Use of fluorine-containing compound-modified chitosan as a drug carrier and a method for preparing the same

Abstract

The application discloses a fluorine-modified chitosan derivative used as a drug carrier, which has the following structure: a fluorine-containing compound is covalently connected to a chitosan main chain, the molecular weight of the chitosan ranges from 5000 to 5000000, and the fluorine-containing compound is a fluorine-containing aliphatic chain shown in the following formula (I) or an aromatic ring functional group shown in the following formula (II), wherein R1 is halogen (fluorine, chlorine, bromine or iodine), halogen-substituted alkane, cycloalkane, aldehyde group, carboxyl group, double bond, acetylene bond, hydroxyl group, sulfonyl chloride, sulfonic acid bond or sulfydryl, which are active groups capable of reacting with primary amino groups. The application aims to provide a novel drug carrier material with obvious drug absorption promotion effect and low toxicity, and the synthesis process of the fluorine-containing compound-modified chitosan is mature, simple to operate, high in synthesis efficiency, short in period, and capable of obtaining a high-yield drug carrier without complicated purification steps, so that the simple synthesis method provides a good foundation for commercialization.

Description

Technical Field

[0001] This invention relates to the fields of polymer chemistry and pharmaceutical biomaterials, specifically to fluoride-modified chitosan polymeric drug carriers, their preparation methods, and applications. Background Technology

[0002] In recent years, hydrophilic cationic polymers such as polyethyleneimine (PEI) and polylysine have demonstrated the ability to bind with nucleic acids, peptides, and proteins to form nanocomposites. These nanocomposites not only facilitate the entry of these macromolecules into cells but also protect drugs from degradation by hydrolytic enzymes in the microenvironment. Their internal tertiary ammonium structures, acting as proton sponges, promote drug escape from cellular endosomes. Furthermore, cationic polymers can increase epithelial permeability by weakening the tight junctions of epithelial cells, thereby enhancing the absorption efficiency of large drug molecules within epithelial cells. However, the high cytotoxicity of cationic polymers during use ultimately limits their clinical application.

[0003] Chitosan, a cationic polysaccharide derived from the deacetylation of chitin, possesses excellent biosafety properties and superior mucosal adhesion, making it widely used in the design of transmucosal drug delivery formulations. Literature reports that chitosan can achieve mucosal adhesion through the interaction of its positive charge with the negative charge on the skin and mucous membrane surfaces, as well as the hydrophobic effect of its hydrophobic groups. This effectively prolongs the retention time of bioactive substances (drugs, peptides, proteins, etc.) in chitosan solutions at the lesion site. Driven by diffusion or subsequent chitosan degradation, the active substances are slowly released from the chitosan solution, achieving a long-lasting sustained-release effect on the skin and mucous membranes. Although chitosan can significantly improve the bioavailability of infused drugs, high concentrations of chitosan may cause severe mucosal and epithelial damage, limiting its clinical application as a drug carrier.

[0004] Therefore, inventing a novel drug carrier material that significantly promotes drug absorption while exhibiting low cytotoxicity is a challenging direction. Summary of the Invention

[0005] The purpose of this invention is to provide a novel drug carrier material that significantly promotes drug absorption and has low toxicity. Furthermore, the fluorine-modified chitosan synthesis process proposed in this invention is mature, easy to operate, highly efficient, and has a short cycle. It can obtain high-yield drug carriers without cumbersome purification steps. Its simple synthesis method provides a good foundation for its commercialization. The fluorine-modified chitosan described in this invention has the potential to be used as a variety of drug carriers.

[0006] This patent provides the following technical solution: a fluorinated chitosan derivative for use as a drug carrier, having the following structure: a fluorinated compound is covalently linked to the chitosan backbone, wherein the chitosan has a molecular weight range of 5000-5000000 and a degree of deacetylation greater than 55%.

[0007] The fluorine-containing compound has the following chemical formula (Ⅰ). The fluorinated aliphatic chain shown, or formula (II), The aromatic ring functional group shown, wherein R1 is a halogen (fluorine, chlorine, bromine, iodine), halogen-substituted alkanes, cycloalkanes, aldehydes, carboxyl groups, double bonds, alkynes, hydroxyl groups, sulfonyl chlorides, sulfonic acid bonds, or thiol groups, which are active groups capable of reacting with primary amino groups.

[0008] This invention provides a fluorinated chitosan derivative for use as a drug carrier, having a chitosan molecular backbone containing primary amino groups as shown in formula (IV):

[0009]

[0010] The linking groups formed between the primary amino groups and the fluorinated functional groups of the chitosan are: -NH-, -N=C-, -NHCH2CH(OH)-, -NHCH2CH(OH)CH2O-, and derived groups;

[0011] The fluorinated functional groups are fluorinated aliphatic chains and aromatic ring functional groups.

[0012] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention: in the formula (Ⅰ), x is an integer from 0 to 3, y is an integer from 0 to 20, z is an integer from 0 to 8, and R2 is CF3, CHF2, CH2F, or CH3 (when y is not 0).

[0013] The fluorinated aliphatic chain compounds refer to fluorinated hydrocarbon groups and their derivatives, including trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid, undecanoic acid, tridecafluoroheptanoic acid, pentafluorooctanoic acid, heptadecafluorononanoic acid, nonafluoroquinic acid, perfluorobutyric anhydride, perfluoroheptanoic anhydride, perfluorodecanoic anhydride, 2,2,3,3,4,4,4-heptafluorobutyl acrylate, 3-(1H,1H,5H octafluoropentoxy)-1,2-epoxypropylene, nonafluorobutyl sulfonamide anhydride and their derivatives.

[0014] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention, in formula (II), R is H, CH3, OH, NO2, O, CF3, F, CH2OH, CN, NCO, or (CF2)aCF3 (a is an integer from 1 to 20), and at least one R is F;

[0015] The fluorinated aromatic ring compounds include 3-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2,3,5,6-tetrafluoro-4-methylbenzoic acid, pentafluorobenzoic acid, 2-fluoro-3-(trifluoromethyl)benzoic acid and their derivatives.

[0016] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention, the chitosan and a fluorinated compound are covalently linked, and the surface of the chitosan molecule is modified to form a drug carrier, the structure of which is shown in formula (V), where b and c are both integers from 20 to 500, and formula (V) is as follows:

[0017]

[0018] In this context, B is a linking group formed by a fluorinated functional group and a primary amino group of chitosan, and C is a fluorinated aliphatic chain and aromatic ring functional group.

[0019] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention, the fluorinated aliphatic chain is a class of fluorinated compounds with active groups that can react with amino groups, including those shown in formula (VI):

[0020]

[0021] Where A is -COOH, The active group that can react with primary amines, where x is an integer from 0 to 3 and y is an integer from 0 to 8.

[0022] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention, the fluorinated aromatic ring compound is a class of fluorinated compounds with active groups that can react with amino groups, including those shown in formula (VII):

[0023]

[0024] As a preferred embodiment of the fluorinated chitosan derivative used as a drug carrier according to the present invention, the fluorinated chitosan derivative serves as a drug carrier for drugs including small molecule drugs, peptides, protein drugs, combinations of different drugs, and combinations of drugs with other pharmaceutical excipients.

[0025] The application of fluorinated chitosan as a drug carrier: The fluorinated chitosan derivatives can be used as drug carriers for small molecule drugs, peptides, protein drugs, combination drugs of different drugs, and combination drugs of drugs and other pharmaceutical excipients.

[0026] A method for preparing fluorinated chitosan derivatives includes the following steps: preparing a chitosan-acetic acid aqueous solution, weighing chitosan and adding it to the acetic acid aqueous solution, stirring to dissolve it completely, then adding sodium hydroxide dropwise, stirring until the solution is clear and the pH is 6.2-6.8;

[0027] Activation of fluorine-containing compounds: Weigh the fluorine-containing compound and dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide. Then add the reaction amount of EDC and NHS in sequence and stir in the dark.

[0028] The activated fluorine-containing compound solution was added dropwise to the rapidly stirred chitosan solution, and stirred in the dark until the reaction was complete.

[0029] The method for preparing fluorinated chitosan derivatives further includes the following steps: slowly adding the fully reacted solution dropwise to a potassium hydroxide ethanol solution and stirring, filtering the precipitate, washing with a large amount of anhydrous ethanol until the filtrate is neutral, washing the precipitate with methanol and ether to remove water, vacuum drying, dissolving the dried precipitate in hydrochloric acid solution, and lyophilizing to obtain fluorinated chitosan hydrochloride.

[0030] A method for preparing fluorinated chitosan with 3-fluorobenzoic acid, comprising the following steps:

[0031] (1) Preparation of chitosan acetic acid aqueous solution: Weigh out fully dried chitosan and add it to acetic acid aqueous solution. Stir to dissolve it completely, then slowly add sodium hydroxide dropwise and stir until the solution is clear and the pH is 6.2-6.8.

[0032] (2) Activation of 3-fluorobenzoic acid: Weigh 3-fluorobenzoic acid, dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide, add the reaction amount of EDC in sequence, and stir thoroughly in the dark with NHS.

[0033] (3) Preparation of 3-fluorobenzoyl chitosan: The activated 3-fluorobenzoic acid solution was slowly added dropwise to the chitosan solution that was being stirred rapidly, and the mixture was stirred in the dark to allow it to react fully.

[0034] The method for preparing 3-fluorobenzoic acid fluorinated chitosan further includes the following steps:

[0035] The fully reacted solution was slowly added dropwise to a potassium hydroxide ethanol solution and stirred thoroughly. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water and then dried under vacuum.

[0036] The dried precipitate was dissolved in hydrochloric acid solution and freeze-dried to obtain 3-fluorobenzoic acid fluorinated chitosan hydrochloride molecules.

[0037] A method for preparing perfluoroheptanoic acid fluorinated chitosan includes the following steps:

[0038] (1) Preparation of chitosan acetic acid aqueous solution: Weigh out fully dried chitosan and add it to acetic acid aqueous solution. Stir to dissolve it completely, then slowly add sodium hydroxide dropwise and stir until the solution is clear and the pH is 6.2-6.8.

[0039] (2) Activation of perfluoroheptanoic acid (13-fluoroheptanoic acid): Weigh perfluoroheptanoic acid, dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide, add an appropriate amount of EDC in sequence, and stir thoroughly in the dark with NHS.

[0040] (3) Preparation of 13F heptanoic acid chitosan: The activated perfluoric acid solution was slowly added dropwise to the chitosan solution that was being stirred rapidly, and the mixture was stirred in the dark to allow it to react fully.

[0041] The method for preparing perfluoroheptanoic acid fluorinated chitosan further includes the following steps:

[0042] The fully reacted solution was slowly added dropwise to a potassium hydroxide ethanol solution and stirred thoroughly. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water. It was then vacuum dried, and the dried precipitate was dissolved in hydrochloric acid solution and freeze-dried to obtain perfluoroheptanoic acid fluorinated chitosan hydrochloride.

[0043] The fluorinated chitosan derivative used as a drug carrier: the fluorinated chitosan derivative is a perfluoroheptanoic acid fluorinated chitosan hydrochloride molecule, and the degree of fluorination modification of the perfluoroheptanoic acid fluorinated chitosan hydrochloride is 18% to 25% or 20% to 22%.

[0044] A drug complex comprising a fluorinated chitosan derivative used as a drug carrier and a drug, said drug including small molecule drugs, peptides, protein drugs, combinations of different drugs, and combinations of drugs with other pharmaceutical excipients.

[0045] Given that the high cytotoxicity of cationic polymer materials during use ultimately limits their clinical application, and that while chitosan can significantly improve the bioavailability of infused drugs, high concentrations of chitosan may cause severe mucosal and epithelial damage, limiting its clinical application as a drug carrier, the inventors have recently explored various technical solutions to improve biocompatibility. For example, they have attached various modifying groups, such as polyethylene glycol, cyclodextrin, amino acids, glycosyl groups, and fluorination, to the surface of these cationic polymers, but with little effect. In a chance experiment, they discovered that fluorinated cationic polymer materials exhibit significantly improved biocompatibility and macromolecular transport efficiency, and demonstrate stronger binding protection for nucleic acid, peptide, and protein drugs.

[0046] Furthermore, the inventors designed and synthesized a series of fluorinated chitosan derivatives. Experimental results showed that fluorinated chitosan (FCS) had a more significant effect on promoting drug penetration and absorption than chitosan. In addition, cell and mouse in vivo safety evaluation results showed that FCS had good biosafety. Even high concentrations of FCS did not have obvious cytotoxicity or mucosal epithelial damage. Its biotoxicity was significantly lower than that of unmodified chitosan (CS).

[0047] The inventors established an in vitro bladder mucosal barrier model using SV-HUC-1 human normal bladder cancer commercial cells. By investigating the effects of FCS on the membrane resistance of SV-HUC-1 monolayer cells, the permeability of fluorescein, the ultrastructure of tight junctions, and tight junction proteins, they elucidated the mechanism by which FCS promotes drug permeation and absorption in the bladder mucosa. Experimental results showed that FCS significantly reduced the membrane resistance of SV-HUC-1 monolayer cells, increased the permeation efficiency of fluorescein, and increased the efficiency of drug molecule uptake via the cellular bypass pathway by regulating tight junctions through alterations in the structure and spatial distribution of tight junction proteins and E-cadherin protein. In other words, FCS can effectively increase the intercellular gaps in biological tissue barriers (such as mucosal epithelial tissue), thereby enabling free drug molecules or drugs carried by FCS to more effectively cross these biological tissue barriers.

[0048] Furthermore, extensive experimental data show that as the length and degree of substitution of the fluorinated fatty chains on the chitosan backbone increase, the drug penetration and absorption promotion capacity of the modified product first increases and then decreases. This indicates that chitosan modification should not be excessively fluorinated, and the effect of fluorinated chitosan on promoting the mucosal penetration and absorption of infused drugs may be the result of the combined action of the positively charged molecular backbone of chitosan and the fluorinated fatty chains.

[0049] Therefore, this patent selects FCS as a novel drug carrier for further research. This patent designs and synthesizes a series of fluorinated chitosan derivatives, whose applications in the pharmaceutical field include, but are not limited to, the following disease models: bladder cancer instillation (or other intracavitary instillation), pulmonary inhalation, transdermal delivery, and oral delivery.

[0050] Bladder cancer is one of the most common urological tumors. Clinically, more than 75% of bladder cancers are non-muscle-invasive bladder cancer (NMIBC). Among these, 30%–80% of NMIBC patients relapse within 5 years after transurethral resection of bladder tumor (TURBT), and 10%–20% of NMIBC patients progress to muscle-invasive bladder cancer. Therefore, adjuvant instillation chemotherapy or immunotherapy after TURBT to inhibit or delay tumor recurrence has become the preferred treatment option in clinical guidelines for bladder cancer. Although adjuvant chemotherapy drugs after TURBT can delay tumor recurrence, due to the physiological characteristics of the bladder and the physiological barrier function of its mucosa, the retention time of the drug solution in the bladder by traditional methods is limited, the duration of action is short, and the bioavailability is low. This prevents time- and concentration-dependent instillation drugs from exerting a significant anti-tumor effect, and thus cannot effectively reduce the absolute risk of bladder cancer recurrence and progression or effectively improve prognosis. This patented technology utilizes FCS as a novel transmucosal drug carrier, which improves the bioavailability of instilled drugs and increases the efficiency of drug delivery into the internal structures of bladder tumors after intracavitary instillation, thereby enhancing the efficacy of bladder instillation therapy. This technology can also be applied to other intracavitary instillation therapies (such as those in the abdominal cavity, pelvis, and pleural cavity).

[0051] Compared to intravenous administration, pulmonary inhalation delivers drugs locally to tumor tissue, requiring significantly lower dosages and resulting in fewer toxic side effects. The unique physiological structure of the lungs determines the characteristics and advantages of pulmonary inhalation: the lungs have a large surface area, abundant capillaries, and a thin alveolar epithelial cell layer, leading to rapid onset of action; the concentrated distribution and low bioactivity of metabolic enzymes in the lungs reduce protein hydrolysis, allowing proteins and peptides to be rapidly absorbed through the alveolar surface, maintaining their bioactivity; and the first-pass effect of the liver is avoided. However, some drawbacks limit its clinical application, such as the rapid clearance of inhaled drugs from the lungs, making effective drug deposition in the lungs uncertain. For lung cancer, although inhaled drugs can reach the alveoli, their efficiency in entering the lung tumor is generally low, severely impacting the efficacy of pulmonary inhalation in treating lung cancer. This patent's technical solution, based on FCS as a novel transmucosal drug carrier, can improve the bioavailability of inhaled drugs, increase the efficiency of drug entry into the lung tumor structure after inhalation, and thus improve the efficacy of pulmonary inhalation therapy.

[0052] Transdermal drug delivery systems refer to formulations that deliver drugs to the skin surface, allowing the drug to penetrate the skin layers at a controlled rate and enter the systemic circulation to produce systemic or local therapeutic effects. As a non-invasive topical drug delivery method, transdermal drug delivery offers numerous advantages, including ease of operation and high patient adaptability. However, transdermal drug delivery is often limited by the lipid barrier of the stratum corneum and the physicochemical properties of the drugs. Improving the ability of drugs to enter the bloodstream or reach subcutaneous lesions (such as skin cancer) through transdermal delivery is a significant challenge for this technology. The technical solution of this patent is based on FCS as a novel transmucosal drug carrier, which can improve the bioavailability of transdermal drugs and significantly enhance their ability to penetrate the skin barrier. This allows drugs to more effectively enter the bloodstream or reach subcutaneous lesions (such as skin cancer) through transdermal delivery, thereby improving the efficacy of transdermal drug delivery therapy.

[0053] This invention provides the application of chitosan modified with fluorine compounds in promoting drug absorption efficiency; and provides chitosan modified with fluorine compounds and its use as a carrier for various drugs.

[0054] The fluorinated compound-modified chitosan described in this patent is covalently linked to the chitosan backbone; the chitosan has a molecular weight range of 5,000-5,000,000, a degree of deacetylation of 55%-100%, and a viscosity of 25-1,000 centipoise (1% acetic acid solution); the drug is a small molecule drug, polypeptide, protein drug, combination drug of different drugs, or combination drug of drug and other pharmaceutical excipients, which can be applied to various related diseases.

[0055] The fluorine-containing compound includes the following chemical formula (Ⅰ). Japanese style (II) The fluorinated aliphatic chains and aromatic ring functional groups shown are shown, wherein R1 is a halogen (F, Cl, Br, I), halogen-substituted alkanes, cycloalkanes, aldehydes, carboxyl groups, double bonds, alkynes, hydroxyl groups, sulfonyl chlorides, sulfonic acid bonds, mercapto groups and other active groups that can react with primary amino groups.

[0056] In formula (Ⅰ), x is an integer from 0 to 3, y is an integer from 0 to 20, z is an integer from 0 to 8, and R2 is CF3, CHF2, CH2F, or CH3 (when y is not 0); the fluorinated aliphatic chain compounds include trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid, undecanoic acid, tridecafluoroheptanoic acid, pentafluorooctanoic acid, heptadecafluorononanoic acid, nonafluoroquinic acid, perfluorobutyric anhydride, perfluoroheptanoic anhydride, perfluorodecanoic anhydride, 2,2,3,3,4,4,4-heptafluorobutylacrylate, 3-(1H,1H,5H octafluoropentoxy)-1,2-epoxypropylene, nonafluorobutyl sulfonamide anhydride and their derivatives, etc.

[0057] In formula (II), R is H, CH3, OH, NO2, O, CF3, F, CH2OH, CN, NCO, or (CF2)aCF3 (a is an integer from 1 to 20), and at least one R is F; the fluorinated aromatic ring compound includes 3-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2,3,5,6-tetrafluoro-4-methylbenzoic acid, pentafluorobenzoic acid, 2-fluoro-3-(trifluoromethyl)benzoic acid and its derivatives.

[0058] The structure of chitosan modified with fluorine-containing compounds is shown in formula (III).

[0059]

[0060] Wherein, A is the chitosan molecular backbone containing primary amino groups, as shown in formula (Ⅳ):

[0061]

[0062] B is a linking group formed by a fluorinated functional group and a primary amino group of chitosan, such as -NH-, -N=C-, -NHCH2CH(OH)-, -NHCH2CH(OH)CH2O-, And derivative groups.

[0063] C is a fluorinated aliphatic chain or aromatic ring functional group.

[0064] The chitosan and fluorinated compound described in this invention are covalently linked. The surface of the chitosan molecule is modified to form a novel chitosan drug carrier modified with fluorinated aliphatic chain or aromatic ring compound. For example, it can be used as a drug carrier to promote the absorption of instilled drugs by the bladder mucosa. Its structure is shown in formula (V), where b and c are both integers from 20 to 500.

[0065] The chitosan is a chitosan with a molecular weight ranging from 5,000 to 5,000,000, a degree of deacetylation of 55% to 95%, and a viscosity of 25 to 1,000 centipoise (in 1% acetic acid solution). Its structure is shown in formula (Ⅳ), where n is an integer from 20 to 2,000, and the surface of this chitosan polymer has primary amine groups.

[0066]

[0067] In this invention, "fluorinated aliphatic chain" refers to fluorinated hydrocarbon groups and their derivatives, such as trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, nonafluoropentanoic acid, undecanoic acid, tridecafluoroheptanoic acid, pentadecanoic acid, heptadecafluorononanoic acid, nonadecanoic acid, perfluorobutyric anhydride, perfluoroheptanoic anhydride, perfluorodecanoic anhydride, 2,2,3,3,4,4,4-heptafluorobutyl acrylate, 3-(1H,1H,5H-octafluoropentoxy)-1,2-epoxypropylene, nonafluorobutyl sulfonamide anhydride, and their derivatives; the fluorinated aliphatic chain is a class of fluorinated compounds with active groups that can react with amino groups. An example structure is shown in formula (VI):

[0068]

[0069]

[0070] Where A is -COOH, Active groups that can react with primary amines, where x is an integer from 0 to 3 and y is an integer from 0 to 8.

[0071] In this invention, "fluorinated aromatic ring compounds" refers to 3-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2,3,5,6-tetrafluoro-4-methylbenzoic acid, pentafluorobenzoic acid, 2-fluoro-3-(trifluoromethyl)benzoic acid, and their derivatives, etc. These fluorinated aromatic ring compounds are a class of fluorinated compounds containing active groups that can react with amino groups. An example structure is shown in formula (VII):

[0072]

[0073]

[0074] This invention proposes a complex comprising chitosan modified with fluorinated compounds and a drug, wherein the drug includes small molecule drugs, peptides, protein drugs, combinations of different drugs, and combinations of drugs with other pharmaceutical excipients, and its use in promoting drug absorption.

[0075] For example, using epirubicin (THP) as a bladder instillation drug, the fluorinated chitosan modified with the present invention serves as a drug delivery carrier, promoting drug entry into the bladder tissue. Experiments show that the present invention has the following advantages: it maintains excellent biocompatibility while significantly improving the absorption efficiency of instilled drugs through the bladder mucosa. In vivo instillation experiments in mice revealed that the fluorinated chitosan modified with the present invention significantly improves the absorption of THP in the bladder mucosa more efficiently than THP aqueous solution and its chitosan solution; simultaneously, the fluorinated chitosan modified with the present invention achieves highly efficient drug absorption through the bladder mucosa at lower concentrations. The bladder instillation drug carrier proposed in this invention combines the advantages of high efficiency, low toxicity, low cost, and simple synthesis.

[0076] likeFigure 21 As shown, Figure 21 Synthetic route for chitosan bladder instillation drug carrier modified with fluorinated carboxylic acid.

[0077] In summary, the fluorinated chitosan drug carrier provided by this patent has the advantages of significantly promoting drug absorption and low toxicity. Furthermore, the fluorinated compound-modified chitosan synthesis process proposed in this invention is mature, easy to operate, highly efficient, and has a short cycle. It can obtain high-yield drug carriers without complicated purification steps. Its simple synthesis method provides a good foundation for its commercialization. The fluorinated compound-modified chitosan described in this invention has the potential to serve as a variety of drug carriers, effectively improving therapeutic effects, and has a wide range of applications, while also being relatively inexpensive. Attached Figure Description

[0078] Figure 1 This figure illustrates the effect of heptafluorobutyric acid-modified chitosan (7FCS) on the distribution and intensity of THP in mouse bladder tissue in Example 5. THP represents epirubicin; CS represents chitosan; and FCS represents fluorinated chitosan. The right figure shows the relative fluorescence intensity analysis of THP corresponding to the left figure.

[0079] Figure 2 This figure shows the effect of tridecylfluoroheptanic acid-modified chitosan (13FCS) on the distribution and intensity of THP in mouse bladder tissue in Example 6, where epirubicin is THP; CS is chitosan; and FCS is fluorinated chitosan. The right figure is the relative fluorescence intensity analysis of THP corresponding to the left figure.

[0080] Figure 3 This is an example of the effect of different fluorinated fatty acid-modified chitosans (7FCS, 13FCS, 19FCS) on the distribution and intensity of THP in mouse bladder tissue. Epirubicin represents THP; CS represents chitosan; and FCS represents fluorinated chitosan. The right figure shows the relative fluorescence intensity analysis of THP corresponding to the left figure.

[0081] Figure 4 a is a comparison diagram showing that 13F-3 exhibits excellent in vitro cell safety in Example 8.

[0082] Figure 4 b indicates that the body weight of mice in the FCS group in Example 8 was not significantly different from that in the blank control group.

[0083] Figure 4 c shows a comparison image of mouse bladder tissue and HE (hematoxylin-eosin) stained sections after perfusion in each group of mice in Example 8, compared with those in the blank control group.

[0084] Figure 5The images shown are comparison images of the immunofluorescence results in Example 8. They show that the chitosan perfusion group of mice experienced severe inflammatory stress and congestion and edema in the bladder, while there was no significant difference between the FCS group and the blank control group. The left image is a fluorescence confocal image of a bladder tissue section, and the right image is a comparison image of the CS, FCS treatment group and the blank control group.

[0085] Figure 6 Images showing the MPI / FPEI ratio measured by transmission electron microscopy in Example 9.

[0086] Figure 7 Images of MPI / PEI obtained by transmission electron microscopy in Example 9.

[0087] Figure 8 In Example 9, the mucosal permeability index of the F-PEI group peptide was significantly higher than that of the PEI group and the blank control group. The horizontal axis represents the mass ratio of the peptide drug MPI to the material PEI or FPEI, and the vertical axis represents the permeability coefficient papp.

[0088] Figure 9 In Example 9, the mucosal permeability index of the F-PEI histone drug was significantly higher than that of the PEI group and the blank control group. The horizontal axis represents the mass ratio of the peptide drug CAT-Ce6 to the material PEI or FPEI, and the vertical axis represents the permeability coefficient papp.

[0089] Figure 10 This is a chart comparing the distribution and intensity of drug fluorescence in the bladder after different infusion times for different MPI drug systems in Example 9. The horizontal axis represents different infusion times, and the vertical axis represents the relative values ​​of peptide drug fluorescence intensity.

[0090] Figure 11 The chart shows the distribution and intensity of drug fluorescence in the bladder tissue after perfusion of different CAT drug systems in Example 9. The left figure shows the fluorescence distribution of different CAT-Ce6 drug systems in bladder tissue, and the right figure shows the fluorescence intensity analysis of the drug.

[0091] Figure 12 This is a transmission electron microscope (TEM) image from Example 10.

[0092] Figure 13 The chart shows the comparison of fluorescence intensity in frozen bladder sections and fluorescence confocal microscopy analysis in Example 10. The left chart, from left to right, shows the fluorescence distribution of the drug CAT-TCPP in transverse and longitudinal sections of bladder tissue; the right chart, from top to bottom, shows the fluorescence intensity of the drug in transverse and longitudinal sections of bladder tissue and in bladder tissue homogenate.

[0093] Figure 14This is a schematic diagram of the detection of dynamic light scattering hydrated particle size and charge in Example 11. FITC is fluorescein isothiocyanate; IgG is immunoglobulin g; FCS is fluoresced chitosan; αPDL1 is a monoclonal antibody against programmed death receptor ligand-1 (αPDL1). The left and right graphs have size on the horizontal axis and quantity on the vertical axis. The middle graph has the ratio of fluorescein isothiocyanate to immunoglobulin g on the horizontal axis and the ZETA potential on the vertical axis.

[0094] Figure 15 This is a schematic diagram of the in vitro transdermal characterization of mouse skin using a vertical diffusion cell in Example 12. The vertical diffusion cell is shown in the diagram.

[0095] Figure 16 The data represents the permeability obtained during the in vitro transdermal characterization process in Example 12. IgG represents immunoglobulin g; FCS represents fluorinated chitosan; and the vertical axis represents the percentage of fluorescence that has been transmitted.

[0096] Figure 17 This image shows the penetration of FCS-IgG into the tumor site in the C57 mouse B16 tumor model using Cy5.5-labeled FCS-IgG, as described in Example 12. (Cy5.5, fluorescent dye; IgG, immunoglobulin g; CS, chitosan; FCS, fluorinated chitosan; DAPI fluorescence signal indicates cell nucleus; Cy5.5 fluorescence signal indicates IgG; Merged indicates overlay; arrow indicates skin layer.)

[0097] Figure 18 Confocal microscopy images obtained for the penetration evaluation of the FCS-αPDL1 antibody in the C57 mouse B16 tumor model in Example 12. FITC (fluorescein isothiocyanate); αPDL1 (anti-programmed death receptor ligand-1 monoclonal antibody); CS (chitosan); FCS (fluorinated chitosan); DAPI fluorescence signal indicating cell nuclei; FITC fluorescence signal indicating αPDL1; Merged indicates overlay.

[0098] Figure 19 The images show fluorescence images of lung tissue in mice during the evaluation of Cy5.5-labeled FCS-IgG lung penetration in Example 13, and ex vivo fluorescence images of the lungs 24 hours after administration. Cy5.5 fluorescence signal indicates IgG.

[0099] Figure 20 The images shown are panoramic images of the lung lobe taken under a Zeiss LSM 800 confocal microscope in Example 13, which obtained images related to the infiltration of FCS-IgG in the lung. Cy5.5 is a fluorescent dye; IgG is immunoglobulin g; CS is chitosan; FCS is fluorinated chitosan; DAPI fluorescence signal indicates cell nuclei; FITC fluorescence signal indicates αPDL1; Merged indicates overlay image.

[0100] Figure 21 Synthetic route for chitosan bladder instillation drug carrier modified with fluorinated carboxylic acid. Detailed Implementation

[0101] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. All variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims.

[0102] Abbreviations: THP (epirubicin); EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); NHS (N-hydroxythiosuccinimide); DMSO (dimethyl sulfoxide); MPI (polypeptide drug Polybia-MPI); MPI-Cy5.5 (fluorescent labeling of the polypeptide drug MPI); PEI (polyethyleneimine); FPEI (fluorinated polyethyleneimine); CAT (protein drug catalase); CAT-Ce6 (complex protein drug labeled with photosensitizer Ce6); CAT-TCPP (complex protein drug labeled with sonosensitive agent TCPP).

[0103] Reference example

[0104] The experimental designers prepared chitosan bladder instillation drug systems by mixing doxorubicin, epirubicin (THP), and the fluorescent dye rhodamine B with chitosan (1% acetic acid aqueous solution). One hour after bladder instillation, bladder tissue was harvested and frozen sections were prepared. Drug fluorescence intensity was analyzed using confocal fluorescence microscopy to examine the drug distribution in the bladder tissue. The results showed that chitosan solutions of equal concentrations (5 mg / ml, 10 mg / ml, 15 mg / ml) exhibited significantly better adhesion and permeability to the bladder mucosa than aqueous solutions containing only the drug. Furthermore, the adhesion and permeability of the instillation system increased with increasing chitosan concentration. To further investigate the biosafety of chitosan as a drug carrier, the researchers administered 15 mg / ml chitosan aqueous solution to mice via bladder instillation. Instillation was stopped after one hour, and the mice were fed under normal conditions, with their weight recorded. The results showed that mice experienced a sharp drop in weight and lethargy the day after chitosan instillation. Mice began dying on the second day after treatment, and all eight mice in the experimental group died within three days. Dissection of their bladders and comparison with those of the control group revealed severe bladder congestion in the chitosan-infused group. HE staining and CD45 and Ki67 immunofluorescence results showed severe inflammatory stress, congestion, and edema in the bladders of the chitosan-infused group. These experimental results indicate that while chitosan can significantly improve the bioavailability of infused drugs in the bladder mucosa, high concentrations of chitosan can also cause severe damage to the bladder mucosa and epithelium, severely limiting its clinical application as a carrier for bladder instillation drugs.

[0105] Example 1: Preparation of chitosan with different degrees of 3-fluorobenzoic acid modification (degree of deacetylation ≥95%, viscosity 100-200 mpa.s), wherein the molar ratio of 3-fluorobenzoic acid to N-glucosamine units was 1:1.1, 1:2.2, 1:4.4, and 1:8.8, respectively.

[0106] Synthesis method: (1) Preparation of chitosan acetic acid aqueous solution: Weigh 200mg of fully dried chitosan and add it to 10ml of 1% acetic acid aqueous solution. Of course, hydrochloric acid aqueous solution can also be used. Stir for 30min to fully dissolve, and then slowly add 1.6ml of 0.5M sodium hydroxide. Stir until the solution is clear and the pH is about 6.5. Considering only the alkalinization of the solution, sodium hydroxide can be replaced by ammonia, triethylamine and other bases. However, from the perspective of product process, the byproduct of using sodium hydroxide is sodium chloride, which is more suitable for industrialization. Prepare 4 portions of chitosan acetic acid aqueous solution in this way. (2) Activation of 3-fluorobenzoic acid: Weigh 5.0mg, 9.8mg, 19.7mg and 40mg of 3-fluorobenzoic acid respectively, dissolve them in an appropriate amount of anhydrous dimethyl sulfoxide, add the reaction amount of EDC in sequence, stir for 1h in the dark under NHS. (3) Preparation of 3-fluorobenzoyl chitosan: The activated 3-fluorobenzoic acid solution was slowly added dropwise to the rapidly stirred chitosan solution, and the reaction was carried out in the dark for 20 h. After the reaction was completed, the solution after the above reaction was slowly added dropwise to 100 ml of 0.5 M potassium hydroxide ethanol solution and stirred for 8 h. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water, and then dried under vacuum for 30 min. The dried precipitate was dissolved in 10 ml of 0.1 M hydrochloric acid solution and lyophilized to obtain 3-fluorobenzoic acid fluorinated chitosan hydrochloride molecules with different degrees of fluorination modification (the products were named 1FCS-1, 1FCS-2, 1FCS-3, 1FCS-4).

[0107] The degree of modification of the fluorinated aliphatic chains on the surface of fluorinated chitosan (FCS) polymer was determined by the ninhydrin reaction method. The ninhydrin reaction method is a simple, rapid, accurate, and reliable method that can accurately detect the number of primary amino groups on the surface of FCS polymer in aqueous solution, thereby calculating the number of fluorinated groups on the FCS surface.

[0108] Example 2: Preparation of chitosan with different degrees of modification of heptafluorobutyric acid (degree of deacetylation ≥95%, viscosity 100-200 mPa·s), wherein the molar ratio of perfluoroheptanoic acid to N-glucosamine units was 1:1.1, 1:2.2, 1:4.4, and 1:8.8, respectively.

[0109] Synthesis method: (1) Preparation of chitosan acetate aqueous solution: Weigh 200mg of fully dried chitosan and add it to 10ml of 1% acetic acid aqueous solution. Stir for 30min to dissolve it completely. Then slowly add 1.6ml of 0.5M sodium hydroxide and stir until the solution is clear and the pH is about 6.5. Prepare 4 portions of chitosan acetate aqueous solution in this way. (2) Activation of heptafluorobutyric acid: Weigh 7.6mg, 15mg, 30mg and 61mg of heptafluorobutyric acid respectively and dissolve them in an appropriate amount of anhydrous dimethyl sulfoxide. Add the reaction amount of EDC in sequence and stir in the dark for 1h under NHS. (3) Preparation of heptafluorobutyric acid chitosan: Slowly add the activated heptafluorobutyric acid solution to the rapidly stirred chitosan solution and stir in the dark for 20h. After the reaction was complete, the reaction mixture was slowly added dropwise to 100 ml of 0.5 M potassium hydroxide ethanol solution and stirred for 8 h. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and diethyl ether to remove water, and then dried under vacuum for 30 min. The dried precipitate was dissolved in 10 ml of 0.1 M hydrochloric acid solution and lyophilized to obtain perfluoroheptanoic acid fluorinated chitosan hydrochloride molecules with different degrees of fluorination modification (the products were named 7FCS-1, 7FCS-2, 7FCS-3, and 7FCS-4).

[0110] The degree of fluorinated aliphatic chains on the surface of the FCS (fluorinated chitosan) polymer was determined using the ninhydrin reaction method. The ninhydrin reaction method is a simple, rapid, accurate, and reliable method that can accurately detect the number of primary amino groups on the surface of the FCS polymer in aqueous solution, thereby calculating the number of fluorinated groups on the FCS surface. The fluorination modification degree of the FCS prepared above, calculated using the ninhydrin reaction method, was as follows: 7FCS-1, 6.9%; 7FCS-2, 10.4%; 7FCS-3, 23.5%; 7FCS-4, 42.3%.

[0111] Example 3: Preparation of chitosan with different degrees of perfluoroheptanoic acid modification (degree of deacetylation ≥95%, viscosity 100-200 mPa·s), wherein the molar ratio of perfluoroheptanoic acid to N-glucosamine units was 1:1.1, 1:2.2, 1:4.4, and 1:8.8, respectively.

[0112] Synthesis method: (1) Preparation of chitosan acetate aqueous solution: Weigh 200 mg of fully dried chitosan and add it to 10 ml of 1% acetic acid aqueous solution. Stir for 30 min to dissolve completely. Then slowly add 1.6 ml of 0.5 M sodium hydroxide and stir until the solution is clear and the pH is about 6.5. Prepare 4 portions of chitosan acetate aqueous solution in this way. (2) Activation of perfluoroheptanoic acid (13-fluoroheptanoic acid): Weigh 13 mg, 26 mg, 51.5 mg and 103 mg of perfluoroheptanoic acid respectively, dissolve them in an appropriate amount of anhydrous dimethyl sulfoxide, add an appropriate amount of EDC in sequence, stir in NHS in the dark for 1 h. (3) Preparation of 13F-heptanoic acid chitosan: Slowly add the above activated perfluoroacid solution to the chitosan solution that is stirred rapidly, and stir in the dark for 20 h. After the reaction was complete, the reaction mixture was slowly added dropwise to 100 ml of 0.5 M potassium hydroxide ethanol solution and stirred for 8 h. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and diethyl ether to remove water, and then dried under vacuum for 30 min. The dried precipitate was dissolved in 10 ml of 0.1 M hydrochloric acid solution and lyophilized to obtain perfluoroheptanoic acid fluorinated chitosan hydrochloride molecules with different degrees of fluorination modification (the products were named 13FCS-1, 13FCS-2, 13FCS-3, and 13FCS-4).

[0113] The degree of fluorination modification of the above-prepared FCSs, calculated by the ninhydrin reaction method, is as follows: 13FCS-1, 5.2%; 13FCS-2, 11.3%; 13FCS-3, 21.4%; 13FCS-4, 42.5%. The linkage efficiency of the 13-fluoroheptane carbonyl group in 13FCS-1 to 13FCS-4 increases from 5.2% to 42.5% with the increase of perfluoroheptanoic acid feed, meaning that on average, 5.2% to 42.5% of the glucose structural units in each chitosan molecule have undergone fluorination modification. The products are named 13FCS-1, 13FCS-2, 13FCS-3, and 13FCS-4.

[0114] Example 4: Preparation of chitosan with different degrees of modification of 19F decanoic acid (degree of deacetylation ≥95%, viscosity 100-200 mPa·s), wherein the molar ratio of 19F decanoic acid to N-glucosamine units was 1:1.1 and 1:2.2, respectively.

[0115] Synthesis method: (1) Preparation of chitosan acetate aqueous solution: Weigh 200mg of fully dried chitosan and add it to 10ml of 1% acetic acid aqueous solution. Stir for 30min to dissolve it completely. Then slowly add 1.6ml of 0.5M sodium hydroxide and stir until the solution is clear and the pH is about 6.5. Prepare two portions of chitosan acetate aqueous solution in this way. (2) Activation of 19F decanoic acid: Weigh 18mg and 36.7mg of 19F decanoic acid respectively and dissolve them in an appropriate amount of anhydrous dimethyl sulfoxide. Add an appropriate amount of EDC in sequence and stir in NHS in the dark for 1h. (3) Preparation of 19F decanoic acid chitosan: Slowly add the above activated 19F decanoic acid solution to the rapidly stirred chitosan solution and stir in the dark for 20h. After the reaction was complete, the reaction mixture was slowly added dropwise to 100 ml of 0.5 M potassium hydroxide ethanol solution and stirred for 8 h. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water, and then dried under vacuum for 30 min. The dried precipitate was dissolved in 10 ml of 0.1 M hydrochloric acid solution and lyophilized to obtain white powders of different degrees of fluorination modification of 19F decanoic acid fluorinated chitosan hydrochloride molecules (products named 19FCS-1, 19FCS-2).

[0116] 19FCS-2 has poor water solubility, making it unsuitable for subsequent characterization and application evaluation. Therefore, the degree of fluorination modification of the prepared 19FCS-1 was calculated using the ninhydrin reaction method as follows: 19FCS-1, 5.2%.

[0117] Example 5: Evaluation of the bladder mucosal permeation enhancement effect of 7FCS: The 7FCS prepared in Example 2 was mixed with THP aqueous solution and instilled into the bladder through the mouse urethra. Frozen sections of mouse bladder were then prepared, and the distribution of THP fluorescence in the tissue was detected to evaluate the drug carrier's efficiency in promoting drug absorption in the bladder mucosa.

[0118] The specific method was as follows: Female C57BL / 6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. A 0.2% THP solution was prepared using 0.5% FCS aqueous solution and instilled into the mouse bladder via a closed intravenous catheter (100 μl). The urethra was clamped for 1 hour, and then the instilled fluid was released from the bladder. The bladder was flushed with 1 ml of ultrapure water. Bladder tissue was harvested and placed in a tissue embedding machine at -80°C, followed by sectioning and examination using a fluorescence confocal microscope. A simple THP aqueous solution of equal concentration or a similarly prepared THP-chitosan aqueous solution served as controls.

[0119] Experimental results: See Figure 1 The left side shows images of mouse bladder tissue sections under microscopes under different conditions, and the right side shows the corresponding data statistics. The horizontal axis on the right represents different drug systems, and the vertical axis represents relative fluorescence intensity.

[0120] like Figure 1 Fluorescence confocal microscopy was used to observe mouse bladder tissue sections. The results showed that the fluorescence distribution area and intensity of the drug in the transverse section of the bladder of the 7FCS group were significantly higher than those of chitosan (CS) and the blank control group (THP aqueous solution only). This indicates that FCS can significantly improve the tissue permeability of drugs in the bladder mucosa, and the permeation-enhancing effect increases with the degree of fluorination. However, when the degree of fluorination reaches a certain level (7FCS, 42.3%), the permeation-enhancing effect of the prepared FCS is most significant. At this level, the 7FCS prepared has lower solubility than fluorinated chitosan, making it less suitable for clinical application. These results indicate that FCS can significantly improve the permeability and absorption performance of drugs, enhancing the absorption efficiency of drugs in the bladder mucosa; however, excessive fluorination may be detrimental to the application of the material.

[0121] Example 6: Effect of 13FCS on bladder mucosal absorption of instilled drugs: The 13FCS prepared in Example 3 and THP aqueous solution were mixed and instilled into the bladder through the urethra of mice. Frozen sections of mouse bladders were then prepared, and the distribution of THP fluorescence in the tissue was detected to evaluate the efficiency of the drug carrier in promoting bladder mucosal absorption.

[0122] The specific method was as follows: Female C57BL / 6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. A 0.2% THP aqueous solution was prepared using 0.5% 13FCS aqueous solution and instilled into the mouse bladder via a closed intravenous catheter (100 μl). The urethra was clamped for 1 hour, and then the instilled fluid was released from the bladder. The bladder was flushed with 1 ml of ultrapure water, and bladder tissue was harvested and placed in a tissue embedding machine at -80°C. Sections were then prepared and examined using a fluorescence confocal microscope. A similarly prepared THP-chitosan aqueous solution was used as a control.

[0123] Experimental results are as follows Figure 2 The fluorescence distribution area and intensity of the drug in the longitudinal section of the bladder of mice in the FCS group were significantly higher than those in chitosan (CS), indicating that 13FCS can significantly improve the permeability of the drug in the bladder mucosa. Meanwhile, 13FCS-3 showed the strongest permeation-enhancing effect, with a fluorination modification degree of 21.4%. These results also suggest that the permeation-enhancing effect of FCS on the bladder mucosa may be the result of the combined action of the chitosan cationic backbone and the fluorinated fatty chains; however, this effect may be due to multiple factors.

[0124] Example 7: In order to screen the fluorinated chitosan with the best effect on promoting bladder mucosal perfusion, 19FCS-1 from Example 4 and the FCS with the best effect among the above-mentioned fluorinated modifications were evaluated in mice.

[0125] The specific method was as follows: Female C57BL / 6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. A 0.2% THP solution was prepared using 0.5% aqueous solutions of 7FCS-4, 13FCS-3, and 19FCS-1, and 100 μl was instilled into the mouse bladder via a closed intravenous catheter. The urethra was clamped for 1 hour, after which the instillation fluid was drained from the bladder, and the bladder was flushed with 1 ml of ultrapure water. Bladder tissue was then collected and placed in a tissue embedding machine at -80°C for sectioning and examination using a fluorescence confocal microscope. A simple aqueous solution of THP at the same concentration or a similarly prepared THP-chitosan aqueous solution served as controls.

[0126] Experimental results: such as Figure 3 The results of observing mouse bladder tissue sections using a fluorescence confocal microscope showed that the distribution area and intensity of drug fluorescence in the transverse section of the bladder of the 19FCS group were significantly different from those of the THP and CS groups, but the perfusion-promoting effect of 13FCS-3 was the most prominent.

[0127] Example 8: In vitro and in vivo safety evaluation of different types of fluorinated chitosan modified in Example 7. The specific experimental protocol is as follows:

[0128] The cytotoxic effect of fluorinated chitosan on SV-HUC-1 normal human bladder epithelial cells was evaluated using the CCK-8 (Cell Counting Kit-8) method (a mature in vitro method for evaluating cell viability), and the in vitro biosafety of fluorinated chitosan was investigated. The specific procedure was as follows: 1 x 10⁴ cells / well T24 cells were seeded into 96-well plates and cultured overnight at 37°C with 5% CO₂. Serum-free medium containing different types of fluorinated chitosan (500 μg / ml) was added, and the cells were cultured for another 24 h. Then, an appropriate amount of CCK-8 was added. Finally, the in vitro safety of fluorinated chitosan was evaluated based on cell viability. The experimental results are as follows: Figure 4 As shown in Figure a, 13F-3 (abbreviation for 13FCS-3) exhibits excellent in vitro cell safety. Combined with the above findings, 13F-3 demonstrates the most significant effect in promoting bladder mucosal absorption of infused drugs, while also exhibiting good in vitro cell safety. To further evaluate the biosafety of FCS (13F-3), further in vivo safety evaluation experiments were conducted in mice.

[0129] Healthy C57BL / 6 mice aged 10-12 weeks were randomly divided into three groups of eight mice each. The experimental groups were perfused with 15 mg / ml fluorinated chitosan or 1% acetic acid chitosan solution for 1 hour, once a week for three weeks. The blank control group was perfused with an equal volume of double-distilled water. The in vivo biosafety of fluorinated chitosan was evaluated by mouse body weight, survival rate, and HE and immunohistochemical analysis (CD45 and Ki67) of bladder sections from mice on day 28 after the first administration.

[0130] The experimental results showed that mice administered chitosan experienced a sharp drop in weight and lethargy on the second day after treatment, and began to die from the second day onwards. Within three days, all eight mice in the experimental group died, while no mice in the fluorinated chitosan group died. Figure 4 As shown in b, there was no significant difference in body weight between the FCS group and the blank control group. Meanwhile, as... Figure 4 As shown in c, a comparison of the bladders of mice in each perfusion group with those in the blank control group revealed that the bladders of mice in the chitosan perfusion group were more severely congested than those in the blank control group, as indicated by HE staining. Figure 5 Immunofluorescence results for CD45 and Ki67 showed that the chitosan-infused mice exhibited severe inflammatory stress, congestion, and edema in their bladders, while there was no significant difference between the FCS group and the blank control group. These experimental results indicate that fluorinated chitosan (13FCS-3) can significantly improve the bioavailability of infused drugs in the bladder mucosa, and high concentrations of fluorinated chitosan do not cause significant damage to the bladder mucosa or epithelium, suggesting its potential as a carrier for bladder infusion drugs.

[0131] Example 9: Application of F-PEI bladder instillation peptide-protein drug carrier, taking peptide drug MPI and protein drug CAT-Ce6 as examples.

[0132] (1) Synthesis of fluorinated polyetherimide (F-PEI)

[0133] The specific procedure is as follows: An appropriate amount of 3-(perfluorohex-1-yl)-1,2-propenoxide was slowly added dropwise to a methanol solution of branched polyetherimide (PEI), and the mixture was stirred at room temperature for 48 hours. The crude product was purified by dialysis with methanol / double-distilled water (MWCO 3500 Da), and then freeze-dried to obtain the final product. The structure of the product was identified by 1H NMR, and the average number of fluorine substitutions in the molecule was calculated using fluorine elemental analysis.

[0134] (2) Preparation and characterization of MPI / F-PEI and CAT-Ce6 / F-PEI nanomedicine systems

[0135] MPI / F-PEI and CAT / F-PEI NPs were obtained by mixing peptide (MPI) and protein (CAT) drugs with an aqueous solution of F-PEI at room temperature for 2 hours. Dynamic light scattering analysis revealed that their hydrated particle size was approximately 200-300 nm, carrying a small amount of positive charge. Transmission electron microscopy (TEM) imaging... Figure 6 ,7) Characterize it as a uniform spherical particle.

[0136] (3) Evaluation of bladder mucosal permeability of MPI / F-PEI NPs and CAT-Ce6 / F-PEI NPs

[0137] The using chamber (also called the Ussing chamber) is a tool for studying transepithelial transport, including ion transport, nutrient transport, and drug transport. By studying transepithelial transport, we can understand drug absorption through the epithelium. This example uses the using chamber to evaluate the mucosal permeability of MPI-cy5.5 / F-PEI NPs and CAT-ce6 / F-PEI NPs prepared with different material ratios. MPI-cy5.5 / PEI NPs and CAT-ce6 / PEI NPs were used as controls. Mice were anesthetized, and the bladder was removed and the bladder mucosa was peeled off on ice and fixed at the interface between the two chambers. 3 ml of either MPI-cy5.5 / F-PEI NPs or CAT-ce6 / F-PEI NPs solution was added to the diffusion chamber, and an equal volume of blank solution was added to the receiving chamber. Every 15 minutes, 0.5 ml of staged solution was taken from the receiving chamber, and an equal volume of blank staged solution was added to the receiving chamber. This process was repeated four times, and the corresponding drug content was detected using a fluorescence spectrophotometer. The experimental results showed that the F-PEI group of peptides ( Figure 8 ) or mucosal penetration of protein drugs ( Figure 9 The permeability index (papp) was significantly higher in the papp group than in the PEI group and the free group (blank group).

[0138] We also investigated its bladder mucosal permeability in mice. After anesthesia, mice were instilled into their bladders with equal amounts of fluorescently labeled peptide or protein drug solutions. Bladder sections were prepared from different drug systems in the MPI group (free MPI-cy5.5, MPI-cy5.5 / PEI, MPI-cy5.5 / F-PEI) at different time points after instillation (15, 30, 60 min). Figure 10 After 1 hour of perfusion with different drug systems in the CAT group (free CAT-ce6, CAT-ce6 / PEI, CAT-ce6 / F-PEI), bladder sections were prepared and the fluorescence intensity was analyzed by fluorescence confocal microscopy. Figure 11 The experimental results showed that, compared with the PEI and free drug groups, the F-PEI group had significantly greater bladder mucosal permeability to peptide and protein drugs, meaning that F-PEI can significantly improve the bladder mucosal permeability of peptide or protein drugs.

[0139] Example 10: Application of FCS bladder instillation protein drug carrier, taking the protein drug CAT-TCPP as an example.

[0140] (1) Preparation and characterization of CAT-TCPP / FCS nanomedicine system

[0141] CAT-TCPP / FCS NPs were obtained by mixing the protein drug (CAT-TCPP) with an aqueous solution of FCS at room temperature for 2 hours. Dynamic light scattering analysis revealed that their hydrated particle size was approximately 200-300 nm, carrying a small amount of positive charge. Transmission electron microscopy (TEM) imaging... Figure 12 It is characterized as a uniform spherical particle.

[0142] (2) Evaluation of bladder mucosal permeability of CAT-TCPP / FCS NPs

[0143] We investigated the bladder mucosal permeability in mice. After anesthesia, mice were instilled into their bladders with equal amounts of fluorescently labeled protein drug solutions. Different drug systems in the CAT-TCPP group (free CAT-TCPP, CAT-TCPP / CS, CAT-TCPP / FCS) were used, and bladder sections were prepared after the same instillation time (60 min). Figure 13 The fluorescence intensity was analyzed using fluorescence confocal microscopy. The results showed that, compared to the chitosan (CS) and free drug groups, the protein drug in the FCS group exhibited significantly greater bladder mucosal retention and permeability, indicating that FCS can significantly improve the bladder mucosal permeability of protein drugs.

[0144] Example 11: (Preparation and characterization of fluorinated chitosan as a protein drug carrier), taking proteins IgG and αPDL1 as examples.

[0145] (1) Preparation of FCS-IgG nanomedicine system

[0146] Immunoglobulins are globulins with antibody activity or chemical structures similar to antibody molecules. Taking immunoglobulin g (IgG) as an example, this study investigates the preparation and characterization of fluorinated chitosan as a drug carrier. FCS-IgG NPs are obtained by stirring an aqueous solution of IgG with an aqueous solution of FCS at room temperature for 1 hour. Figure 14 As shown in the left figure, the horizontal axis represents particle size, and the vertical axis represents quantity. The dynamic light scattering hydrated particle size of FCS-IgG is approximately 200 nm. Taking the anti-programmed death receptor ligand-1 (αPDL1) monoclonal antibody as an example, the preparation and characterization of fluorinated chitosan as a drug carrier were studied. Figure 14 As shown in the left figure, the horizontal axis represents particle size and the vertical axis represents quantity. The dynamic light scattering hydrated particle size of FCS-αPDL1 is approximately 200 nm.

[0147] (2) Synthesis and characterization of FCS-IgG NPs with different mass ratios

[0148] FCS-IgG NPs were obtained by stirring IgG aqueous solution and FCS aqueous solution at room temperature for 1 hour, with a dynamic light scattering hydrated particle size of approximately 200 nm. The final concentration of FITC-labeled IgG was 0.2 mg / ml. FCS aqueous solutions with final concentrations of 0.2 mg / ml, 0.1 mg / ml, and 0.05 mg / ml were added to prepare FCS:IgG NPs at ratios of 1:1, 1:2, and 1:4 (m / m). The dynamic light scattering hydrated particle size and charge were then measured. Figure 14 The higher the FCS ratio, the more positive the charge.

[0149] Example 12: Application of fluorinated chitosan as a transdermal drug carrier, taking protein IgG and αPDL1 as examples.

[0150] Use a vertical diffusion cell ( Figure 15 This device was used to characterize mouse skin transdermally in vitro. It consisted of two cup-shaped ground glass containers joined together, with the skin sample sandwiched between them for in vitro permeability studies. A 1.5cm x 1.5cm piece of C57 mouse skin was cut and placed in the sample layer (the effective permeability area of ​​the sample layer was 1.13cm²). 2 After leak testing, PBS buffer was gradually added to the sampling chamber until it came into contact with the skin, and the volume of liquid added was recorded. 1 mL of PBS solution containing FITC-labeled FCS-IgG in different proportions was added to the sampling chamber, sealed to prevent evaporation, stirred at 37°C, and samples were taken from the sampling chamber at different time points to detect the fluorescence intensity of the samples. An equal amount of PBS was added to maintain the volume of the sampling chamber.

[0151] The fluorescence transmittance is calculated as follows:

[0152]

[0153] Where P is the transmittance of fluorescence, and V i The volume of the i-th sampling is 3 * 100 μL by default. i F represents the average fluorescence intensity of the i-th sample. n Vs represents the average fluorescence intensity of the nth sample, Vs is the sampling cell volume, approximately 19 mL (slightly varying between different sampling cells), V0 is the injection cell volume, and F0 is the average fluorescence intensity of the sample in the injection cell.

[0154] Obtain the corresponding transmittance data, such as Figure 16 Fluorinated chitosan, when used as a transdermal drug carrier, can help drugs successfully penetrate mouse skin. The preferred mass ratio of drug to chitosan is 1:1.

[0155] (2) Evaluation of tumor penetration rate in mice

[0156] a) Evaluation of the penetration of Cy5.5-labeled FCS-IgG in the B16 tumor model of C57 mice

[0157] Equal mass When the ointment is mixed with FCS-IgG aqueous solution, the translucent ointment turns milky white. After thorough mixing, apply to an area of ​​approximately 100 mm. 3 In mouse B16 tumor sites, using Tegaderm TM The mice were covered with film to prevent the ointment from being destroyed. After 24 hours, the subcutaneous tumors of the mice were removed, embedded in OCT gel, and frozen overnight at -80°C. 10mm sections were prepared, and panoramic images of the tumors were captured using a Zeiss LSM 800 confocal microscope to obtain the FCS-IgG penetration status of the tumor site. Figure 17 In the figure, arrows represent the superficial skin, DAPI signals represent cell nuclei, Cy5.5 fluorescence signals represent fluorescently labeled IgG, and Merged indicates the superposition of both. Free IgG and ordinary chitosan carriers remain only on the skin surface, while IgG, with the help of fluorinated chitosan, can reach the tumor interior. The results indicate that fluorinated chitosan, as a transdermal drug carrier, can help drugs successfully penetrate mouse skin.

[0158] b) Evaluation of FCS-αPDL1 antibody penetration in the C57 mouse B16 tumor model

[0159] To prevent errors caused by dye shedding, we evaluated the penetration of FCS-αPDL1 antibody in a C57 mouse B16 tumor model. Six hours after application, we removed subcutaneous tumors from the mice, embedded them, sectioned them, and simultaneously stained them with FITC-Rat anti-mouse-IgG to specifically label mouse αPDL1 within the tumor. Confocal microscopy imaging is shown below. Figure 18 The arrows indicate the surface skin, and the FITC fluorescence signal indicates fluorescently labeled IgG. The results show that fluorinated chitosan, as a transdermal drug carrier, can help drugs successfully penetrate mouse skin.

[0160] Example 13: Application of fluorinated chitosan as an inhaled drug carrier, taking protein IgG and αPDL1 as examples.

[0161] (1) Preparation of FCS-IgG nanomedicine system

[0162] FCS-IgG NPs can be obtained by stirring IgG aqueous solution and FCS aqueous solution at room temperature for 1 hour. The dynamic light scattering hydrated particle size is about 200 nm.

[0163] (2) Evaluation of lung penetration of Cy5.5-labeled FCS-IgG in mice

[0164] 24 hours after administration via pulmonary inhalation, mice were dissected and lung tissue was collected for observation of Cy5.5-labeled drug retention using a small animal imaging system. Figure 19 In the diagram, 1 represents free IgG-Cy5.5, 2 represents CS-IgG-Cy5.5, and 3 represents FCS-IgG-Cy5.5. Fluorescent signals indicate fluorescently labeled IgG. Fluorinated chitosan exhibited optimal drug retention as a drug carrier for pulmonary administration.

[0165] Twenty-four hours after lung administration, lung tissue was dissected, embedded in OCT gel, and frozen overnight at -80°C to prepare 10mm sections. Panoramic lung lobe imaging was performed using a Zeiss LSM 800 confocal microscope to obtain the lung's FCS-IgG penetration status. Figure 20 The Cy5.5 fluorescence signal indicates fluorescently labeled IgG. Free IgG is difficult to retain effectively in the lungs, and ordinary chitosan carriers mainly remain near the bronchial openings. Fluorinated chitosan, as a drug carrier for pulmonary drug delivery, exhibits the best drug penetration and diffusion effects.

[0166] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art. The invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Claims

1. A fluorinated chitosan derivative for use as a drug carrier, having the following structure: a fluorinated compound is covalently linked to the chitosan backbone, wherein the molecular weight of the chitosan is in the range of 5000-5000000. The fluorinated compound is a fluorinated aliphatic chain compound or a fluorinated aromatic ring compound. The fluorinated aliphatic chains include trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, nonafluorovalerate, undecanoic acid, tridecafluoroheptanoic acid, pentafluorooctanoic acid, heptadecafluorononanoic acid, nonafluoroquinic acid, perfluorobutyric anhydride, perfluoroheptanoic anhydride, perfluorodecanoic anhydride, 2,2,3,3,4,4,4-heptafluorobutylacrylate or nonafluorobutyl sulfonamide anhydride. The fluorinated aromatic ring compounds include 3-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2,3,5,6-tetrafluoro-4-methylbenzoic acid, pentafluorobenzoic acid, or 2-fluoro-3-(trifluoromethyl)benzoic acid; The chitosan derivative has a chitosan molecular backbone containing primary amino groups as shown in Formula IV: Formula IV; The linking group formed between the primary amino group and the fluorinated functional group of the chitosan is: -NH-, or ; The fluorinated functional group is a fluorinated aliphatic chain or aromatic ring functional group; The chitosan derivative is administered as a drug carrier via intracavitary infusion, pulmonary inhalation, or transdermal delivery.

2. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, characterized in that: The fluorinated chitosan derivative serves as a drug carrier for drugs including small molecule drugs, peptides, protein drugs, combinations of different drugs, and combinations of drugs with other pharmaceutical excipients.

3. The application of chitosan modified with fluorine-containing compounds as a drug carrier, characterized in that: The fluorinated chitosan derivatives according to any one of claims 1 to 2 can be used as drug carriers for small molecule drugs, peptides, protein drugs, combination drugs of different drugs, and combination drugs of drugs and other pharmaceutical excipients.

4. A method for preparing the fluorinated chitosan derivative according to claim 1, comprising the following steps: To prepare a chitosan-acetic acid aqueous solution, weigh out chitosan and add it to the acetic acid aqueous solution. Stir until it is fully dissolved, then add sodium hydroxide dropwise and stir until the solution is clear and the pH is between 6.2 and 6.

8. Activation of fluorinated compounds: Weigh the fluorinated compound and dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide. Then, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (NHS) in the reaction mixture and stir in the dark. The activated fluorine-containing compound solution was added dropwise to the rapidly stirred chitosan solution, and stirred in the dark until the reaction was complete.

5. The method for preparing the fluorinated chitosan derivative according to claim 4 further comprises the following steps: slowly adding the fully reacted solution dropwise to a potassium hydroxide ethanol solution and stirring, filtering the precipitate, washing with a large amount of anhydrous ethanol until the filtrate is neutral, washing the precipitate with methanol and diethyl ether to remove water, vacuum drying, dissolving the dried precipitate in hydrochloric acid solution, and freeze-drying to obtain fluorinated chitosan hydrochloride.

6. The method for preparing the fluorinated chitosan derivative according to claim 4, comprising the following steps: (1) Preparation of chitosan acetic acid aqueous solution: Weigh out fully dried chitosan and add it to acetic acid aqueous solution. Stir to dissolve it completely, then slowly add sodium hydroxide dropwise and stir until the solution is clear and the pH is 6.2-6.8; (2) Activation of 3-fluorobenzoic acid: Weigh 3-fluorobenzoic acid, dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide, add the reaction amount of EDC in sequence, and stir thoroughly in the dark with NHS. (3) Preparation of 3-fluorobenzoyl chitosan: The activated 3-fluorobenzoic acid solution was slowly added dropwise to the chitosan solution that was being stirred rapidly, and the mixture was stirred in the dark to allow it to react fully.

7. The preparation method according to claim 6 further comprises the following steps: The fully reacted solution was slowly added dropwise to a potassium hydroxide ethanol solution and stirred thoroughly. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water and then dried under vacuum. The dried precipitate was dissolved in hydrochloric acid solution and freeze-dried to obtain 3-fluorobenzoic acid fluorinated chitosan hydrochloride molecules.

8. The method for preparing the fluorinated chitosan derivative according to claim 4, comprising the following steps: (1) Preparation of chitosan acetic acid aqueous solution: Weigh out fully dried chitosan and add it to acetic acid aqueous solution. Stir to dissolve it completely, then slowly add sodium hydroxide dropwise and stir until the solution is clear and the pH is 6.2-6.8; (2) Activation of perfluoroheptanoic acid: Weigh perfluoroheptanoic acid, dissolve it in an appropriate amount of anhydrous dimethyl sulfoxide, add an appropriate amount of EDC in sequence, and stir thoroughly in the dark with NHS. (3) Preparation of 13F heptanoic acid chitosan: The activated perfluoric acid solution was slowly added dropwise to the chitosan solution that was being stirred rapidly, and the mixture was stirred in the dark to allow it to react fully.

9. The preparation method according to claim 8, further comprising the following steps: The fully reacted solution was slowly added dropwise to a potassium hydroxide ethanol solution and stirred thoroughly. The precipitate was filtered, washed with a large amount of anhydrous ethanol until the filtrate was neutral, and the precipitate was washed with methanol and ether to remove water. It was then vacuum dried, and the dried precipitate was dissolved in hydrochloric acid solution and freeze-dried to obtain perfluoroheptanoic acid fluorinated chitosan hydrochloride.

10. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, characterized in that: The fluorinated chitosan derivative is a perfluoroheptanoic acid fluorinated chitosan hydrochloride molecule, and the degree of fluorination modification of the perfluoroheptanoic acid fluorinated chitosan hydrochloride is 18%~25%.

11. The fluorinated chitosan derivative for use as a drug carrier according to claim 10, characterized in that: The degree of fluorination modification of the perfluoroheptanoic acid fluorinated chitosan hydrochloride is 20%~22%.

12. A pharmaceutical complex, characterized in that: The invention includes fluorinated chitosan derivatives used as drug carriers as described in any one of claims 1 to 2, and drugs, wherein the drugs include small molecule drugs, peptides, protein drugs, combinations of different drugs, and combinations of drugs with other pharmaceutical excipients.

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