying the chitosan amino group with a fatty chain containing 12 carbon atoms is named CCS-1.
  • R1 is a fatty chain containing 11 carbon atoms, and this structurally modified chitosan is named CCS-12; the modified chitosan obtained by covalently modifying the chitosan amino group with a fatty chain containing 16 carbon atoms, R1 is a fatty chain containing 15 carbon atoms, and this structurally modified chitosan is named CCS-16.
  • Table 2 Statistical table of product properties and mucosal penetration-enhancing effects of chitosan modified with fatty chains of different lengths.
  • the fatty chains with different carbon number can modify chitosan to obtain modified chitosan
  • the modified chitosan with different substitution degrees can be obtained by adjusting the feed ratio, wherein the feed ratio refers to the molar ratio of the modified molecule to chitosan in the raw material.
  • the modified chitosan obtained by reacting the fatty chains of different lengths as the modified molecules with chitosan has a substitution degree of no more than 40%.
  • the product has good solubility and can be further used for the preparation of the composite.
  • Embodiment A4 A complex comprising the modified chitosan described in Embodiment A3 and bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC).
  • BSA-FITC fluorescein isothiocyanate
  • the preparation method is similar to that of Embodiment A2, except that the raw material CCS-7 is replaced with CCS-8, CCS-12 or CCS-16 obtained in Embodiment A3.
  • Several modified chitosans prepared with different degrees of substitution and different modified molecules can form stable complexes with proteins and can be further used in transdermal preparations.
  • Example 1 A modified chitosan having a structure of CS-X 1 -R 2 -R 3.
  • the chitosan having this structure is named CS-S08.
  • Chitosan with a molecular weight of 50KDa and a deacetylation degree of >90% was used as the raw material, and sodium 8-(2-hydroxybenzamido)octanoate (SNAC) was selected as the modified molecular intermediate.
  • the chitosan derivative with a CS-X 1 -R 2 -R 3 structure was synthesized through an acylation reaction between the amino group on the chitosan and the carboxyl group of the modified molecule.
  • the modification schematic and product structure are as follows:
  • the specific synthesis method is as follows: accurately weigh 1g of chitosan, dissolve it in 100mL of 0.1M hydrochloric acid solution, and adjust the pH to between 5 and 7 with 1M sodium hydroxide aqueous solution; separately weigh 0.001-0.0025mol of SNAC and dissolve it in 20mL of anhydrous dimethyl sulfoxide, add 0.005-0.0125mol of carbodiimide (EDC) and 0.005-0.0125mol of N-hydroxysuccinimide (NHS) in sequence (the molar ratio of carboxyl to EDC and NHS is 1:5:5), react at room temperature and magnetic stirring for 1 hour, and maintain the pH between 5 and 7 during the reaction.
  • EDC carbodiimide
  • NHS N-hydroxysuccinimide
  • the chitosan solution is mixed with the reaction solution of the activated intermediate, and the reaction is continued at room temperature and magnetic stirring for 48 hours. After the reaction is completed, a final reaction solution is obtained. The final reaction solution is filtered through anhydrous ethanol sedimentation and then dried in vacuo to obtain a modified chitosan (CS-S08) having a CS-X 1 -R 2 -R 3 structure.
  • X1 is an amide bond
  • R2 is an alkyl chain containing 7 carbon atoms
  • R3 is a phenolic hydroxyl group connected to R2 via an amide bond.
  • the input amount of SNAC is different, and the amino substitution degree of chitosan in the obtained product is different.
  • Example 2 A composite comprising the modified chitosan having a CS-X 1 -R 2 -R 3 structure prepared in Example 1 and bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC), the preparation method of which is as follows:
  • Example 3 In vitro mucosal permeation and in vitro permeation experiments of modified chitosan with CS-X 1 -R 2 -R 3 structure were performed to verify its permeation effect.
  • the experimental method is as follows:
  • a complex solution of CS-S08 or CS and bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was prepared according to the method described in Example 2 (the concentration of BSA-FITC was 0.2 mg/mL), and a free BSA-FITC solution of the same concentration (0.2 mg/mL) was prepared as a control example;
  • the fresh ex vivo mouse skin is a complete skin tissue that can be used as a representative of the skin barrier.
  • the diffusion cell system is adjusted to maintain a constant temperature of 37°C. After 12 hours, the total fluorescence in the receiving pool is detected to calculate the protein permeability.
  • Fluorescent isothiocyanate labeling has a fluorescent signal, and the distribution, concentration and other information of the protein can be identified by the intensity of the fluorescent signal.
  • Protein transmittance (total fluorescence of protein in receiving pool)/(total fluorescence of original solution)*%
  • CS-S08 As the degree of substitution of CS-S08 increased, the whole skin permeability of BSA-FITC in mice increased accordingly; when the degree of substitution of CS-S08 was 15.2%, its transdermal penetration enhancement effect could reach 10.05%, which greatly improved the permeability of the protein compared with the control example 2.
  • This embodiment shows that CS-S08 can achieve a better penetration enhancement effect by adjusting the degree of substitution, and has a certain controllable degree of substitution range for penetration enhancement carriers with different needs, further illustrating that the modified chitosan of the present invention has an ideal ability to promote the penetration of macromolecular active ingredients and the potential to improve the therapeutic effect.
  • the degree of substitution in the above embodiment is the result of one sample in each group of experiments. Each group of experiments is repeated more than 5 times, the degree of substitution is close, and the mouse full skin permeability data is close, which has good repeatability. Many experiments have found that the products obtained with the same feed ratio have similar degrees of substitution. When the feed ratio is 12%, the degree of substitution is about 8% to 10%; when the feed ratio is 15%, the degree of substitution is about 11% to 13%; when the feed ratio is 18, the degree of substitution is about 13% to 16%, and when the feed ratio is 20%, the degree of substitution does not exceed 20%.
  • the feed ratio of the raw materials for preparing modified chitosan can be no more than 20%, and its degree of substitution does not exceed 20%.
  • the preparation method obtains modified chitosan, which has the characteristics of high reaction efficiency, high yield, and stable reaction, which is helpful to achieve mass production.
  • the modified chitosan with a higher degree of substitution has a better permeation-enhancing effect.
  • the modified chitosan that can promote mucosal permeation can also promote the permeation of macromolecules through the skin, and can increase the permeability of macromolecules by more than 20 times.
  • Example B1 Composite of modified chitosan and protein active ingredient
  • a complex was prepared by mixing CS-S08 with a feed ratio of 15% and a substitution degree of about 12.5% with active proteins of different molecular weights.
  • CS-S08 can form complexes with proteins of different molecular weights, indicating that CS-S08 has good universality and can load active proteins with molecular weights ranging from 5kD to 240kD.
  • BSA is a protein with a molecular weight of about 66kD. It is speculated that after CS-S08 forms complexes with active proteins of 5kD to 240kD, they are expected to pass through the skin barrier and achieve the effect of transdermal delivery.
  • the complex formed by CS-S08 and protein has a relatively uniform average particle size. Overall, the uniformity of the complex in each embodiment is good.
  • the modified chitosan of the present invention can form a complex with uniform particle size and good dispersibility with proteins of different molecular weights.
  • the uniform particle size of the complex indicates that the product of modified chitosan loaded with active protein has good controllability and has the potential for batch production of the complex, which is conducive to further industrial application.
  • Table 4 The complexes obtained in different examples and their states.
  • Figure 1 is a CD value spectrum of each complex in Example B1, taking the group with a carrier to protein ratio of 1:1. It can be seen from the figure that compared with the protein that did not form a complex, the CD spectrum of the protein in the complex did not change, indicating that the process of CS-S08 and protein forming a complex will not cause the secondary structure of the protein to change and become inactive. It proves from the microstructural level that after the modified chitosan and protein form a complex, the protein can maintain its activity and can be further activated. One step to play its function and achieve the expected effect.
  • Example B2 An in vitro transdermal absorption experiment was conducted using a Franz diffusion cell to demonstrate that modified chitosan can promote protein penetration into the skin non-invasively.
  • nude mouse skin was used as the test object
  • BSA-FITC was used as the target protein
  • the initial protein content was consistent (1.6 mg)
  • the content of the carrier added in each group was consistent (1.6 mg)
  • the sample was added to one side of the Franz diffusion cell.
  • the protein content in the receiving cell was measured as a proportion of the initial experimental dose
  • the protein content retained in the mouse skin was measured as a proportion of the initial experimental dose, to verify the efficiency of different carriers in promoting protein penetration.
  • the experimental groups are as follows:
  • Control Example B2.2 Complex of chitosan and BSA-FITC
  • Control Example B2.4 A complex of chitosan, SNAC and BSA-FITC physically mixed, wherein the molar ratio of SNAC to chitosan was 15%, and the mass ratio of the carrier to the protein was 1:1;
  • Example B2.5 The complex of CS-S08 and BSA-FITC prepared in Example 3.3, the ratio of carrier to protein is 1:1.
  • the modified chitosan described in the present invention in Example B2.5 can promote the protein component to pass through the skin surface barrier, enter the skin and pass through the skin to the greatest extent.
  • the raw material SNAC of the modified chitosan is directly mixed with chitosan without chemical grafting modification, it cannot promote the protein to pass through the skin barrier.
  • Further analysis suggests that it is difficult for the physically mixed SNAC and chitosan in the control example B2.4 to form a stable complex with the protein, and no effective loading is formed, so that the effect of promoting penetration cannot be played.
  • modified chitosan can increase the retention of proteins in the skin, which can greatly improve the application effect of active ingredients in the field of medical cosmetology.
  • Increasing the retention time of active ingredients in the skin can better play the role of active ingredients in improving cells or metabolism in the skin.
  • it improves the utilization rate of active ingredients, and on the other hand, it can improve the efficacy of medical cosmetology products.
  • Example B3 The technical effect was also verified in the complex of CS-S08 and immunoglobulin IgG (molecular weight 150kD), and the specific experimental groups are as follows:
  • Control Example B3.2 Mixed solution of chitosan and IgG
  • Control Example B3.3 Mixed solution of SNAC and IgG;
  • Control Example B3.4 a mixed solution of chitosan, SNAC and IgG;
  • Example B3.5 The complex of CS-S08 and IgG obtained by the preparation method of Example A2, the ratio of the carrier to the protein is 2:1.
  • Figure 3 is a statistical diagram of the relative content of protein retention in mouse skin for about 12 hours in the in vitro transdermal experiment of different groups in Example B3.
  • the results show that the modified chitosan can significantly increase the retention of IgG in the skin and prolong the residence time of the protein in the skin.
  • the protein stays in the skin for a long time and can better act on the cells in the skin or subcutaneous tissue to play a therapeutic and improvement role.
  • This beneficial effect shows that the modified chitosan of the present invention is helpful for the loading and delivery of active ingredient proteins or vaccine active ingredients in the field of medical cosmetology.
  • Example B4 Application of the complex formed by CS-S08 and collagen in wound repair
  • mice (6-8 weeks old) were selected for the experiment.
  • the hair of healthy mice was shaved and then pentobarbital was used to Mice were anesthetized with sodium bital, and after routine disinfection with 75% ethanol, the full-thickness skin of the mice was surgically removed to create a full-thickness skin wound with a diameter of 5 mm.
  • an acrylic splint was fixed on the back wound of the mouse to prevent the natural contraction of the mouse skin and better simulate the characteristics of human wound healing.
  • hemostasis and disinfection were performed, and the mice were divided into groups for drug treatment. After treatment, photos were taken and recorded, and the wound healing of the mice was calculated using Image J.
  • the specific experimental groups are as follows:
  • Example B4.2 Collagen and vaseline were fully mixed to form an ointment, which was applied to the wound model. The dosage was once a day, 100 ⁇ g/each time;
  • Example B4.3 The complex of CS-S08 and collagen prepared as described in Example B1.1.2 was thoroughly mixed with vaseline to form an ointment, and applied to the wound model. The dosage was once a day, 100 ⁇ g per wound each time.
  • Figure 4 is a statistical chart of the relative wound area of mice after treatment.
  • the results show that after a single dose of treatment, the wound surface of the mice in Example D1.3 was significantly reduced and basically completely healed 10 days after administration.
  • Collagen as the main component of human tissue, can stimulate cell proliferation and migration through interaction with surrounding tissue cells, thereby accelerating the healing process of the wound surface.
  • the above results show that the modified chitosan described in this application can successfully deliver collagen into the skin, thereby shortening the wound healing time.
  • the modified chitosan described in the present application can load collagen and help collagen enter the skin.
  • the complex of modified chitosan and protein described in the present application can efficiently deliver collagen and ensure protein activity, thereby accelerating the skin damage repair process.
  • the CS-S08 carrier has the antibacterial effect of chitosan. It acts on the skin surface in the form of a dressing to keep the wound clean and inhibit wound infection, thereby further accelerating the wound repair process.
  • Example B5 Application study of the complex formed by CS-S08 and collagen for skin tightening
  • mice (6-8 weeks old) were selected for the experiment.
  • the complex was directly applied to the skin of the nude mice once a day for three times. On the 4th day, the mice were killed and the back skin was collected. The changes in the epidermal thickness and collagen content of the mice were analyzed by sectioning.
  • the specific experimental groups are as follows:
  • Example B5.1 Blank control, only vaseline was applied without any treatment
  • Example B5.2 Apply a mixed ointment of collagen and vaseline to the back skin of nude mice, once a day, and repeat three times;
  • Example B5.3 The complex prepared as described in Example B2.5 was thoroughly mixed with vaseline to prepare an ointment, which was applied to the back skin of nude mice once a day, and the above process was repeated 3 times.
  • Figure 5 is an electron microscopic result of H&E staining sections of mouse back skin.
  • the black line segment indicates the thickness of mouse epidermis.
  • the thickness of mouse epidermis in Example B5.3 is significantly increased.
  • the dotted line part is located in the dermal cell area.
  • the section shows that the density of dermal cells in the dotted area is significantly increased, and the intercellular space becomes smaller, indicating that collagen can help the mouse skin restore its plump and firm state.
  • Figure 6 is an electron microscopic result of a Masson-stained section of the mouse back skin, wherein the dotted line indicates the collagen fiber coloring area, and the result shows that the content of collagen fibers in the mouse skin of Example B5.3 is significantly increased.
  • the above results all indicate that the complex described in the present application can successfully deliver collagen (COL) to the dermis, thereby stimulating the synthesis of new collagen fibers, while increasing the thickness of the epidermis and the density of dermal cells. Since the process of skin sagging and aging is closely related to the loss of collagen, the supplementation of collagen can increase the firmness of the skin, thereby effectively delaying the process of skin aging.
  • the above results show the application prospects of the modified chitosan described in the present application as a protein carrier in medical beauty anti-aging products.
  • Example B6 The complex of CS-S08 and adalimumab is used in the treatment experiment of psoriasis.
  • Adalimumab (ADA) is a fully humanized tumor necrosis factor monoclonal antibody that has been approved for the treatment of psoriasis by intradermal injection.
  • ADA Adalimumab
  • adalimumab is used by intradermal injection, which can cause pain and even the risk of local infection, and poor patient compliance.
  • adalimumab is combined with CS-S08 described in this application to form a complex for psoriasis treatment experiments.
  • imiquimod was used to induce the Balb/c mouse psoriasis model, and 5% imiquimod was applied to the mouse skin every day for 5 consecutive days.
  • the damage to the mouse skin was recorded by taking pictures every day to evaluate the severity of psoriasis; in addition, the first use of imiquimod was counted as day 0, and mouse skin tissue samples were taken on the 3rd and 5th days, and the cytokine content was detected after lysis to evaluate the level of inflammation; and on the 5th day, the skin samples of the affected part of the mouse were taken, and the mouse skin thickness was counted by H&E sectioning and ImageJ to determine the degree of inflammation of the mouse skin.
  • Control B6.1 healthy mice, not stimulated with imiquimod, and not treated with any other drugs
  • Control Example B6.3 Vaseline was mixed with adalimumab, and the dosage per mouse was 150 ⁇ g/time, based on the weight of adalimumab, and the drug was administered once on the first day and the third day respectively;
  • Control Example B6.4 On day 1, adalimumab was subcutaneously injected into the affected area of mice at a dose of 300 ⁇ g;
  • Example B6.5 A complex of CS-S08 (with a degree of substitution of approximately 12.1%) and adalimumab (the mass ratio of CS-S08 to adalimumab is 2:1) was prepared according to the method of Example 2, and mixed with an equal amount of vaseline of control example C1.1.
  • the dosage per mouse was 150 ⁇ g/time, based on the mass of adalimumab, and the drug was administered once on the 1st day and once on the 3rd day.
  • Figure 7 is a photo of the affected part of the mouse skin in Example B6.1 to Example B6.5
  • Table 5 is a severity score table of the affected part scales and erythema of the mice on days 1-5 in Example B6.1 to Example B6.5.
  • Control Example B6.2 the affected part of the mouse skin showed severe psoriasis symptoms, indicating that the psoriasis modeling was successful
  • Control Example B6.4 is a subcutaneous adalimumab treatment regimen currently used in clinical practice, which can effectively prevent the occurrence of psoriasis
  • Control Example B6.3 of transdermal administration has almost no effect in preventing the onset of the disease, and the mouse skin symptoms are similar to those of Control Group B6.2, while Example B6.5 can effectively alleviate psoriasis symptoms.
  • the photo of the mouse on Day 1 of Control Example B6.1 in Figure 7 is damaged, so it is not given, but the photos of the following days can still illustrate the problem, which does not affect the effect judgment of the
  • Table 5 Scoring statistics of the severity of scaling and erythema in the affected area of mice on days 1-5
  • Figure 8 is a microscopic picture of H&E sections of mouse skin tissue obtained on the 5th day in Examples B6.1 to B6.5, and the black line segments in the figure indicate the surface thickness of the mouse skin.
  • Figure 9 is the ImageJ quantitative analysis result of the slice data in Figure 8. The greater the thickness of the skin surface, the more severe the inflammation of the skin tissue. According to the ImageJ quantitative analysis results, the thickness of the mouse skin in Example B6.5 is thinner, reflecting that the complex of CS-S08 and adalimumab can improve keratinocyte proliferation and relieve psoriasis symptoms through transdermal administration.
  • the modified chitosan described in the present invention can promote adalimumab to penetrate the skin barrier and protect the activity of adalimumab, indicating that the modified chitosan described in the present invention can promote adalimumab to penetrate the skin to play a role in treating psoriasis, and has the potential to be applied to transdermal administration. That is, the modified chitosan described in the present application can be used as a carrier of macromolecular active ingredients for transdermal administration.
  • Example B7 Study on the effectiveness of CS-S08 loaded with antigen protein as an anti-tumor vaccine.
  • the complex of CS-S08 and chicken ovalbumin (OVA) was prepared according to the method described in Example 2, except that PolyIC was also added during the preparation process, wherein the mass ratio of CS-S08, OVA, and PolyIC was 8:4:1.
  • the specific experimental method is: OVA and PolyIC were given to healthy C57 mice multiple times by subcutaneous injection and application, respectively. The first administration time was recorded as day 0, and the OVA antibody titer in the mice was detected on days 8, 21, and 28. Spleen tissue samples were taken on day 28 to detect the relative content of immune cells.
  • the experimental groups and experimental conditions are as follows:
  • Control B7.1 healthy mice, without any treatment
  • Control Example B7.2 On day 0, a mixed solution of OVA and PolyIC was subcutaneously injected, with an OVA dose of 60 ⁇ g/mouse and a PolyIC dose of 15 ⁇ g/mouse;
  • Example B7.3 On day 0, day 7, and day 14, the complex was mixed with vaseline and applied to the exposed skin surface of mice.
  • the dosage of OVA was 20 ⁇ g/mouse/time
  • the dosage of PolyIC was 5 ⁇ g/mouse/time.
  • Enzyme-linked immunosorbent assay was used to detect the antibody titer in the mouse serum, and the titer endpoints of the two groups of mice in Examples B7.2-B7.3 were evaluated with the mice in Control Example B7.1 as the background, and the statistical results are shown in Figure 10. The results showed that the antibody titer level in the mice in Example B7.3 was close to that in Control Example B7.2, indicating that transdermal administration achieved a vaccination effect similar to that of subcutaneous injection.
  • Figure 11 is a statistical chart of the relative content of IFN- ⁇ + CD4 + T cells and CD8 + T cells in the spleen of mice in Example B7.
  • IFN- ⁇ + indicates the expression of gamma interferon in T cells. Gamma interferon can promote the activity of natural killer cells. The higher the content of IFN- ⁇ + T cells, the better the anti-tumor immune response can be triggered; CD4 + T cells can regulate the activity of other immune cells to exert anti-tumor effects, and CD8 + T cells can directly kill tumor cells.
  • the results show that the proportions of IFN- ⁇ + CD4 + T cells and CD8 + T cells in Control Example B7.2 and Example B7.3 increased, indicating that both can secrete more IFN- ⁇ , thereby enhancing the anti-tumor immune response.
  • CS-S08 can promote the penetration of antigen proteins through the skin and induce immune memory effects, indicating that the modified chitosan of the present invention can be used as an antigen delivery carrier for the preparation of vaccine preparations, and further used in the fields of disease prevention, tumor prevention and treatment.
  • the most common vaccination methods in clinical practice are invasive methods such as subcutaneous injection and intramuscular injection. It is not friendly to vaccine recipients, especially infants and young children.
  • the local application method replaces subcutaneous injection and intramuscular injection, which is of great significance. It has the beneficial effects of alleviating patient pain, improving patient compliance, and reducing consumables costs.
  • Example B8 CS-S08 loaded with catalase for removing intradermal reactive oxygen species
  • CS-S08 is loaded with catalase to remove intradermal reactive oxygen species.
  • the complex of CS-S08 and catalase was prepared according to the method described in Example 2, and the mass ratio of CS-S08 to catalase was 5:1.
  • Imiquimod was applied to the exposed skin of mice for 5 consecutive days to establish a psoriasis model.
  • the first use of imiquimod was regarded as day 0.
  • day 4 the skin tissues of the affected areas of the mice were obtained, and immunofluorescence staining sections were prepared to observe the content of reactive oxygen species.
  • the experimental groups and experimental conditions are as follows:
  • Control B8.1 healthy mice
  • Control B8.2 Psoriasis mice without any relief treatment
  • Example B8.3 Vaseline was mixed with the complex and applied to the affected area on the 1st, 2nd and 3rd day respectively.
  • the dosage of catalase was 100 ⁇ g/mouse.
  • Figure 12 is an immunofluorescence staining signal diagram of mouse skin tissue sections in Example B8, which was obtained by laser confocal microscopy.
  • the results show that there is no active oxygen signal in the skin of healthy mice in Control Example B8.1, and there is an obvious active oxygen signal in the skin of psoriasis mice in Control Example B8.2, indicating that the active oxygen content in the skin of psoriasis is much higher than that in normal tissues, and in Example B8.3, after three interventions, the active oxygen level is significantly reduced, indicating that the complex can enter the skin through the epidermis, especially the skin, and catalase plays a role in removing active oxygen.
  • the modified chitosan of the present invention can help large molecular weight proteins enter the skin and exert a therapeutic effect.
  • the modified chitosan of the present invention has the potential to prepare relevant active ingredient preparations.
  • Example B of the specification the function of vaseline is to ensure the residence time of the sample on the skin surface.
  • 3M patch is used for fixation to avoid sample loss and experimental result deviation due to mouse activity.
  • Vaseline itself has limited effect on the transdermal penetration of active ingredients and can be further used as a thickener, adhesive and other auxiliary materials in transdermal drug preparations.
  • auxiliary materials can be added to help the complex further form a product.
  • Example C Application of modified chitosan (alkyl chain modified chitosan) and protein complex in penetrating ocular barrier
  • BSA-FITC bovine serum albumin labeled with fluorescein isothiocyanate
  • mice were anesthetized, and 5 ⁇ L of the drug was administered to each eye.
  • the mice were kept in a dark environment and sacrificed 6 hours later.
  • the eyeballs were rinsed with PBS neutral buffer, and the eyeballs were removed to prepare slice samples.
  • the central longitudinal section samples of the eyeballs were photographed using a confocal microscope, and the distribution and intensity of the fluorescent signal in the retina of the mouse eyeballs were observed. The results are shown in Figure 13 and Table 6.
  • the fluorescently labeled signal in Figure 13 represents the distribution of the active ingredient of the protein in the posterior segment of the eye. The farther the signal is from the ocular surface, the more complete the protein penetration is. The stronger the fluorescent signal is, the higher the amount of penetration is.
  • the labeling of the cell nucleus mainly shows the location of tissues such as the retina, choroid and sclera, which is convenient for distinguishing that the fluorescent signal is a signal in the tissue section rather than an invalid distribution of unpenetrated tissue.
  • Example C1.1 only a small part of the protein in Example C1.1 is on the surface of the sclera, indicating that it is difficult for free protein to penetrate the ocular surface barrier; the protein in Example C1.2 can penetrate more ocular surface barriers, such as the cornea and conjunctival barriers, and the distribution area of the fluorescent signal in the retina is larger, but the stronger fluorescent signal stays on the surface of the ocular barrier;
  • Example C1.2 In Example C1.3, Example C1.4, and Example C1.5, chitosan modified with different lengths of fat chains, after being compounded with proteins, can help proteins better penetrate the eye barrier to reach the fundus and accumulate in the retina and choroid, and the protein penetration of these three groups of samples increases with the increase of fat chains, and the fluorescence signal of chitosan modified with palmitic acid is the strongest.
  • Free protein is used as a control to calculate the relative fluorescence signal intensity of protein markers in each group of samples.
  • the results in Table 6 show that the efficiency of chitosan modified with palmitic acid (hexadecanoic acid) in promoting protein penetration is more than 26 times that of free protein.
  • Table 6 Statistics of fluorescence signal intensity of protein markers in the retina and choroid of mouse eyeball slice samples 6 hours after eye drop administration.
  • Example C2 Quantitative detection of modified chitosan to promote protein penetration through the ocular barrier
  • PBS eye drop matrix phosphate buffer
  • mice were anesthetized, and then 5 ⁇ L (IgG concentration was 2 mg/mL) was quantitatively administered to each eyeball of each mouse. After 6 hours, the mice were sacrificed, the surface of the eyeballs was rinsed with a neutral PBS solution, the eyeballs were removed, and the excess tissue on the eye surface was removed. The weight of each eyeball was weighed and recorded, and the eyeball samples were frozen and ground in a liquid nitrogen environment to obtain tissue powder, and the tissue powder was dispersed using WB and IP cell lysis buffers, and treated at 4°C overnight. The supernatant was obtained after high-speed centrifugation, and the concentration of IgG in the supernatant was detected using an ELISA detection kit. The results are shown in Table 7.
  • Example C1 intuitively show the distribution of the active ingredient in the eye after penetrating the ocular barrier.
  • Example C2 quantitatively analyze the efficiency of chitosan carrier materials in promoting protein penetration. Combined with the in vivo ocular barrier penetration experiments of Example C1 and Example C2, it is shown that the cationic polymer modified with aliphatic chains has the effect of effectively promoting protein molecules to penetrate the ocular barrier, and has excellent potential for use as a penetration enhancer in ocular drug delivery.
  • Example C3 The method of Example C2 was followed, except that rabbits were used instead of mice. The different structural layers of the rabbit eyes were separated by dissection, and the IgG content in different parts was quantitatively detected to evaluate the ability of palmitic acid-modified chitosan (CCS-16) to promote IgG penetration.
  • CCS-16 palmitic acid-modified chitosan
  • the tissues were separated in the order of aqueous humor, cornea, lens, vitreous body and retina. The weight of each tissue was weighed and each sample was cryo-ground. Then, each eye tissue was lysed with WB and IP cell lysis buffer. The supernatant was collected after high-speed centrifugation and the IgG concentration in the sample was detected using an ELISA kit. Table 8 is a statistical table of IgG content in different tissue structures of rabbit eyes.
  • Example C3.2 had a higher distribution in each structural layer, indicating that CCS-16 can effectively promote the penetration of proteins.
  • the groups with CCS-16 as a penetration enhancer all had higher protein content.
  • more active ingredients were enriched in the retina, which is the target site for the treatment of most posterior ocular diseases, further verifying the potential of this composition of chitosan modified by alkyl chains as a penetration enhancer in the treatment of posterior ocular diseases.
  • Example C4 Using artificial tear matrix, the effects of traditional eye drop formulation adjuvants on the eye drop penetration of modified chitosan and protein complex were investigated.
  • Artificial tear matrix components povidone (20 g/L), polyvinyl alcohol (14 g/L), hypromellose (10 g/L), dextran (2 g/L) and hypromellose (10 g/L).
  • Example C3.2 The complex in Example C3.2 was mixed with different artificial tear matrix components to obtain five different eye drop preparations.
  • the IgG content in the retinal tissue of rabbit eyes of different groups was quantitatively evaluated according to the experimental method of Example C2.
  • Table 9 shows the IgG content in the retinal tissue of rabbit eyes of different groups.
  • Example C4.1 in addition to povidone, polyvinyl alcohol, hydroxypropyl methylcellulose, and the combination of dextran and hydroxypropyl methylcellulose all promoted the eye penetration of alkylated chitosan, especially the compound artificial tears of dextran and hydroxypropyl methylcellulose, and the IgG content in the retinal tissue increased by more than 3 times.
  • povidone showed an inhibitory effect on eye penetration, and the penetration efficiency was reduced, which may be because povidone has the effect of promoting close binding between epithelial cells, restoring the epithelial barrier, and reducing permeability, thus affecting the penetration effect of modified chitosan.
  • These artificial tear matrix components are commonly used excipients in commercial eye drop preparations, which are used to increase the time that the active ingredients stay in the eye and thus increase the amount of active ingredients that pass through the eye barrier. These excipients themselves do not have the ability to promote the penetration of macromolecular active ingredients through the eye barrier. The ability of the material barrier is helpful for the preparation of the final formulation.
  • Example C5 Safety of modified chitosan in eye drops
  • a composition of dextran and hydroxypropyl methylcellulose was prepared as an eye drop base, and mixed with the complexes of Example C2.2, Example C2.3, and Example C2.4 to prepare eye drops, which were then administered to the rabbit ocular surface with an IgG concentration of 2 mg/mL. 200 ⁇ L was administered to each eye twice a day for four weeks. The rabbit eyes were observed for any abnormalities after each eye drop, and the rabbit eye status was observed four weeks later. The results showed that the rabbit eyes did not show the aforementioned abnormalities, indicating that the modified chitosan does not have short-term and long-term eye irritation and has good safety.
  • Example C6 Experiment on the treatment of macular degeneration using a complex of modified chitosan and anti-VEGFA.
  • the macula is an important area of the retina, located in the posterior segment of the eye. It is mainly related to visual functions such as fine vision and color vision. Lesions in the macula can cause symptoms such as decreased vision, dark shadows in front of the eyes, and visual distortion, affecting the patient's visual function.
  • the causes of macular lesions are diverse, and heredity, aging, inflammation, etc. may all lead to macular lesions.
  • the late symptoms of patients with macular lesions are due to abnormal proliferation of blood vessels in the eyeball, breaking through the choroid or even the retina.
  • the abnormal blood vessel walls are fragile and prone to vascular leakage, which aggravates the patient's blurred vision and aggravates the condition of macular lesions.
  • the active ingredients used in the clinical treatment of macular lesions are mainly angiogenic factor antibodies such as ranibizumab, conbercept, aflibercept, etc. These large-molecule antibody active ingredients have good short-term efficacy in clinical trials.
  • mice with choroidal neovascularization were randomly divided into four groups:
  • Example C6.4 Intravitreal injection of anti-VEGFA PBS solution, concentration 10 mg/mL, injection volume 5 ⁇ L, as a positive control
  • FIG. 14 shows the images of mouse optical coherence tomography and fundus fluorescence angiography.
  • An increase in the area of choroidal neovascularization indicates aggravation of the disease, whereas an increase in the area of choroidal neovascularization indicates improvement, i.e., the active ingredient is effective.
  • choroidal neovascularization leakage was significantly reduced.
  • Example C6.1 and Example C6.2 had no improvement on the choroidal neovascularization leakage area, and the choroidal neovascularization area increased, while Example C6.3 and Example C6.4 as a positive control mice effectively inhibited the choroidal neovascularization leakage area after treatment, which was about 40% before treatment.
  • Example C7 Study on the eye-penetration-enhancing effect of modified chitosan
  • PBS eye drop matrix phosphate buffer
  • mice were anesthetized, and then 5 ⁇ L was quantitatively administered to each eyeball of each mouse using a quantitative dosing device.
  • the mice were fed for 6 hours, and then the mice were sacrificed, the surface of the eyeball was rinsed with a neutral PBS solution, the eyeball was removed, and the excess tissue on the eye surface was removed.
  • the eyeball sample was frozen and ground in a liquid nitrogen environment to obtain tissue powder, and the tissue powder was dispersed using WB and IP cell lysis buffer, and treated overnight at 4°C.
  • Example D Treatment of eye diseases
  • Example D1 The modified chitosan (CS-S08) and protein macromolecular active ingredient complex described in Example B1 is used to treat wet age-related macular degeneration in mice.
  • Aflibercept is a fully human recombinant fusion protein that can inhibit vascular endothelial growth factor and placental growth factor.
  • the protein molecular weight of aflibercept is about 96kD, and the molecular weight after glycosylation is about 115kD.
  • modified chitosan CS-S08 and aflibercept are used to form a complex, and the active ingredient is delivered by eye drops for the treatment of wet age-related macular degeneration.
  • Example D1.1 No treatment was performed
  • Example D1.2 Intravitreal injection of aflibercept solution, calculated by the mass of aflibercept, the dose is 80 ⁇ g/eye, only Medicine once;
  • Example D1.3 Aflibercept dissolved in artificial tears, calculated by the mass of aflibercept, the dose is 10 ⁇ g/day/eye, and the administration is continued for 28 days;
  • Example D1.4 A complex dissolved in artificial tears, wherein the complex is prepared according to the method of Example A2, wherein the modified chitosan is CS-S08, and the protein macromolecular active ingredient is aflibercept.
  • the mass ratio of modified chitosan CS-S08 and aflibercept is 2:1. Calculated based on the mass of aflibercept, the dosage is 10 ⁇ g/day/eye, and the administration is continuous for 28 days.
  • Relative lesion area spot leakage area on the Xth day / spot leakage area on the 7th day.
  • FIG. 15 is an angiographic image of the mouse eye in Examples D1.1 to 1.4
  • FIG. 16 is a statistical graph of the relative lesion area of the choroid of the mouse eye in Examples D1.1 to 1.4.
  • Example D1.3 directly dissolved Aflibercept with artificial tears for eye drops, which could not effectively control neovascularization, indicating that the direct eye drops of macromolecular active ingredients without the help of carriers could not pass through the eye barrier and reach the lesion site to exert therapeutic effects.
  • Example D1.4 modified chitosan was used to form a complex with Aflibercept, and then dispersed with artificial tears and then eye drops were used, which could inhibit neovascularization, and as the treatment time increased, the final treatment effect was close to the treatment effect of Example D1.2, which was administered by a one-time intravitreal injection.
  • modified chitosan can help large molecular active ingredients pass through the ocular barrier and exert their therapeutic effects to treat posterior ocular diseases, and has the potential to be used in the preparation of eye drops.
  • numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about”, “approximately” or “substantially” in some examples. Unless otherwise specified, “about”, “approximately” or “substantially” indicates that the number is allowed to vary within a range of 0.5%-10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which can be changed according to the desired features of individual embodiments.

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Abstract

本申请提供了一种促大分子活性成分透生物屏障的改性壳聚糖及其制备方法。与传统适用于小分子活性成分的促渗剂相比,本申请所述的改性壳聚糖可以与多种大分子活性成分(如分子量较大的的治疗性蛋白活性成分等)产生结合作用,并将这些大分子活性成分高效递送穿透皮肤屏障、眼部屏障、黏膜屏障等多种宏观生物屏障,从而改变大分子活性成分的给药途径,且具有普适性。并且由改性壳聚糖和活性成分分子形成的复合物,其透过生物屏障的方式是非侵入性的,能够临时打开细胞间隙,安全性更高,其制备方法简便,便于转化应用。

Description

一种改性壳聚糖及其在大分子活性成分递送中的应用 技术领域
本发明涉及大分子活性成分递送领域,尤其涉及一种改性壳聚糖及其在大分子活性成分递送中的应用。
背景技术
大分子活性成分主要包括多肽、蛋白质、抗体、核酸活性成分等,近年来市场扩展迅速,随着研究的不断深入和技术的不断发展,大分子活性成分的临床应用已扩展到多个治疗领域。但是,大分子活性成分本身会易于被胃酸以及消化道中的酶代谢分解导致活性成分失效,此外蛋白等大分子活性成分由于其分子量大往往难以通过各种黏膜屏障而被吸收(如肠道黏膜等),现阶段临床批准使用的大分子活性成分多为注射使用。注射给药的患者依从性较差,特别是对于需要持续给药的患者而言尤其如此。此外,注射还会给患者带来恐惧和疼痛,存在操作不便的问题。因此,生物大分子活性成分新的非侵入性的给药方式及其相应剂型,是现在乃至未来大分子活性成分制剂研发的难点。
非侵入性的递送方式包括透皮、滴眼、经粘膜、口服等方式,这类递送途径的关键在于保护递送成分的活性和使活性成分穿过人体生物屏障。与小分子相比,蛋白等生物大分子由于其更大的分子量和尺寸,导致其渗透性差,很难穿透生物屏障,且易受环境影响发生聚集或降解。如何在保护生物大分子活性的同时促进其透过生物屏障,是现阶段研究的重点。
鉴于生物大分子在透生物屏障递送中的诸多不利因素,需要有合适的辅料保护其结构和活性不受破坏。现阶段有不同的辅料被开发以针对特定的递送方式帮助生物大分子(现有产品研发管线多为多肽类活性成分,例如分子量约4.1kD的索马鲁肽等)经生物屏障给药,比如MannKind开发的Technosphere技术,可以将辅料组装得到装载胰岛素的微球,冻干后制备为肺部吸入粉剂;Emisphere的Eligen技术,利用SNAC和多肽的非共价结合,保护胰岛素免受胃酸的破坏,进入肠道被胃肠黏膜吸收后,在血液中释放活性成分;或者在口服制剂中添加蛋白酶抑制剂,降低生物大分子活性成分在胃肠中的降解。但是这些辅料功能单一,以保护活性为主,依然需要在制剂中添加多种传统辅料帮助有效成分透过生物屏障,同时这类制剂的制备复杂,仅能够针对某种或几种特定的活性成分进行递送,并且不能实现蛋白类活性成分(例如分子量为几十KD甚至更高的抗体)的递送,不具有普适性。
研发在生物大分子活性成分制剂(如治疗性蛋白活性成分、抗体等)中具有促渗透功能的保护型辅料,是生物大分子活性成分透生物屏障给药技术中的难题。
发明内容
为了解决上述问题,本申请提供了一种促大分子活性成分透生物屏障的改性壳聚糖及其制备方法。与传统小分子促渗剂相比,本申请所述的改性壳聚糖的活性成分装载量可控性好,促渗效率高,与多种生物大分子活性成分能够产生结合作用,更具有普适性。并且由改性壳聚糖和活性成分形成的复合物,其透过生物屏障的方式是非侵入性的,能够临时打开细胞间隙,不会破坏细胞屏障和粘膜系统,安全性更高,制备方法简便,便于转化应用。为生物大分子透生 物屏障的应用提供了新载体。
本发明的第一个目的在于,提供一种改性壳聚糖,由壳聚糖或壳聚糖的衍生物与修饰分子反应生成的产物,改性壳聚糖具有通式Ⅰ:CS-X1-R1,所述CS为壳聚糖或壳聚糖的衍生物,所述X1选自酰胺键、磺酰胺键、席夫碱基、仲胺、烷基胺、氨基甲酸酯、酯键、醚、磷酸酯、磺酸酯、脲、磷酰胺、脒键中的任意一种,所述R1来自于所述修饰分子。
具体地,所述壳聚糖或壳聚糖的衍生物的分子量范围在1000-5000000,脱乙酰度不低于85%。
或所述X1选自-NH-CO-、-NH-SO2-、-N=C-、-NH-、-NH-CH2-、-NH-CH2-CH(OH)-、-NH-CO-O-、-O-CO-、-O-CH2-O-CH2-、-O-PO-(OR1)2、-O-SO2-、-NH-CO-NH-、-NH-PO-(OR1)2、-NH-C(=NH2+)-中的任意一种。
具体地,所述壳聚糖或壳聚糖的衍生物选自壳聚糖、壳寡糖、甲壳素、羟乙基壳聚糖、羟丙基壳聚糖、羧甲基壳聚糖、乙二醇壳聚糖、N-三甲基壳聚糖、三甲基铵乙二醇壳聚糖碘化物、壳聚糖季铵盐、壳聚糖盐酸盐、壳聚糖乳酸盐、壳聚糖硝酸盐、壳聚糖谷氨酸盐、壳聚糖壬二酸盐、中的一种或多种。
具体地,所述修饰分子选自脂肪族化合物。
具体地,所述脂肪族化合物选自脂肪酸、脂肪醛、脂肪酰卤、脂肪酸酐、脂肪族聚酯。
进一步地,所述脂肪族化合物含有n个碳原子的脂肪链,其中3≤n≤21。
进一步地,所述所n=7,n=8,n=12,n=16。
进一步地,通式Ⅰ中,所述X1是酰胺键,所述R1是含有6个碳原子的脂肪链、含有7个碳原子的脂肪链、含有11个碳原子的脂肪链、含有15个碳原子的脂肪链中的任意一种。
进一步地,所述改性壳聚糖上修饰分子的取代度不超过40%。
本发明的第二个目的在于,提供一种改性壳聚糖,所述改性壳聚糖具有通式Ⅱ:
CS-X1-R2-R3
所述CS为壳聚糖或壳聚糖的衍生物;
所述X1选自酰胺键、磺酰胺键、席夫碱基、仲胺、烷基胺、氨基甲酸酯、酯键、醚、磷酸酯、磺酸酯、脲、磷酰胺、脒键中的任意一种。
或所述X1选自-NH-CO-、-NH-SO2-、-N=C-、-NH-、-NH-CH2-、-NH-CH2-CH(OH)-、-NH-CO-O-、-O-CO-、-O-CH2-O-CH2-、-O-PO-(OR)2、-O-SO2-、-NH-CO-NH-、-NH-PO-(OR2)2、-NH-C(=NH2+)-中的任意一种。
所述R2为含有n个碳原子的脂肪链,其中2≤n≤20;
所述R3含有酚羟基或其衍生物、邻苯二酚基团或其衍生物、邻苯三酚基团或其衍生物中的一种或多种;
所述R3通过连接基团与R2相连接。
所述连接基团选自酰胺键、磺酰胺键、席夫碱基、仲胺、烷基胺、氨基甲酸酯、酯键、醚、磷酸酯、磺酸酯、脲、磷酰胺、脒键中的任意一种。
进一步地,所述壳聚的分子量范围在1000-5000000,脱乙酰度不低于55%
进一步地,所述酚羟基与壳聚糖的摩尔比不超过20%。
进一步地,所述酚羟基与壳聚糖的摩尔比为8%~18%。
进一步地,所述改性壳聚糖上修饰分子的取代度不超过20%。
所述CS-X1-R2-R3可以通过中间体与壳聚糖或其衍生物反应得到,所述中间体选自以下结构:
具体地,所述中间体与壳聚糖的摩尔比不大于20%。
进一步地,所述中间体与壳聚糖的投料比为1%~18%。
进一步地,所述中间体与壳聚糖的投料比不小于10%,不大于20%。
所述壳聚糖或壳聚糖的衍生物的分子量范围在1000-5000000,脱乙酰度不低于55%。
进一步地,所述壳聚糖或壳聚糖的衍生物的分子量范围在1000-5000000,脱乙酰度不低于85%。
本发明的第三个目的在于,提供一种改性壳聚糖CS-X1-R1的制备方法,其特征在于,通过修饰分子与壳聚糖或壳聚糖的衍生物反应。
所述修饰分子含有能与壳聚糖伯氨基反应的活性基团。
所述修饰分子含有以下活性基团:羧酸基团、磺酸基团、酸酐、碳酸盐、酰卤基团、磺酰氯、酰氯、NHS酯、亚氨酸酯、五氟苯酯、醛基、异氰酸酯、异氰酸盐、环氧基团、双键、炔基、羟甲基膦、羧酸根。
具体地,所述修饰分子与壳聚糖的摩尔比不大于40%。
进一步地,所述修饰分子与壳聚糖的投料比为1%~40%。
进一步地,所述修饰分子与壳聚糖的投料比不小于10%,不大于40%。
本发明的第四个目的在于,提供一种透生物屏障的复合物,包括改性壳聚糖和活性成分。
进一步地,所述改性壳聚糖和活性成分以物理或化学方式结合形成所述透生物屏障的复合物。
进一步地,所述活性成分选自小分子活性成分和/或大分子活性成分。
进一步地,所述改性壳聚糖和活性成分的质量比为1:0.1~10。
进一步地,所述改性壳聚糖和活性成分的质量比为1:0.2~5。
具体地,所述生物屏障包括皮肤屏障、口腔黏膜、阴道黏膜、膀胱黏膜、眼部屏障、泪液屏障、角膜/结膜屏障、血房水屏障、血视网膜屏障中的任一种。
进一步地,所述活性成分的分子量为5~260kD。
进一步地,所述活性成分的分子量约为5kD、10kD、20kD、40kD、60kD、140kD、180kD、200kD、240kD、260kD。
具体地,所述活性成分选自抗肿瘤活性成分、抗生素、免疫调节剂、镇痛药、眼病治疗活性成分、心血管系统药物、抗感染药物、内分泌系统代谢药物、消化系统药物、免疫系统药物、神经系统药物、精神障碍类药物、血液系统药物、皮肤疾病活性成分、除性激素和胰岛素外的 全身激素类药物、抗寄生虫、杀虫药、驱虫药、电解质、酸碱平衡及营养药、口腔病药物、糖尿病用药、抗感染药、其他疾病治疗药物或活性成分、靶向抑制剂、抗衰老成分、抗菌成分、抗敏成分、抗皱成分、抗氧化成分中的一种或多种。
具体地,所述大分子活性成分选自治疗性疫苗、预防性疫苗、mRNA疫苗、单特异性抗体、双特异性抗体、双抗体偶联物、抗体类融合蛋白、多特异性抗体、其他抗体、白蛋白类、免疫球蛋白类、凝血因子类、酶、细胞因子类、非抗体类融合蛋白、放射性核素偶联活性成分、siRNA、mRNA、ASO、其他核酸、多肽、溶瘤病毒、蛋白降解靶向嵌合体、诊断试剂、伴随诊断试剂、外泌体中的一种或多种。
具体地,所述大分子活性成分选自蛋白类活性成分、治疗糖尿病的活性成分、治疗肥胖症的类多肽活性成分、其他多肽类活性成分、激素类活性成分、抗体类活性成分、生长因子、核酸物质中的一种或多种。
具体地,所述眼病治疗活性成分选自雷珠单抗、贝伐珠单抗、VEGF抗体、 MP0112、ARC1905、FCFD4514S、康柏西普、阿柏西普、补体途径抑制剂、视觉周期抑制剂、雷帕霉素靶点抑制剂、丝氨酸/苏氨酸蛋白激酶抑制剂、LIM激酶抑制剂、非甾体类EP2受体激动剂中的一种或多种。
具体地,生长因子选自血小板类生长因子、表皮生长因子类、成纤维细胞生长音字、类胰岛素生长因子、神经生长因子、白细胞介素类生长因子、红细胞生长素、集落刺激因子、纤维母细胞生长因子、肝细胞生长因子、类胰岛素生长因子、生长激素释放抑制因子中的一种或几种。
具体地,核酸物质选自siRNA、mRNA、shRNA、lnc RNA、pDNA、poly IC、CpG或环状二核苷酸中的一种或几种。
具体地,所述蛋白类活性成分选自抗体、单特异性抗体、双特异性抗体、血管内皮生长因子抗体、过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶中的一种或多种。
进一步地,所述大分子活性成分选自胶原蛋白、神经酰胺、高分子量的透明质酸钠、银耳多糖或其他黏多糖、聚谷氨酸(PGA)、硫酸软骨素、纤维素、天然胶体、蓝铜胜肽、芋螺肽、乙酰基六肽、九肽-1、棕榈酰三肽-8、棕榈酰四肽-7、棕榈酰五肽-4、棕榈酰三肽-5、过氧化氢酶中的一种或多种。
进一步地,所述小分子活性成分选自光甘草定、虾青素、水杨酸、阿魏酸、苯乙基间苯二酚、白藜芦醇;传明酸、烟酰胺、维生素C、麦角硫因、小分子肽、氨基丁酸、脱氧核糖核酸、玻色因或依克多因、精氨酸/赖氨酸多肽、泛醌、二肽二氨基丁酰苄基酰胺二乙酸盐、伊维菌素中的一种或多种。
本发明的第五个目的在于,提供一种改性壳聚糖或透生物屏障的复合物在制备疾病治疗药物或医疗美容产品中的应用。
具体地,所述疾病选自肿瘤、眼部疾病、代谢疾病、疼痛、炎症、免疫性疾病、神经系统疾病、精神性疾病、生殖系统疾病、骨骼疾病、口腔疾病、激素紊乱、呼吸系统疾病中的任一种。
本发明的第六个目的在于,提供一种透皮制剂,包括改性壳聚糖和活性成分的复合物。
进一步地,所述透皮制剂还包括增稠剂、赋形剂、抗过敏成分、保湿成分、抑菌剂、结晶 抑制剂、增溶剂中的一种或多种。
本发明的第七个目的在于,提供一种滴眼制剂,包括改性壳聚糖和活性成分的复合物。
进一步地,所述滴眼制剂还包括分散剂、赋形剂、增稠剂、保湿成分、抑菌剂、结晶抑制剂、防腐剂中的一种或多种提供一种在疾病治疗制剂制备中的应用。
根据本发明提供的改性壳聚糖与现有技术相比,有如下有益效果:
1)改性壳聚糖具有原料易得、制备简便,能够根据需求得到合适取代度的产物进一步用于大分子活性成分的透皮递送。
2)该改性壳聚糖能够高效地装载大分子活性成分,实现大分子活性成分透生物屏障,从而实现一些传统活性成分的非侵入性给药方式,大大增加临床使用便利性。同时,由于该改性壳聚糖具有较高的促渗效率,有助于提高活性成分的利用率。
3)改性壳聚糖打开生物屏障具有非破坏性的特点,与现有技术相比,提高了应用安全性和舒适性。
4)提供了一种平台技术,由于其水溶性好、稳定性好等特点,非剂型原因,不需要添加过多辅料助剂,适用于多种大分子量的蛋白、多肽以及小分子活性成分的透生物屏障递送,尤其突破了大分子活性成分的透生物屏障递送障碍,普适性更好,化学试剂的引入更少,制剂组分更简单,制备方法更简捷,有助于提高生产效率和安全性,增加使用便利性,且具有更大的剂量使用空间。
5)另外,在人类皮肤中,皮下(指真皮以下的疏松结缔组织)存在许多抗原呈递细胞,可吞噬、摄取、处理抗原,通过淋巴通道迁移到局部淋巴结,激活T细胞,引发免疫应答,特别是表皮中的朗格汉斯细胞和真皮中的间质树突状细胞。抗原停留时间长,有助于刺激免疫应答的强度,因此本申请所述改性壳聚糖能够帮助活性成分更多地停留在皮下,从而更好地的引发免疫应答,或者帮助活性成分(例如胶原蛋白)在皮下发挥其功能。
6)本申请提供了一种平台技术,本发明所述改性壳聚糖具有良好的水溶性,能够帮助各种分子量的活性成分透过生物屏障,提供了一种新的经皮递送以及滴眼给药的载体,应用潜力大。
附图说明
图1是实施B1中各复合物的CD值图谱;
图2是实施例B2中经计算得到的12h后BSA-FITC的透皮率和皮内滞留率;
图3是实施例B3中不同组别体外透皮实验中,12小时左右小鼠皮肤内IgG蛋白滞留的相对含量统计图;
图4是实施例B4中治疗后小鼠的相对伤口面积统计图;
图5是实施例B5中小鼠背部皮肤的H&E染色切片的电镜结果图;
图6是实施例B5中小鼠背部皮肤的马松(Masson)染色切片的电镜结果图。
图7是实施例B6中小鼠皮肤患处照片;
图8是实施例B6中第5天获取的小鼠皮肤组织H&E切片的显微图片;
图9是实施例B6中中小鼠皮肤组织H&E切片数据(即图3)的ImageJ定量分析结果;
图10是实施例B7中给药不同天数后小鼠血清中的OVA抗体滴度统计图;
图11是实施例B7中小鼠脾脏中IFN-γ+的CD4+T细胞、CD8+T细胞相对含量统计图;
图12是实施例B8中小鼠皮肤组织切片的免疫荧光染色信号图;
图13是实施例C1中经不同滴眼处理后蛋白渗透至小鼠眼球视网膜部位的荧光信号分布;
图14是实施例C2中小鼠脉络膜新生血管成像,通过进入血液的荧光素钠荧光信号显示血管位置,显示小鼠脉络膜新生血管模型经过不同手段治疗后的新生血管生长情况;
图15是实施例D1中小鼠眼部造影成像图;
图16是实施例D1中小鼠眼脉络膜的相对病变面积统计图。
具体实施方式
实施例A1:一种改性壳聚糖,具有CS-X1-R1的结构,其中CS是壳聚糖,X1是酰胺键,由庚酸的羧基和壳聚糖上的游离氨基反应得到,R1是含有6个碳原子的脂肪链,将此结构改性壳聚糖命名为CCS-7。
以分子量为50KDa,脱乙酰度>90%的壳聚糖为原料,选择庚酸作为脂肪酸修饰分子,通过化学偶联反应合成系列庚酸改性的壳聚糖(CCS-7)。记录所得壳聚糖衍生物的水溶性(定义为:以1mg/mL的浓度溶解于pH=5-7的去离子水中),并采用茚三酮法测定所得壳聚糖衍生物的氨基取代度,以计算疏水脂肪链的接枝率。
庚酸改性的壳聚糖(CCS-7)的合成:
以碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)为耦合剂,通过壳聚糖上的氨基与庚酸的羧基之间的缩合反应,制备系列庚酸改性的壳聚糖衍生物。具体的合成方法为:准确称取1g壳聚糖,溶于100mL 0.1M盐酸溶液中,以1M氢氧化钠水溶液调节pH至5-7之间,加入0.005-0.0125mol的N-羟基琥珀酰亚胺(NHS)溶解后,在室温和磁力搅拌的条件下反应1小时;另分别称取0.001-0.0025mol的庚酸溶于20mL的无水二甲基亚砜,加入0.005-0.0125mol的碳二亚胺(EDC)(羧基与EDC、NHS的摩尔比为1:5:5),在室温和磁力搅拌的条件下反应1小时。在上述两反应液各反应1小时后,分别将壳聚糖的反应液与庚酸的反应液混合,继续在室温、磁力搅拌的条件下反应48小时。反应结束,向终反应液加入大量无水乙醇,低温沉降得到絮状产物,抽滤后,真空干燥,得到系列庚酸改性的壳聚糖衍生物(CCS-7)。
庚酸改性的壳聚糖(CCS-7)的合成路线图:
茚三酮法测定CCS-7氨基取代度:
取不同量的壳聚糖(0.5-4mg),精密称量,分别溶于2mL的醋酸/醋酸钠缓冲液(0.2M,pH=5.4)中,加入0.30wt%茚三酮的乙二醇甲醚溶液1mL和1.7wt%还原茚三酮的乙二醇甲醚溶液1mL,于100℃孵育10min。冷却后加入2mL 60%的乙醇溶液,摇匀,测定570nm波 长处的吸收值,制备标准曲线。取上述庚酸改性的壳聚糖衍生物2mg溶于醋酸/醋酸钠缓冲液,相同操作,测定570nm波长处的吸收值,按标准曲线计算庚酸改性的壳聚糖衍生物的氨基取代度。
实验结果如表1所示,按照上述制备步骤得到的CCS-7的实际氨基取代度随着庚酸投料比的增加而增加,且产物均可以很好的溶解于偏酸性的水溶液中(1mg/mL,pH=5-7),但是,当投料比增加到一定程度时,CCS-7(庚酸投料比增加至50%)的产物出现了在水相中溶解不完全的现象,不能进一步用于实际组合物制备的应用中。投料比在10%~40%时,能够实现修饰,并且产物可溶,投料比在40%时,其取代度不小于36%。
在实验设计的投料比下,CCS-7的氨基取代度(即碳氢链的接枝率)从最低的9.1%增加到最高的36.4%。两亲性聚合物载体的疏水链段所占比例对其与天然大分子活性成分的组装效率以及组装稳定性有因果关系,因此,CCS-7具有较宽的碳氢链接枝效率的调整空间,可以为后续应用提供更多的选择。
表1:不同投料比的烷基化壳聚糖对应取代度统计表
(投料比是指制备时投入的脂肪酸的羧基占壳聚糖的氨基摩尔百分比)
经过多次实验,发现该方法制备得到的产物具有较好的均一性,同样投料比制备得到的改性壳聚糖,取代度接近,投料比为40%时,取代度约为36~38%,一方面说明制备方法具有可重复性,另一方面,具备反应效率高,产率高的特点。
实施例A2:一种复合物(CCS-7/BSA),包括改性壳聚糖和蛋白质,所述改性壳聚糖是实施例A1得到的CCS-7,所述蛋白质是牛血清白蛋白(简称BSA),用于说明改性壳聚糖和蛋白质能够形成复合物,并且具有明显的促渗效果。
选择实施例A1.1至实施例A1.4中得到的CCS-7的水溶性产物作为载体,与牛血清白蛋白(BSA)制备改性壳聚糖和蛋白质的复合物,然后通过体外黏膜促渗实验以及体外透皮促渗实验进行促渗效果验证。
改性壳聚糖和蛋白质的复合物的制备方法:
称取一定质量的实施例A1.1至实施例A1.4中得到的CCS-7以及BSA粉末分别溶于去离子水,制备载体母液与蛋白母液,然后按照质量比为1:1的比例将各取代度的CCS-7分别与BSA混合均匀之后,再加入与混合液等体积的磷酸缓冲液(PB,0.01M,pH=7)调节体系的pH值,该过程中BSA与CCS-7之间通过静电作用以及疏水相互作用相互结合,可得到CCS-7/BSA的复合物。
实施例A3:一种改性壳聚糖,具有CS-X1-R1的结构,其中CS是壳聚糖,X1是酰胺键,由不同碳链长度的修饰分子和壳聚糖上的游离氨基反应得到。由含有8个碳原子的脂肪链与壳聚糖氨基反应,共价修饰得到的改性壳聚糖,R1是含有7个碳原子的脂肪链,将此改性壳聚糖命名为CCS-8;由含有12个碳原子的脂肪链与壳聚糖氨基反应,共价修饰得到的改性壳聚 糖,R1是含有11个碳原子的脂肪链,将此结构改性壳聚糖命名为CCS-12;由含有16个碳原子的脂肪链与壳聚糖氨基反应,共价修饰得到的改性壳聚糖,R1是含有15个碳原子的脂肪链,将此结构改性壳聚糖命名为CCS-16。
CCS-8、CCS-12、CCS-16制备方法与实施例A1相同,区别在于使用不同的修饰分子。
使用实施例A1中茚三酮法测定CCS-8、CCS-12、CCS-16的氨基取代度。结果如表2所示。
表2:不同长度的脂肪链修饰壳聚糖的产物性质及黏膜促渗效果统计表。
结果显示,不同碳原子数的脂肪链均能够修饰壳聚糖得到改性壳聚糖,并且通过调节投料比能够得到不同取代度的改性壳聚糖,其中投料比是指原料中修饰分子与壳聚糖的摩尔比。综合实施例A1和A3,经过多次重复实验,不同长度的脂肪链作为修饰分子,与壳聚糖反应得到的改性壳聚糖,其取代度不超过40%。产物溶解性良好,能够进一步用于复合物的制备。
实施例A4:一种复合物,包括实施例A3所述的改性壳聚糖和异硫氰酸荧光素标记的牛血清白蛋白(BSA-FITC)。制备方法与实施例A2相似,区别在于将原料CCS-7替换为实施例A3得到的CCS-8、CCS-12或CCS-16。不同取代度、不同修饰分子制备得到的几种改性壳聚糖均能够与蛋白形成稳定的复合物,能够进一步用于透皮制剂。
实施例1:一种改性壳聚糖,具有CS-X1-R2-R3的结构,将此结构改性壳聚糖命名为CS-S08。
以分子量为50KDa,脱乙酰度>90%的壳聚糖为原料,选取8-(2-羟基苯甲酰胺基)辛酸钠(SNAC)为修饰分子中间体,通过壳聚糖上的氨基与修饰分子的羧基基团之间的酰化反应,合成具有CS-X1-R2-R3结构的壳聚糖衍生物。改性示意及产物结构式如下:
具体的合成方法为:准确称取1g壳聚糖,溶于100mL 0.1M盐酸溶液中,以1M氢氧化钠水溶液调节pH至5-7之间;另分别称取0.001-0.0025mol的SNAC溶于20mL的无水二甲基亚砜,依次加入0.005-0.0125mol的碳二亚胺(EDC)和0.005-0.0125mol的N-羟基琥珀酰亚胺(NHS)(羧基与EDC、NHS的摩尔比为1:5:5),在室温和磁力搅拌的条件下反应1小时,反应过程中维持pH在5-7之间。随后,将壳聚糖溶液与活化后的中间体的反应液混合,继续在室温、磁力搅拌的条件下反应48小时。反应结束,得到终反应液,终反应液经无水乙醇沉降过滤后,真空干燥,即可得到具有CS-X1-R2-R3结构的改性壳聚糖(CS-S08)。
其中X1为酰胺键,R2为含有7个碳原子的烷基链,R3为酚羟基,通过酰胺键与R2相连接,SNAC的投入量不同,得到的产物中壳聚糖的氨基取代度不同。
实施例2:一种复合物,包括实施例1制备得到的具有CS-X1-R2-R3结构的改性壳聚糖和异硫氰酸荧光素标记的牛血清白蛋白(BSA-FITC),制备方法如下:
称取一定质量的实施例1中得到的CS-S08以及BSA-FITC粉末分别溶于去离子水,制备载体母液与蛋白母液,然后按照质量比为1:1的比例将各取代度的CS-S08分别与BSA-FITC混合均匀之后,再加入与混合液等体积的磷酸缓冲液(PB,0.01M,pH=7)调节体系的pH值,该过程中BSA-FITC与CS-S08之间通过静电作用以及疏水相互作用相互结合,可得到CS-S08/BSA-FITC的复合物。
实施例3:具有CS-X1-R2-R3结构的改性壳聚糖的体外黏膜促渗和体外透皮实验,验证其促渗效果。实验方法如下:
按实施例2所述方法准备CS-S08或CS与异硫氰酸荧光素标记的牛血清白蛋白(BSA-FITC)的复合物溶液(BSA-FITC的浓度为0.2mg/mL),同时准备等浓度(0.2mg/mL)的游离BSA-FITC溶液作为对照例;
将处理干净的新鲜离体小鼠全皮置于竖式透皮扩散池中(小鼠皮内表面朝向加样池,外表面朝向接收池),将游离/复合BSA-FITC溶液(0.2mg/mL)使用于离体小鼠全皮外表面,新鲜离体小鼠全皮是一种完整皮肤组织,可以作为皮肤屏障的代表。调节扩散池体系保持在37℃恒温,12小时后,检测接收池中的荧光总量,以此计算蛋白透过率。
异硫氰酸荧光素标记具有荧光信号,可以通过荧光信号的强度辨别蛋白的分布、浓度等信息。
蛋白透过率=(接收池中蛋白的荧光总量)/(原溶液的荧光总量)*%
实验结果如表3所示,其中投料比指的是改性原料中SNAC与壳聚糖的摩尔百分比,取代度是指采用茚三酮法测定产物中壳聚糖支链上的氨基被【-X1-R2-R3】取代的比例。各取代度的CS-S08与BSA-FITC复合后,提高了BSA-FITC在小鼠全皮的透过率,随着CS-S08取代度的增加,其帮助BSA-FITC的小鼠全皮透过率随之增加;当CS-S08取代度为15.2%,其透皮促渗效果可达10.05%,相较于对照例2,大大提高了蛋白的渗透率。该实施例说明CS-S08能够通过调节取代度达到较好的促渗透效果,具有一定的可控取代度范围以用于不同需求的促渗载体,进一步说明本发明改性壳聚糖具有理想的促进大分子活性成分渗透的能力和提高治疗效果的潜力。
得到具有CS-X1-R2-R3结构的改性壳聚糖和异硫氰酸荧光素标记的牛血清白蛋白(BSA-FITC)的复合物。实验结果如表3所示。
表3:具有CS-X1-R2-R3结构的改性壳聚糖的取代度及促渗效果
由于高分子的修饰过程具有不确定性,因此上述实施例的取代度是各组实验中的一个样品的结果,每组实验均有5次以上重复,取代度接近,小鼠全皮透过率数据接近,具有较好的重复性。多次实验发现,相同投料比得到的产物,其取代度接近,当投料比为12%时,取代度约为8%~10%;投料比为15%时,取代度约为11%~13%;当投料比为18时,取代度约为13%~16%,投料比为20%时,取代度不超过20%。综上,制备改性壳聚糖的原料的投料比可以不大于20%,其取代度不超过20%。该制备方法得到改性壳聚糖,其具有反应效率高,产率高,反应稳定的特点,有助于实现量产。
通过表3的小鼠全皮透过率分析,取代度更高的改性壳聚糖,其促渗效果更好,能够促进黏膜渗透的改性壳聚糖同样能够促进大分子渗透皮肤,能够使大分子渗透透过率提高20倍以上。
实施例B1:改性壳聚糖和蛋白活性成分的复合物
根据实施例2的方法,将投料比为15%,取代度为12.5%左右的CS-S08与不同分子量的活性蛋白制备得到复合物。
其中,对CS-S08和原料活性蛋白的起始浓度进行调整,检测所得复合物的粒径分布情况,结果如表4所示。
结果显示,首先,CS-S08能够与不同分子量的蛋白形成复合物,说明CS-S08具有较好的普适性,能够装载分子量在5kD~240kD的活性蛋白。结合实施例3的数据,BSA为分子量约66kD的蛋白,推测CS-S08与5kD~240kD的活性蛋白形成复合物后,均有望透过皮肤屏障,达到经皮递送的效果。
其次,CS-S08与蛋白形成的复合物具有较为均一的平均粒径,整体来看,各实施例中复合物的均一度较好,本发明所述的改性壳聚糖能够和不同分子量的蛋白形成粒度均匀的复合物,分散性良好。粒度均匀的复合物说明改性壳聚糖装载活性蛋白的产物可控性好,有批量生产复合物的潜力,从而有助于进一步产业化应用。
表4:不同实施例得到的复合物及其状态。
此外,除上表中列出的实验参数外,还考察了相同质量比,不同浓度的CS-S08与蛋白形成复合物的实验,结果表明,当控制CS-S08和蛋白的质量比例在5:1及以下时,均能够形成复合物。CS-S08本身含有大量正电荷,能够与蛋白所携带的负电荷之间形成较为稳定的作用力,当CS-S08或蛋白的含量过高时,由于电荷失衡会破坏体系的稳态,产生粒径较大的颗粒。如果活性蛋白本身携带的负电荷较多,那么进一步提高CS-S08相较于蛋白的比例,依然能够和蛋白形成较稳定和均一性良好的复合物。
使用圆二色谱测定复合物的CD值,图1是实施例B1中各复合物的CD值图谱,取载体与蛋白比例为1:1的组别。从图中可以看出,与未形成复合物的蛋白相比,复合物中蛋白的CD谱未发生改变,说明CS-S08和蛋白结合形成复合物的过程,不会导致的蛋白二级结构变化而失活,从微观结构层面证明改性壳聚糖和蛋白形成复合物后,蛋白能够保证活性,能够进 一步发挥功能并达到预期效果。
实施例B2:采用Franz扩散池进行体外透皮吸收实验,证明改性壳聚糖能够非侵入性地促进蛋白渗透皮肤。
具体地,以裸鼠皮肤为测试对象,以BSA-FITC为目标蛋白,起始蛋白含量一致(1.6mg),各组别中添加的载体的含量一致(1.6mg),通过Franz扩散池的一侧添加样品,12小时后检测接收池内的蛋白含量占起始实验剂量的比例以及滞留在小鼠皮肤内的蛋白含量占起始实验剂量的比例,验证不同载体促进蛋白渗透的效率。实验分组如下:
对照例B2.1:游离的BSA-FITC;
对照例B2.2:壳聚糖与BSA-FITC的复合物;
对照例B2.3:SNAC与BSA-FITC的复合物;
对照例B2.4:壳聚糖、SNAC与BSA-FITC经物理混合的复合物,SNAC和壳聚糖的摩尔比例为15%,载体与蛋白质量比为1:1;
实施例B2.5:实施例3.3制备得到的CS-S08与BSA-FITC的复合物,载体与蛋白比例为1:1。
检测结果如图2所示。综合透过率与皮内滞留率,实施例B2.5中使用本发明所述的改性壳聚糖能够最大程度地促进蛋白组分透过皮肤表层屏障,进入皮内和透过皮肤。并且与对照例B2.4相比,如果将改性壳聚糖的原料SNAC与壳聚糖直接进行混合,而不进行化学接枝改性,并不能促进蛋白通过皮肤屏障。进一步分析,推测对照例B2.4中物理混合的SNAC和壳聚糖与蛋白之间难以形成稳定的复合物,没有形成有效的装载,从而无法发挥促进渗透的作用。
此外,我们还意外地发现,改性壳聚糖能够增加蛋白在皮肤内的滞留,该特点能够大大提高活性成分在医疗美容领域的应用效果。增加活性成分在皮肤内滞留的时间,则能够更好地发挥活性成分对皮肤内细胞或代谢的改善作用,一方面,提高了活性成分的利用率,另一方面,能够提高医疗美容产品的功效。
实施例B3:该技术效果在CS-S08和免疫球蛋白IgG(分子量150kD)的复合物中也得到了验证,具体实验分组如下:
对照例B3.1:IgG溶液;
对照例B3.2:壳聚糖和IgG的混合溶液;
对照例B3.3:SNAC和IgG的混合溶液;
对照例B3.4:壳聚糖、SNAC和IgG的混合溶液;
实施例B3.5:实施例A2制备方法得到的CS-S08和IgG的的复合物,载体与蛋白质量比为2:1。
图3是实施例B3中不同组别体外透皮实验中,12小时左右小鼠皮肤内蛋白滞留的相对含量统计图。结果显示,改性壳聚糖能够显著提高IgG在皮肤内的滞留量,延长蛋白在皮肤内的停留时间,蛋白在皮肤内停留时间久,能够更好地作用与皮内或皮下的细胞,发挥治疗、改善等作用,该有益效果说明,本发明所述改性壳聚糖有助于医疗美容领域活性成分蛋白或疫苗活性成分的装载和递送。
实施例B4:CS-S08和胶原蛋白形成的复合物用于创面修复的应用研究
实验方法:选用Balb/c小鼠(周龄6-8周)进行实验。先刮除健康小鼠的被毛,使用戊巴 比妥钠对小鼠进行麻醉,用75%乙醇进行常规消毒后通过手术切除小鼠的全层皮肤制造直径为5mm的全层皮肤创面,切除后将丙烯酸酯夹板固定在小鼠的背部伤口上来阻止小鼠皮肤的自然收缩,更好的模拟人类伤口愈合的特性。术后止血、消毒并分组给药治疗。在治疗后拍照记录并用Image J计算小鼠的创面愈合情况。实验具体分组如下:
实施例B4.1:不治疗;
实施例B4.2:胶原蛋白与凡士林充分混匀制成软膏,涂抹于伤口造模处,给药量为每天涂抹一次,每次100μg/只;
实施例B4.3:按实施例B1.1.2所述制得的CS-S08和胶原蛋白的复合物与凡士林充分混匀制成软膏,并涂覆于伤口造模处,给药量为每天涂抹一次,每次100μg/只。
图4是治疗后小鼠的相对伤口面积统计图。结果表明经过单次给药治疗,实施例D1.3中的小鼠创面明显减小,在给药后10天基本完全愈合。胶原蛋白作为人体组织的主要成分,可以通过与周围组织细胞的相互作用刺激细胞的增殖和迁移,从而加速创面的愈合过程,以上结果表明,本申请所述改性壳聚糖能够成功将胶原蛋白递送到皮内,从而缩短创面愈合时间。
综上所述,本申请所述改性壳聚糖能够装载胶原蛋白并帮助胶原蛋白进入皮肤,针对医美术后受损皮肤的细小创面,本申请所述改性壳聚糖和蛋白的复合物能够高效递送胶原蛋白,并保证蛋白活性,从而加速皮肤损伤修复过程,与此同时CS-S08载体兼具了壳聚糖的抗菌作用,通过敷料的形式作用到皮肤表面能够保持伤口清洁,抑制伤口感染,从而进一步加速创面的修复过程。
实施例B5:CS-S08和胶原蛋白形成的复合物用于紧致皮肤的应用研究
实验方法:选用Balb/c裸鼠(周龄6-8周)进行实验。直接对裸鼠皮肤进行复合物的涂抹,每天一次,涂抹三次,在第4天处死小鼠并收集背部皮肤,通过切片分析小鼠的表皮厚度和胶原的含量变化。具体的实验分组如下:
实施例B5.1:空白对照,仅涂抹凡士林,不做任何处理;
实施例B5.2:涂抹胶原蛋白和凡士林的混合软膏,涂抹于裸鼠背部皮肤,每天涂抹一次,重复三次;
实施例B5.3:按实施例B2.5所述制得的复合物与凡士林充分混匀制成软膏,涂抹于裸鼠背部皮肤,每天1次,重复上述过程3次。
图5是小鼠背部皮肤的H&E染色切片的电镜结果图。如图所示,黑色线段指示小鼠表皮厚度,实施例B5.3的小鼠表皮厚度明显增加,虚线部分位于真皮细胞区,切片显示虚线区域内真皮细胞的密度明显提升,细胞间隙变小,说明胶原蛋白能够帮助小鼠皮肤恢复饱满、紧致的状态。
图6是小鼠背部皮肤的马松(Masson)染色切片的电镜结果图。其中虚线指示区域为胶原纤维显色区域,结果显示,实施例B5.3的小鼠皮肤中的胶原纤维的含量明显增加。
以上结果均表明本申请所述复合物能够成功实现将胶原蛋白(COL)递送至真皮层,从而刺激了新生胶原纤维的合成,同时提高了表皮厚度和真皮细胞密度。由于皮肤松弛衰老的过程与胶原蛋白的流失是息息相关的,胶原蛋白的补充能够增加皮肤的紧致度,从而有效的延缓皮肤衰老的过程。上述结果表明了本申请所述改性壳聚糖作为蛋白载体在医美抗衰产品的应用前景。
实施例B6:CS-S08和阿达木单抗的复合物用于银屑病的治疗实验。
银屑病是一种常见的慢性炎症性自身免疫性疾病,阿达木单抗(ADA)是全人源化肿瘤坏死因子单克隆抗体,已获批通过皮内注射对银屑病进行治疗,目前阿达木单抗的使用方式为皮内注射,会导致疼痛甚至局部感染的风险,患者依从性差。本实施例中,将阿达木单抗与本申请所述CS-S08形成复合物,用于银屑病治疗实验。
具体地,使用咪喹莫特诱导Balb/c小鼠银屑病模型,每天使用5%的咪喹莫特涂抹小鼠皮肤,连续5天。实验期间,每天通过拍照记录小鼠皮肤的损伤情况,评估银屑病严重程度;此外,以初次使用咪喹莫特计为第0天,在第3天和第5天取小鼠皮肤组织样本,裂解后检测细胞因子含量以评估炎症水平;并且在第5天,取小鼠患处皮肤样本,通过H&E切片和ImageJ统计小鼠皮肤厚度,以判断小鼠皮肤的炎症程度。
实验分组和样品如下:
对照例B6.1:健康小鼠,未进行咪喹莫特刺激,未经过任何治疗;
对照例B6.2:仅进行咪喹莫特刺激建模;
对照例B6.3:凡士林混合阿达木单抗,以阿达木单抗质量计,每只小鼠给药量为150μg/次,第1天和第3天各给药一次;
对照例B6.4:第1天在小鼠患处皮下注射阿达木单抗,剂量为300μg;
实施例B6.5:按照实施例2的方法制备CS-S08(取代度约为12.1%)和阿达木单抗的复合物(CS-S08和阿达木单抗的质量比为2:1),与对照例C1.1等量的凡士林混合,以阿达木单抗质量计,每只小鼠给药量为150μg/次,第1天和第3天各给药一次。
图7是实施例B6.1~实施例B6.5中小鼠皮肤患处照片,表5是实施例B6.1~实施例B6.5中小鼠第1-5天的患处鳞屑和红斑的严重程度评分表。结合图7和表5分析,首先,对照例B6.2中,小鼠患处皮肤呈现严重的银屑病病症,说明银屑病建模成功;其次,对照例B6.4是目前临床使用的皮下注射阿达木单抗治疗方案,能够有效阻止银屑病的发生;透皮给药的对照例B6.3几乎没有阻止发病的作用,小鼠皮肤病症与对照组B6.2近似,而实施例B6.5则能够有效缓解银屑病病症。图7中对照例B6.1第1天的小鼠照片损坏,因此未给出,但根据其后续几天的照片依然能够说明问题,不影响本申请所述技术方案的效果判断。
表5:小鼠第1-5天的患处鳞屑和红斑严重程度的评分统计表

图8是实施例B6.1~实施例B6.5中第5天获取的小鼠皮肤组织H&E切片的显微图片,图中的黑色线段标示小鼠皮肤的表层厚度。图9是图8切片数据的ImageJ定量分析结果,皮肤表层的厚度越大,说明皮肤组织炎症越严重,根据ImageJ定量分析结果,实施例B6.5中小鼠皮肤厚度较薄,反映出CS-S08和阿达木单抗的复合物经过透皮给药能改善角质细胞增生,缓解银屑病病症。
综合实施例B6中的多类实验数据,从表观层面到机理层面,均说明本发明所述的改性壳聚糖能够促进阿达木单抗透过皮肤屏障,并且保护阿达木单抗的活性,说明本发明所述的改性壳聚糖能够促进阿达木单抗透皮发挥治疗银屑病的作用,具有应用于经皮给药的潜力。即本申请所述改性壳聚糖能够作为大分子活性成分的载体,用于透皮给药。
实施例B7:CS-S08装载抗原蛋白作为抗肿瘤疫苗的有效性研究。
CS-S08装载抗原蛋白作为抗肿瘤疫苗的有效性研究。
按照实施例2所述的方法制备CS-S08和鸡卵清白蛋白(OVA)的复合物,区别在于,制备过程还加入了PolyIC。其中,CS-S08、OVA、PolyIC的质量比为8:4:1。
具体实验方法为:分别通过皮下注射和涂抹的方式多次给与健康的C57小鼠OVA和PolyIC,以第一次给药时间记为第0天,于第8天、第21天、第28天检测小鼠体内的OVA抗体滴度,并于第28天取脾脏组织样本检测免疫细胞的相对含量。
实验分组和实验条件如下:
对照例B7.1:健康小鼠,未经过任何治疗;
对照例B7.2:第0天进行皮下注射OVA和PolyIC的混合溶液,OVA的使用剂量为60μg/只,PolyIC的使用剂量为15μg/只;
实施例B7.3:第0天、第7天、第14天,将复合物与凡士林混合后涂抹于小鼠裸露的皮肤表面,OVA的使用剂量为20μg/只/次,PolyIC的使用剂量为5μg/只/次。
采取酶联免疫分析(ELISA)检测小鼠血清内的抗体滴度,以对照例B7.1小鼠为背景评估实施例B7.2-B7.3两组小鼠的滴度终点,统计结果如图10所示。结果显示,实施例B7.3中小鼠体内抗体滴度水平接近对照例B7.2,说明经皮给药达到了皮下注射给药近似的疫苗接种效果。
此外,图11是实施例B7中小鼠脾脏中IFN-γ+的CD4+T细胞、CD8+T细胞相对含量统计图。IFN-γ+表示T细胞中γ干扰素的表达,γ干扰素能够促进自然杀伤细胞的活性,IFN-γ+的T细胞含量越高说明能够引发更好的抗肿瘤免疫反应;CD4+T细胞能够调节其他免疫细胞的活性来发挥抗肿瘤作用,CD8+T细胞能够直接杀伤肿瘤细胞。结果显示对照例B7.2和实施例B7.3中IFN-γ+的CD4+T细胞、CD8+T细胞比例均上升,说明二者能够分泌更多的IFN-γ,进而增强抗肿瘤免疫反应。
综上,CS-S08能够促进抗原蛋白透过皮肤并引发免疫记忆效应,说明本发明所述改性壳聚糖能够作为抗原递送载体,用于疫苗制剂的制备,进一步用于疾病预防、肿瘤预防治疗等领域。
现阶段临床常见的疫苗接种方式多为皮下注射、肌肉注射这类侵入性给药方式,对于接 种者尤其是婴幼儿接种者并不友好,局部涂抹的方式取代皮下注射、肌肉注射意义重大,具有减轻患者痛苦,提高患者依从性,降低耗材成本等有益效果。
实施例B8:CS-S08装载过氧化氢酶用于清除皮内活性氧
CS-S08装载过氧化氢酶用于清除皮内活性氧。
按照实施例2所述的方法制备CS-S08和过氧化氢酶的复合物,CS-S08和过氧化氢酶的质量比为5:1。
用咪喹莫特连续5天涂抹小鼠裸露皮肤,建立银屑病模型,以第一次使用咪喹莫特为第0天,第4天获取小鼠患处皮肤组织,制作免疫荧光染色切片,观察活性氧含量。
实验分组和实验条件如下:
对照例B8.1:健康小鼠;
对照例B8.2:银屑病小鼠,未经过任何缓解处理;
实施例B8.3:在第1、2、3天分别将凡士林与复合物混合后涂抹于患处,过氧化氢酶每次给药剂量为100μg/只。
图12是实施例B8中小鼠皮肤组织切片的免疫荧光染色信号图,通过激光共聚焦显微镜拍摄得到,结果显示,对照例B8.1健康小鼠皮肤内没有活性氧信号,对照例B8.2银屑病小鼠皮肤内有明显的活性氧信号,说明银屑病患处皮肤内活性氧含量远高于正常组织,而实施例B8.3中,经过三次干预,活性氧水平明显下降,说明复合物能够经过表皮进入皮肤,尤其是皮内,过氧化氢酶发挥作用,清除活性氧。即说明本发明所述改性壳聚糖能够帮助大分子量的蛋白进入皮内,发挥治疗效果,对于一些由皮内异常导致的疾病,本发明所述改性壳聚糖具有制备相关活性成分制剂的潜力。
说明书实施例B中,凡士林的功能为保证样品在皮肤表面的停留时间,涂抹给药后均使用3M敷贴进行固定,避免因小鼠活动导致样品损失、实验结果偏差,凡士林本身对活性成分透皮的影响有限,可进一步作为经皮给药制剂中增稠剂、粘附剂等辅料使用。此外,为制剂成药需要,还可添加多种辅料,以帮助复合物进一步形成产品。
实施例C:改性壳聚糖(烷基链修饰的壳聚糖)和蛋白复合物在透眼部屏障的应用
实施例C1:按照实施例A2的方法制备改性壳聚糖(实施例A3.2、实施例A3.6、实施例A3.10)和异硫氰酸荧光素标记的牛血清白蛋白(BSA-FITC)的复合物,改性壳聚糖与蛋白的比例为4:1,与滴眼液基质磷酸盐缓冲液(PBS,0.01M,pH=7.4)混合备用,BSA-FITC浓度为1mg/mL。
将小鼠麻醉,给每只眼睛定量给药5μL,在避光条件下饲养小鼠,6小时后牺牲小鼠,用PBS中性缓冲液冲洗眼球,摘取眼球以制作切片样品,然后使用共聚焦显微镜拍摄眼球中央纵切面样本,观察小鼠眼球视网膜部位的荧光信号分布及强度,结果如图13和表6所示。
图13中荧光标记的信号代表蛋白活性成分在眼后段组织中的分布情况,信号距离眼表越远,说明蛋白的渗透越充分,荧光信号越强,说明渗透的量越高,细胞核的标记主要显示出视网膜,脉络膜以及巩膜等组织的位置,便于辨别荧光信号是组织切片中的信号而非未渗透组织的无效分布。从图13中可以看出,实施例C1.1中仅有很小一部分蛋白在巩膜表面,说明游离的蛋白很难透过眼表屏障;实施例C1.2中的蛋白能够更多的穿透眼表屏障,如角膜,结膜屏障,其视网膜部位的荧光信号分布的面积更大,但是更强的荧光信号停留在眼部屏障表面;实 施例C1.3、实施例C1.4、实施例C1.5中,不同长度的脂肪链改性的壳聚糖,与蛋白复合后,能够帮助蛋白更好的渗透眼部屏障到达眼底并累积在视网膜和脉络膜部位,并且这三组样品的蛋白渗透量随着脂肪链的增加而增加,软脂酸改性的壳聚糖的荧光信号最强。将游离的蛋白作为对照,计算各组样品中蛋白标记的相对荧光信号强度,表6结果显示,软脂酸(十六烷酸)改性的壳聚糖,其促进蛋白透过的效率是游离蛋白的26倍以上。
该实施例的结果表明,一定取代度和多种脂肪链长度修饰的阳离子聚合物均能有效促进蛋白分子透过眼部屏障,具有进一步应用于大分子活性成分滴眼递送的潜力。
表6:滴眼给药后6小时,小鼠眼球切片样品视网膜与脉络膜处蛋白标记的荧光信号强度统计表。
实施例C2:定量检测改性壳聚糖促进蛋白渗透眼部屏障
按照实施例A2的方法制备改性壳聚糖(CCS-8、CCS-12、CCS-16)和人源免疫球蛋白(IgG)的复合物,与滴眼液基质磷酸盐缓冲液(PBS,0.01M,pH=7.4)混合备用,IgG浓度为2mg/mL。
同时制备壳聚糖和人源免疫球蛋白的复合物,与滴眼液基质磷酸盐缓冲液(PBS,0.01M,pH=7.4)混合备用,IgG浓度为2mg/mL。
另制备人源免疫球蛋白与滴眼液基质磷酸盐缓冲液(PBS,0.01M,pH=7.4)混合溶液,IgG浓度为2mg/mL。
将小鼠麻醉,然后给每只小鼠的每只眼球定量给药5μL(IgG浓度为2mg/mL),6小时后牺牲小鼠,用中性PBS溶液冲洗眼球表面,摘取眼球,去除眼表多余的组织,称量每个眼球重量并记录,将眼球样品在液氮环境下冷冻研磨得到组织粉末,并使用WB及IP细胞裂解液分散组织粉末,并在4℃条件下处理过夜,高速离心后取上清液,用ELISA检测试剂盒检测上清液中IgG的浓度,结果如表7所示。
由于未渗透眼部屏障进入眼部的蛋白会在解剖前经PBS冲洗后除去,因此样品中检测得到的蛋白浓度越高,说明渗透眼部屏障并进入眼球内部的样品越多,即该组别中的渗透促进剂效果越好。随着改性壳聚糖的脂肪链长度的增加,促进蛋白渗透的效率提高,有更多的蛋白透过角膜进入眼部。通过计算相较于游离蛋白的透过率,结果显示软脂酸改性的壳聚糖,其促进蛋白渗透的效率达到游离蛋白的16倍以上,说明改性壳聚糖能够大大提高蛋白渗透眼部屏障的效率。
表7:不同组别中小鼠眼球中IgG含量统计表

实施例C1的结果直观的表明了活性成分渗透过眼部屏障后在眼睛部位的分布,实施例C2的结果定量地分析了壳聚糖载体材料促进蛋白渗透的效率,综合实施例C1和实施例C2的活体促眼部屏障渗透实验,说明脂肪链修饰的阳离子聚合物具有有效促进蛋白分子透过眼部屏障的效果,有极好的作为渗透促进剂应用于眼部给药的潜力。
实施例C3:按照实施例C2的方法,区别在于用兔取代小鼠,通过解剖分离兔眼不同结构层,定量检测不同部位中的IgG含量,从而评估软脂酸改性的壳聚糖(CCS-16)促进IgG渗透的能力。
按照房水、角膜、晶状体、玻璃体、视网膜的顺序分离各组织,称量各组织重量后冷冻研磨各个样品后用WB及IP细胞裂解液裂解各眼部组织,高速离心后取上清液,施用ELISA试剂盒检测样品中IgG的浓度,表8是兔眼不同组织结构中的IgG含量统计表。
结果显示,五个主要结构中,实施例C3.2中蛋白在各个结构层均有更高的分布量,说明CCS-16能够有效的促进蛋白的渗透,在眼中各个部位,有CCS-16作为渗透促进剂的组别,均有更高的蛋白含量。并且,更多的活性成分富集在视网膜部位,是大多数眼后端疾病治疗的靶部位,进一步验证了这种由烷基链修饰的壳聚糖作为渗透促进剂的组合物在治疗眼后段疾病的潜力。
表8:兔眼不同部位的IgG含量统计表
实施例C4:使用人工泪液基质,探究传统滴眼剂型助剂对改性壳聚糖和蛋白复合物滴眼渗透的影响。
人工泪液基质成分:聚维酮(20g/L)、聚乙烯醇(14g/L)、羟丙甲纤维素(10g/L)、葡聚糖(2g/L)和羟丙甲纤维素(10g/L)。
将实施例C3.2中的复合物与不同人工泪液基质成分分别混合,得到五种不同的滴眼制剂,按照实施例C2的实验方法定量评价不同组别兔眼视网膜组织中的IgG含量,表9为不同组别兔眼视网膜组织中的IgG含量。
结果显示,相比较实施例C4.1,除了聚维酮之外,聚乙烯醇、羟丙甲纤维素以及葡聚糖和羟丙甲纤维素的组合物均对烷基化壳聚糖的滴眼促渗具有促进作用,尤其以葡聚糖和羟丙甲纤维素的复方人工泪眼效果最佳,视网膜组织中的IgG含量提高了3倍以上。而聚维酮对滴眼促渗表现出抑制作用,促渗效率降低,可能是因为聚维酮具有促进上皮细胞间紧密结合,恢复上皮屏障,降低通透性的作用,因此影响了改性壳聚糖的促渗作用。
这些人工泪液基质成分均为市售滴眼制剂中常用的辅料,用于增加活性成分在眼部停留的时间从而增加透过眼部屏障的活性成分量,这些辅料本身不具有促进大分子活性成分透过生 物屏障的能力,对于最终制剂的制备有帮助。
表9:实施例C4中不同组别兔眼视网膜组织中的IgG含量
实施例C5:改性壳聚糖在滴眼应用中的安全性
以眼部状态变化作为观测指标,记录实验中兔子眼部是否出现眼角膜浑浊、虹膜纹理不清晰、结膜充血、水肿、异常分泌物等情况,在给药后短期及长期观测中,如无前述异常情况出现,说明该组别的复合物安全性良好。
制备葡聚糖和羟丙甲纤维素的组合物作为滴眼基质,和实施例C2.2、实施例C2.3、实施例C2.4的复合物混合制备眼药水,然后对兔子眼表进行滴眼给药,IgG浓度为2mg/mL,每只眼睛给药200μL,每天给药两次,持续四周,观察每次滴眼后,兔子眼部是否出现异常,并观察四周之后兔子眼部状态,结果发现,兔子眼部均未出现前述异常情况,说明改性壳聚糖不具有短期和长期的眼部刺激性,安全性较好。
实施例C6:使用改性壳聚糖和anti-VEGFA的复合物对黄斑病变的治疗实验。
黄斑区是位于视网膜的一个重要区域,位于眼后段,主要与精细视觉、色觉功能等视功能有关,黄斑区的病变会引起视力下降、眼前黑影、视物变形等症状,影响患者视觉功能。黄斑病变的诱因多样,遗传、年老、炎症等均可能导致黄斑病变。黄斑病变患者的晚期病征是因眼球内的血管异常增生,突破了脉络膜甚至是视网膜,异常的血管壁脆弱,易出现血管渗漏,加重病人视力模糊感,并加重黄斑病变病情。及时在该阶段实施有效的临床治疗,能够降低失明的几率。临床用于治疗黄斑病变的活性成分主要是血管生成因子抗体如雷珠单抗,康柏西普,阿柏西普等,这些大分子抗体活性成分在临床试验中具有较好的短期疗效。
建立激光诱导的小鼠脉络膜新生血管模型,模拟黄斑病变疾病。患有脉络膜新生血管的小鼠被随机分为四组:
实施例C6.1:施用无活性成分的PBS(浓度0.01M,pH=7.0)
实施例C6.2:滴眼给药方式,使用PBS(浓度0.01M,pH=7.0)溶解anti-VEGFA得到滴眼液,anti-VEGFA浓度为2mg/mL,每天进行滴眼治疗一次,给药体积为5μL;
实施例C6.3:滴眼给药方式,使用PBS(浓度0.01M,pH=7.0)溶解实施例A3.11制备得到的CCS-16和anti-VEGFA的复合物得到滴眼液,使用人工泪液溶解,anti-VEGFA的浓度为2mg/mL,每天进行滴眼治疗一次,给药体积为5μL,载体和蛋白的质量比为5:1;
实施例C6.4:玻璃体腔注射anti-VEGFA的PBS溶液,浓度10mg/mL注射体积5μL,作为阳性对照
经过两周的治疗,利用光学相干断层扫描以及眼底荧光造影技术对给药前后患有脉络膜新生血管的小鼠进行脉络膜新生血管严重程度的评价,图14是小鼠光学相干断层扫描以及眼底荧光造影图像。脉络膜新生血管面积增加说明病情加重,反之说明好转,即活性成分起效。 从图中可以看出,经过CCS-16/anti-VEGFA滴眼液以及作为阳性对照的游离anti-VEGFA玻璃体腔注射的两周治疗后,脉络膜新生血管渗漏明显减少。此外,对脉络膜新生血管面积统计,并计算脉络膜新生血管面积%(表10)发现,实施例C6.1和实施例C6.2对脉络膜新生血管渗漏面积无改善,脉络膜新生血管面积增加,而实施例C6.3以及作为阳性对照的实施例C6.4的小鼠在治疗后有效抑制脉络膜新生血管渗漏面积,约为治疗前的40%。说明改性壳聚糖和抗体活性成分的复合物能够促进滴眼方式给药的活性成分渗透率,并且活性成分能够有效发挥作用,从而达到和玻璃腔体注射类似的治疗效果,而玻璃体注射给药是目前临床上必要的治疗手段,因其侵入性导致患者依从性差,如果能够通过滴眼的方式实现治疗,将具有重要意义。
表10:脉络膜新生血管面积变化统计表
实施例C7:改性壳聚糖的滴眼促渗效果研究
实施例B1的改性壳聚糖和蛋白的复合物滴眼渗透效果实验。
按照实施例A2的方法制备改性壳聚糖(实施例A10.2、实施例A11.2、实施例A13.2)和人源免疫球蛋白(IgG)的复合物,载体和蛋白的比质量比为2:1,与滴眼液基质磷酸盐缓冲液(PBS,0.01M,pH=7.4)混合备用,IgG浓度为2mg/mL。
同时制备单独的IgG与滴眼基质酸盐缓冲液(PBS,0.01M,pH=7.4)混合溶液,IgG浓度为2mg/mL。将小鼠麻醉,然后使用定量给药器给每只小鼠的每只眼球定量给药5μL,饲养小鼠6小时,然后牺牲小鼠,用中性PBS溶液冲洗眼球表面,摘取眼球,去除眼表多余的组织,称量每个眼球重量后将眼球样品在液氮环境下冷冻研磨得到组织粉末,并使用WB及IP细胞裂解液分散组织粉末,并在4℃条件下处理过夜,高速离心后取上清液,用ELISA检测试剂盒检测上清液中IgG的浓度。结果显示,几种改性壳聚糖均能够促进IgG渗透眼部屏障,进入眼球,预期可实现分子量与IgG接近或更小的活性成分的渗透。
实施例D:眼部疾病治疗
实施例D1:实施例B1所述改性壳聚糖(CS-S08)和蛋白大分子活性成分复合物用于小鼠湿性年龄相关黄斑变性的治疗。
湿性年龄相关黄斑变性的特点是眼底形成异常新生血管,是危害视力、致盲的主要原因,目前临床可使用玻璃体腔内注射阿柏西普进行治疗,阿柏西普是一种全人类的重组融合蛋白,能够抑制血管内皮生长因子和胎盘生长因子,阿柏西普的蛋白质分子量约96kD,糖基化后分子量约115kD。
本实施例中使用改性壳聚糖CS-S08和阿柏西普形成复合物,通过滴眼的给药方式进行有效成分的递送,用于湿性年龄相关黄斑变性的治疗。
具体地,采用激光光凝术构建小鼠黄斑变性模型,建模当天记为第0天,第7天起,进行分组治疗,实验分组及实验方法如下:
实施例D1.1:不进行任何治疗;
实施例D1.2:玻璃体内注射阿柏西普溶液,以阿柏西普质量计算,剂量为80μg/眼,仅给 药一次;
实施例D1.3:人工泪液溶解的阿柏西普,以阿柏西普质量计算,剂量为10μg/天/眼,连续给药28天;
实施例D1.4:人工泪液溶解的复合物,所述复合物按照实施例A2的方法制备得到,改性壳聚糖为CS-S08,蛋白大分子活性成分为阿柏西普,改性壳聚糖CS-S08和阿柏西普的质量比为2:1,以阿柏西普质量计算,剂量为10μg/天/眼,连续给药28天。
在第7天通过荧光素眼底血管造影术验证小鼠黄斑变性模型的成功建立并分组给药治疗,在第14天、第21天、第28天、第35天通过荧光素眼底血管造影术进行疗效评价,并通过ImageJ对光斑渗漏面积进行测量和统计,具体公式如下:
相对病变面积=第X天的光斑渗漏面积/第7天光斑渗漏面积。
图15是实施例D1.1~1.4小鼠眼部造影成像图,图16是实施例D1.1~1.4小鼠眼脉络膜的相对病变面积统计图。
结果显示,第14天,实施例D1.2中通过玻璃体内注射阿柏西普已表现出抑制新生血管生成的效果,说明阿柏西普的现有给药方式能够达到治疗效果。同时,根据统计数据,实施例D1.3用人工泪液直接溶解阿柏西普进行滴眼,不能有效控制新生血管生成,说明大分子活性成分在没有载体帮助时直接滴眼递送不能透过眼部屏障,到达病变部位,发挥疗效。而实施例D1.4中,用改性壳聚糖与阿柏西普形成复合物,再使用人工泪液分散后滴眼,能够抑制新生血管生成,且随着治疗时间的增加,最终治疗效果接近玻璃体一次性注射给药的实施例D1.2的治疗效果。
综合来看,改性壳聚糖能够帮助大分子活性成分透过眼部屏障,并发挥疗效,对眼后区疾病进行治疗,具备制备滴眼制剂的潜力。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。
尽管上述披露中通过各种示例讨论了一些目前认为有用的发明实施例,但应当理解的是,该类细节仅起到说明的目的,附加的权利要求并不仅限于披露的实施例,相反,权利要求旨在覆盖所有符合本申请实施例实质和范围的修正和等价组合。
同理,应当注意的是,为了简化本申请披露的表述,从而帮助对一个或多个发明实施例的理解,前文对本申请实施例的描述中,有时会将多种特征归并至一个实施例、附图或对其的描述中。但是,这种披露方法并不意味着本申请对象所需要的特征比权利要求中提及的特征多。实际上,实施例的特征要少于上述披露的单个实施例的全部特征。
一些实施例中使用了描述成分、属性数量的数字,应当理解的是,此类用于实施例描述的数字,在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明,“大约”、“近似”或“大体上”表明所述数字允许有在指定数值以上或以下0.5%-10%的范围内变动,例如在指定数值以上或以下0.5%、1%、1.5%、2%、2.5%、3%、3.5%、4%、4.5%、5%、5.5%、6%、6.5%、7%、7.5%、8%、8.5%、9%、9.5%、或10%的范围内变动。相应地,在一些实施例中,说明书和权利要求中使用的数值参数均为近似值,该近似值根据个别实施例所需特点可以发生改变。
最后,应当理解的是,本申请中所述实施例仅用以说明本申请实施例的原则。其他的变形也可能属于本申请的范围。因此,作为示例而非限制,本申请实施例的替代配置可视为与本 申请的教导一致。相应地,本申请的实施例不仅限于本申请明确介绍和描述的实施例。

Claims (20)

  1. 一种改性壳聚糖,其特征在于,
    所述改性壳聚糖具有通式Ⅰ:CS-X1-R1
    所述CS为壳聚糖或壳聚糖的衍生物,所述壳聚的分子量范围在1000-5000000,脱乙酰度不低于55%;
    所述X1选自酰胺键、磺酰胺键、席夫碱基、仲胺、烷基胺、氨基甲酸酯、酯键、醚、磷酸酯、磺酸酯、脲、磷酰胺、脒键中的任意一种;
    所述R1来自于所述修饰分子;
    所述修饰分子选自脂肪族化合物。
  2. 根据权利要求1所述的改性壳聚糖,其特征在于,所述脂肪族化合物选自脂肪酸、脂肪醛、脂肪酰卤、脂肪酸酐、脂肪族聚酯。
  3. 根据权利要求1所述的改性壳聚糖,其特征在于,所述X1是酰胺键,所述R1是含有6个碳原子的脂肪链、含有7个碳原子的脂肪链、含有11个碳原子的脂肪链、含有15个碳原子的脂肪链中的任意一种。
  4. 如权利要求1所述的改性壳聚糖,其特征在于,所述改性壳聚糖的取代度不超过40%。
  5. 一种改性壳聚糖,其特征在于,所述改性壳聚糖具有通式Ⅱ:
    CS-X1-R2-R3
    所述CS为壳聚糖或壳聚糖的衍生物,所述壳聚的分子量范围在1000-5000000,脱乙酰度不低于55%;
    所述X1选自酰胺键、磺酰胺键、席夫碱基、仲胺、烷基胺、氨基甲酸酯、酯键、醚、磷酸酯、磺酸酯、脲、磷酰胺、脒键中的任意一种;
    或所述X1选自-NH-CO-、-NH-SO2-、-N=C-、-NH-、-NH-CH2-、-NH-CH2-CH(OH)-、-NH-CO-O-、-O-CO-、-O-CH2-O-CH2-、-O-PO-(OR2)2、-O-SO2-、-NH-CO-NH-、-NH-PO-(OR2)2、-NH-C(=NH2+)-中的任意一种;
    所述R2为含有n个碳原子的脂肪链,其中2≤n≤20;
    所述R3含有酚羟基或其衍生物、邻苯二酚基团或其衍生物、邻苯三酚基团或其衍生物中的一种或多种;
    所述R3通过连接基团与R2相连接。
  6. 如权利要求5所述的改性壳聚糖,其特征在于,所述改性壳聚糖具有如下化学结构式:
    x、y、z均为大于零的整数。
  7. 如权利要求5所述的改性壳聚糖,其特征在于,所述改性壳聚糖的取代度不超过20%。
  8. 一种权利要求1或5所述的改性壳聚糖的制备方法,其特征在于,通过修饰分子与壳聚糖或壳聚糖的衍生物反应;
    所述修饰分子含有以下基团中的一种或多种:羧酸基团、磺酸基团、酸酐、酰卤基团、磺酰氯、酰氯、NHS酯、亚氨酸酯、五氟苯酯、醛基、异氰酸酯、环氧基团、双键、炔基、羟甲基膦。
  9. 一种透生物屏障的复合物,其特征在于,包括权利要求1或5任一项所述的改性壳聚糖和被所述改性壳聚糖装载递送的活性成分;
    所述改性壳聚糖和所述活性成分以物理或化学方式结合;
    所述活性成分选自小分子活性成分和/或大分子活性成分。
  10. 根据权利要求9所述的透生物屏障的复合物,其特征在于,所述生物屏障包括皮肤屏障、口腔黏膜、阴道黏膜、膀胱黏膜、眼部屏障、泪液屏障、角膜/结膜屏障、血房水屏障、血视网膜屏障中的任一种。
  11. 根据权利要求9所述的透生物屏障的复合物,其特征在于,所述大分子活性成分的分子量不超过240kD。
  12. 根据权利要求9所述的透生物屏障的复合物,其特征在于,所述大分子活性成分选自蛋白类活性成分、其他多肽类活性成分、激素类活性成分、抗体类活性成分、生长因子、核酸物质中的一种或多种。
  13. 根据权利要求9所述的透生物屏障的复合物,其特征在于,所述小分子活性成分选自抗肿瘤活性成分、抗生素、免疫调节剂、镇痛药、眼病治疗活性成分、抗感染活性成分、靶向抑制剂中的一种或多种。
  14. 根据权利要求9所述的透生物屏障的复合物,其特征在于,所述活性成分选自抗衰老成分、抗菌成分、抗敏成分、抗皱成分、抗氧化成分中的一种或多种。
  15. 根据权利要求1所述的改性壳聚糖或权利要求9所述的透生物屏障的复合物在制备疾病治疗活性成分或医疗美容产品中的应用。
  16. 根据权利要求15所述的应用,其特征在于,所述疾病选自肿瘤、眼部疾病、代谢疾病、疼痛、炎症、免疫性疾病、神经系统疾病、精神性疾病、生殖系统疾病、骨骼疾病、口腔疾病、激素紊乱、呼吸系统疾病中的任一种。
  17. 一种透皮制剂,其特征在于,包括权利要求9所述的复合物。
  18. 根据权利要求17所述的透皮制剂,其特征在于,还包括增稠剂、赋形剂、抗过敏成分、保湿成分、抑菌剂、结晶抑制剂、增溶剂、防腐剂中的一种或多种。
  19. 一种滴眼制剂,其特征在于,包括权利要求9所述的复合物。
  20. 根据权利要求19所述的滴眼制剂,其特征在于,所述制剂还包括分散剂、赋形剂、增稠剂、保湿成分、抑菌剂、结晶抑制剂、防腐剂中的一种或多种。
PCT/CN2023/123053 2022-09-30 2023-09-30 一种改性壳聚糖及其在大分子活性成分递送中的应用 WO2024067874A1 (zh)

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