CN117186503B - Preparation method and application of chitosan material suitable for mucosal administration - Google Patents
Preparation method and application of chitosan material suitable for mucosal administration Download PDFInfo
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- CN117186503B CN117186503B CN202311079119.8A CN202311079119A CN117186503B CN 117186503 B CN117186503 B CN 117186503B CN 202311079119 A CN202311079119 A CN 202311079119A CN 117186503 B CN117186503 B CN 117186503B
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention relates to the field of new marine medicine biological materials, and discloses a preparation method and application of a chitosan material suitable for mucous membrane administration, wherein the chitosan material comprises the following steps: s1, pretreatment of chitosan, S2, modification of chitosan and S3, physical crosslinking of chitosan. The invention fully utilizes ocean resources, and the prepared chitosan new material has a plurality of excellent characteristics, enhanced adhesion in saliva, small water absorption expansion coefficient, large drug storage quantity and strong drug release capability, can be used as a drug storage agent and an adhesive agent simultaneously to prepare a long-acting release drug preparation for being applied to buccal mucosa, simplifies the structure of the patch and reduces the manufacturing cost of the patch. The long-acting antibacterial peptide patch is particularly suitable for long-acting antibacterial patches of the oral cavity, slowly releases antibacterial peptide, has small side effect, is convenient to use, has good compliance after long-term use, and is particularly suitable for patients with canker sore or tumor with low immunity.
Description
The application requires priority, and the application number of the prior application is: CN202211428293.4, name: a preparation method and application of chitosan material suitable for mucosa administration are provided, which is 11/15/2022 at priority date.
Technical Field
The invention belongs to the field of new marine medicine biological materials, and particularly discloses a preparation method and application of a chitosan material suitable for mucosal administration.
Background
At present, the clinical administration routes are various. Common routes of administration include subcutaneous injection, intravenous injection, oral administration, and application. In essence, the administration route of the drug has very close relation with the treatment effect of various clinical symptoms, and the drug effect of the same drug is very different in some cases if the administration routes are different. Along with the development of science and technology, the pharmaceutical dosage form brings more convenience to the use, and doctors need to reasonably select the medication mode according to the pharmaceutical property and the treatment purpose. The physical and chemical properties of the medicine provide basis for the application compatibility of the medicine in the preparation of pharmaceutical excipients. The pharmaceutical excipients can be dissolved, suspended, thickened, diluted, emulsified, stabilized, protected, colored, flavored or modified to form an effective and suitable pharmaceutical formulation. Biopharmaceuticals consider that the distribution of drugs is very closely related to therapeutic effects and also to accumulation of drugs in tissues and adverse reactions. For example, the Bioadhesive Drug Delivery System (BDDS) is a new branch of modern drug delivery formulations, and can be divided into various formulations such as oral adhesive formulations, nasal adhesive formulations, gastrointestinal oral adhesive formulations, ocular adhesive formulations, uterine adhesive formulations, vaginal adhesive formulations, rectal adhesive formulations and the like according to the difference of the formulations acting on the tissue parts of human bodies. Different routes of administration can affect the amount and rate of drug absorption, as compared to the following: intravenous injection, inhalation injection, intramuscular injection, subcutaneous injection, mucous membrane, oral administration, skin.
Oral mucosa is mainly divided into buccal mucosa absorption and sublingual mucosa absorption as systemic administration routes. The sublingual mucosa has strong permeability, quick drug absorption and convenient administration, and the bioavailability of many drugs with strong oral first pass effect or easy degradation in gastrointestinal tract, such as steroid hormone, nitroglycerin and isosorbide dinitrate sublingual administration is obviously improved. The susceptibility to saliva washout and short retention time are major drawbacks of sublingual administration. Thus sublingual tablets require fast drug dissolution, small dosage and strong action. The buccal mucosa can avoid the first pass effect of the liver, avoid enzymolysis and acidolysis in the gastrointestinal tract, is little influenced by the flushing effect of saliva in the oral cavity, can be kept on the mucosa for a quite long time, is favorable for the absorption of polypeptide and protein medicines and is favorable for the release of the controlled release preparation.
Currently, the drug forms developed for the buccal mucosa route are mainly buccal mucosa adhesive films (mucoadhesive buccal films, MBFs), spray agents, liposomes, nanoemulsions and the like. MBFs can improve compliance on the basis of avoiding gastrointestinal tract first pass effect and improving bioavailability, can terminate administration at any time, has low preparation difficulty, and becomes a cheek mucosa administration system with the most industrialization prospect.
The film-forming material acts as a drug carrier in MBFs and provides adhesion properties in a wet environment. At present, the theory of the adhesiveness of the film-forming material is electronic theory, diffusion theory, adsorption theory, wetting theory, dehydration theory and the like. The 1 st generation adhesive polymer, such as cellulose, gum and the like, adheres to mucus in a non-covalent mode under the action of hydrogen bonds, hydrophobic force and electrostatic force in a wet environment, and depends on the turnover rate of the mucus, so that the adhesive time is short. The 2 nd generation adhesion polymer, such as new polymer of grafted lectin and mannitol, adheres to the cell surface through the action of receptor-ligand, so as to enhance the adhesion performance. Anionic polymers such as sodium carboxymethylcellulose (carboxymethylcellulose sodium, CMC-Na), pectin, polyacrylic acid, etc. have become the most widely used MBFs carrier due to high adhesion and low toxicity. At present MBFs has the problems of low drug permeability, short adhesion time of an application part, poor in-vivo and in-vitro relativity and the like, and needs to be solved.
The chitosan is a product of removing partial acetyl from natural polysaccharide chitin, and chitin substances exist in the cell walls of shells of marine arthropods such as shrimps, crabs and the like, shells and bones of insects, mollusks and annelids, fungi and few seaweeds. Chitin is widely distributed in nature, the reserve is only behind cellulose, the second largest natural polymer is the chitin biosynthesis amount of about 100 hundred million tons per year, the chitin is a recyclable renewable resource, the natural polymer is mainly distributed in coastal areas, and chitosan is commercially produced. Chitin (chitin), chitosan and cellulose have similar chemical structures, the cellulose is hydroxyl at the C2 position, the chitin and the chitosan are respectively replaced by an acetamido group and an amino group at the C2 position, and the chitin and the chitosan have various unique properties such as biodegradability, cell affinity, biological effect and the like, and especially the chitosan containing free amino groups is the only alkaline polysaccharide in natural polysaccharide. The amino group in the chitosan molecular structure has stronger reactivity than the acetamido group in the chitin molecule, so that the polysaccharide has excellent biological functions and can carry out chemical modification reaction. Thus, chitosan is considered as a functional biomaterial with greater application potential than cellulose.
The chitosan resource in the ocean is so abundant, and most of the prior patents use chitosan in oral preparations, such as CN202111183969.3, an ionic emulsifier chitosan nanoparticle modified quercetin oral sustained release preparation and a preparation method thereof; or intestinal mucosa repair agent such as CN202111347477.3, a medicine for repairing intestinal mucosa and improving mucosa immunity, and its preparation method; vaccine delivery systems such as CN201710942135.3 mannosylated chitosan delivery systems assembled tuberculosis mucosal gene vaccines via nasal drops, spray or bronchoscopy drops. The prior patent for applying chitosan to the buccal mucosa adhesive membrane is less, how to utilize and enhance the physiological activity of chitosan, and the defects of low drug permeability, small drug storage amount, small adhesive force at the application part and short adhesive time of MBFs are solved, and further research is needed.
Disclosure of Invention
In order to solve the technical problems, a first object of the present invention is to provide a preparation method of a chitosan material suitable for mucosal administration, wherein the chitosan material has the advantages of large drug loading capacity, small expansion coefficient in saliva, reduced adhesion force attenuation in saliva, and long adhesion time, and has the potential of being a candidate new material for oral mucosa administration.
Another object of the present invention is to disclose the use of the chitosan material described above for the preparation of a long-acting release pharmaceutical formulation for application to the buccal mucosa, in particular an antibacterial patch suitable for buccal mucosa administration.
In order to achieve the first object, the present invention adopts the following technical scheme:
a method for preparing a chitosan material suitable for mucosal administration, comprising the following steps:
S1, pretreatment of chitosan: weighing chitosan, putting the chitosan into a sodium hydroxide solution with the mass fraction of 1-3% for ultrasonic cleaning for 10-20min, then putting the chitosan into a glacial acetic acid solution with the mass fraction of 0.5-1%, carrying out suction filtration on the chitosan solution, then precipitating and washing with ethanol with the concentration of more than 95%, and drying to obtain a chitosan raw material, wherein the deacetylation degree of the chitosan raw material is 90-95%, and the average molecular weight is 20000-25000Da;
S2, modifying chitosan: adding chitosan raw materials and 1-3% hydrochloric acid solution by mass fraction into a reaction container, wherein the mass volume ratio of the chitosan raw materials to the hydrochloric acid solution is 1g:25-50mL; controlling the reaction temperature in ice water bath at 0-5 ℃; adding a proper amount of modified soybean phospholipid and modified dialdehyde starch under stirring; removing the ice water bath after the dripping is finished, and continuously stirring and reacting for 2-4 hours at room temperature; standing, precipitating, suction filtering, drying and recrystallizing to obtain modified chitosan; wherein the mass ratio of the chitosan raw material to the modified soybean phospholipid to the modified dialdehyde starch is 1: (0.1-0.2): (0.02-0.3);
S3, chitosan crosslinking: dissolving the modified chitosan in acetic acid solution to prepare 60-100mg/mL modified chitosan solution, and regulating the pH value to 5.5-6.5; dropwise adding 3-6mg/mL of sodium hexametaphosphate solution into the modified chitosan solution according to the volume ratio of 1 (1.0-1.5), and adding a stabilizing agent with the final concentration of 2-20 mg/mL; the stabilizer is selected from one or more of xanthan gum, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose and hydroxypropyl cellulose, and forms a crosslinked stable chitosan solution; and standing, precipitating, filtering, drying and recrystallizing to obtain the chitosan material suitable for mucosal administration.
The swelling rate and the water absorbability which are too high can cause discomfort of patients when the oral mucosa is used for drug delivery, and simultaneously, the drug release is too fast, and the mechanical property of the gel layer is reduced and dissolved, so that the technical scheme is used for mixing the chitosan and gelatin after improving the chitosan, the swelling rate and the water absorbability are reduced, the aperture is reduced, the mechanical strength is improved, and the oral mucosa is more suitable for the oral mucosa administration.
Preferably, the recrystallization in the step S2 or S3 means that the solid obtained by suction filtration is dried and then recrystallized with absolute ethanol. The boiling point of the ethanol is not very high, the ethanol can be removed easily after crystallization, and the ethanol is low in price.
A chitosan material suitable for mucosal administration is prepared by the method.
The application of the chitosan material suitable for mucous membrane administration in preparing a long-acting release pharmaceutical preparation for being applied to the mucous membrane of the oral cavity and the cheek.
Preferably, the long-acting release pharmaceutical preparation is selected from one of an oral antibacterial pharmaceutical preparation, a blood sugar stabilizing pharmaceutical preparation and an anti-schizophrenia pharmaceutical preparation. Oral antibacterial pharmaceutical formulations, stable glycemic formulations, and anti-schizophrenia pharmaceutical formulations all require a stable and slow release of the drug, and are well suited to the characteristics of the present invention.
According to a second object of the present invention, there is provided an antibacterial patch suitable for buccal mucosa administration, comprising the above chitosan material and an antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide comprises the amino acid sequence shown in SEQ ID NO: 1= RRFPSRLSSSPLGLRLFILLSWLLF.
Preferably, the antimicrobial peptide is fused at the N-terminus to a polypeptide as set forth in SEQ ID NO: 2= GRRRRRRRRRPPQ, and the complete sequence of the final antibacterial peptide is shown in SEQ ID NO: 3= GRRRRRRRRRPPQRRFPSRLSSSPLGLRLFILLSWLLF. After the membrane penetrating peptide is added, the membrane penetrating capability of the antibacterial peptide is increased, and the antibacterial effect on eukaryotic bacteria is increased.
Preferably, the antibacterial peptide is subjected to point mutation at N3, N14, N21 and N29 of the complete sequence site, wherein the mutation is R3-H3, R14-K14, S21-K21 and L29-R29 respectively; finally obtaining the antibacterial peptide mutant sequence shown as SEQ ID NO: 4= GRHRRRRRRRPPQKRFPSRLKSSPLGLRRFILLSWLLF. The mutation of the above sites, combined with the structural biology knowledge of the inventors, through computer prediction, exposes the active site SPLG, which in some embodiments can be observed to have enhanced bacteriostatic properties.
Furthermore, the invention also discloses a preparation method of the antibacterial patch suitable for buccal mucosa administration, which comprises the following steps: preparing gelatin solution with the mass fraction of 20-25%, heating to 60-70 ℃, continuously stirring, adding chitosan material into the gelatin solution according to the mass ratio of 1:5-10, cooling to below 50 ℃, adding maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch. The preparation is convenient and mild and simple, and various auxiliary materials or flavoring agents suitable for medical use can be added simultaneously.
Preferably, the mass ratio of the chitosan material to the maleic anhydride grafting compatilizer PP-g-ST to the antibacterial peptide is 1 (0.2-0.4): (0.02-0.03). The maleic anhydride grafted compatilizer PP-g-ST has high polarity and reactivity by introducing a strong polar reactive group, can be grafted on chitosan well, and adsorbs antibacterial peptide.
The invention has the following beneficial effects:
Compared with unmodified chitosan, the gel layer of the antibacterial patch has the advantages of reduced swelling rate and water absorption and increased mechanical strength. In contrast to the general belief that the swelling rate is high, the excessively high swelling rate and water absorption can cause discomfort to patients when the oral mucosa is used, and simultaneously cause the drug release to be excessively fast, and the mechanical property of the gel layer to be reduced and dissolved, so that the chitosan is mixed with gelatin after being improved, the swelling rate and water absorption are reduced, the pore diameter is reduced, and the mechanical strength is improved, so that the chitosan is more suitable for oral mucosa administration. The antibacterial patch prepared from the chitosan has strong antibacterial capability on common oral streptococcus, bacillus caligenes and candida albicans in the oral cavity, is firmly applied, has slow and durable release rate of antibacterial peptide, can meet the requirement of once-a-day administration, and has great medicinal potential.
Drawings
FIG. 1 is a comparative gel layer performance test chart;
FIG. 2 Minimum Inhibitory Concentration (MIC) of the antimicrobial peptides prepared in examples 4-6;
FIG. 3 shows the release rate of the antimicrobial peptide from the antimicrobial patch;
Fig. 4 evaluation of clinical use of the antibacterial patch.
Detailed Description
The method of the present invention will be further described with reference to examples, and the experimental methods without specifying the specific conditions in examples can be generally performed under conventional conditions, such as those described in "molecular cloning laboratory Manual" written in Cruze Schweit Rick (Klaus Schwetlick) et al, and "molecular cloning laboratory Manual" written in J. The present invention may be better understood and appreciated by those skilled in the art by reference to the examples. The method of implementing the invention should not be limited to the specific method steps described in the embodiments of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Modified soybean phospholipid: available from the company Siam Darling Biotech, inc., as hydroxylated lecithin (modified soybeanphospholipids), cat# OP-10;
Modified dialdehyde starch: purchased from wuhank biomedical technologies, inc., english name: DIALDEHYDE STARCH CAS number 9047-50-1;
Chitosan: purchased from Jiangsu Orfu biosciences Inc., CAS number 9012-76-4.
Maleic anhydride grafting compatibilizer PP-g-ST was purchased from Nanjing bermuda Biotechnology Co., ltd., product number HD900P, 25kg in size.
Example 1
Preparation of chitosan material
A method for preparing chitosan material suitable for mucosal administration, comprising the following steps:
s1, pretreatment of chitosan: weighing chitosan (purchased from aladdin-e reagent net, medium viscosity, 200-400mPa.s, product number C105802-500 g), putting into sodium hydroxide solution with mass fraction of 1%, and ultrasonically cleaning for 10min, wherein the mass volume ratio of chitosan to hydrochloric acid solution is 1g:20mL; mixing the solution with an equal volume of 0.5% glacial acetic acid solution, carrying out suction filtration on the chitosan solution, then precipitating and washing with 96% ethanol, and drying to obtain a chitosan raw material, wherein the deacetylation degree of the chitosan raw material is 90%, and the average molecular weight of the chitosan raw material is 20000Da;
S2, modifying chitosan: adding 100g of the chitosan raw material and a hydrochloric acid solution with the mass fraction of 1% into a reaction container, wherein the volume of the hydrochloric acid solution is 2500mL; controlling the reaction temperature in ice water bath at 0-5 ℃;10 g of modified soybean phospholipid and 2g of modified dialdehyde starch are added under stirring; removing the ice water bath after the dripping is finished, and continuously stirring and reacting for 2 hours at room temperature; standing, precipitating, suction filtering, drying and recrystallizing to obtain modified chitosan;
s3, chitosan crosslinking: dissolving the modified chitosan in acetic acid solution to prepare 60mg/mL modified chitosan solution, and regulating the pH value to 5.5-6.5; dropwise adding 3mg/mL of sodium hexametaphosphate solution into the modified chitosan solution according to the volume ratio of 1:1, and adding stabilizer xanthan gum with the final concentration of 2mg/mL to form a crosslinked chitosan solution; and (5) standing, precipitating, filtering, drying and recrystallizing to obtain 85g of the chitosan material suitable for mucosal administration.
Example 2
Preparation of chitosan material
A method for preparing chitosan material suitable for mucosal administration, comprising the following steps:
S1, pretreatment of chitosan: taking chitosan ((purchased from aladdin-e reagent net, medium viscosity, 200-400mPa.s, product number C105802-500 g)) and putting the chitosan into a sodium hydroxide solution with mass fraction of 2% for ultrasonic cleaning for 15min, wherein the mass volume ratio of the chitosan to the hydrochloric acid solution is 1g:15mL; then placing the chitosan into glacial acetic acid solution with the volume of 0.75%, carrying out suction filtration on the chitosan solution, then precipitating and washing with 96% ethanol, and drying to obtain a chitosan raw material, wherein the deacetylation degree of the chitosan raw material is 92%, and the average molecular weight of the chitosan raw material is 23000Da;
S2, modifying chitosan: adding 100g of the chitosan raw material and 1-3% hydrochloric acid solution with the mass fraction into a reaction container, wherein the volume of the hydrochloric acid solution is 4000mL; controlling the reaction temperature in ice water bath at 0-5 ℃; 15g of modified soybean phospholipid and 3g of modified dialdehyde starch are added under stirring; removing the ice water bath after the dripping is finished, and continuously stirring and reacting for 3 hours at room temperature; standing, precipitating, suction filtering, drying and recrystallizing to obtain modified chitosan;
S3, chitosan crosslinking: dissolving the modified chitosan in acetic acid solution to prepare 80mg/mL modified chitosan solution, and regulating the pH value to 6; dropwise adding 4.5mg/mL of sodium hexametaphosphate solution into the modified chitosan solution according to the volume ratio of 1:1.2, and adding 5mg/mL of stabilizer hydroxypropyl methylcellulose to form a crosslinked chitosan solution; 91g of chitosan material suitable for mucosa administration is obtained through standing, precipitation, suction filtration, drying and recrystallization, and compared with the chitosan raw material before modification, the chitosan material after modification and crosslinking has more pores, smaller and denser single pore diameter and more overall porosity through observation of a microscope.
The recrystallization in the step S2 or S3 refers to that the solid obtained by suction filtration is dried and then recrystallized by absolute ethyl alcohol.
Example 3
Preparation of chitosan material
A method for preparing chitosan material suitable for mucosal administration, comprising the following steps:
S1, pretreatment of chitosan: weighing chitosan ((purchased from aladdin-e reagent net, medium viscosity, 200-400mPa.s, product number C105802-500 g)) and putting into a sodium hydroxide solution with mass fraction of 3% for ultrasonic cleaning for 20min, wherein the mass volume ratio of chitosan to hydrochloric acid solution is 1g:10mL; then placing the chitosan into glacial acetic acid solution with the volume of 1%, carrying out suction filtration on the chitosan solution, then precipitating and washing with 96% ethanol, and drying to obtain a chitosan raw material, wherein the deacetylation degree of the chitosan raw material is 95%, and the average molecular weight of the chitosan raw material is 25000Da;
S2, modifying chitosan: adding 100g of chitosan raw material and 3% hydrochloric acid solution with the mass fraction into a reaction container, wherein the volume of the hydrochloric acid solution is 5000mL; controlling the reaction temperature in ice water bath at 0-5 ℃; adding 20g of modified soybean phospholipid and 4g of modified dialdehyde starch under stirring; removing the ice water bath after the dripping is finished, and continuously stirring and reacting for 2-4 hours at room temperature; standing, precipitating, suction filtering, drying and recrystallizing to obtain modified chitosan;
S3, chitosan crosslinking: dissolving the modified chitosan in acetic acid solution to prepare 100mg/mL modified chitosan solution, and regulating the pH value to 6.5; dropwise adding 6mg/mL of sodium hexametaphosphate solution into the modified chitosan solution according to the volume ratio of 1:1.5, and adding a stabilizing agent (mixing hypromellose and sodium carboxymethyl cellulose 1:1) with the final concentration of 20mg/mL to form a crosslinked chitosan solution; and (3) standing, precipitating, filtering, drying and recrystallizing to obtain 98g of the chitosan material suitable for mucosal administration.
Example 4
Preparation of antibacterial patch
An antibacterial patch suitable for buccal mucosa administration comprises the chitosan material and antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide sequence is =seq ID NO: 1= RRFPSRLSSSPLGLRLFILLSWLLF;
The preparation method of the antibacterial patch suitable for buccal mucosa administration comprises the following steps: preparing a gelatin solution with the mass fraction of 20%, heating to 60 ℃, continuously stirring, adding the chitosan material prepared in the embodiment 2 into the gelatin solution according to the mass ratio of 1:5, cooling to below 50 ℃, adding a maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch;
the mass of the chitosan material, the mass of the maleic anhydride grafting compatilizer PP-g-ST and the mass of the antibacterial peptide are respectively 10g, 2g and 0.2g.
Example 5
Preparation of antibacterial patch
An antibacterial patch suitable for buccal mucosa administration comprises the chitosan material and antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide sequence is SEQ ID NO: 3= GRRRRRRRRRPPQRRFPSRLSSSPLGLRLFILLSWLLF;
Preparing a gelatin solution with the mass fraction of 23%, heating to 65 ℃, continuously stirring, adding a chitosan material into the gelatin solution according to the mass ratio of 1:8, cooling to below 50 ℃, adding a maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch;
The mass ratio of the chitosan material to the maleic anhydride grafting compatilizer PP-g-ST to the antibacterial peptide is 10g, 3g and 0.25g.
Example 6
Preparation of antibacterial patch
An antibacterial patch suitable for buccal mucosa administration comprises the chitosan material and antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide sequence is SEQ ID NO: 4= GRHRRRRRRRPPQKRFPSRLKSSPLGLRRFILLSWLLF; namely, the sequence in the embodiment 5 is subjected to point mutation, the antibacterial peptide is subjected to point mutation on N3, N14, N21 and N29 of the complete sequence site, the mutation is respectively dynamic 3D modeling of constructing polypeptide by using tLEaP tools in AmberTools for R3H, R14K, S, K, L R, the penetrating peptide is folded closer to the antibacterial peptide in a hydrophilic environment, and the active site SPLG of the antibacterial peptide is exposed through mutation, so that the antibacterial capability of the antibacterial peptide can be theoretically improved.
Preparing gelatin solution with mass fraction of 25%, heating to 70 ℃, continuously stirring, adding chitosan material into the gelatin solution according to mass ratio of 1:10, cooling to below 50 ℃, adding maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch;
The mass ratio of the chitosan material to the maleic anhydride grafting compatilizer PP-g-ST to the antibacterial peptide is 10g, 4g and 0.3g.
Comparative example 1
Preparation of antibacterial patch
An antibacterial patch suitable for buccal mucosa administration comprises chitosan (chitosan is the chitosan raw material prepared in step S1 in example 2) and antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide sequence is SEQ ID NO: 4= GRHRRRRRRRPPQKRFPSRLKSSPLGLRRFILLSWLLF;
Preparing a gelatin solution with the mass fraction of 23%, heating to 65 ℃, continuously stirring, adding chitosan into the gelatin solution according to the mass ratio of 1:8, cooling to below 50 ℃, adding a maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch;
The mass ratio of the chitosan material to the maleic anhydride grafting compatilizer PP-g-ST to the antibacterial peptide is 10g, 3g and 0.25g.
Comparative example 2
Preparation of antibacterial patch
An antibacterial patch suitable for buccal mucosa administration comprises chitosan (chitosan is modified chitosan prepared in step S2 in example 2) and antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide sequence is SEQ ID NO: 4= GRHRRRRRRRPPQKRFPSRLKSSPLGLRRFILLSWLLF; preparing a gelatin solution with the mass fraction of 23%, heating to 65 ℃, continuously stirring, adding chitosan into the gelatin solution according to the mass ratio of 1:8, cooling to below 50 ℃, adding a maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer, forming a gel layer, and drying to obtain the antibacterial patch;
the mass ratio of the chitosan to the maleic anhydride grafting compatilizer PP-g-ST to the antibacterial peptide is 10g, 3g and 0.25g.
Test example 1
Swelling ratio and Water absorption Performance test of gel layer of antibacterial Patch
The gel layers (0.5 mm) of the antibacterial patches for buccal mucosa administration prepared in examples 4 to 6 and comparative examples 1 to 2 were subjected to experimental comparisons of swelling rate and water absorption in artificial saliva (purchased from AMEKO cat No. RC 56877) and mechanical strength, the experiments being carried out at 36 degrees celsius simulated human body, the test methods being as follows: preparation of chitosan microgels with different molecular weights, research on swelling property, report of the university of Chifeng (Nature science edition), and national standard for gel strength measurement. The results are shown in Table 1 below and in FIG. 1.
Table 1 gel layer performance comparative test
As can be seen from the data of Table 1, the gel layer of the antibacterial patch of the present invention has a lower swelling rate and lower water absorption and has an increased mechanical strength than the unmodified chitosan after modification. In contrast to the general belief that the swelling rate is high, the excessively high swelling rate and water absorption can cause discomfort to patients when the oral mucosa is used, and simultaneously cause the drug release to be excessively fast, and the mechanical property of the gel layer to be reduced and dissolved, so that the chitosan is mixed with gelatin after being improved, the swelling rate and water absorption are reduced, the pore diameter is reduced, and the mechanical strength is improved, so that the chitosan is more suitable for oral mucosa administration.
Test example 2
Antibacterial test on the antibacterial peptides prepared in examples 4 to 6
The antibacterial effect of the sample is tested by a micro broth dilution method, and the specific method is as follows: preparing antibacterial peptide samples with the concentration of 4mg/mL, adding 100 mu L of broth into each of 96-well plates in advance, adding 100 mu L of sample stock solution into a first row of wells, uniformly mixing the first row of solutions, taking out 100 mu L of solutions, adding the solutions into a second row of wells, uniformly mixing, taking out 100 mu L of solutions from the second row, adding the solutions into a third row, pushing the solutions, sucking out the surplus 100 mu L of solutions after uniformly mixing until a tenth row, adding bacterial solution with the concentration of 10 5 CFU/mL into each well after the samples are diluted step by step, diluting the bacterial solution with broth in advance, adding 200 mu L of broth into a11 th row, and adding 100 mu L of broth and 100 mu L of bacterial solution without adding the samples into a 12 th row, and taking positive control. After the 96-well plates with the bacterial liquid are placed into a constant temperature incubator at 37 ℃ for culturing for 12-16 hours, 20 mu L of resazurin indicator with the concentration of 0.625mg/mL is added into each well plate, and the well plates are put back into the incubator after being added, so that the minimum antibacterial concentration (minimuminhibitory concentration, MIC) can be obtained when obvious color change exists after culturing for 2-4 hours. The measurement results are shown in the following table 2 and fig. 2: the data in the table show that the antibacterial polypeptide has obviously enhanced capability of resisting streptococcus stomatitis, bacillus cassieri and candida albicans after fusion and point mutation, and has great potential for being clinically used as a medicine in the antibacterial patch.
TABLE 2 Minimum Inhibitory Concentration (MIC) of antibacterial peptides prepared in examples 4-6
Test example 3
The antibacterial patches for buccal mucosa administration prepared in examples 4 to 6 and comparative examples 1 to 2 were immersed in artificial saliva at 37℃for 0h, 1h, 2h, 4h, 8h, 16h, 32h to test their release rates of antibacterial peptides. The results are shown in Table 3 and FIG. 3. The antibacterial patch prepared by the chitosan has slow and durable release rate of the antibacterial peptide, can meet the requirement of once-a-day administration, and has great medicinal potential.
TABLE 3 Release Rate of antimicrobial peptides from antimicrobial Patches
Test example 4
Stomatitis patient experiment.
The stomatocace volunteers were selected at the hospital clinic and divided into 6 groups of 30 persons, and each group was applied to the buccal mucosa of examples 4 to 6, comparative examples 1 to 2, negative control (prepared by the process of example 4, without antibacterial peptide), and the patch area of 2x2 cm. Positive control (commercial miconazole patch-norgestrel, instruction clinical dose) after 1 day of use, the total number of oral colonies (streptococcus stomatitis, bacillus californicus, candida albicans) in saliva of each group of patients was analyzed. The results are shown in Table 4 and FIG. 4.
Table 4 evaluation of clinical use of antibacterial patches
As can be seen from the results of Table 4 above, the antibacterial patches prepared in examples 4 to 6 of the present invention are firmly applied, are superior to the positive control, and are superior to comparative examples 1 to 2 in terms of their antibacterial properties, and the addition of the transmembrane peptide of example 5 and the point mutation of example 6, both enhance the antibacterial ability of the antibacterial peptide.
As can be seen from the above examples, the gel layer of the antibacterial patch of the present invention has a decreased swelling rate and water absorption and an increased mechanical strength compared to the unmodified chitosan. In contrast to the general belief that the swelling rate is high, the excessively high swelling rate and water absorption can cause discomfort to patients when the oral mucosa is used, and simultaneously cause the drug release to be excessively fast, and the mechanical property of the gel layer to be reduced and dissolved, so that the chitosan is mixed with gelatin after being improved, the swelling rate and water absorption are reduced, the pore diameter is reduced, and the mechanical strength is improved, so that the chitosan is more suitable for oral mucosa administration. The antibacterial patch prepared from the chitosan has strong antibacterial capability on common oral streptococcus, bacillus caligenes and candida albicans in the oral cavity, is firmly applied, has slow and durable release rate of antibacterial peptide, can meet the requirement of once-a-day administration, and has great medicinal potential.
The foregoing is only illustrative of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims and their equivalents.
Claims (10)
1. A method for preparing a chitosan material suitable for mucosal administration, comprising the following steps:
S1, pretreatment of chitosan: weighing chitosan, putting the chitosan into a sodium hydroxide solution with the mass fraction of 1-3% for ultrasonic cleaning for 10-20min, then putting the chitosan into a glacial acetic acid solution with the mass fraction of 0.5-1%, carrying out suction filtration on the chitosan solution, then precipitating and washing with ethanol with the mass fraction of more than 95%, and drying to obtain a chitosan raw material, wherein the deacetylation degree of the chitosan raw material is 90-95%, and the average molecular weight of the chitosan raw material is 20000-25000Da;
S2, modifying chitosan: adding chitosan raw materials and 1-3% hydrochloric acid solution by mass fraction into a reaction container, wherein the mass volume ratio of the chitosan raw materials to the hydrochloric acid solution is 1g:25-50mL; controlling the reaction temperature in ice water bath at 0-5 ℃; adding a proper amount of modified soybean phospholipid and modified dialdehyde starch under stirring; removing the ice water bath after the dripping is finished, and continuously stirring and reacting for 2-4 hours at room temperature; standing, precipitating, suction filtering, drying and recrystallizing to obtain modified chitosan; wherein the mass ratio of the chitosan raw material to the modified soybean phospholipid to the modified dialdehyde starch is 1: (0.1-0.2): (0.02-0.3);
S3, chitosan crosslinking: dissolving the modified chitosan in acetic acid solution to prepare 60-100mg/mL modified chitosan solution, and regulating the pH value to 5.5-6.5; dropwise adding 3-6mg/mL of sodium hexametaphosphate solution into the modified chitosan solution according to the volume ratio of 1 (1.0-1.5), and adding a stabilizing agent with the final concentration of 2-20mg/mL to form a crosslinked stable chitosan solution; standing, precipitating, suction filtering, drying and recrystallizing to obtain the chitosan material suitable for mucosa administration;
the modified soybean lecithin is hydroxylated lecithin;
The CAS number of the modified dialdehyde starch is 9012-76-4.
2. The method for preparing a chitosan material suitable for mucosal administration according to claim 1, wherein the recrystallization in step S2 or S3 means that the solid obtained by suction filtration is dried and then recrystallized with absolute ethanol.
3. A chitosan material suitable for mucosal administration, characterized by being prepared by the method of claim 2.
4. Use of a chitosan material suitable for mucosal administration according to claim 3 for the preparation of a long-acting release pharmaceutical formulation for application to the buccal mucosa.
5. The use according to claim 4, wherein the long-acting release pharmaceutical formulation is selected from one of an oral antibacterial pharmaceutical formulation, a blood glucose stabilizing pharmaceutical formulation, an anti-schizophrenia pharmaceutical formulation.
6. An antibacterial patch suitable for buccal mucosa administration, comprising the chitosan material of claim 3 and an antibacterial peptide grafted on the surface of the chitosan material; the antibacterial peptide comprises the amino acid sequence shown in SEQ ID NO: 1= RRFPSRLSSSPLGLRLFILLSWLLF.
7. An antimicrobial patch for buccal mucosal administration according to claim 6, wherein said antimicrobial peptide is fused at the N-terminus to a peptide as set forth in SEQ ID NO: 2= GRRRRRRRRRPPQ, and the complete sequence of the final antibacterial peptide is shown in SEQ ID NO:
3= GRRRRRRRRRPPQRRFPSRLSSSPLGLRLFILLSWLLF.
8. An antimicrobial patch for buccal mucosal administration according to claim 7, wherein said antimicrobial peptide is subjected to point mutations at the N3, N14, N21, N29 of the complete sequence site, said mutations being R3H, R14K, S21K, L R, respectively; finally obtaining the antibacterial peptide mutant sequence shown as SEQ ID NO:
4= GRHRRRRRRRPPQKRFPSRLKSSPLGLRRFILLSWLLF.
9. A method of preparing an antibacterial patch for buccal mucosal administration according to any one of claims 6 to 8, comprising the steps of: preparing gelatin solution with the mass fraction of 20-25%, heating to 60-70 ℃, continuously stirring, adding chitosan material into the gelatin solution according to the mass ratio of 1:5-10, cooling to below 50 ℃, adding maleic anhydride grafting compatilizer PP-g-ST and antibacterial peptide, continuously stirring to normal temperature for solidification, coating on a backing layer to form a gel layer, and drying to obtain the antibacterial patch.
10. The method for preparing an antibacterial patch for buccal mucosa administration according to claim 9, wherein the mass ratio of the chitosan material, maleic anhydride grafting compatibilizer PP-g-ST and the antibacterial peptide is 1 (0.2-0.4): (0.02-0.03).
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| CN117186503A (en) | 2023-12-08 |
| CN115819853A (en) | 2023-03-21 |
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