USE OF FLUORINE-CONTAINING COMPOUND-MODIFIED CATIONIC POLYMER AS DRUG CARRIER AND PREPARATION METHOD

Abstract

The invention provides a fluorinated chitosan derivative as a drug carrier, which has the following structure: a fluorine-containing compound covalently attached to the backbone of chitosan. The chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps. The fluorine-containing compound is a fluorine-containing aliphatic chain shown by the following chemical formula (I)

##STR00001##

or an aromatic ring with functional groups shown by the following formula (II)

##STR00002##

wherein R.sub.1 is halogen (fluorine, chlorine, bromine, iodine), or a halogen-substituted alkane, cycloalkane, aldehyde group, carboxyl group, alkenyl group, alkynyl group, hydroxyl group, sulfonyl chloride, sulfonic acid bond or mercapto group, for interacting with a primary amino group. The compounds of the present invention can be universally bound to a variety of drugs to promote drug absorption and bioavailability, and reduce toxicity.

Claims

1. A fluorinated chitosan derivative for use as a drug carrier, having the following structure: a fluorine-containing compound is covalently attached to the backbone of chitosan, wherein the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; and the fluorine-containing compound is a fluorine-containing aliphatic chain shown by the following chemical formula (I) ##STR00015## or an aromatic ring with functional groups shown by the following formula (II) ##STR00016## wherein R.sub.1 is an active group capable of reacting with a primary amino group, selected from halogen (fluorine, chlorine, bromine, iodine), a halogen-substituted alkane, cycloalkane, aldehyde group, carboxyl group, alkenyl group, alkynyl Group, hydroxyl group, sulfonyl chloride, sulfonic acid bond or mercapto group.

2. A fluorinated chitosan derivative for use as a drug carrier, having a molecular skeleton of chitosan which contains a primary amino group, as shown in formula (IV) ##STR00017## wherein a linking group formed between the primary amino group of the chitosan and a fluorine-containing functional group is: ##STR00018## and a derivative group thereof, the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; the fluorine-containing functional group is a fluorine-containing aliphatic chain or an aromatic ring with functional groups.

3. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein: in the formula (I), x is an integer of 0-3, y is an integer of 0-20, z is an integer of 0-8, and R.sub.2 is CF.sub.3, CHF.sub.2, CH.sub.2F, or CH.sub.3 (when y is not 0); the fluorine-containing aliphatic chain compound refers to a fluorine-containing hydrocarbon compound and derivatives thereof, comprising trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, nonafluorovaleric acid, undecafluorohexanoic acid, tridecafluoroheptanoic acid, pentadecafluorooctanoic acid, heptadecafluorononanoic acid, nonadecafluorodecanoic acid, heptafluorobutyric anhydride, perfluoroheptanoic anhydride, nonadecafluorodecanoic anhydride, 2,2,3,3,4,4,4-heptafluorobutyl acrylate, 3-(1H, 1H, 5H octafluoropentyloxy)-1,2-epoxypropane, nonafluorobuanesulphonic anhydride and derivatives thereof.

4. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein in the formula (II), R is H, CH.sub.3, OH, NO.sub.2, O, CF.sub.3, F, CH.sub.2OH, CN, NCO, or (CF.sub.2).sub.aCF.sub.3 (a is an integer of 1-20), or the like, and at least one R is F; the fluorine-containing aromatic ring compound comprises 3-fluorobenzoic acid, 3,5-difluorobenzoic acid, 2,3,5,6-tetrafluoro-4-methylbenzoic acid, pentafluorobenzoic acid, 2-fluoro-3-(trifluoromethyl)benzoic acid and derivatives thereof.

5. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein: the chitosan and the fluorine-containing compound are covalently linked and the chitosan is surface-modified, to form a drug carrier having a structure as shown in formula (V) below, wherein b and c are both an integer of 20-500: ##STR00019## wherein B is a linking group formed by a fluorine-containing functional group and a primary amino group of the chitosan, and C is a fluorine-containing aliphatic chain or an aromatic ring of functional groups.

6. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein: the fluorine-containing aliphatic chain is a class of fluorine-containing compounds with an active group capable of reacting with an amino group, and comprises those as shown in formula (VI): ##STR00020## wherein A is —COOH or ##STR00021## as an active group capable of reacting with a primary amino group, x is an integer of 0-3, and y is an integer of 0-8.

7. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein: the fluorine-containing aromatic ring compound is a class of fluorine-containing compounds with an active group capable of reacting with an amino group, and comprises those as shown in formula (VII): ##STR00022##

8. The fluorinated chitosan derivative for use as a drug carrier according to claim 1, wherein: the fluorinated chitosan derivative serves as a drug carrier of a drug, and the drug is selected from a small molecule drug, a polypeptide, a protein drug, a combined drug of different drugs, and a combined drug of a drug and other pharmaceutical excipients.

9. Use of a fluorine-containing compound-modified chitosan as a drug carrier, wherein: the fluorinated chitosan derivative of claim 1 is used as a drug carrier of a small molecule drug, a polypeptide, a protein drug, a combined drug of different drugs, and a combined drug of a drug and other pharmaceutical excipients.

10. A drug composite, comprising the fluorinated chitosan derivative for use as a drug carrier according to claim 1 and a drug, wherein the drug is selected from a small molecule drug, a polypeptide, a protein drug, a combined drug of different drugs, and a combined drug of a drug and other pharmaceutical excipients.

11. A transdermal administration preparation prepared from the fluorinated chitosan derivative for use as a drug carrier according to claim 1, comprising a transdermal preparation component (a), wherein the component (a) is a fluorine-containing compound-modified cationic polymer; the fluorine-containing compound-modified cationic polymer is a fluorinated chitosan; the fluorine-containing compound is covalently attached to the backbone of the chitosan; the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps.

12. A transmucosal administration preparation prepared from the fluorinated chitosan derivative for use as a drug carrier according to claim 1, comprising a transmucosal preparation component (a), wherein the component (a) is a fluorine-containing compound-modified cationic polymer; the fluorine-containing compound-modified cationic polymer is a fluorinated chitosan; the fluorine-containing compound is covalently attached to the backbone of the chitosan; the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; the mucosa comprises nasal mucosa, lung mucosa, vaginal mucosa, oral mucosa, and gastrointestinal mucosa.

13. An ocular barrier-penetrating administration preparation prepared from a fluorinated chitosan derivative for use as a drug carrier according to claim 1, comprising an ocular barrier-penetrating preparation component (a), wherein the component (a) is a fluorine-containing compound-modified cationic polymer; the fluorine-containing compound-modified cationic polymer is a fluorinated chitosan; the fluorine-containing compound is covalently attached to the backbone of the chitosan; the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; the ocular barrier is a tear barrier, a corneal/conjunctival barrier, and a blood-aqueous barrier, or a blood-retinal barrier.

14. A transdermal vaccine carrier prepared from a fluorinated chitosan derivative for use as a drug carrier according to claim 1, comprising a transdermal vaccine carrier (a), wherein the transdermal vaccine carrier (a) is a fluorine-containing compound-modified cationic polymer; the fluorine-containing compound-modified cationic polymer is a fluorinated chitosan; the fluorine-containing compound is covalently attached to the backbone of the chitosan; the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; the transdermal vaccine carrier has three antigen penetration pathways: transcellular penetration, paracellular penetration and transappendageal penetration.

15. A carrier for cosmetic and health care products prepared from a fluorinated chitosan derivative for use as a drug carrier according to claim 1, comprising a carrier for cosmetic and health care products (a), wherein the carrier for cosmetic and health care products (a) is a fluorine-containing compound-modified cationic polymer; the fluorine-containing compound-modified cationic polymer is a fluorinated chitosan; the fluorine-containing compound is covalently attached to the backbone of the chitosan; the chitosan has a molecular weight in the range of 1000-5000000, a degree of deacetylation of not less than 55%, and a viscosity in the range of 25-1000 cps; the carrier for cosmetic and health care products is suitable for hair growth drugs and hair care drugs, cosmetic drugs, and health care drugs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0199] FIG. 1-1 shows the effect of heptafluorobutyric acid modified chitosan (7FCS) on the distribution and intensity of THP in mouse bladder tissue in Example 1-5. THP, epirubicin; CS, chitosan; FCS, fluorinated chitosan; the right panel shows the relative fluorescence intensity analysis of THP corresponding to the left panel.

[0200] FIG. 1-2 shows the effect of tridecafluoroheptanoic acid-modified chitosan (13FCS) on the distribution and intensity of THP in mouse bladder tissue in Example 1-6. THP, epirubicin; CS, chitosan; FCS, fluorinated chitosan; the right panel shows the relative fluorescence intensity analysis of THP corresponding to the left panel.

[0201] FIG. 1-3 shows the effect of different fluorinated fatty acid-modified chitosans (7FCS, 13FCS, 19FCS) on the distribution and intensity of THP in mouse bladder tissue in Example 1-7. THP, epirubicin; CS, chitosan; FCS, fluorinated chitosan; the right panel shows the relative fluorescence intensity analysis of THP corresponding to the left panel.

[0202] FIG. 1-4a is a comparison diagram showing that 13F-3 has good in vitro cell safety in Example 1-8.

[0203] FIG. 1-4b shows that there is no significant difference between the body weight of mice in the FCS group and the blank control group in Example 1-8.

[0204] FIG. 1-4c is a comparison diagram of mouse bladder tissues and HE (hematoxylin-eosin) stained sections after perfusion in each group and those of the blank control group in Example 1-8.

[0205] FIG. 1-5 is a comparison diagram of immunofluorescence results in Example 1-8, showing that the bladder of mice in the chitosan perfusion group has severe inflammatory stress and congestion and edema, while there is no significant difference between the FCS group and the blank control group. The left panel is a confocal fluorescence image of the bladder tissue sections, and the right panel shows the results of the CS and FCS treatment groups and the blank group.

[0206] FIG. 1-6 is a related image of MPI/FPEI measured by transmission electron microscopy in Example 1-9.

[0207] FIG. 1-7 is a related image of MPI/PEI measured by transmission electron microscopy in Example 1-9.

[0208] FIG. 1-8 shows that the mucosal permeability index of a polypeptide in the F-PEI group is significantly higher than that of the PEI group and the blank control group in Example 1-9, where the abscissa is the feed weight ratio of the polypeptide drug MPI to the material PEI or FPEI, and the ordinate is the permeability coefficient papp.

[0209] FIG. 1-9 shows that the mucosal permeability index of a protein drug in the F-PEI group is significantly higher than that of the PEI group and the blank control group in Example 1-9, where the abscissa is the feed weight ratio of the polypeptide drug CAT-Ce6 to the material PEI or FPEI, and the ordinate is the permeability coefficient papp.

[0210] FIG. 1-10 is a comparison diagram showing distribution and intensity of drug fluorescence in tissue when frozen sections of the bladder are prepared at different times after drug perfusion with different drug systems of MPI in Example 1-9, where the abscissa is the perfusion time, and the ordinate is the relative value of the fluorescence intensity of the polypeptide drug.

[0211] FIG. 1-11 is a comparison diagram showing distribution and intensity of drug fluorescence in tissue when frozen sections of the bladder are prepared after perfusion with different drug systems of CAT in Example 1-9, where the left panel is the fluorescence distribution of different drug systems of the drug CAT-Ce6 in the bladder tissue, and the right panel is the fluorescence intensity analysis of the drug.

[0212] FIG. 1-12 is a transmission electron microscope (TEM) image in Example 1-10.

[0213] FIG. 1-13 is a comparison diagram of frozen sections of the bladder and the fluorescence intensity analyzed by a fluorescent confocal microscope in Example 1-10. The left panel shows the fluorescence distribution of the drug CAT-TCPP in the transverse and longitudinal sections of the bladder tissue, from left to right; the right panel shows the fluorescence intensity of the drug in the homogenate of bladder tissue after transverse section and longitudinal section of the bladder, from top to bottom.

[0214] FIG. 1-14 shows a synthetic route diagram of a fluorine-containing carboxylic acid-modified chitosan as a bladder perfusion drug carrier.

[0215] FIG. 2-1 shows a schematic diagram of the transdermal mechanism of a fluorine-containing compound-modified cationic polymer.

[0216] FIG. 2-2 shows changes in particle size of perfluoroheptanoic acid-modified chitosan and a drug in different ratios in an aqueous solution before and after the reaction, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0217] FIG. 2-3 shows changes in potential of perfluoroheptanoic acid-modified chitosan and a drug in different ratios in an aqueous solution before and after the reaction, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0218] FIG. 2-4 shows the difference of transdermal effect of perfluoroheptanoic acid-modified chitosan and a drug in different ratios, where the right panel is a schematic diagram of a transdermal diffusion cell, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0219] FIG. 2-5 shows a real photo and a SEM photo of a gel, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0220] FIG. 2-6 shows release of a drug from a gel, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0221] FIG. 2-7 shows the therapeutic effect of a drug at the living body level, namely, the fluctuation of blood sugar after the application of a patch, take perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0222] FIG. 2-8 shows the particle size and potential of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) in different ratios in an aqueous solution.

[0223] FIG. 2-9 shows cumulative penetration to the skin of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) in different ratios at different time points, in which the ratios of perfluoroheptanoic acid-modified chitosan-immunoglobulin G are 1:0.25, 1:0.5, 1:1, 1:2, 1:4 and 0:1 (pure immunoglobulin G).

[0224] FIG. 2-10 shows dynamic analysis of in vivo skin penetration of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (hereinafter referred to as FCS-IgG), in which the first five are the fluorescence intensity (white) of mouse tumor tissue (gray) sections at different time points, and the sixth one is quantitative analysis of fluorescence intensity detected after tumor tissue lysis at different time points in mice.

[0225] FIG. 2-11 shows comparison of transdermal efficiency in in vivo tumor tissue of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody, chitosan-programmed cell death-ligand 1 antibody and pure programmed cell death-ligand 1 antibody: from the top to the bottom, pure programmed cell death-ligand 1 antibody (free aPDL1), chitosan-programmed cell death-ligand 1 antibody (CS-aPDL1), and perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody (FCS-aPDL1) group; from left to right, the fluorescence intensity of DAPI channel (gray, indicating tumor tissue), FITC fluorescence channel (white, indicating programmed cell death-ligand 1 antibody) and mixed channel.

[0226] FIG. 2-12 shows the transdermal mechanism of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG): the influence of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) on the cell resistance of a dense cell monolayer, where the left panel is a schematic diagram of a measurement method, and the right panel is the resistance change after adding perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG).

[0227] FIG. 2-13 shows the transdermal mechanism of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG): an immunofluorescence staining image of the influence of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) on related tight junction proteins of a dense cell monolayer. The right panel is a semi-quantitative analysis graph of fluorescence intensity.

[0228] FIG. 2-14 shows the transdermal mechanism of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG): Western Blotting analysis of the influence of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) on related tight junction proteins of a dense cell monolayer.

[0229] FIG. 2-15 shows in vivo subcutaneous tumor treatment with perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody, divided into a blank group (blank), a pure programmed cell death-ligand 1 antibody (free aPDL1) group, a chitosan-programmed cell death-ligand 1 antibody (CS-aPDL1) group, and a perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody (FCS-aPDL1) group, where the left panel is mouse tumor growth curve, and the right panel is the mouse survival rate curve (defining mouse tumors greater than 1500 cubic millimeters as dead).

[0230] FIG. 3-1 is a schematic diagram of the intestinal mucosa.

[0231] FIG. 3-2 is a schematic diagram showing the opening of the epithelial cell barrier of intestinal mucosa by perfluoroheptanoic acid-modified chitosan.

[0232] FIG. 3-3 shows changes in particle size of perfluoroheptanoic acid-modified chitosan and a drug in different ratios in an aqueous solution before and after the reaction in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0233] FIG. 3-4 shows changes in potential of perfluoroheptanoic acid-modified chitosan and a drug in different ratios in an aqueous solution before and after the reaction in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0234] FIG. 3-5 shows changes in particle size of a perfluoroheptanoic acid-modified chitosan and drug composite before and after lyophilization in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0235] FIG. 3-6 shows the difference of transmucosal effect of perfluoroheptanoic acid-modified chitosan and a drug in different ratios in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0236] FIG. 3-7 shows the effect of transmucosal drug delivery of a drug-loaded capsule in gastric juice and intestinal juice in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0237] FIG. 3-8 shows blood glucose fluctuation of mice after oral delivery of a drug-loaded capsule in Example 3-1, taking perfluoroheptanoic acid-modified chitosan-insulin (FCS-Insulin) as an example.

[0238] FIG. 3-9 shows cumulative penetration to the intestinal mucosa of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) in different ratios at different time points in Example 3-2. The abscissa is the injection time, and the ordinate is the penetration rate of the cumulative penetration amount divided by the total injection amount. Compared with pure macromolecular protein immunoglobulin G, different molecular weights of perfluoroheptanoic acid-modified chitosan-immunoglobulin G all can increase the efficiency of immunoglobulin G permeating the intestinal mucosa of rats. Preferably, a perfluoroheptanoic acid-modified chitosan-immunoglobulin G composite with a ratio of 1:1 is obtained.

[0239] FIG. 3-10 shows that perfluoroheptanoic acid-modified chitosan-bovine serum albumin-cyanine dye Cy5.5 is at the site of the digestive tract at 3 h and at 5 h in Example 3-2, where white represents the cyanine dye Cy5.5 labeled bovine serum albumin, indicating that the capsule can be targeted to the colorectal release, so the capsule is selected for subsequent experiments.

[0240] FIG. 3-11 is a schematic diagram of the activity of programmed cell death-ligand 1 antibody before and after lyophilization in Example 3-2. After a lyoprotectant is added for lyophilization, the activity of the programmed cell death-ligand 1 antibody after turning into a lyophilized powder keeps unchanged. It shows that the perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles can be freeze-dried and can be used for filling of oral capsules.

[0241] FIG. 3-12 shows the binding activity of FCS/a PD-L1 and PD-L1 on CT-26 cell surface before and after lyophilization in Example 3-2.

[0242] FIG. 3-13 shows changes in the distribution of tight junction proteins in epithelial cells of human colorectal cancer before and after treatment with perfluoroheptanoic acid-modified chitosan and perfluoroheptanoic acid-modified chitosan/immunoglobulin G in Example 3-2.

[0243] FIG. 3-14 shows the effect of a capsule containing freeze-dried powders of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody in the treatment of colorectal cancer in Balb/c mice in Example 3-2, where the black color indicates the bioluminescence intensity of colorectal cancer cells in mice.

[0244] FIG. 3-15 shows the transmucosal effect of different molecular weights of perfluoroheptanoic acid-modified chitosan/immunoglobulin G in Example 3-2.

[0245] FIG. 3-16 is immunofluorescence section of the lungs of mice in Example 3-3. The fluorescence signal indicates the retention of a drug in the lungs.

[0246] FIG. 3-17 shows photos of mouse brain in each experimental group in Example 3-4, in which the white arrows indicate the fluorescence signal of Cy5.5 in the mouse brain.

[0247] FIG. 3-18 shows laser confocal photos of mouse brain sections in Example 3-4, in which the white arrows indicate the fluorescence signal of Cy5.5 in the tumor site in the mouse brain.

[0248] FIG. 4-1 is a schematic diagram of the eyeball structure.

[0249] FIG. 4-2 is a schematic diagram of membrane permeation of a fluorine-containing drug eye drop.

[0250] FIG. 4-3 shows the cumulative permeation percentage of perfluoroheptanoic acid-modified chitosan in different ratios through the cornea in vitro in Example 4-1.

[0251] FIG. 4-4 is an immunofluorescence staining image of a macromolecular drug bovine serum albumin in the eyeball in Example 4-1, where the left panel is an immunofluorescence staining image of membrane penetration of perfluoroheptanoic acid-modified chitosan, and the right panel is an immunofluorescence staining image of membrane penetration of the free drug.

[0252] FIG. 4-5 is an immunofluorescence staining image of a small molecule drug Rhodamine B in the eyeball in Example 4-1, where the left panel is an immunofluorescence staining image of membrane penetration of perfluoroheptanoic acid-modified chitosan, and the right panel is an immunofluorescence staining image of membrane penetration of the free drug.

[0253] FIG. 4-6 shows the eye tissue content of albumin of perfluoroheptanoic acid-modified chitosan after eye instillation in Example 4-1.

[0254] FIG. 4-7 shows immunofluorescence staining of perfluoroheptanoic acid-modified chitosan at different times after eye instillation in local cornea in Example 4-1.

[0255] FIG. 4-8 shows photos of corneal fluorescence staining of perfluoroheptanoic acid-modified chitosan at various time points in mice to detect the safety of perfluoroheptanoic acid-modified chitosan in Example 4-1.

[0256] FIG. 4-9 shows bioluminescence images of malignant choroidal melanoma in situ before treatment and one week after treatment in Example 4-2.

[0257] FIG. 4-10 shows bioluminescence quantitative analysis of malignant choroidal melanoma in situ one week after treatment in Example 4-2.

[0258] FIG. 5-1 shows Flow cytometry processing graph and statistical graph of the percentage of mature dendritic cells in total dendritic cells after in vitro stimulation with a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite in Example 5-1. Mature dendritic cells can effectively present antigens, activate T cells, and initiate immune response. The blank is a naturally mature negative control group without treatment, the lipopolysaccharide group is a positive control group added with lipopolysaccharide, and the perfluoroheptanoic acid-modified chitosan-chicken ovalbumin group serves as an experimental group.

[0259] FIG. 5-2 shows changes in major histocompatibility complex class II (MHC II) and cluster of differentiation 40 (CD40), which are the key proteins for the expression and presentation of antigens by dendritic cells, after in vitro stimulation with a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite in Example 5-1. The stronger the intensity of the allophycocyanin dye, the more CD40 protein is expressed on the surface of dendritic cells, and the stronger the intensity of the phycoerythrin dye indicates the more MHC II is expressed on the surface of dendritic cells.

[0260] FIG. 5-3 shows in vivo imaging of the accumulation of a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite in lymph nodes over time, after a fluorescent perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite and an ointment are mixed and applied to the back of mice in Example 5-1. The white arrow indicates one of the lymph nodes in mice.

[0261] FIG. 5-4 shows tumor growth over time after implanting a melanoma expressing chicken ovalbumin on the cell surface (B16-OVA) on the back of mice, after vaccination for mice implanted with a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin vaccine complex using a transdermal patch and for mice implanted with a chicken ovalbumin vaccine using a transdermal patch in Example 5-2.

[0262] FIG. 6-1 shows the in vitro transdermal delivery efficiency of perfluoroheptanoic acid-modified chitosan and a small molecule drug UK5099 with hair growth treatment effect in different ratios at different time points in Example 6-1.

[0263] FIG. 6-2 shows the in vitro transdermal cumulative permeation of perfluoroheptanoic acid-modified chitosan and a small molecule drug UK5099 with hair growth treatment effect in different ratios at different time points in Example 6-1.

[0264] FIG. 6-3 shows the hair growth on the back of mice at different time points when treated with different treatments in Example 6-1. FCS-metformin group is an experimental group in which metformin is modified with perfluoroheptanoic acid-modified chitosan for transdermal administration, and metformin group is a free drug group.

[0265] FIG. 6-4 shows the results of Example 6-2. The left panel shows the efficiency of a mixture of perfluoroheptanoic acid-modified chitosan and poly(I.C) for the production of retinoic acid in mouse fibroblasts. The right panel shows the effect of the mixture of perfluoroheptanoic acid-modified chitosan and poly(I:C) when applied to the scars of mice.

[0266] FIG. 6-5 shows that in a rat skin permeation experiment with a diffusion cell, the cumulative permeation percentage of tranexamic acid through rat skin [(cumulative permeation/theoretical total permeation)×100%] within three hours using a mixture of perfluoroheptanoic acid-modified chitosan and tranexamic acid and pure tranexamic acid in Example 6-3.

DETAILED DESCRIPTION OF THE INVENTION

[0267] The following describes the present invention in further detail with reference to specific examples and drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages that those skilled in the art can think of are all included in the present invention, and the scope of protection is subject to the attached claims.

[0268] Description of abbreviations: THP (epirubicin); EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysulfosuccinyl imine); DMSO (dimethyl sulfoxide); MPI (a polypeptide drug Polybia-MPI); MPICy5.5 (fluorescence labeling of polypeptide drug MPI); PEI (polyethylene imine); FPEI (fluorinated polyethylene imine); CAT (a protein drug catalase); CAT-Ce6 (a composite protein drug labeled with photosensitizer Ce6); CAT-TCPP (a composite protein drug labeled with sonosensitizer TCPP).

[0269] In view of the fact that there are many examples and drawings in the present document, in order to distinguish between different groups of examples and different drawings, the naming of the examples adopts the following rules: the first series of examples is Example 1-1, Example 1-2, Example 1-3, and so on, the second series of examples are Example 2-1, Example 2-2, and so on. Similar rules are adopted for the naming of the drawings: the first series of drawings are FIG. 1-1, FIG. 1-2, and FIG. 1-3, and so on, the second series of drawings are FIG. 2-1, FIG. 2-2, and so on.

Reference Example

[0270] Experimental designers used adriamycin, epirubicin (THP) and fluorescent dye rhodamine B as a bladder perfusion drug respectively to mix with chitosan (1% acetic acid aqueous solution) to prepare chitosan bladder perfusion drug systems. After 1 h of bladder perfusion, the bladder tissue was removed, frozen sections were prepared, the fluorescence intensity of the drug was analyzed with a confocal fluorescence microscope, and the distribution of the drug in the bladder tissue was investigated. The experimental results show that the adhesion and penetration ability of the chitosan (5 mg/ml, 10 mg/ml, 15 mg/ml) solution of the bladder perfusion drug of equal concentration in the bladder mucosa is significantly better than that in the case of perfusion of the aqueous solution of the pure drug. As the concentration of chitosan increases, the adhesion and penetration ability of the perfusion system is stronger. In order to further investigate the biological safety of chitosan as a drug carrier for perfusion, the designers of the project perfused the mouse bladder with a 15 mg/ml chitosan aqueous solution, stopped the perfusion after 1 h, continued to raise the mice under normal feeding conditions, and recorded the mice weight. The results of the experiment show that the weight of the mice dropped sharply on the second day after perfusion of chitosan, and the mice were sluggish in activity. From the second day after treatment, the mice began to die, and all 8 mice in the experimental group died within 3 days. After dissection, the bladder of the mice in the experimental group was compared with that of the mice in the control group. It was found that the bladder of mice in the chitosan perfusion group was more congested compared with that in the blank group, and the results of HE staining and CD45 and Ki67 immunofluorescence showed that the bladder of the mice in the chitosan perfusion group had severe inflammatory stress and congestion and edema. The above experimental results show that although chitosan can significantly improve the bioavailability of the perfusion drug in the bladder mucosa, high concentrations of chitosan can also cause serious bladder mucosal and epithelial damage, which severely limits its clinical use as a drug carrier for bladder perfusion.

[0271] Example 1-1: Preparation of chitosan with different modification degrees of 3-fluorobenzoic acid (degree of deacetylation ≥95%, viscosity 100-200 MPa.Math.s), in which the feed molar ratios of 3-fluorobenzoic acid and N-glucosamine unit were 1:1.1, 1:2.2, 1:4.4, and 1:8.8.

[0272] Synthesis method: (1) preparation of a solution of chitosan in an aqueous acetic acid solution: 200 mg of fully dried chitosan was weighed and added into 10 mol % of an aqueous acetic acid solution (of course, an aqueous hydrochloric acid solution could also be used) and stirred for 30 min to be fully dissolved, and then 1.6 ml of 0.5M sodium hydroxide was slowly added dropwise and stirred until a clear solution was obtained and the pH was about 6.5. From the perspective of simply considering the alkalizing solution, sodium hydroxide may be replaced by alkali such as ammonia or triethylamine, but from the perspective of product technology, the by-product is sodium chloride when sodium hydroxide is used, which is more suitable for industrialization. In this way, 4 parts of aqueous solutions of chitosan in acetic acid were prepared. (2) activation of 3-fluorobenzoic acid: 5.0 mg, 9.8 mg, 19.7 mg and 40 mg of 3-fluorobenzoic acid were weighed respectively and dissolved in an appropriate amount of anhydrous dimethyl sulfoxide, and the reaction amounts of EDC and NHS were sequentially added, and stirred for 1 h under protection from light. (3) preparation of 3-fluorobenzoyl chitosan: the above-mentioned activated 3-fluorobenzoic acid solutions were slowly added dropwise respectively to the rapidly stirring chitosan solution, and stirred for 20 h under protection from light. After the reaction was complete, the reacted solutions above were slowly added dropwise successively to 100 ml of a 0.5M solution of potassium hydroxide in ethanol and stirred for 8 h, a precipitate was filtered and washed with a large amount of absolute ethanol until a filtrate was neutral, the precipitate was dehydrated by washing with methanol and diethyl ether and dried in vacuum for 30 min. The dried precipitate was dissolved in 10 ml 0.1M of a hydrochloric acid solution and lyophilized to obtain 3-fluorobenzoic acid fluorinated chitosan hydrochloride with different degrees of fluorination modification in the appearance of white powder (the products were named 1FCS-1, 1FCS-2, 1FCS-3, and 1FCS-4).

[0273] The materials obtained from the above reactions were tested for the degree of modification of the fluorinated fatty chains on the surface of the fluorinated chitosan (FCS) polymer by means of the ninhydrin reaction method. The ninhydrin reaction method is a simple, fast, accurate and reliable method, which can accurately detect the number of primary amino groups on the surface of FCS polymer in the aqueous solution, to calculate the number of fluorinated groups on the surface of FCS.

[0274] Example 1-2: Preparation of chitosan with different modification degrees of heptafluorobutyric acid (degree of deacetylation ≥95%, viscosity 100-200 MPa.Math.s), in which the feed molar ratios of heptafluorobutyric acid and N-glucosamine unit were 1:1.1, 1:2.2, 1:4.4, and 1:8.8.

[0275] Synthesis method: (1) preparation of a solution of chitosan in an aqueous acetic acid solution: 200 mg of fully dried chitosan was weighed and added into 10 mol % of an aqueous acetic acid solution and stirred for 30 min to be fully dissolved, and then 1.6 ml of 0.5M sodium hydroxide was slowly added dropwise and stirred until a clear solution was obtained and the pH was about 6.5. In this way, 4 parts of aqueous solutions of chitosan in acetic acid were prepared. (2) activation of heptafluorobutyric acid: 7.6 mg, 15 mg, 30 mg, and 61 mg of heptafluorobutyric acid were weighed respectively and dissolved in an appropriate amount of anhydrous dimethyl sulfoxide, and the reaction amounts of EDC and NHS were sequentially added, and stirred for 1 h under protection from light. (3) preparation of heptafluorobutyric acid chitosan: the above-mentioned activated heptafluorobutyric acid solutions were slowly added dropwise respectively to the rapidly stirring chitosan solution, and stirred for 20 h under protection from light. After the reaction was complete, the reactions were slowly added dropwise successively to 100 ml of a 0.5M solution of potassium hydroxide in ethanol and stirred for 8 h, a precipitate was filtered and washed with a large amount of absolute ethanol until a filtrate was neutral, the precipitate was dehydrated by washing with methanol and diethyl ether and dried in vacuum for 30 min. The dried precipitate was dissolved in 10 ml 0.1M of a hydrochloric acid solution and lyophilized to obtain heptafluorobutyric acid fluorinated chitosan hydrochloride with different degrees of fluorination modification in the appearance of white powder (the products were named 7FCS-1, 7FCS-2, 7FCS-3, and 7FCS-4).

[0276] The materials obtained from the above reactions were tested for the degree of modification of the fluorinated fatty chains on the surface of the fluorinated chitosan (FCS) polymer by means of the ninhydrin reaction method. The ninhydrin reaction method is a simple, fast, accurate and reliable method, which can accurately detect the number of primary amino groups on the surface of FCS polymer in the aqueous solution, to calculate the number of fluorinated groups on the surface of FCS. By the ninhydrin reaction method, the degrees of fluorination modification of FCS prepared above were calculated to be: 7FCS-1, 6.9%; 7FCS-2, 10.4%; 7FCS-3, 23.5%; 7FCS-4, 42.3%.

[0277] Example 1-3: Preparation of chitosan with different modification degrees of perfluoroheptanoic acid (degree of deacetylation ≥95%, viscosity 100-200 MPa.Math.s), in which the feed molar ratios of perfluoroheptanoic acid and N-glucosamine unit were 1:1.1, 1:2.2, 1:4.4, and 1:8.8.

[0278] Synthesis method: (1) preparation of a solution of chitosan in an aqueous acetic acid solution: 200 mg of fully dried chitosan was weighed and added into 10 mol % of an aqueous acetic acid solution and stirred for 30 min to be fully dissolved, and then 1.6 ml of 0.5M sodium hydroxide was slowly added dropwise and stirred until a clear solution was obtained and the pH was about 6.5. In this way, 4 parts of aqueous solutions of chitosan in acetic acid were prepared. (2) activation of perfluoroheptanoic acid (13-fluoroheptanoic acid): 13 mg, 26 mg, 51.5 mg and 103 mg of perfluoroheptanoic acid were weighed respectively and dissolved in an appropriate amount of anhydrous dimethyl sulfoxide, and appropriate amounts of EDC and NHS were sequentially added, and fully stirred for 1 h under protection from light. (3) preparation of 13F-heptanoic acid chitosan: the above-mentioned activated perfluoroheptanoic acid solutions were slowly added dropwise respectively to the rapidly stirring chitosan solution, and stirred for 20 h under protection from light. After the reaction was complete, the reactions were slowly added dropwise successively to 100 ml of a 0.5M solution of potassium hydroxide in ethanol and stirred for 8 h, a precipitate was filtered and washed with a large amount of absolute ethanol until a filtrate was neutral, the precipitate was dehydrated by washing with methanol and diethyl ether and dried in vacuum for 30 min. The dried precipitate was dissolved in 10 ml 0.1M of a hydrochloric acid solution and lyophilized to obtain heptafluorobutyric acid fluorinated chitosan hydrochloride with different degrees of fluorination modification in the appearance of white powder (the products were named 13FCS-1, 13FCS-2, 13FCS-3, and 13FCS-4). By the ninhydrin reaction method, the degrees of fluorination modification of FCS prepared above were calculated to be: 13FCS-1, 5.2%; 13FCS-2, 11.3%; 13FCS-3, 21.4%; 13FCS-4, 42.5%. The linking efficiency of 13FCS-1 to 13FCS-413 fluoroheptacarbonyl group is 5.2% to 42.5% with the increase of perfluoroheptanoic acid feed, that is, on average, 5.2% to 42.5% of the glucose structural units in each chitosan molecule have been fluorinated and the products are named 13FCS-1, 13FCS-2, 13FCS-3, 13FCS-4.

[0279] Example 1-4: Preparation of chitosan with different modification degrees of 19F-capric acid (degree of deacetylation ≥95%, viscosity 100-200 MPa.Math.s), in which the feed molar ratios of 19F-capric acid and N-glucosamine unit were 1:1.1, 1:2.2.

[0280] Synthesis method: (1) preparation of a solution of chitosan in an aqueous acetic acid solution: 200 mg of fully dried chitosan was weighed and added into 10 mol % of an aqueous acetic acid solution and stirred for 30 min to be fully dissolved, and then 1.6 ml of 0.5M sodium hydroxide was slowly added dropwise and stirred until a clear solution was obtained and the pH was about 6.5. In this way, 2 parts of aqueous solutions of chitosan in acetic acid were prepared. (2) activation of 19F-capric acid: 18 mg and 36.7 mg of 19F-capric acid were weighed respectively and dissolved in an appropriate amount of anhydrous dimethyl sulfoxide, and appropriate amounts of EDC and NHS were sequentially added, and fully stirred for 1 h under protection from light. (3) preparation of 19F-capric acid chitosan: the above-mentioned activated 19F-capric acid solutions were slowly added dropwise respectively to the rapidly stirring chitosan solution, and stirred for 20 h under protection from light. After the reaction was complete, the reactions were slowly added dropwise successively to 100 ml of a 0.5M solution of potassium hydroxide in ethanol and stirred for 8 h, a precipitate was filtered and washed with a large amount of absolute ethanol until a filtrate was neutral, the precipitate was dehydrated by washing with methanol and diethyl ether and dried in vacuum for 30 min. The dried precipitate was dissolved in 10 ml 0.1M of a hydrochloric acid solution and lyophilized to obtain 19F-capric acid fluorinated chitosan hydrochloride with different degrees of fluorination modification in the appearance of white powder (the products were named 19FCS-1 and 19FCS-2).

[0281] The water solubility of 19FCS-2 was relatively poor, and subsequent characterization and application evaluation could not be carried out. Therefore, the degree of fluorination modification of 19FCS-1 prepared above was calculated by the ninhydrin reaction method as follows: 19FCS-1, 5.2%.

[0282] Example 1-5: Evaluation of bladder mucosa penetration promoting effect of prepared 7FCS: The 7FCS prepared in Example 1-2 was mixed with an aqueous THP solution, and perfused into the bladder through the mouse urethra. Then, frozen sections of the mouse bladder were prepared, and the promotion of the drug carrier on absorption efficiency of the drug in the bladder mucosa was evaluated by detecting the distribution of THP fluorescence in the tissue.

[0283] The specific method was as follows: female C57BL/6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. 0.2% THP solution was prepared with 0.5% aqueous FCS solution, and 100 μl of the solution was perfused into the mouse bladder with a closed intravenous indwelling needle. The urethra was clamped for 1 h, and then the perfusion solution in the bladder was released. The bladder was flushed with 1 ml ultrapure water, the bladder tissue was removed and placed in a tissue embedding machine at −80° C., and then sections were made and examined with a fluorescent confocal microscope. An aqueous pure THP solution of equal concentration or an aqueous THP chitosan solution prepared in the same way was used as a control.

[0284] Experimental results: referring to FIG. 1-1, the left side is microscope pictures of mouse bladder tissue sections under different conditions, and the right side is the corresponding data statistics, where the right abscissa is different drug systems, and the ordinate is relative fluorescence intensity.

[0285] As shown in FIG. 1-1, a confocal fluorescence microscope was used to observe the mouse bladder tissue sections. The results showed that the distribution area and intensity of the drug fluorescence on the cross section of the bladder of the mice in the 7FCS group were significantly higher than those of chitosan (CS) and the blank control group (aqueous pure THP solution), indicating that FCS can significantly improve the tissue permeability of the drug in the bladder mucosa, and the penetration promoting effect increases with the increase of the degree of fluorination, However, when the degree of fluorination reaches a certain level (7FCS, 42.3%), the prepared FCS has the most significant penetration promoting effect, and at this time, the prepared 7FCS has poorer solubility than low-fluorination substituted chitosan, which is not conducive to clinical use. The above results indicate that FCS can significantly improve the penetration and absorption of drugs and enhance the absorption efficiency of drugs in the bladder mucosa, but excessive fluorination substitution may not be conducive to the application of materials.

[0286] Example 1-6: Promotion of 13FCS on absorption of a bladder perfusion drug in the bladder mucosa: The 13FCS prepared in Example 1-3 was mixed with an aqueous THP solution, and perfused into the bladder through the mouse urethra. Then, frozen sections of the mouse bladder were prepared, and the efficiency of the drug carrier on promoting bladder mucosal absorption of the drug was evaluated by detecting the distribution of THP fluorescence in the tissue.

[0287] The specific method was as follows: female C57BL/6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. 0.2% aqueous THP solution was prepared with 0.5% aqueous 13FCS solution, and 100 μl of the solution was perfused into the mouse bladder with a closed intravenous indwelling needle. The urethra was clamped for 1 h, and then the perfusion solution in the bladder was released. The bladder was flushed with 1 ml ultrapure water, the bladder tissue was removed and placed in a tissue embedding machine at −80° C., and then sections were made and examined with a fluorescent confocal microscope. An aqueous THP chitosan solution prepared in the same way was used as a control.

[0288] The experimental results are shown in FIG. 1-2. The distribution area and intensity of the drug fluorescence on the longitudinal section of the mouse bladder in the FCS group were significantly higher than that of chitosan (CS), indicating that 13FCS can significantly improve the permeability of the drug in the bladder mucosa; and also, 13FCS-3 has the strongest penetration promoting effect, and its degree of fluorination modification is 21.4%. The above results also indicate that the promoting effect of FCS on penetration of the perfusion drug into the bladder mucosa may be the result of the combined effect of the chitosan cationic skeleton and the fluorinated fatty chain. Of course, this reason may also be diverse.

[0289] Example 1-7: In order to screen fluorinated chitosan with the best promoting effect on penetration of a perfusion drug into the bladder mucosa, 19FCS-1 in Example 1-4 and FCS with the best effect among the above-mentioned fluorination modification types were subjected to in vivo evaluation in mice.

[0290] The specific method was as follows: female C57BL/6 mice aged 10-12 weeks were anesthetized with pentobarbital solution. 0.2% THP solution was prepared with 0.5% aqueous solution of 7FCS-4, 13FCS-3 and 19FCS-1 respectively, and 100 μl of the solution was perfused into the mouse bladder with a closed intravenous indwelling needle. The urethra was clamped for 1 h, and then the perfusion solution in the bladder was released. The bladder was flushed with 1 ml ultrapure water, the bladder tissue was removed and placed in a tissue embedding machine at −80° C., and then sections were made and examined with a fluorescent confocal microscope. An aqueous pure THP solution of equal concentration or an aqueous THP chitosan solution prepared in the same way was used as a control.

[0291] Experimental results: As shown in FIG. 1-3, the mouse bladder tissue sections were observed with a fluorescent confocal microscope. The results showed that the distribution area and intensity of drug fluorescence on the cross-section of the mouse bladder in the 19FCS group were significantly different than those in the THP and CS groups. However, 13FCS-3 has the most prominent effect of promoting the permeability of the perfusion drug.

[0292] Example 1-8: In vitro and in vivo safety evaluations of different types of fluorinated fluorinated chitosan in Example 1-7 were carried out. The specific experimental protocol was as follows:

[0293] The CCK-8 (Cell Counting Kit-8) method (a mature in vitro evaluation method for evaluating cell activity) was used to evaluate the cytotoxic effect of fluorinated chitosan on SV-HUC-1 human normal bladder epithelial cells. In vitro biological safety of fluorinated chitosan was investigated, and the specific operation was as follows: T24 cells were seeded into a 96-well plate at 1×10.sup.4 cells/well, and incubated overnight at 37° C., 5% CO.sub.2. Serum-free medium with different types of fluorinated chitosan (500 ug/ml) were added for further 24 h culture. Then, an appropriate amount of cck-8 was added. Finally, the in vitro safety of fluorinated chitosan was evaluated by cell viability. The experimental results are shown in FIG. 1-4a. 13F-3 (abbreviation of 13FCS-3) has good cell safety in vitro. Combined with the above research results, it is shown that 13F-3 has the most significant effect of promoting the absorption of the perfusion drug into the bladder mucosa, and also has good cell safety in vitro. In order to further evaluate the biosafety of FCS (13F-3), further safety evaluation experiment in vivo in mice was carried out.

[0294] Healthy C57BL/6 mice aged 10-12 weeks were divided into three groups, 8 mice/each group. The experimental group was perfused with 15 mg/ml fluorinated chitosan or 1% chitosan in aqueous acetic acid solution for 1 h, once a week for three weeks, and the blank control group was perfused with equal volume of double distilled water. The mouse body weight, survival rate, and HE and immunohistochemical analysis (CD45 and Ki67) results of mouse bladder sections on day 28 after the first administration were used to evaluate the in vivo biological safety of fluorinated chitosan.

[0295] The experiment results showed that the weight of the mice dropped sharply on the second day after perfusion of chitosan, the mice were sluggish in activity, and the mice began to die from the second day after the treatment, and all 8 mice in the experimental group died within 3 days, while no death occurred in the fluorinated chitosan group. As shown in FIG. 1-4b, there was no significant difference between the body weight of the FCS group and the blank control group. In addition, as shown in FIG. 1-4c, the mouse bladder after perfusion in each group was compared with that of the mice in the control group. It was found that the bladder of mice in the chitosan perfusion group was more congested compared with that in the blank group, and the results of HE staining and CD45 and Ki67 immunofluorescence in FIG. 1-5 showed that the bladder of the mice in the chitosan perfusion group had severe inflammatory stress and congestion and edema, while There was no significant difference between the FCS group and the blank control group. The above experimental results show that fluorinated chitosan (13FCS-3) can significantly improve the bioavailability of the perfusion drug in the bladder mucosa, and high concentration of fluorinated chitosan will not cause obvious bladder mucosal and epithelial damage, and has the potential as a drug carrier for bladder perfusion.

Example 1-9: Application of F-PEI Bladder Perfusion Polypeptide Protein Drug Carrier, Taking Polypeptide Drug MPI and Protein Drug CAT-Ce6 as Examples

[0296] (1) Synthesis of Fluorinated Polyether Imide (F-PEI)

[0297] The specific operation was 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 stirred at room temperature for 48 h. The crude reaction product was purified by dialysis with methanol/double distilled water (MWCO 3500 Da), and 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 by fluorine element analysis.

[0298] (2) Preparation and Characterization of MPI/F-PEI, CAT-Ce6/F-PEI Nano Drug Systems

[0299] MPI/FPEI, CAT/F-PEI NPs could be obtained by mixing polypeptide (MPI), protein (CAT) drug and aqueous F-PEI solution for 2 h at room temperature. The hydrated particle size measured by a dynamic light scattering instrument was about 200-300 nm with a small amount of positive charge. It was characterized by transmission electron microscopy (TEM) imaging (FIGS. 1-6, 1-7) as uniform spherical particles.

[0300] (3) Evaluation of Bladder Mucosa Permeability of MPI/F-PEI NPs, CAT-Ce6/F-PEI NPs

[0301] The Ussing chamber (also called Ussing perfusion chamber) is a tool for studying transepithelial transport, and can be used for research including ion transport, nutrient transport, and drug transport. Through the study of transepithelial transport, we can understand the absorption of epithelial drugs through the epithelium. In this example, the Ussing chamber was used 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 respectively used as control. The mice were anesthetized, the bladder was removed and placed on ice, and the bladder mucosa was peeled off and fixed at the interface between the two chambers. 3 ml of MPI-cy5.5/F-PEI NPs or CAT-ce6/F-PEI NPs bench-top solution was added into the diffusion chamber, and an equal volume of blank bench-top solution was added into the receiving chamber. 0.5 ml of the bench-top solution was taken from the receiving chamber every 15 min and simultaneously, an equal volume of the blank bench-top solution was added in the receiving chamber for four consecutive times, and the corresponding drug content was detected by a fluorescence spectrophotometer. The experimental results showed that the mucosal permeability index papp of polypeptide (FIG. 1-8) or protein drug (FIG. 1-9) in the F-PEI group was significantly higher than that of the PEI group and the free group (blank group).

[0302] At the same time, we also investigated the permeability of the bladder mucosa in mice. After the mice were anesthetized, the bladder was perfused with solutions containing the same amount of fluorescently labeled polypeptide or protein drug respectively, and for different drug systems in the MPI group (free MPI-cy5.5, MPI-cy5.5/PEI, MPI-cy5.5/F-PEI), frozen sections were prepared from the bladder at different times (15, 30, 60 min) after drug perfusion (FIG. 1-10); for different drug systems in the CAT group (free CAT-ce6, CAT-ce6/PEI, CAT-ce6/F-PEI), frozen sections were prepared from the bladder after 1 h of perfusion, and the fluorescence intensity was analyzed by a fluorescent confocal microscope (FIG. 1-11). The experimental results show that the polypeptide and protein drugs in the F-PEI group have more significant bladder mucosal permeability compared with the PEI and free drug groups, that is, F-PEI can significantly improve the bladder mucosal permeability of polypeptide or protein drugs.

Example 1-10: Application of FCS Bladder Perfusion Protein Drug Carrier, Taking the Protein Drug CAT-TCPP as an Example

[0303] (1) Preparation and Characterization of CAT-TCPP/FCS Nano Drug System

[0304] CAT-TCPP/FCS NPs could be obtained by mixing the protein drug (CAT-TCPP) with an aqueous solution of FCS for 2 h at room temperature. The hydrated particle size measured by a dynamic light scattering instrument was about 200-300 nm with a small amount of positive charge. It was characterized by transmission electron microscopy (TEM) imaging (FIG. 1-12) as uniform spherical particles.

[0305] (2) Evaluation of Bladder Mucosa Permeability of CAT-TCPP/FCS NPs

[0306] We also investigated the permeability of the bladder mucosa in mice. After the mice were anesthetized, the bladder was perfused with solutions containing the same amount of fluorescently labeled protein drug respectively. For different drug systems (free CAT-TCPP, CAT-TCPP/CS, CAT-TCPP/FCS) in the CAT-TCPP group, frozen sections were prepared from the bladder at the same time point (60 min) after drug perfusion (FIG. 1-13), and the fluorescence intensity was analyzed by a fluorescent confocal microscope. The experimental results show that the protein drugs in the FCS group have more significant bladder mucosal retention capacity and permeability compared with the chitosan (CS) and the free drug group, that is, FCS can significantly improve the bladder mucosal permeability of the protein drug.

[0307] Unless otherwise specified, the perfluoroheptanoic acid-modified chitosan used in all the examples of the present invention is fluorinated chitosan in Example 1-3 where the feed molar ratio of perfluoroheptanoic acid to N-glucosamine unit is 1:4.2.

[0308] The following is a schematic diagram of the structure of fluorinated chitosan in Example 1-3.

##STR00011##

[0309] wherein A is a chitosan molecular skeleton containing primary amino groups, and the structural formula is as follows:

##STR00012##

[0310] wherein B is a linking group formed by a fluorine-containing functional group and a primary amino group of the chitosan, and is herein an amide bond, namely,

##STR00013##

[0311] wherein C is a fluorine-containing aliphatic chain or an aromatic ring functional group; perfluoroheptanoic acid is used herein, and the structural formula is as follows:

##STR00014##

[0312] Example 2-1: A transdermal patch was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and insulin was transdermally delivered for the treatment of diabetes. For the specific preparation process of fluorinated chitosan in this example, refer to Example 2-5.

[0313] Specific Method:

[0314] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-insulin composite: perfluoroheptanoic acid-modified chitosan and insulin were respectively dissolved in a weak acid solution environment until uniformly dissolved. A weak base solution was dropwise added during stirring after uniform mixing, the pH was adjusted to 6-7, and under neutral conditions, perfluoroheptanoic acid-modified chitosan and insulin were bound to together due to electrostatic adsorption to form a stable composite, in which the preferred reaction weight ratio of perfluoroheptanoic acid-modified chitosan to insulin was 1:0.25-4, more preferably 1:0.5-2. After the reaction was complete, it was pre-added with a cryoprotectant for lyophilization, to obtain a perfluoroheptanoic acid-modified chitosan-insulin lyophilized powder. The particle size (FIG. 2-2) and the potential (FIG. 2-3) were analyzed by dynamic light scattering.

[0315] As shown in FIG. 2-2, reaction of perfluoroheptanoic acid-modified chitosan and insulin in different ratios all could generate composites. When the weight ratio of perfluoroheptanoic acid-modified chitosan to insulin was 1:0.5-2, the particle size was relatively uniform.

[0316] As shown in FIG. 2-3, the different ratios of perfluoroheptanoic acid-modified chitosan to insulin affect the overall potential of the composite.

[0317] 2. In vitro transdermal kinetic analysis: the insulin in step 1 was replaced with fluorescently labeled insulin, and optimization and transdermal kinetic analysis of the prepared perfluoroheptanoic acid-modified chitosan-insulin (hereinafter referred to as FCS-Insulin) in different ratios were performed. First, perfluoroheptanoic acid-modified chitosan-insulin in different weight ratios was synthesized and put into the injection cell of the Franz vertical diffusion cell, and then the fluorescence intensity of perfluoroheptanoic acid-modified chitosan-insulin that enters the sampling pool through the sandwiched mouse skin was tested to characterize its transdermal effect at different time points. The ratios of perfluoroheptanoic acid-modified chitosan-insulin are 1:0.25, 1:0.5, 1:1, 1:2, 1:4 and 0:1 (pure immunoglobulin G). The abscissa is the penetration time, and the ordinate is the cumulative penetration calculated from fluorescence. The results are shown in FIG. 2-4, and the preferred ratio is 1:1.

[0318] 3. Preparation of a transdermal patch of a perfluoroheptanoic acid-modified chitosan-insulin composite: perfluoroheptanoic acid-modified chitosan—insulin lyophilized powder was reconstituted and a hydrogel matrix prepared in advance was added and uniformly mixed to obtain a transdermal patch of a perfluoroheptanoic acid-modified chitosan-insulin composite, as shown in FIG. 2-5 on the left. The SEM characterization of the gel is shown in FIG. 2-5 on the right.

[0319] 4. Measurement of drug release from the gel: the perfluoroheptanoic acid modified perfluoroheptanoic acid-modified chitosan—insulin lyophilized powder was reconstituted and different concentrations of a hydrogel matrix prepared in advance was added and uniformly mixed to obtain a transdermal patch of a perfluoroheptanoic acid-modified chitosan-insulin composite. The concentration of the gel affects the release behavior of the drug from the gel. The gel patch was soaked in a buffer solution and the supernatant of the solution was taken at different time points. According to the Coomassie brilliant blue staining, the cumulative insulin penetration was calculated, as shown in FIG. 2-6.

[0320] The abscissa is the time, and the ordinate is the cumulative insulin penetration calculated according to Coomassie Brilliant Blue staining.

[0321] 5. Evaluation of the penetration enhancing effect of a drug-loaded transdermal patch by changes in blood glucose in mice: female C57BL/6 mice aged 10-12 weeks were anesthetized with isoflurane, and a drug-loaded transdermal patch was applied to the depilated skin on the back of the mouse, and fixed with an elastic bandage, and then the blood glucose fluctuation of the mouse was measured at different time points, and a blank patch was used as a control. FIG. 2-7 shows the blood glucose fluctuations of mice, where the abscissa is the time of action after the patch is applied, and the ordinate is the blood glucose concentration.

[0322] As shown in FIG. 2-7, the blood glucose of the mice was monitored by a blood glucose meter, and it is found that compared with the blank control group, hyperglycemia in the mice applied with perfluoroheptanoic acid-modified chitosan drug-loaded transdermal patch is significantly suppressed and remains stable for a long time, which indicates that perfluoroheptanoic acid-modified chitosan can significantly improve the permeability of the drug in the skin, and promote the drug to enter the blood to maintain a stable blood drug concentration, achieving a sustained role. The above results collectively indicate that the fluorine-containing compound-modified cationic polymer can successfully achieve the transdermal delivery of the drug, and has great medical value and transformation value.

[0323] Example 2-2: A transdermal ointment was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and programmed cell death-ligand 1 antibody was transdermally delivered for the treatment of surface melanoma.

[0324] Specific Method:

[0325] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-immunoglobulin G composite (hereinafter referred to as FCS-IgG): Since immunoglobulin G and programmed cell death-ligand 1 antibody have the same structure, at the material level, immunoglobulin G was used as a template to study the optimal ratio of binding of perfluoroheptanoic acid-modified chitosan and programmed cell death-ligand 1 antibody. Different amounts of immunoglobulin G were added to an aqueous solution of perfluoroheptanoic acid-modified chitosan and stirred at room temperature for 1 h to form a stable composite. Herein, the reaction weight ratio of perfluoroheptanoic acid-modified chitosan and immunoglobulin G was 1:0.25-4, and was further preferably 1:1 through particle size analysis and potential analysis by dynamic light scattering. The results of particle size distribution and potential distribution are shown in FIG. 2-8.

[0326] An immunoglobulin refers to a globulin that has the activity or chemical structure of an antibody and is similar to an antibody molecule. The immunoglobulin G used in the present experiment is not specific. The antibody is an immunoglobulin that specifically binds to an antigen. The programmed cell death-ligand 1 antibody is also a type of immunoglobulin G, but the light chain end is specific, so immunoglobulin G can be used to simulate the behavior of programmed cell death-ligand 1 antibody in non-therapeutic experiments.

[0327] Experimental results: referring to FIG. 2-8, the left panel shows the particle size distribution of perfluoroheptanoic acid-modified chitosan-immunoglobulin G in different ratios in an aqueous solution, and the right panel shows the potential distribution. Herein, the 1:1 group that has a better particle size and maintains a higher positive charge is preferred.

[0328] 2. Preparation of a transdermal ointment of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: In the same way, a perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody composite was obtained. An aqueous solution of the obtained perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody was mixed with a blank ointment at a weight ratio of 1:1 to form a transdermal ointment of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody, in which the main component of the ointment was petrolatum.

[0329] 3. In vitro transdermal kinetic analysis of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: with fluorescently labeled immunoglobulin G as a template to simulate transdermal kinetics of programmed cell death-ligand 1 antibody, optimization and transdermal kinetic analysis of the prepared perfluoroheptanoic acid modified chitosan-immunoglobulin G (hereinafter referred to as FCS-IgG) were performed. First, perfluoroheptanoic acid-modified chitosan-immunoglobulin G in different weight ratios was synthesized and put into the injection cell of the Franz vertical diffusion cell, and then the fluorescence intensity of perfluoroheptanoic acid-modified chitosan-immunoglobulin G that enters the sampling pool through the sandwiched mouse skin was tested to characterize its transdermal effect at different time points. The ratios of perfluoroheptanoic acid-modified chitosan-immunoglobulin G are 1:0.25, 1:0.5, 1:1, 1:2, 1:4 and 0:1 (pure immunoglobulin G). In each group, samples were taken at 2 h, 4 h, 8 h, 12 h and 24 h after drug application, and the cumulative penetration was calculated. The results are shown in FIG. 2-9.

[0330] Experimental results: FIG. 2-9 shows cumulative penetration of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) in different ratios at different time points. The abscissa is the injection time, and the ordinate is the penetration rate of the cumulative penetration amount divided by the total injection amount. Compared with pure macromolecular protein immunoglobulin G, perfluoroheptanoic acid-modified chitosan-immunoglobulin G in different ratios of all can penetrate the skin of mice to varying degrees. Preferably, a perfluoroheptanoic acid-modified chitosan-immunoglobulin G composite with a ratio of 1:1 is obtained, and used for subsequent experiments.

[0331] 4. In vivo transdermal kinetic analysis of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: with fluorescently labeled immunoglobulin G as a template to simulate transdermal kinetics of programmed cell death-ligand 1 antibody, dynamic analysis of in vivo skin penetration of the prepared perfluoroheptanoic acid modified chitosan-immunoglobulin G (hereinafter referred to as FCS-IgG) were performed. First, C57 mice were subcutaneously injected with B16 melanocyte suspension (about 1*10{circumflex over ( )}6 cells/mouse). When the tumor volume was about 60 cubic millimeters, the surface of the mouse tumor was smeared with a perfluoroheptanoic acid modified chitosan-immunoglobulin G ointment in a weight ratio of 1:1, and fixed with a transdermal patch to prevent falling. The mice were killed at different time points, the tumor tissue was removed, the remaining ointment on the surface was wiped off and the epidermis was removed. The tumor tissue was divided into two, half of which was lysed and the fluorescence intensity in the tissue was measured, and the other half of which was sliced for fluorescence imaging under a confocal microscope. The results are shown in FIG. 2-10.

[0332] Experimental results: Refer to FIG. 2-10, showing fluorescence imaging with a confocal fluorescence microscope and fluorescence content in tumor after lysis for the tumor tissues dissected respectively at 0 h, 4 h, 8 h, 12 h and 24 h of the perfluoroheptanoic acid-modified chitosan-immunoglobulin G ointment. It can be clearly found that the penetration rate of the ointment reaches a peak at 12 h and begins to decrease at 24 h. This may be due to the degradation of perfluoroheptanoic acid-modified chitosan-immunoglobulin G that enters the tumor.

[0333] 5. Comparison of in vivo transdermal efficiency of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: according to the kinetic analysis, the time point of applying the ointment for 12 h was preferred, and comparison of transdermal efficiency of pure programmed cell death-ligand 1 antibody, chitosan-programmed cell death-ligand 1 antibody and perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody was performed. The same amounts of pure programmed cell death-ligand 1 antibody, chitosan-programmed cell death-ligand 1 antibody and perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody were applied to the tumor site of the tumor-bearing mice, and fixed with transparent films. After 12 h, the mice were sacrificed and the tumor tissues were removed and subjected to immunofluorescence staining and confocal microscopy fluorescence imaging. The results are shown in FIG. 2-4.

[0334] Experimental results: see FIG. 2-11. From left to right are DAPI, FITC, and mixed channel fluorescence. DAPI is a nuclear dye that is indicative of the nucleus, and FITC is a fluorescent secondary antibody label for programmed cell death-ligand 1 antibody. It can be seen that the perfluoroheptanoic acid modified chitosan-programmed cell death-ligand 1 antibody group has the strongest transdermal efficiency under the same dosage of programmed cell death-ligand 1 antibody.

[0335] 7. Transdermal mechanism study of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: with immunoglobulin G as a template to simulate programmed cell death-ligand 1 antibody, the transdermal mechanism of perfluoroheptanoic acid-modified chitosan-immunoglobulin G was analyzed. The first was the verification of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) for the transdermal effect in a dense cell monolayer. The human skin epithelial cells Hacat were incubated in a Transwell plate as shown in FIG. 2-12. A cell resistance tester was used to detect the resistance changes every 1 day until the growth reached the plateau in which the resistance no longer changed, and at this time, perfluoroheptanoic acid-modified chitosan-immunoglobulin G was added, and the detection of resistance changes was continued. The results are shown in FIG. 2-5.

[0336] Experimental results: see FIG. 2-12. The formation of a dense cell monolayer was observed on day 10, and FCS-IgG was added on day 11. The resistance change was detected 24 h later, and a sudden decrease in resistance was seen, indicating that the dense cell monolayer was opened, and then the resistance slowly recovered to a plateau, indicating that the material is temporary for opening tight junctions between cells.

[0337] 8. Transdermal mechanism study of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: with immunoglobulin G as a template to simulate programmed cell death-ligand 1 antibody, changes of related proteins that regulate tight junctions between cells after adding perfluoroheptanoic acid modified chitosan-immunoglobulin G (FCS-IgG) were further analyzed. The human skin epithelial cells Hacat were incubated in a special petri dish for a confocal microscope, and allowed to grow into a dense cell monolayer. Then, 24 h after adding FCS-IgG, the cells in the petri dish were subjected to immunofluorescence staining, and tight junction related proteins were detected respectively: Occludin, Claudin-1, E-Cadherin and ZO-1. The results are shown in FIG. 2-13.

[0338] Experimental results: see FIG. 2-13. After adding FCS-IgG, the protein fluorescence content in each group (the white band around the white cells in the figure) decreased significantly. It shows that FCS-IgG can open the tight junctions of cells by changing the distribution of tight junction proteins in the cell membrane, to penetrate the skin through the intercellular space.

[0339] 9. Transdermal mechanism study of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: with immunoglobulin G as a template to simulate programmed cell death-ligand 1 antibody, the mechanism of tight junction protein distribution changes was further studied. The human skin epithelial cells Hacat were incubated in a 25 mm2 petri dish and allowed to grow into a dense cell monolayer. Then, after the cells were lysed, the whole cell proteins were determined by Western Blotting. First, the changes in the content of the four tight junction proteins were detected, and then the changes in the phosphorylation level of actin were detected. The tight junction proteins are Occludin, Claudin-1, E-Cadherin and ZO-1; actin is MLC, phosphated actin is p-MLC; GAPDH is glyceraldehyde-3-phosphate dehydrogenase, which has relatively stable expression in various tissues, and is used here as an internal control. The results are shown in FIG. 2-14.

[0340] Experimental results: Refer to FIG. 2-14. The left panel shows the changes in the content of the four tight junction proteins. It can be seen that after adding FCS-IgG, the protein content has no significant change, indicating that FCS-IgG only affects the distribution of the tight junction proteins on the cell membrane surface, rather than reducing their expression, further indicating that this effect is only temporary. The right panel shows the changes in phosphorylation level of actin. It can be seen that there is significant phosphorylation of actin, indicating that FCS-IgG stimulates the paracellular transport by stimulating actin phosphorylation, thereby further opening the intercellular space and promoting the material to penetrate the skin through the intercellular space.

[0341] 10. In vivo subcutaneous tumor treatment with perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: C57 mice were equally divided into 4 groups, a blank group, a group with intravenous injection of programmed cell death-ligand 1 antibody alone, a chitosan-programmed cell death-ligand 1 antibody group, and a perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody group, respectively. Mice were injected subcutaneously with B16 cell suspension (approximately 1*10{circumflex over ( )}6 pcs), the tumor size was monitored every day, and the tumor volume was calculated by the following formula: volume=0.5*tumor length*tumor width{circumflex over ( )}2. When the initial tumor size was about 10 cubic millimeters, application of an ointment or intravenous administration was started, and the treatment was performed every other day for a total of 4 treatments, and the tumor size was recorded every other day. The results are shown in FIG. 2-8.

[0342] Experimental results: see FIG. 2-15. The left panel is a mouse tumor growth curve, and the right panel is a broken line graph of mouse survival rate. The tumor size of 1500 cubic millimeters serves as the standard of mouse death. It can be found that due to the small initial volume, the intravenous injection therapy conventionality used in clinical practice also has a certain inhibitory effect on mouse tumors, but in contrast, the therapeutic effect of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody is much higher than that of the other groups because the group has an antibody penetration rate of more than 50%.

[0343] The fluorine-containing compound-modified cationic polymers in the present invention, especially fluorinated chitosans involves drugs including but not limited to diabetes treatment drugs, anti-tumor drugs (see Table 2-1 below for details), immunomodulators, antiviral drugs, anti-inflammatory drugs, analgesic and anesthetic drugs, medical cosmetic drugs, and the like and derivatives thereof in various dosage forms.

TABLE-US-00006 TABLE 2-1 Hormone Estrogen drugs steroid estrogens such as quinestrol, ethinylestradiol, nilestriol, drugs estradiol benzoate, estradiol 3,17-dipropionate, estradiol valerate, estradiol 17-cyclopentanoate, menstrand, and derivatives thereof non-steroidal hormones and derivatives thereof such as diethylstilbestrol and derivatives thereof anti-estrogens such as fulvestrant, aminoglutethimide, formestane, anastrozole, letrozole, exemestane, clomiphene, tamoxifen, toremifene, and derivatives thereof Androgen androgen drugs such as methyltestosterone, testosterone propionate, drugs and derivatives thereof anabolic hormones such as oxymetholone, stanozolol, nandrolone phenylpropionate, metandienone, and derivatives thereof anti-androgen drugs such as flutamide, spironolactone, finasteride, abiraterone, and derivatives thereof Progestogen progesterone drugs such as progesterone and medroxyprogesterone drugs acetate, and derivatives thereof testosterone drugs such as levonorgestrel, and derivatives thereof progestogen antagonist drugs such as mifepristone, and derivatives thereof Adrenal hydrocortisone, corticosterone, aldosterone, triamcinolone, corticosteroids prednisolone, dexamethasone acetate, methylprednisolone aceponate and the like and derivatives thereof Prostaglandins misoprostol and the like and derivatives thereof Peptide insulin, calcitonin, oxytocin and the like hormones Signal Tyrosine imatinib, dasatinib (Sprycel), nilotinib and the like and derivatives transduction protein kinase thereof inhibitors inhibitors Cyclooxygenase aspirin, acetaminophen, non-specific cyclooxygenase inhibitors such inhibitors as antipyrine, metamizole sodium, phenylbutazone, oxyphenbutazone, mefenamic acid, indometacin, sulindac, diclofenac sodium, ibuprofen, naproxen, piroxicam, meloxicam and derivatives thereof specific COX-2 inhibitor drugs such as celecoxib, and derivatives thereof Calcium ion nifedipine, verapamil, diltiazem, flunarizine, prenylamine and the like channel and derivatives thereof blockers Phosphatidyl puquitinib mesylate and the like and derivatives thereof alcohol kinase inhibitors Serine protein SP1NK6 antibody (serine protein kinase inhibitor antibody) and the kinase like and derivatives thereof inhibitors Antitumor Bioalkylating chlormethine hydrochloride, chlorambucil, melphalan, prenimustine, drugs agents cyclophosphamide, thiotepa, carmustine, busulfan, cisplatin, carboplatin and the like and derivatives thereof Anti-metabolic fluorouracil, cytarabine, mercaptopurine, methotrexate and the like drugs and derivatives thereof Antitumor actinomycin D, doxorubicin, zorubicin, mitoxantrone and the like and antibiotics derivatives thereof Effective natural medicine active ingredients such as 10-hydroxycamptothecin, components of vinblastine sulfate, paclitaxel, docetaxel, and derivatives thereof traditional Chinese Angiogenesis Monoclonal bevacizumab, ranibizumab and the like and derivatives thereof inhibitors antibodies Small molecule sorafenib, sunitinib, vandetanib, vatalanib and the like and derivatives inhibitors thereof Angiopoietin Angiopoietin signaling pathway inhibitors and derivatives thereof signaling Immunotherapy Checkpoint CTLA4 monoclonal antibody (Ipilimumab ®), PD-1 monoclonal drugs inhibitors antibody (Pembrolizumab ®, Nivolumab ®, Arezolizumab ®, Avelumab ®, Durvelumab ®), PD-LI monoclonal antibody (Atezolizumab ®, Avelumab ®, Durvalumab ®), LAG-3 (lymphocyte activation gene 3) monoclonal antibody, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) monoclonal antibody, TIGIT (T cell immunoglobulin and ITIM domain protein) monoclonal antibody, costimulatory factors B7-H3, B7-H4 and B7-H5 monoclonal antibodies and the like and derivatives thereof. Cytokines IFNa2b, IFNa2a, IL-2, etc. Oncolytic recombinant human papgm-csf activated HSV genes, etc. viruses Bispecific CD19 and CD3b specific antibodies, etc.

[0344] The drugs may be immunomodulators, including, but not limited to cytokines, BCG, immune checkpoint blocking antibodies, and the like. Cytokines are a class of small molecular proteins with a wide range of biological activities synthesized or secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NiK cells, etc.) and some non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokines include, but are not limited to, interleukins (ILs), interferons (IFNs), tumor necrosis factors (TNFs), colony stimulating factors (CSFs), chemokine family, growth factors (GFs), transforming growth factor-β family (TGF-β family). The interleukins include, but are not limited to, IL-1-IL-38. The colony-stimulating factors include, but are not limited to, G (granulocyte)-CSF, M (macrophage)-CSF, GM (granulocyte, macrophage)-CSF, Multi (multiple)-CSF (IL-3), SCF, EPO etc. The interferons include, but are not limited to, IFN-α, IFN-β, and IFN-γ. The tumor necrosis factors include, but are not limited to, TNF-α and TNF-β. The transforming growth factor-β family includes, but is not limited to, TGF-β1, TGF-β2, TGF-β3, TGFβ1β2, and bone morphogenetic proteins (BMPs). The growth factors include, but are not limited to, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncostatin M (OSM), platelet-derived endothelial cell growth factor (PDECGF), transforming growth factor-α (TGF-α), vascular endothelial cell growth factor (VEGF). The chemokine family includes, but is not limited to, four subfamilies: (1) CXC/a subfamily, mainly chemotactic neutrophils, the main members of which are IL-8, melanoma growth stimulating activity (GRO/MGSA), platelet factor-4 (PF-4), platelet basic protein, proteolysis-derived products CTAP-III and β-thromboglobulin, inflammatory protein 10 (IP-10), ENA-78. (2) CC/3 subfamily, mainly chemotactic monocytes, the members of which include macrophage inflammatory protein 1α (MIP-1α), MIP-1β, RANTES, monocyte chemotactic protein-1 (MCP-1/MCAF), MCP-2, MCP-3 and I-309. (3) Type C subfamily, the representative of which is lymphotactin. (4) CX3C subfamily, Fractalkine, which is a CX3C type chemokine and has a chemotactic effect on monocytes-macrophages, T cells and NK cells.

[0345] The cytokines include, but are not limited to, cytokines used to treat cancer and cytokines that reduce the side effects of cancer treatment. They play an important role in the normal immune response of the human body and the ability of the immune system to respond to cancer. The cytokines used to treat cancer include, but are not limited to, interferons and interleukins. The cytokines may also be hematopoietic growth factors, which reduce the side effects of cancer treatment by promoting the growth of blood cells destroyed by chemotherapy. The cytokines that reduce the side effects of cancer treatment include, but are not limited to, erythropoietin, IL-11, granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF). BCG Vaccine is a live vaccine made from a suspension of attenuated Mycobacterium bovis, which can increase the activity of macrophages, improve the body's cellular immunity, and be used to treat bladder cancer. Immunomodulatory drugs include, but are not limited to thalidomide (Thalomid®), lenalidomide (Revlimid®), pomalidomide (Pomalyst®), imiquimod (Aldara®, Zyclara®). Immune checkpoint blocking antibodies include but are not limited to CTLA4 monoclonal antibody (Lpilimumab®), PD-1 monoclonal antibody (Pembrolizumab®, Nivolumab®, Arezolizumab®, Avelumab®, Durvelumab®), PD-L1 monoclonal antibody (Atezolizumab®, Avelumab®, Durvalumab®), LAG-3 (lymphocyte activation gene 3) monoclonal antibody, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) monoclonal antibody, TIGIT (T cell immunoglobulin and ITIM domain protein) monoclonal antibody, co-stimulatory factors B7-H3, B7-H4 and B7-H5 monoclonal antibodies and the like and derivatives thereof.

[0346] The drugs may be anesthetic drugs. Example drugs of general anesthesia include but are not limited to ketamine hydrochloride, propofol, sodium thiopental, etomidate, midazolam and sodium γ-hydroxybutyrate. Local anesthetics include, but are not limited to, aromatic acid esters, aromatic amides, amino ketones, amino ethers, carbamates, hydroxyprocaine, chloroprocaine, tetracaine, butacaine, thiocaine, procainamide, bupivacaine, articaine, etidocaine, ropivacaine, mepivacaine, clonin and the like and derivatives thereof.

[0347] The drugs may be diabetes treatment drugs including, but not limited to, sulfonylureas such as sulfambutamide, tolbutamide, chlorpropamide, acetohexamide, gliclazide, glipyride, glimepiride, and derivatives thereof; non-sulfonylureas such as repaglinide, nateglinide, and derivatives thereof, thiazolidinediones such as rosiglitazone, pioglitazone, and derivatives thereof; biguanides such as phenformin, metformin, and derivatives thereof; α-glucosidase inhibitors such as acarbose, voglibose, miglitol, and derivatives thereof; dipeptidyl peptidase-IV drugs such as glucagon-like peptide, DPP-IV inhibitors, sitagliptin, vildagliptin, and saxagliptin, and insulin and the like and derivatives thereof.

[0348] Diabetes is a metabolic endocrine disease mainly characterized by hyperglycemia, and the clinical dosage form is generally insulin injection. Patients need to endure the pain of repeated injections, and long-term medication can also cause side effects such as inflammation and induration at the injection site. The fluorine-containing compound-modified chitosan drugs can penetrate the skin, carry hypoglycemic drugs into the blood, and improve the bioavailability of the drugs. As described in Example 2-1, the fluorine-containing compound-modified chitosan drugs can be used as a drug carrier to deliver hypoglycemic drugs, which can be administered in the form of drug patches for the treatment of diabetes. By use of transdermal administration with fluorine-containing compound-modified chitosan drug patches, the effective concentration of the drugs is maintained for a long time, and the degree of action and maintenance time can be adjusted according to the area of application and the application time, which has the advantages of flexibility and convenience. In addition, it can also be prepared into a more flexible lotion, liniment, smear and other dosage forms.

[0349] The drugs may be anti-tumor drugs (see Table 2-1 for details). Although transdermal delivery is a non-invasive way of delivery, it brings great convenience, the stratum corneum barrier of the skin often prevents the drug from entering the subcutaneous lesion or even entering the blood vessel. Melanoma is a malignant tumor that originates from cells that can produce melanin. It is easy to metastasize, has strong drug resistance, poor prognosis, and extremely high mortality. Melanoma chemotherapy drugs are mainly delivered by oral and injection methods, but this often causes many adverse reactions. It even leads to organ damage, and also it is unable to deliver drugs efficiently, accurately and controllably. The transdermal delivery mode has unique advantages for the treatment of subcutaneous melanoma, but higher requirements for the efficiency of transdermal delivery is put forward. As described in b2-2, the fluorine-containing compound-modified chitosan can be used as a drug carrier to deliver anti-tumor drugs, which can be administered in the form of ointments for the treatment of tumor diseases.

[0350] The fluorinated chitosan in each of Examples 2-1 to 2-6 of the present invention can be used as a transdermal preparation, and is useful as a transdermal administration preparation for diabetes treatment drugs, tumor disease drugs, and anti-inflammatory drugs. Also, it can also be used as a transdermal administration preparation in the preparation of a medical cosmetic drug, a topical drug preparation, a topical preparation for medical devices, and a cosmetic skin care product.

[0351] Example 3-1: An oral drug was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and insulin was orally delivered for the treatment of treat diabetes.

[0352] Specific Method:

[0353] 1. Preparation of perfluoroheptanoic acid-modified chitosan-insulin capsules: perfluoroheptanoic acid-modified chitosan and insulin were dissolved in a weak acid solution until uniformly dissolved, a weak base solution was dropwise added under stirring after uniform mixing, the pH was adjusted to 6-7, and under neutral conditions, the perfluoroheptanoic acid-modified chitosa and the insulin bound together due to electrostatic adsorption to form stable nanoparticles. After the reaction was complete, it was pre-added with a cryoprotectant for lyophilization, to obtain a perfluoroheptanoic acid-modified chitosan-insulin lyophilized powder. The lyophilized powder was filled into capsules and wrapped in enteric coating.

[0354] 2. The perfluoroheptanoic acid-modified chitosan—insulin lyophilized powder was reconstituted, and the particle size (FIG. 3-3) and the potential (FIG. 3-4) were analyzed by dynamic light scattering.

[0355] 3. The perfluoroheptanoic acid-modified chitosan-insulin before and after lyophilization was subjected to particle size analysis by dynamic light scattering, and as shown in FIG. 3-5, there was no significant change before and after lyophilization.

[0356] 4. In vitro transdermal kinetic analysis: the insulin in step 1 was replaced with fluorescently labeled insulin, and optimization and transdermal kinetic analysis of the prepared perfluoroheptanoic acid-modified chitosan-insulin (hereinafter referred to as FCS-Insulin) in different ratios were performed. First, perfluoroheptanoic acid-modified chitosan-insulin in different weight ratios was synthesized and put into the donor chamber of the Franz vertical diffusion cell, and then the fluorescence intensity of perfluoroheptanoic acid-modified chitosan-insulin that enters the receptor chamber through the mouse mucosa was tested to characterize its transdermal effect at different time points. The results are shown in FIG. 3-6, and the preferred ratio is 1:0.5-2.

[0357] 5. The drug-loaded capsule was put into simulated gastric juice or simulated intestinal juice, and the releasing effect in simulated gastric juice or simulated intestinal juice was characterized by the fluorescence intensity of the perfluoroheptanoic acid-modified chitosan-insulin released in the solution. As shown in FIG. 3-7, the left side is the release behavior in simulated gastric juice, and the right side is the release behavior in intestinal juice. The capsule can remain stable and is not released for a long time in the simulated gastric juice, while the drug can be released in the simulated intestinal juice.

[0358] 6. Evaluation of the administration effect by changes in blood glucose of mice: perfluoroheptanoic acid-modified chitosan-insulin capsules were lavaged, and then blood glucose fluctuations in mice were measured at different time points, and a blank capsule was used as a control.

[0359] FIG. 3-8 shows the blood glucose fluctuations of mice, where the abscissa is the time after capsules were lavaged, and the ordinate is the blood glucose concentration.

[0360] As shown in FIG. 3-8, the blood glucose of the mice was monitored by a blood glucose meter, and it is found that compared with the blank control group, hyperglycemia in the mice lavaged with perfluoroheptanoic acid-modified chitosan insulin capsules is significantly suppressed and remains stable for a long time, which indicates that perfluoroheptanoic acid-modified chitosan can significantly improve the retention and penetration of the drug in the intestine, and promote the drug to enter the blood to maintain a stable blood drug concentration, achieving a sustained role. The above results collectively indicate that the fluorine-containing compound-modified cationic polymer can successfully achieve the oral delivery of the drug, and has good medical and transformation values.

[0361] Example 3-2: An oral capsule was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, programmed cell death-ligand 1 antibody was orally delivered, and the mucous membrane penetrating effect of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles in different ratios was observed.

[0362] Specific Method:

[0363] 1. Preparation of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles: 0.2 mg of perfluoroheptanoic acid-modified chitosan was weighted and dissolved in 0.5 mL of ultrapure water to obtain 0.4 mg/mL of perfluoroheptanoic acid-modified chitosan, and gradient dilution was performed to finally obtain 0.4 mg/mL, 0.2 mg/mL, 0.1 mg/mL, 0.05 mg/mL, and 0.0025 mg/mL solutions of perfluoroheptanoic acid-modified chitosan. Fluorescently labeled immunoglobulin G was used as a template to replace programmed cell death-ligand 1 antibody, and 0.5 mL 0.1 mg/mL of FITC-labeled immunoglobulin G in 0.02M potassium phosphate buffer at pH=7.2 was added dropwise under constant stirring, and stirred at room temperature for 30 min.

[0364] 2. In vitro kinetic analysis through the intestinal mucosa of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: the rat intestine was removed of the fascia layer, and fixed at the upper part of the transdermal diffusion cell. Perfluoroheptanoic acid-modified chitosan-FITC-immunoglobulin G particles with different weight ratios were added at the upper part of the transdermal diffusion cell, and a PBS solution was added at the lower part of the transdermal diffusion cell. The liquids were taken at the lower part of the transdermal diffusion cell at 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 180 min, and 210 min, the fluorescence intensity was measured, and the cumulative penetration rate was calculated. The results are shown in FIG. 3-9.

[0365] Experimental results: FIG. 3-9 shows cumulative penetration of perfluoroheptanoic acid-modified chitosan-immunoglobulin G (FCS-IgG) in different ratios at different time points. The abscissa is the injection time, and the ordinate is the penetration rate of the cumulative penetration amount divided by the total injection amount. Compared with pure macromolecular protein immunoglobulin G, perfluoroheptanoic acid-modified chitosan-immunoglobulin G in different ratios of all can penetrate the skin of mice to varying degrees. Preferably, perfluoroheptanoic acid-modified chitosan-immunoglobulin G nanoparticles with a ratio of 1:1 are obtained, and used for subsequent experiments.

[0366] 3. Preparation of lyophilized powder of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles: 0.1 mg of perfluoroheptanoic acid-modified chitosan was weighted and dissolved in 1 mL of ultrapure water to obtain 0.1 mg/mL of a perfluoroheptanoic acid-modified chitosan solution. 0.5 mL 0.1 mg/mL of cy5.5-labeled immunoglobulin G in 0.02M potassium phosphate buffer at pH=7.2 was added dropwise under constant stirring, and stirred at room temperature for 30 min. After the stirring was complete, a lyoprotectant was added, and the solution was pre-cooled in a refrigerator at −20° C. and then dried in a freeze dryer.

[0367] 4. Preparation of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody capsules and targeted release properties thereof: a certain amount of lyophilized powder was weighed and filled into dedicated capsules for mice, and coating was performed with Eudragit S100. The mice were fed the above capsules with an applicator, and the mice were sacrificed at 3 h and 5 h, the intestines were removed, and small animal imaging was used to photograph the fluorescence distribution of cy5.5 in the intestines to determine the distribution of the capsules.

[0368] Experimental results: Refer to FIG. 3-10. Fluorescence can be observed in the colorectal area at 3 h and 5 h, indicating that the capsule can be targeted to the colorectal release. Therefore, the capsule was selected for subsequent experiments.

[0369] 5. Activity determination of lyophilized powder of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles before and after lyophilization: the frozen programmed cell death-ligand 1 was diluted in a carbonate buffer to different concentrations, and added to an enzyme-linked immunosorbent assay (ELISA) plate, and the plate was placed at 4° C. overnight. After the coating was complete, it was washed with PBST three times, and a BSA solution was added and blocking was performed for 2 h at room temperature. After the blocking was complete, it was washed with PBST three times, and the programmed cell death-ligand 1 antibody before and after lyophilization was added and incubated at room temperature for two hours. After the incubation was complete, it was washed with PBST three times, and horseradish peroxidase-labeled goat anti-rat secondary antibody was added and incubated at room temperature for one hour. After the incubation was complete, the color-developing agent TMB was added, and then a stop solution was added to stop the color development. The absorbance of the ELISA plate at OD450 was determined to calculate the antibody affinity before and after lyophilization.

[0370] Experimental results: Refer to FIG. 3-11. After a lyoprotectant is added for lyophilization, the activity of the programmed cell death-ligand 1 antibody after turning into a lyophilized powder keeps unchanged. It shows that the perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles can be freeze-dried and can be used for filling of oral capsules.

[0371] 6. Binding activity of FCS/a PD-L1 and PD-L1 on CT-26 cell surface before and after lyophilization

[0372] (1) 500 ng of IFN-γ was added to 10 mL of 1640 medium to serve as a culture medium for CT-26 cells, and co-incubated with CT-26 cells for 36 h.

[0373] (2) the adherent CT-26 was scraped off with a cell scraper, and washed three times with FACS buffer (50 mL PBS+0.5 mL serum), and the supernatant was discarded.

[0374] (3) 1*10.sup.6 cells were added to each 1.5 mL centrifuge tube and FCS/α PD-L1 was added with the content of α PD-L1 being 1 μg, incubation was performed at room temperature for 0.5 h, the supernatant was discarded, and the cells were washed three times with FACS buffer, and then the supernatant was discarded.

[0375] (4) 0.5 μL of PE anti PD-L1 antibody was added to each 1.5 mL centrifuge tube and incubated for 30 min at room temperature, the supernatant was discarded, and the cells were washed 3 times with FACs buffer. Finally, 200 μL FACS buffer was added.

[0376] (5) Binding activity of FCS/a PD-L1 and PD-L1 on CT-26 cell surface after lyophilization was tested with a flow cytometer. The results are shown in FIG. 3-13.

[0377] The experimental results are shown in FIG. 3-12. It can be seen that the binding capacity of FCS/α PD-L1 and PD-L1 on CT-26 cell surface after lyophilization is not much different from the binding activity of FCS/α PD-L1 before lyophilization. FCS/a PD-L1 can be used to further make capsules.

[0378] 7. Changes in tight junction proteins in epithelial cells of human colorectal cancer before and after treatment with perfluoroheptanoic acid-modified chitosan and perfluoroheptanoic acid-modified chitosan/immunoglobulin G.

[0379] (1) 0.1 mg/mL FCS/IgG or FCS solution in medium was formulated, and then placed in a confocal small dish with CaCO-2 monolayer cells cultured, and incubated in a constant-temperature incubator at 37° C. for 5 h.

[0380] (2) the culture medium was removed, and the cells were washed three times with PBS for 5 min each time.

[0381] (3) 4% paraformaldehyde solution was added to each well, and the cells were fixed on ice for 20 min, and washed three times with PBS for 5 min each time.

[0382] (4) 0.1% Triton x-100 solution was added to each well, and the cells were left for 15 min, and washed three times with PBS for 5 min each time.

[0383] (5) 2% BSA solution was added to each small dish, and the cells were blocked at room temperature for 1 h.

[0384] (6) ZO-1 antibody solution was added at 1:200 or E-Caclherin antibody was added at 1:1000, and the temperature was maintained at 4° C. overnight; the cells were removed and washed three times with PBS for 5 min each time.

[0385] (7) FITC Goat anti rabbit was added to each well, and the cells were incubated for 1 h at room temperature, and washed three times with PBS.

[0386] (8) DAPI solution was added, and the cells were incubated for 5 min, and washed three times with PBS for 5 min each time.

[0387] (9) Pictures were taken with a confocal microscope, and the experimental results are shown in FIG. 3-13.

[0388] The experimental results are shown in FIG. 3-13. It can be seen that after FCS/IgG is added, the tight junction proteins in the cells become loose and the cytoskeleton rearranges.

[0389] 8. Treatment of colorectal cancer in mice with perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody

[0390] (1) 1 mg of perfluoroheptanoic acid-modified chitosan was weighted and dissolved in 1 mL of ultrapure water to obtain 1 mg/mL of a perfluoroheptanoic acid-modified chitosan solution. 1 mL of 1 mg/mL programmed cell death-ligand 1 antibody was added dropwise under constant stirring, and stirred at room temperature for 30 min. After the stirring was complete, a lyoprotectant was added, and the solution was pre-cooled in a refrigerator at −20° C. and then dried in a freeze dryer.

[0391] (2) Lyophilized powder was weighed and filled into dedicated capsules for mice, and coating was performed with Eudragit S100. The prepared capsules were stored for future use.

[0392] (3) Balb/c mice were anesthetized with 1% sodium pentobarbital. The mice were fixed with the abdomen facing up, a small opening was made on the right side of the abdomen, the cecum was removed, and 500,000 CT-26 cells transfected with luciferase were injected into the wall of the cecum. Then, the cecum was put back, the wound was sutured, and treatment was started four days later.

[0393] (4) The mice were fed the capsules prepared in step 2 with an applicator, and then 100 μL of metoclopramide hydrochloride was gavaged to promote gastric emptying of the mice. The capsules were given on day 4, 7, 12, and 16 after the tumor was re-implanted.

[0394] (5) On the third day after administration, Balb/c mice were anesthetized with 1% sodium pentobarbital, and each mouse was injected with a bioluminescent substrate. Ten mins later, the mice were imaged and the tumor growth was observed. The experimental results are shown in FIG. 3-14.

[0395] The experimental results are shown in FIG. 3-14. It can be seen that after treatment with the capsules containing perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody, the tumor growth trend can be significantly suppressed. This indicates that perfluoroheptanoic acid-modified chitosan can significantly improve the penetration of drugs in the colon, and promote the entry of drugs into tumor tissues, thereby achieving a sustained role.

[0396] 9. The effect through the intestinal mucosa with different molecular weights of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody

[0397] (1) Preparation of different perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody particles: perfluoroheptanoic acid-modified chitosan with molecular weights of 10 Kda, 50 Kda, 100 Kda, 300 Kda, and 400 Kda were weighted and dissolved in ultrapure water to obtain 0.4 mg/mL of perfluoroheptanoic acid-modified chitosans. Fluorescently labeled immunoglobulin G was used as a template to replace programmed cell death-ligand 1 antibody, and 0.5 mL 0.1 mg/mL of FITC-labeled immunoglobulin G in 0.02M potassium phosphate buffer at pH=7.2 was added dropwise under constant stirring, and stirred at room temperature for 30 min.

[0398] (2) In vitro kinetic analysis through the intestinal mucosa of perfluoroheptanoic acid-modified chitosan-programmed cell death-ligand 1 antibody: the rat intestine was removed of the fascia layer, and fixed at the upper part of the transdermal diffusion cell. Perfluoroheptanoic acid-modified chitosan-FITC-immunoglobulin G particles with different weight ratios were added at the upper part of the transdermal diffusion cell, and a PBS solution was added at the lower part of the transdermal diffusion cell. The liquids were taken at the lower part of the transdermal diffusion cell at 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min, 180 min, and 210 min, the fluorescence intensity was measured, and the cumulative penetration rate was calculated. The results are shown in FIG. 2-7.

[0399] As shown in FIG. 3-15, the perfluoroheptanoic acid-modified chitosans of different molecular weights have different effects on increasing the penetration of drugs through the mucosa. This indicates that the perfluoroheptanoic acid-modified chitosans can promote the drugs to enter tumor tissues.

[0400] Example 3-3: perfluoroheptanoic acid-modified chitosan-immunomodulator particles were prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and administered as sprays/inhalants, and the ability of drug delivery to the lungs was investigated.

[0401] Specific Method:

[0402] 1. Preparation of perfluoroheptanoic acid-modified chitosan-antibody particles: the programmed cell death-ligand 1 antibody was added to the aqueous solution of perfluoroheptanoic acid-modified chitosan and stirred for 1 h at room temperature to form stable nanoparticles.

[0403] 2. The perfluoroheptanoic acid-modified chitosan-antibody particles were delivered as aerosol via a pulmonary administration needle, tissue sections were made 24 h after delivery, and the retention of fluorescent signals in the lung tissue was observed. The results are shown in FIG. 3-16.

[0404] The experimental results are shown in FIG. 3-16. Perfluoroheptanoic acid-modified chitosan-antibody particles can stay in the lungs.

[0405] Example 3-4: With bovine serum albumin (BSA-Cy5.5) labeled with Cy5.5 fluorescent molecule as a model protein, and perfluoroheptanoic acid-modified chitosan (FCS) as a transnasal mucosa carrier, perfluoroheptanoic acid-modified chitosan-bovine serum albumin nanoparticles were prepared, and administered as nasal drops, and the delivery efficiency of BSA through the nose to the brain in a mouse glioma model was investigated by the fluorescence signal of Cy5.5.

[0406] Specific Method:

[0407] 1. Preparation of FCS/BSA-Cy5.5 nanoparticles: perfluoroheptanoic acid-modified chitosan was dissolved in 1% acetic acid until completely dissolved, and a weak base was added dropwise to adjust the pH of the solution to 6-7. Then, it was uniformly mixed with the BSA-Cy5.5 solution, and stirred at 4° C. for 1 h to form stable nanoparticles as nasal drops. Herein, the preferred reaction weight ratio of perfluoroheptanoic acid-modified chitosan and BSA was 1:1, and the final bovine serum albumin concentration was 2.5 mg/mL.

[0408] 2. Construction of a glioma model: male C57BL/6 mice (about 20 g/mouse) aged 7-8 weeks were intra-cerebrally injected with EGFP-expressing glioma cells (5000 cells/mouse) to construct a glioma model.

[0409] 3. Administration with nasal drops: the successfully modeled C57BL/6 mice were anesthetized, and fixed on a heating pad in a supine position. A total volume of 20 μL of FCS/BSA-Cy5.5 solution (with free BSA-Cy5.5 solution as a control group) was slowly dripped into the mouse nostrils with a pipette, and after the addition was completed, the supine position of the mouse was further maintained for 30 min.

[0410] 4. 72 h after nasal drip, the mice were anesthetized and perfused with formalin, then the brain tissue was removed, lyophilized and sectioned, and the fluorescence signal of Cy5.5 at the tumor tissue in the mouse brain was observed by a confocal microscope.

[0411] The experimental results are shown in FIG. 3-17. Compared with the control groups free BSA-Cy5.5 and CS/BSA-Cy5.5, the experimental group FCS/BSA-Cy5.5 has the best brain fluorescence intensity at each time point, indicating that FCS has the best auxiliary effect for BSA delivery through the nasal mucosa to the brain.

[0412] Referring to FIG. 3-18, the left panel represents the EGFP signal of brain tumor in mouse brain tissue, and the right panel represents the Cy5.5 signal of BSA delivered to the brain via the nose. It can be seen from the results on the right panel that compared with the control groups free BSA-Cy5.5 and CS/BSA-Cy5.5, in the experimental group FCS/BSA-Cy5.5, the intact mouse brain tissue removed after nasal drip operation has the highest Cy5.5 fluorescence intensity, indicating that FCS/BSA-Cy5.5 has the best nasal to brain delivery efficiency.

[0413] Referring to FIG. 3-19, EGFP represents tumor tissue and Cy5.5 represents IgG. By comparing the Cy5.5 signal, it can be seen that the Cy5.5 signal of the brain tumor site in the FCS group is the strongest, indicating that FCS has the highest nasal to brain delivery efficiency of BSA.

[0414] Example 4-1: Taking bovine serum albumin labeled with Cy5.5 fluorescent substance and small molecule fluorescent substance Rhodamine B as examples, fluorinated chitosan was used to encapsulate the drugs, and length and depth of penetration in the eye were observed.

[0415] Specific Method:

[0416] 1. The experimental rabbit eyes were removed for dissection, the rabbit cornea was separated and fixed on the Franz diffusion cell, and compared to the free protein, the transmembrane effect of the protein at different time points after mixing with different proportions of perfluoroheptanoic acid-modified chitosan was monitored. Herein, the ratios of protein to perfluoroheptanoic acid-modified chitosan are 1:0.25, 1:0.5, 1:1, and 1:4. In each group, samples were taken at 5 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h after drug application. Finally, the cumulative penetration was calculated by detecting the fluorescence of the label on the protein. The results are shown in FIG. 4-3. Finally, the ratio of protein and perfluoroheptanoic acid-modified chitosan of 1:1 was selected for animal experiments.

[0417] 2. Preparation of fluorine-containing chitosan eye drops: the normal pH of tear fluid is 6.4-7.7, so a phosphate buffer solution with pH=7.4 was used to dissolve the solid powder of fluorine-containing chitosan, with a concentration of 2 mg/mL. A high concentration of bovine serum albumin labeled with Cy5.5 was added dropwise under constant stirring to form a stable composite, and the final concentration of bovine serum albumin was 2 mg/mL. The eye drops of perfluoroheptanoic acid-modified chitosan and small molecule rhodamine B were consistent with the above preparation.

[0418] 3. To test the ability of perfluoroheptanoic acid-modified chitosan to penetrate the eye barrier, the degrees of penetration of the protein with perfluoroheptanoic acid-modified chitosan and the free protein into the inside of the eye were compared. The mice were anesthetized and administered on the ocular surface with a dosing device, 5 μL per eye, and the mice were treated under protection from light for 12 h. The control group was bovine serum albumin labeled Cy5.5 without fluorine-containing chitosan, and the concentration and dosage were the same as the experimental group. 12 h later, the mice were sacrificed by cervical dislocation, and the surface of the eyeball was rinsed with PBS at pH=7.4, the eyeball was removed, the excess tissue was disposed, and the section preparation was made, and the central longitudinal section of the eyeball was photographed. The specific implementation method of the small molecule drug rhodamine B was the same as that of the large molecule drug. The membrane penetration effect of large molecule bovine serum albumin is shown in FIG. 4-4, and the membrane penetration effect of small molecule Rhodamine B is shown in FIG. 4-5. It is obvious from the figures that the protein and small molecule fluorescent substance rhodamine B with perfluoroheptanoic acid-modified chitosan can enter the inside of the eyeball, and that the fluorescence intensity of the protein label and the fluorescence of the small molecule are much higher than that of free protein and free small molecule in the eyes. It is concluded that the perfluoroheptanoic acid-modified chitosan can help a series of drugs to penetrate the eye barriers and enter the eyes to achieve the purpose of treatment.

[0419] 4. In order to evaluate the protein drug concentrations in the eye at different time points, drug concentrations in each part of the dissected eyeball (cornea, lens, vitreous, retina) 3 h and 6 h after administration with the 1:1 ratio of perfluoroheptanoic acid-modified chitosan to protein and the free protein were compared. After the eyeball was dissected, it was broken by a tissue disrupter, and then lysed with a lysis solution, and centrifuged to remove the broken pieces. The supernatant is obtained, the fluorescence of the protein label was detected and the fluorescence intensity was calculated, as shown in FIG. 4-6. First, the protein fluorescence intensities of the drug with perfluoroheptanoic acid-modified chitosan and the free drug in the four tissue parts of the eye were compared. It is obvious that the group with perfluoroheptanoic acid-modified chitosan is significantly higher than the free protein group. Secondly, according to the accumulation of time, the group with perfluoroheptanoic acid-modified chitosan protein at 6 h has higher fluorescence intensity than that at 3 h. It can be seen that the perfluoroheptanoic acid-modified chitosan may have the effect of adhering to the ocular surface and slowly releasing into the eye.

[0420] 5. In order to evaluate the penetrating ability for the cornea, the cornea was subjected to immunofluorescence staining at different time points to observe the penetrating ability. Samples were taken 5 min, 15 min, 30 min, and 60 min after the drug with perfluoroheptanoic acid-modified chitosan was applied. Frozen sections were prepared, and the nuclei of the sections were stained and observed under a confocal microscope as shown in FIG. 4-7. Herein, the red is the fluorescence of the protein label, and the blue is the nucleus of the eye tissue. It can be clearly seen from the figure that after 30 min, the group with perfluoroheptanoic acid-modified chitosan has clearly penetrated into the corneal epithelial cells, while the free protein substantially does not enter. It can be seen that the perfluoroheptanoic acid-modified chitosan can open the corneal barrier for targeted treatment of cornea related diseases.

[0421] 6. In order to evaluate the biological safety of perfluoroheptanoic acid-modified chitosan, 20 Balb/c mice aged 6-8w were selected, regardless of gender, and the mice were divided into a perfluoroheptanoic acid-modified chitosan group, a saline group, a PBS group, and a blank control group, 5 mice in each group. The eye drop frequency was 4 times/day. The sodium fluorescein staining of the corneal epithelium was recorded by a slit-lamp biomicroscope at 24 h, 48 h and 72 h respectively after administration, and the evaluation was carried out according to the clinical evaluation criteria in Table 4-3. The evaluation pictures are shown in FIG. 4-8. In the perfluoroheptanoic acid-modified chitosan group, 24 h after administration, the defect areas are reduced, and the defect areas are all <30%; after 48 h, there are defects in the corneal epithelium of 2 mice, and the epitheliums of the other 3 mice are completely healed; after 72 h, the corneal epitheliums of 5 mice are completely healed. In the saline group: 24 h after administration, the defect areas are reduced, the defect areas in the corneal epithelium of 2 mice are between 30% and 70%, and those in the other three mice are all <30%; after 48 h, there is a defect in the corneal epithelium of 1 mouse, and the defect area is <30%, and the epitheliums of 4 mice are completely healed; after 72 h, there is a defect in the corneal epithelium of 1 mouse, and the defect area is <30%, and the epitheliums of 4 mice are completely healed. In the PBS group: 24 h after administration, the defect areas are reduced, and the defect areas are all <30%; after 48 h, there is punctate staining in the corneal epithelium of 1 mice, and the epitheliums of 4 mice are completely healed; after 72 h, the corneal epitheliums of 5 mice are completely healed. In the blank control group, 24 h after administration, the defect areas are reduced, and the defect areas are all <30%; after 48 h, the corneal epitheliums of 5 mice are completely healed. Under the test of this clinical method, the perfluoroheptanoic acid-modified chitosan has extremely high safety to the eyes. The cornea stained with sodium fluorescein under the slit lamp in FIG. 8 is stained if there is a corneal defect, and should appear gray in black and white mode. Table 4-4 shows the evaluation results of FIG. 4-8.

TABLE-US-00007 TABLE 4-3 Clinical Laboratory Reference Grading Criterion  0% trauma Epithelial defect healed, fluorescein staining turned negative 20% trauma Repair of defect area >70%, fluorescein staining ++ {circumflex over ( )} +, or ++, +{circumflex over ( )}very little ± 80% trauma Repair of defect area 30%-70%, corneal staining +++{circumflex over ( )}++, or ++ {circumflex over ( )} 100% trauma  Repair of defect area <30%, no significant change in corneal staining Note: cure and markedly improvement are summed to be effective, and the effectiveness rate is calculated; improvement and failure are summed to be ineffective, and the ineffectiveness rate is calculated.

TABLE-US-00008 TABLE 4-4 0 h 24 h 48 h 72 h Fluorinated 100% trauma 100% trauma 80% trauma 0% trauma chitosan group Saline group 100% trauma  80% trauma 20% trauma 20% trauma  Phosphate 100% trauma 100% trauma 20% trauma 0% trauma buffer solution group Blank control 100% trauma  0% trauma  0% trauma 0% trauma group

[0422] It is obvious from the evaluation results in FIG. 4-8 and Table 4-4 that the cornea on the surface of the eyeballs of the experimental group and the control group has no visible gray areas showing corneal epithelial defects. It can be seen that perfluoroheptanoic acid-modified chitosan has substantially no effect on the cornea and is highly safe.

[0423] Example 4-2: Anti-PDL1 as an immunotherapeutic drug and perfluoroheptanoic acid-modified chitosan were prepared into eye drops for the treatment of malignant choroidal melanoma, to demonstrate that perfluoroheptanoic acid-modified chitosan has a delivery effect.

[0424] Specific Method:

[0425] 1. Animal model: B16 melanoma in logarithmic phase transfected with bioluminescence gene was intra-ocularly injected into the choroid in the eyeball of the right eye of Balb/c mice at 1×10.sup.5 cells/each eye. After the injection, they were incubated for 4 days, and the tumor size was expressed by the bioluminescence intensity through a bioluminescence imaging system. FIG. 4-9 shows the formation and size of ocular tumors in each group before administration.

[0426] 2. Preparation of perfluoroheptanoic acid-modified chitosan/anti-PDL1 eye drops: The preparation method was the same as that of item 2 in Example 4-1.

[0427] 3. Evaluation method: the rightmost side in FIG. 4-9 is a diagram indicating the change in bioluminescence intensity. In the legend, the larger the spot, the darker the color, the more serious the tumor.

[0428] Treatment method: the successfully modeled mice were divided into groups, one without administration as a control group, and one given with eye drops as an experimental group, with three mice in each group. The treatment was started on the fourth day after the model was established. The experimental group was instilled once a day, with 2.5 μL each time, and the drug concentration was 2 mg/mL. After one week of treatment, bioluminescence imaging was performed. The results are shown in FIG. 4-9 and FIG. 4-10, and in FIG. 4-9, the upper three mice are in the control group, and the lower three are in the experimental group. It is obvious from the figures that the immune bioluminescence of the mice with perfluoroheptanoic acid-modified chitosan is weaker than the control group after one week of treatment, and the in situ bioluminescence quantitative analysis of malignant choroidal melanoma after one week of treatment shows that the autoluminescence intensity after treatment is one-fourth that of the control group, indicating a significant therapeutic effect.

Example 5-1

[0429] I. A fluorinated chitosan-chicken ovalbumin composite was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and incubated with bone marrow-derived dendritic cells, and the ability of the composite to stimulate the maturation of dendritic cells was investigated.

[0430] Specific Method:

[0431] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite: 0.9 mg of perfluoroheptanoic acid-modified chitosan was weighed and dissolved in 900 μL of ultrapure water under stirring. Under constant stirring, 100 μL (20 mg/mL) of chicken ovalbumin was added dropwise, and continuously stirred for one hour to obtain a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite.

[0432] 2. 10 μL of the above-prepared composite was added to a 24-well plate, and 1 mL of a cell suspension containing 1 million dendritic cells was added. They were incubated for 24 h in a 37° C. incubator, the dendritic cells were stained with FITC-CD11c, PE-CD86 and APC-CD80, the fluorescence signal of FITC was analyzed with flow cytometry, the dendritic cells were selected and then the fluorescence signals of PE and APC in the dendritic cells were analyzed, and the results of cell maturity were determined, as shown in FIG. 1-1.

[0433] Experimental results: As shown in FIG. 5-1, the perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite can stimulate the maturation of dendritic cells, compared with the group without treatment.

[0434] II. A perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and incubated with bone marrow-derived dendritic cells, and how the composite stimulates dendritic cells to express histocompatibility complex class II (MHC II) and CD40 protein was investigated.

[0435] Specific Method:

[0436] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite: 0.9 mg of perfluoroheptanoic acid-modified chitosan was weighed and dissolved in 900 μL of ultrapure water with stirring. Under constant stirring, 100 μL (20 mg/mL) of chicken ovalbumin was added dropwise, and continuously stirred for one hour to obtain a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite.

[0437] 2. 10 μL of the above-prepared composite was added to a 24-well plate, and 1 mL of a cell suspension containing 1 million dendritic cells was added. They were incubated for 24 h in a 37° C. incubator, the dendritic cells were stained with FITC-CD11c, PE-MHC II and APC-CD40, the fluorescence signal of FITC was analyzed with flow cytometry, the dendritic cells were selected and then the fluorescence signals of PE and APC in the dendritic cells were analyzed, and the changes of histocompatibility complex class II (MHC II) and CD40 protein were determined. The results are shown in FIG. 5-2.

[0438] Experimental results: As shown in FIG. 5-2, under the stimulation of the perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite, the expression levels of histocompatibility composite class II (MHC II) and CD40 protein in the dendritic cells increase significantly, indicating that the composite can effectively stimulate dendritic cells to present antigens.

[0439] III. A perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and applied to the back of mice as patches, and the accumulation of the composite in the lymph nodes was investigated.

[0440] Specific Method:

[0441] 1. Preparation of cy5.5-labeled chicken ovalbumin: 10 mg of chicken ovalbumin was weighed, and dissolved in 1 mL of PBS solution, and 20 μL (20 mg/mL) cy5.5 was added and placed at 4° C. overnight. Free cy5.5 was removed through G25-molecular sieve gel. Quantification was performed by BSA, and OVA was concentrated by ultrafiltration to 20 mg/mL.

[0442] 2. Preparation of a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite: 1 mg of perfluoroheptanoic acid-modified chitosan was weighed and dissolved in 450 μL PBS with stirring. Under constant stirring, 50 μL (20 mg/mL) PBS dissolved with cy5.5-labeled chicken ovalbumin was added dropwise, and continuously stirred for one hour to obtain a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA-cy5.5) composite.

[0443] 3. 12.5 μL of the above FCS-OVA-cy5.5 composite solution was mixed into 12.5 μg of an ointment to prepare an FCS-OVA-cy5.5 vaccine ointment, and the above ointment was applied to the back of C57 mice. Using small animal imaging, fluorescence distribution on the front of mice was photographed at 0 h, 3 h, 7 h, 11 h, 24 h, and the aggregation of FCS-OVA-cy5.5 in the lymph nodes was observed. The results are shown in FIG. 5-3.

[0444] 4. Experimental results: As shown in FIG. 5-3, fluorescence appears at 11 h and 24 h in the lymph nodes of the mice, which means that the perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite fixed on the back of the mice as patches can penetrate the skin into the lymph nodes of the mice.

[0445] IV. A perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite was prepared with perfluoroheptanoic acid-modified chitosan as a carrier, and applied to the skin with a patch as a vaccine implant, and three weeks later, B16-OVA tumor was implanted and the tumor growth was observed.

[0446] Specific Method:

[0447] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite: 1 mg of perfluoroheptanoic acid-modified chitosan was weighed and dissolved in 450 μL PBS with stirring. Under constant stirring, 50 μL (20 mg/mL) PBS dissolved with chicken ovalbumin was added dropwise, and continuously stirred for one hour to obtain a perfluoroheptanoic acid-modified chitosan-chicken ovalbumin (FCS-OVA) composite.

[0448] 2. 12.5 μL of the above FCS-OVA composite solution was mixed into 12.5 μg of an ointment to prepare an FCS-OVA vaccine ointment, and 12.5 μL of the OVA (2 mg/mL) solution was mixed into 12.5 μg of an ointment to prepare an OVA vaccine ointment. The above ointments were separately applied to the back of C57 mice, and fixed with patches for 12 h. This operation was repeated twice a week for three weeks.

[0449] 3. Each mouse was implanted with 1×10.sup.5 B16-OVA cells and the tumor growth was observed. The results are shown in FIG. 5-4.

[0450] Experimental results: As shown in FIG. 5-4, the tumor growth rate of mice implanted with perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite as a patch vaccine is slower than that of mice implanted with chicken ovalbumin alone, indicating that the perfluoroheptanoic acid-modified chitosan-chicken ovalbumin composite as a patch has the effect of being a vaccine.

[0451] The fluorinated chitosan in each of Examples 1-1 to 1-4 of the present invention can be used as a vaccine preparation, and is useful as various vaccination preparations.

[0452] Example 6-1: Taking the currently known small molecule drug UK5099 with a certain hair growth therapeutic effect as an example, fluorinated chitosan was used to encapsulate the drug, and the in vitro transdermal ability of perfluoroheptanoic acid-modified chitosan and the drug in different ratios was studied. Taking the small molecule drug metformin as an example, fluorinated chitosan was used to encapsulate the drug, and the actual hair growth effect in animals was studied.

[0453] Specific Method:

[0454] 1. In order to observe the membrane penetration effect of different proportions of perfluoroheptanoic acid-modified chitosan, an in vitro diffusion experiment was performed. The back skin of mice was removed of fat, and fixed on the Franz diffusion cell. The ratios of perfluoroheptanoic acid-modified chitosan to UK5099 were 1:1, 2.5:1, 5:1, and 10:1. In each group, samples were taken at 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, 7 h, 10 h, and 24 h after drug application. The concentrations of UK5099 in the recipients at different time points were detected, the cumulative penetration percent was calculated, and the transmembrane effect was compared. The experimental results are shown in FIG. 6-1 and FIG. 6-2. Finally, the ratio of protein and perfluoroheptanoic acid-modified chitosan of 1:1 was selected for a series of animal experiments.

[0455] 2. Preparation of a perfluoroheptanoic acid-modified chitosan-metformin hair growth liquid: first, perfluoroheptanoic acid-modified chitosan solid powder was dissolved, and a liquid dissolved with metformin was added dropwise under constant stirring, so that the final metformin concentration was 2 mg/mL, and the concentration of perfluoroheptanoic acid-modified chitosan was 2 mg/mL.

[0456] 3. Balb/c mice at week 6 in the hair prohibition period were selected, and on the day before treatment, the mice in the perfluoroheptanoic acid-modified chitosan-metformin group, free metformin group, and blank control group were removed of the equivalent hair on the back and photographed as shown in the figure. Each mouse was uniformly sprayed with the drug according to its own treatment method, and the drug was given once every two days, 100 microliters each time. The photos on day 11, day 13, and day 17 after treatment are shown in FIG. 6-3 and Table 6-1.

[0457] Experimental results: It can be seen from the figure that although there are individual differences in mice, compared with the free drug group and the control group, the mice in the group with perfluoroheptanoic acid-modified chitosan have an obvious hair growth trend.

TABLE-US-00009 TABLE 6-1 Fluorinated chitosan- metformin group Metformin group Control group Day 0 No hair No hair No hair No hair No hair No hair Day 11 No hair Slightly No hair No hair No hair No hair hairy Day 13 Slightly Obviously No hair No hair No hair No hair hairy Day 17 Obviously Obviously No hair No hair No hair No hair

[0458] Example 6-2: A scar cream with perfluoroheptanoic acid-modified chitosan (FCS) and polyinosinic polycytidylic acid (poly (I:C)) as the main body was prepared, and the production of endogenous retinoic acid was induced, thereby improving scars with retinoic acid.

[0459] Specific Method:

[0460] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-insulin mixture: perfluoroheptanoic acid-modified chitosan and poly (I:C) were separately dissolved in ultrapure water until uniformly dissolved. After both was mixed, the perfluoroheptanoic acid-modified chitosan and insulin bound together due to electrostatic adsorption, where they were mixed in a weight ratio of 1:1 to form stable nanoparticles.

[0461] 2. Feasibility analysis on scar removal effect in animal experiments through an in vitro retinoic acid stimulation experiment with L929 (mouse fibroblast): L929 was plated in a 6-well plate for cell culture, and the medium volume per well was set to 3 mL. Poly(I:C), perfluoroheptanoic acid-modified chitosan and a mixture of poly(I:C) and perfluoroheptanoic acid-modified chitosan were added to the medium, so that the final concentrations of both poly(I:C) and perfluoroheptanoic acid-modified chitosan were 1 μg/mL. After L929 was cultured for 48 h, the culture medium and the cells were collected, and the supernatant was collected by centrifugation. The supernatant was uniformly mixed with a mixture of diethyl ether and acetone (ether:acetone=1:8) in a ratio of 1:5, and the upper oil phase was collected. The oil phase was concentrated with nitrogen, and then HPLC (High Performance Liquid Chromatography) was used to detect the content of retinoic acid secreted by the cells.

[0462] 3. Preparation of a scar cream of perfluoroheptanoic acid-modified chitosan and poly(I:C):poly(I:C) and perfluoroheptanoic acid-modified chitosan were pre-mixed at a ratio of 1:1, and then uniformly mixed with an equal volume of Aquaphor® ointment to obtain a scar cream of perfluoroheptanoic acid-modified chitosan and poly(I:C).

[0463] 4. Pre-judgment of scar removal effect of the perfluoroheptanoic acid-modified chitosan and poly(I:C) scar cream by morphological changes of mouse scars: Female Balb/c mice aged 10-12 weeks were anesthetized with isoflurane, 3 mm×10 mm of the skin was cut off on the back and applied with iodophor, an open wound was made, the skin of the mice was healed with the scabs removed within one week, and the scar removal experiment was started two months later. The perfluoroheptanoic acid-modified chitosan and poly(I:C) scar cream were applied on the scars every day, and the morphology of the mouse scars was observed and evaluated on a weekly basis.

[0464] Experimental results: see FIG. 6-4. The left panel shows the efficiency of a mixture of perfluoroheptanoic acid-modified chitosan and poly(I:C) for the production of retinoic acid in mouse fibroblasts (the ordinate Mass of RA means the production efficiency of retinoic acid). The right panel shows the effect of the mixture of perfluoroheptanoic acid-modified chitosan and poly(I:C) when applied to the scars of mice.

[0465] As shown in FIG. 6-4, by HPLC detection and analysis, it is found that compared with the blank group, perfluoroheptanoic acid-modified chitosan alone and poly(I:C) alone, the mixture of perfluoroheptanoic acid-modified chitosan and poly(I:C) can significantly increase the production of retinoic acid in mouse fibroblasts in vitro. After 7 days of smearing, the scars of the mice are obviously smoothed. The above results collectively indicate that the delivery of poly(I:C) with perfluoroheptanoic acid-modified chitosan as a carrier can successfully achieve the production of retinoic acid in cells in vitro, and has obvious effects in living scar models and possesses transformational value.

[0466] Example 6-3: Taking tranexamic acid, a drug for treating melasma as an example, fluorinated chitosan was used to encapsulate tranexamic acid, and its transdermal ability was explored using a diffusion cell.

[0467] Specific Method:

[0468] 1. Preparation of a perfluoroheptanoic acid-modified chitosan-tranexamic acid solution: perfluoroheptanoic acid-modified chitosan solid powder was dissolved in PBS, and mixed with tranexamic acid dissolved in PBS at a ratio of 1:1, and vortexed and shaken for 5 min, so that both were bound together by an electrostatic force. The final concentrations of perfluoroheptanoic acid-modified chitosan and tranexamic acid in the final solution were both 1 mg/mL.

[0469] 2. In vitro diffusion cell construction: the abdomen skin of mice was removed, the fat layer was shaved off, and the skin was fixed on a diffusion cell. 7.5 mL PBS was added to the diffusion cell below the skin, and 0.75 mL perfluoroheptanoic acid-modified chitosan-tranexamic acid and the same concentration of pure tranexamic acid solution were respectively added to the diffusion cell above the skin.

[0470] 3. Measurement of tranexamic acid penetration rate: 500 μL of PBS was removed from the bottom of the diffusion cell at 0, 1, and 3 h and the same volume of PBS was supplemented. The content of tranexamic acid was detected under the following HPLC conditions: methanol and 0.05 mol/L KH.sub.2PO.sub.4-0. 2% H.sub.3PO.sub.4 solution (volume ratio 5:95) as mobile phase, flow rate 1.0 mL/min, and detection wavelength 210 nm, and the cumulative penetration was calculated. The results are shown in the figure. The abscissa is time and the ordinate means the cumulative penetration.

[0471] Experimental results: As shown in FIG. 6-5, in the first three hours, tranexamic acid mixed with perfluoroheptanoic acid-modified chitosan can reach a cumulative penetration of 30%, while pure tranexamic acid substantially does not penetrate. It is proved that the perfluoroheptanoic acid-modified chitosan—tranexamic acid has better skin penetration ability and can be used for the treatment of skin disease melasma.

[0472] The above description of the disclosed examples enables those skilled in the art to implement or use the present invention. Various modifications to these examples will be apparent to those skilled in the art. The present invention will not be limited to the examples shown herein, but should conform to the widest scope consistent with the principles and features disclosed herein.