CN112006978A - Targeted chitosan nano-silver gel and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeting chitosan nano silver gel and a preparation method and application thereof, belonging to the technical field of biomedicine. The method uses chitosan and dimethyl imidazole-4,5-dicarboxylate to prepare imidazole cross-linked chitosan, and uses hyaluronic acid coating to give it targeting; using the cross-linked gel, silver nitrate and sodium borohydride, in situ reduction synthesis of targeted nanosilver gels. The nano silver prepared by the invention has good dispersibility and stability, has obvious inhibitory effect on fungi such as Candida albicans, exhibits obvious inhibitory effect on Hela and other tumor cells, and also has potential inhibitory effect on bacteria, viruses, etc. Broad application prospects.

Description

Targeted chitosan nano silver gel and preparation method and application thereof

technical field

The invention relates to a targeted chitosan nano-silver gel and a preparation method and application thereof, in particular to a surface-coated hyaluronic acid, imidazole cross-linked chitosan nano-silver gel antibacterial and tumor suppressor material The material and the preparation method and application thereof belong to the technical field of biomedicine.

Background technique

Nanogels have the advantages of high stability, good biocompatibility, and large drug loading, which can prevent drug degradation and clearance from the circulatory system. Loading various molecules such as deoxyribonucleic acid, ribonucleic acid, proteins, and inorganic materials into nanogel structures helps to improve the residence time of drugs in the systemic circulation and their biocompatibility, so nanogels can be used as a new type of delivery vector.

Metal-based drugs such as nano-silver have unique structures and functions. By acting on targets such as mitochondria, they can not only inhibit the cell division of bacteria or fungi, but also promote wound healing and inhibit the proliferation of cancer cells. However, there are still some problems in the application of nano-silver in the field of biomedicine, such as poor stability, low cellular uptake, high biological toxicity, and low targeting. Studies have shown that the use of carriers to load nano-silver can significantly improve the biocompatibility of nano-silver, improve its bioavailability, and obtain better therapeutic effects.

Chitosan (CS) is a chitosan derivative composed of β-1,4-linked glucosamine and a small amount of N-acetylglucosamine, which has reactive functional groups, biocompatibility, and biodegradation It is widely used in the field of medicine due to its advantages of non-toxicity, non-toxicity and high charge density. The composite nanogels formed by chitosan and nanosilver can improve the antibacterial properties of the gels. Yan Luke and others dispersed silver nitrate in chitosan double network gel, and obtained nano-silver composite gel by ultraviolet light irradiation, which can further prolong the inhibition time of Escherichia coli.

Hyaluronic acid (HA) is a linear mucopolysaccharide composed of alternately repeated N-acetylglucosamine and glucuronic acid disaccharide, is the main part of the extracellular matrix, and has the CD44 receptor targeting function; The surface modification of drug delivery nanogels with HA can significantly enhance the targeting performance of drug delivery.

Imidazolyl metal complexes are widely used in clinical treatment of various diseases. On the one hand, the imidazole ring can interact with a variety of metal ions to form imidazole-based metal drugs through coordination bonds, hydrogen bonds, π-π stacking, etc., which can make the metal obtain a better affinity/hydrophobicity ratio and reduce the metal Inherent toxicity and drug resistance, improving its biocompatibility and bioavailability. On the other hand, metals can protect imidazole ligands from enzymatic degradation before reaching the target. The imidazolyl group can also enhance cellular uptake through surface charge reversal in the tumor microenvironment (pH=6.8), and its strong proton sponge effect can promote endosomal escape.

Using imidazole-containing bifunctional molecules to cross-link chitosan, complex silver and other metal ions and in situ reduction to construct nano-silver and other metal-based drugs, and coat with hyaluronic acid to enhance their circulation stability, cell targeting and Intracellular endosomal escape properties. In this way, through the biocompatibility and targeting of chitosan and hyaluronic acid, combined with the biological function of imidazole, it is expected to achieve safe and efficient metal-based drug delivery. cancerous material. From the survey results of the current literature, there has not been any report on the targeted chitosan nanogel material and its application in the field of antibacterial and tumor suppression.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defects of the existing nano-silver reagents, and to provide a material containing imidazole cross-linked chitosan, an in-situ complex reduction nano-silver structure and a hyaluronic acid targeting structure and a preparation method thereof.

The purpose of the present invention is to provide a novel CD44 targeting chitosan nano-silver gel antibacterial and tumor suppressor material, and a preparation method and application thereof.

The invention provides a preparation method of targeted chitosan nano-silver gel. Specifically, after preparing the imidazole cross-linked chitosan gel, nano-silver is loaded on the surface of the imidazole cross-linked chitosan gel, and hyaluronic acid is applied to the surface of the imidazole cross-linked chitosan gel. Coating to obtain targeted chitosan nano-silver gel.

Further, in the above technical solution, the preparation method of the targeted chitosan nano-silver gel includes the following steps:

(1) Preparation of formyl chitosan: 1.0～5.0g chitosan was added to NaAc/HAc buffer solution with pH=4.5, 0.1～0.5g sodium periodate was added under nitrogen protection, and stirred under ice bath After reacting for a period of time, ethylene glycol was added to stop the reaction; dialyzed, freeze-dried to obtain formyl chitosan;

(2) Preparation of imidazole-4,5-dicarboxylic acid dicarboxylic acid hydrazide: 0.1-0.5 g of dimethyl imidazole-4,5-dicarboxylate was added with 2-50 mL of methanol, and under reflux conditions, 1-4 mL of 60 %～80% hydrazine hydrate is refluxed for a period of time, washed with methanol, centrifuged, and dried in vacuum to obtain imidazole-4,5-dicarboxylic acid dicarboxyhydrazide;

(3) Preparation of imidazolium hydrazine cross-linked chitosan nanogels: add 0.01-0.1 mg/mL imidazole-4,5-dicarboxylic acid dicarboxylic acid dropwise to 0.1-0.5 mg/mL formyl chitosan aqueous solution Formic hydrazide/dimethyl sulfoxide solution, heated to reflux for a period of time, dialyzed with deionized water, freeze-dried to obtain imidazole cross-linked chitosan gel;

(4) Preparation of targeted chitosan nano-silver gel: add 10-30 mL of deionized water to 5-20 mg of imidazole cross-linked chitosan gel, dissolve by ultrasonic in a water bath, and add 1 ～5mL of 1.0～2.0mg/mL silver nitrate solution, after sonicating for 1～10min, adjust the pH value of 0.1～1M sodium hydroxide solution to 8～9, add dropwise 0.1～1mg/mL sodium borohydride solution, continue to sonicate for 30～120min , and then dropwise add 0.1-1 mg/mL hyaluronic acid solution with a volume ratio of 1:1-3:1 cross-linked chitosan gel with imidazole, continue to ultrasonic for 10-30 min, dialyze, freeze-dry, and obtain targeted chitosan Nano silver gel.

Further, in the above technical solution, the stirring reaction in step (1) is carried out under ice bath for 24-48 h; the amount of ethylene glycol added is 1-10 mL.

Further, in the above technical solution, the temperature of the reflux in step (2) is 50-80°C; the reflux time is 1-5h.

Further, in the above technical solution, the reflux temperature in step (3) is 50-80° C.; the reflux time is 4-12 h.

The preparation method according to claim 1, wherein the temperature of the water bath in step (4) is 40-70°C.

Further, in the above technical solution, the molecular weight of the hyaluronic acid is Mw=10-100kDa.

Further, in the above technical solution, the molecular weight of the chitosan is Mw=1～100kDa.

The present invention also provides a targeted chitosan nano-silver gel, which is prepared according to the above-mentioned preparation method, and has the structure of imidazole cross-linked chitosan and in-situ complex reduced nano-silver and a hyaluronic acid target to the structure.

The invention also provides the application of the targeted chitosan nano-silver gel in the preparation of fungi, bacteria, viruses or tumor cell inhibitors.

Compared with the prior art, the present invention has the following advantages:

The bifunctional imidazole cross-linked chitosan material is used to limit the in-situ reduction of nano-silver to obtain nano-silver gel with good biocompatibility and high stability, which can effectively control the exudation of highly toxic silver ions, and give the material good The pH buffering capacity makes it easier to escape from the cell lysosome to exert a therapeutic effect. The material can be used in antibacterial and tumor suppressor fields to avoid the resistance of conventional drugs.

The nano silver prepared by the invention has good dispersibility and stability, has obvious inhibitory effect on fungi such as Candida albicans, exhibits obvious inhibitory effect on Hela and other tumor cells, and also has potential inhibitory effect on bacteria, viruses, etc. Broad application prospects

Description of drawings

Fig. 1 is the ¹ HNMR spectrum of imidazole-4,5-dicarboxylic acid dicarboxyhydrazide prepared in Example 1, in the figure, a is imidazole-4,5-dicarboxylate dimethyl ester, b is imidazole- 4,5-Dicarboxylic acid dicarboxyhydrazide.

Figure 2 shows the imidazole cross-linked chitosan CFCS and the targeted imidazole cross-linked chitosan prepared in Example 2

FTIR spectrum of HCFCS.

3 is a TEM image of the targeted imidazole cross-linked chitosan HCFCS prepared in Example 2 and the targeted nanosilver gel HCFCS@Ag prepared in Example 3.

4 is the XRD result of the targeted nano-silver gel HCFCS@Ag prepared in Example 3.

FIG. 5 is the detection result of the cellular uptake of the targeted chitosan nanogel HCFCS prepared in Example 2. FIG.

Figure 6 is the antibacterial performance evaluation results of the targeted chitosan nanogel HCFCS prepared in Example 2 and the targeted nanosilver gel HCFCS@Ag prepared in Example 3; in the figure, A is Candida albicans, B is Candida albicans

is Staphylococcus aureus, 1 is solvent, and 2 is. Targeted chitosan nanogel (HCFCS), 3 is targeted nanosilver gel (HCFCS@Ag), and 4 is nanosilver.

FIG. 7 is the evaluation result of the inhibitory effect of Hela tumor cell activity of the targeted chitosan nanogel HCFCS prepared in Example 2 and the targeted nanosilver gel HCFCS@Ag prepared in Example 3. FIG.

Detailed ways

The present invention will be described in detail below through specific embodiments and accompanying drawings. It should be understood that the specific embodiments and accompanying drawings described herein are only used to explain the present invention, but not to limit the present invention.

Example 1

Put 0.5 g of dimethyl imidazole-4,5-dicarboxylate in a 100 mL three-neck round-bottom flask, add 10 mL of methanol, and heat under reflux at 70°C until the dimethyl imidazole-4,5-dicarboxylate is completely dissolved. After that, 2 mL of 80% hydrazine hydrate was added to reflux for 3 h, washed with methanol, centrifuged twice, and the obtained sample was vacuum-dried to obtain the product imidazole-4,5-dicarboxylic acid dicarboxyhydrazide. Figure 1 shows the ¹ HNMR spectrum of imidazole-4,5-dicarboxylic acid dicarboxyhydrazide. Dimethyl imidazole-4,5-dicarboxylate (Figure 1a): delta 3.8 (CH3), delta 7.9 (CH), delta 13.5 (NH). Imidazole-4,5-dicarboxylic acid dicarboxyhydrazide (Fig. 1b): After hydrazinolysis, the proton resonance peak of its methoxy group δ3.8 disappeared, and the new resonance peaks of δ9.7 and δ12.0 were assigned to the hydrazide group Group NH peak, δ4.7 is assigned to hydrazide group NH2 peak, indicating that the hydrazinolysis was successful and the target product was obtained.

Example 2

Weigh 1.7760 g of chitosan (MW=5KDa), put it in a 250 mL three-neck round bottom flask, dissolve it in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicate for 30 min to dissolve it. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan.

Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, 1% hyaluronic acid aqueous solution (HA) with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and the ultrasonic wave was continued. The resulting solution was dialyzed and lyophilized to obtain the target. To chitosan nanogels (HCFCS). The FTIR spectra of the imidazole cross-linked chitosan gel (CFCS) and the targeted chitosan nanogel (HCFCS) prepared in this example are shown in Figure 2, wherein the wavenumbers of 1640 cm ^-1 and 1519 cm ^-1 are classified as amides The -CO-NH- vibrational absorption peak.

Example 3

Weigh 15mg of formyl chitosan (MW=5kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise 10% boron 0.5 mL of sodium hydride solution, then dropwise add 1% hyaluronic acid solution with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS), and continue to sonicate for 20 min, dialyze the product solution (MWCO=2000kDa), freeze After drying, a targeted nanosilver gel (HCFCS@Ag) was obtained. Figure 3 shows the TEM images of the targeted chitosan nanogel (HCFCS) prepared in Example 2 and the targeted nanosilver gel (HCFCS@Ag) prepared in this example. The particle size distribution of the targeted gel is 50-70 nm; the particle size distribution of the targeted nano-silver gel is 50-80 nm; the particle size of the nano-silver embedded in the targeted nano-gel is about 10 nm. Figure 4 shows the XRD pattern of the targeted nano-silver gel (HCFCS@Ag) prepared in this example. In the spectrum, the targeted nano-silver gel has nano-silver crystal diffraction peaks at 2θ of 38.5°, 44.5°, 64.8° and 77.5°, with obvious diffraction peaks and no impurity peaks, indicating that the nano-silver in the prepared nano-silver gel has (111), (200), (220), (311) face-centered three-dimensional structures.

Example 4

1.7760 g of chitosan (MW=1KDa) was weighed, placed in a 250 mL three-necked round bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. Add imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution dropwise to the single-necked flask of chitosan solution, heat under reflux at 70°C for 6 hours, take out the product solution after the reaction, and dialyze the solution with deionized water for two days (MWCO=500Da) , the dialysate is frozen and then freeze-dried to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued, and the resulting solution was dialyzed and freeze-dried to obtain targeted chitosan Carbohydrate Nanogels (HCFCS). The correct structure was verified by FTIR spectra.

Example 5

1.7760 g of chitosan (MW=10KDa) was weighed, placed in a 250 mL three-neck round-bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued, and the resulting solution was dialyzed and freeze-dried to obtain targeted chitosan Carbohydrate Nanogels (HCFCS). The correct structure was verified by FTIR spectra.

Example 6

Weigh 1.7760 g of chitosan (MW=5KDa), put it in a 250 mL three-neck round bottom flask, dissolve it in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicate for 30 min to dissolve it. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued, and the resulting solution was dialyzed and lyophilized to obtain targeted chitosan Carbohydrate Nanogels (HCFCS). The correct structure was verified by FTIR spectra.

Example 7

1.7760 g of chitosan (MW=1KDa) was weighed, placed in a 250 mL three-necked round bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued, and the resulting solution was dialyzed and lyophilized to obtain targeted chitosan Carbohydrate Nanogels (HCFCS). The correct structure was verified by FTIR spectra.

Example 8

1.7760 g of chitosan (MW=10KDa) was weighed, placed in a 250 mL three-neck round-bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued, and the resulting solution was dialyzed and lyophilized to obtain targeted chitosan Carbohydrate Nanogels (HCFCS). The correct structure was verified by FTIR spectra.

Example 9

1.7760 g of chitosan (MW=3KDa) was weighed, placed in a 250 mL three-neck round-bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and continued ultrasonication. The resulting solution was dialyzed and lyophilized to obtain targeted chitosan Nanogel (HCFCS). The correct structure was verified by FTIR spectra.

Example 10

1.7760 g of chitosan (MW=3KDa) was weighed, placed in a 250 mL three-neck round-bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued. The resulting solution was dialyzed and freeze-dried to obtain targeted chitosan Nanogel (HCFCS). The correct structure was verified by FTIR spectra.

Example 11

1.7760 g of chitosan (MW=20KDa) was weighed, placed in a 250 mL three-neck round bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and continued ultrasonication. The resulting solution was dialyzed and lyophilized to obtain targeted chitosan Nanogel (HCFCS). The correct structure was verified by FTIR spectra.

Example 12

1.7760 g of chitosan (MW=20KDa) was weighed, placed in a 250 mL three-neck round bottom flask, dissolved in 50 mL of pH=4.5 NaAc/HAc buffer solution, and sonicated for 30 min to dissolve. Weigh 0.2246 g of sodium periodate and dissolve it in 20 mL of NaAc/HAc buffer solution with pH=4.5. The two solutions are placed in an ice bath respectively, filled with nitrogen, degassed for 20 min, mixed, and stirred for 24 h in an ice bath. After that, 10 mL of ethylene glycol was added to stop the reaction. Take out the product liquid, pour it into a petri dish, put it in a refrigerator to freeze at -20°C, take out the coagulated sample and place it in a freeze dryer to freeze dry to obtain the product formyl chitosan. Weigh 0.05g of formyl chitosan in a single-necked flask and add 20mL of deionized water to dissolve, dissolve 0.0055g of imidazole-4,5-dicarboxylic acid hydrazide with 10mL of dimethyl sulfoxide, and add formyl hydrazide to the flask. The imidazole-4,5-dicarboxylic acid dicarboxyhydrazide solution was added dropwise to the single-necked flask of the chitosan solution, heated to reflux at 70 °C for 6 hours, and the product solution was taken out after the reaction was completed. After dialysis with deionized water for two days, the dialysate was frozen. Lyophilized to obtain the product imidazole cross-linked chitosan gel (CFCS). Under ultrasonic conditions, a 1% hyaluronic acid aqueous solution with a volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) was added dropwise, and ultrasonication was continued. The resulting solution was dialyzed and freeze-dried to obtain targeted chitosan Nanogel (HCFCS). The correct structure was verified by FTIR spectra.

Example 13

Weigh 15mg of formyl chitosan (MW=5kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise 10% boron 0.5 mL of sodium hydride solution was added dropwise to 1% hyaluronic acid solution with a volume ratio of 1:1, and continued ultrasonication for 20 min. The product liquid was dialyzed (MWCO=2kDa) and lyophilized to obtain targeted nano-silver gel (HCFCS@ Ag).

Example 14

The preparation method of formyl chitosan used in this example is the same as that in Example 2, except that the chitosan with MW=1kDa is used as the raw material for preparation.

Weigh 15mg of formyl chitosan (MW=1kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 1:1, and continued to sonicate for 20 minutes, the product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 15

The preparation method of formyl chitosan used in this example is the same as that in Example 2, except that the chitosan with MW=10kDa is used as the raw material for preparation.

Weigh 15 mg of formyl chitosan (MW=10 kDa) in 30 mL of deionized water, heat and sonicate in a water bath at 70 °C, until the dissolution is complete, and slowly add 5 mL of 0.6 mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 1:1, and continued to sonicate for 20 minutes, the product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 16

Weigh 15mg of formyl chitosan (MW=5kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 3:1, and continued to sonicate for 20 min. The product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 17

Weigh 15mg of formyl chitosan (MW=1kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 3:1, and continued to sonicate for 20 min. The product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 18

Weigh 15 mg of formyl chitosan (MW=10 kDa) in 30 mL of deionized water, heat and sonicate in a water bath at 70 °C, until the dissolution is complete, and slowly add 5 mL of 0.6 mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 3:1, and continued to sonicate for 20 min. The product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 19

The preparation method of formyl chitosan used in this example is the same as that of Example 2, the difference is only that chitosan with MW=3kDa is used as the raw material for preparation.

Weigh 15mg of formyl chitosan (MW=3kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 1:1, and continued to sonicate for 20 minutes, the product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 20

Weigh 15mg of formyl chitosan (MW=3kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time. Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 3:1, and continued to sonicate for 20 min. The product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 21

The preparation method of formyl chitosan used in this example is the same as that in Example 2, except that the chitosan with MW=20kDa is used as the raw material for preparation.

Weigh 15mg of formyl chitosan (MW=20kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 1:1, and continued to sonicate for 20 minutes, the product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Example 22

Weigh 15mg of formyl chitosan (MW=20kDa) in 30mL of deionized water, heat and sonicate in a water bath at 70°C, until the dissolution is complete, and slowly add 5mL of 0.6mg/mL imidazole-4,5-dicarboxylate dropwise at the same time Acid dicarboxylic acid hydrazide solution, after sonicating for 20min, add 2mL of 1.7mg/mL silver nitrate solution dropwise to the reaction solution, continue to ultrasonicate for 10min, adjust the pH value to 8-9 with sodium hydroxide solution, add dropwise sodium borohydride solution, and then dropwise added a 1% hyaluronic acid solution with a volume ratio of 3:1, and continued to sonicate for 20 min. The product solution was dialyzed and freeze-dried to obtain a targeted nano-silver gel (HCFCS@Ag).

Application Example 1 Evaluation of Cell Uptake

Using Hela cervical cancer cells (Shanghai Cell Bank, Chinese Academy of Sciences), imidazole cross-linked chitosan gel (CFCS), targeted chitosan nanogel (HCFCS) and targeted nanosilver gel (HCFCS@Ag) were evaluated cellular uptake properties. Cells cultured to a confluency of >80% were prepared into a cell suspension and plated in a 24-well plate. When the cell confluence reached 80%, 10 μL of FITC-labeled gel material (gel material and FITC were added to each well) were added to each well. The molar ratio was 3:1 and the reaction was stirred at 40°C for 24h to construct FITC-labeled gel material), after culturing for 4h, the medium was aspirated, 0.04% Trypan Blue was added, washed 3 times with 200 μL PBS, and added to each well. 200 μL of 0.25% trypsin was digested, the trypsin was discarded after 1 min, and 500 μL of high-glucose DMEM (containing serum and double antibody) was added to each well, and the cells were made into a cell suspension and detected by flow cytometry. The results are shown in Fig. 5, which showed that the cellular uptake of the targeted nanosilver gel reached 70%, which was significantly higher than that of the imidazole-crosslinked nanogel.

Application example 2 Antibacterial performance evaluation

Staphylococcus aureus and Candida albicans were respectively inoculated on LB solid medium and SCDLP solid medium for recovery, cultured at 37°C for 1 day and at 28°C for 2 days. Pick colonies with an inoculating loop to prepare bacterial suspensions in LB liquid medium and SCDLP liquid medium, respectively, and count them with a cell counting plate, so that the concentration of the prepared cell suspension is 10 ⁴ cfu/mL to 10 ⁵ cfu/mL. Take 3mL of bacterial suspension and inoculate it in 50mL LB liquid medium and 50mL SCDLP liquid medium respectively, pour the plate, add Oxford cup, and add PBS control, nanosilver, targeted chitosan nanogel (HCFCS) to the Oxford cup respectively. ) and 100 μL of targeted nanosilver gel (HCFCS@Ag) were placed in incubators at 37°C and 28°C, respectively. After a certain period of time, the size of the inhibition zone was observed. The results are shown in Figure 6. The inhibition zone of the targeted nano-silver gel on Candida albicans is larger than that of the nano-silver, indicating that the targeted nano-silver gel has higher antibacterial performance. The inhibitory effect of targeted nano-silver gel on the growth of Candida albicans was higher than that of Staphylococcus aureus, which was attributed to the overexpression of CD44 receptor on the surface of Candida albicans cell membrane and the increased uptake of targeted nano-silver gel.

Application Example 3 Evaluation of Cancer Inhibitory Effect

Hela cells in logarithmic growth phase were taken, digested with 0.25% trypsin, and added to DMEM high-glucose medium (containing serum and double antibody) to prepare cell suspension. After counting with a cytometer, cells were seeded in a 96-well plate (5×10 ³ cells/well) so that the final volume was 100 μL/well. Then, the plate was placed in a cell culture incubator at 37°C and 5% CO ₂ for 18-24 h, so that the cell density reached about 80%. Remove the medium, respectively dilute the nanosilver, targeted chitosan nanogel (HCFCS) and targeted nanosilver gel (HCFCS@Ag) with medium to 100 μL, so that the final drug concentration is 15, 30, 45 μg/mL, 3 duplicate wells were set for each concentration, and were added to each well. After the addition of samples, the plates were placed in a cell culture incubator at 37°C and 5% CO ₂ for 24 hours. The 96-well plate was taken out and placed on an ultra-clean workbench, 10 μL of MTT was added to each well, and after shaking gently, it was placed in an incubator for further incubation for 4 h. The OD value was detected with a multifunctional microplate reader, and the detection wavelength was 570 nm. The results are shown in Figure 7. The targeted nanogel has no significant cell proliferation inhibitory effect, indicating its good biological safety. When the concentration of targeted nano-silver was 45 μg/mL (silver loading rate was 5.94 μg/mL), the inhibitory effect on cell proliferation was comparable to that of nano-silver (30-45 μg/mL), indicating that the targeted nano-silver gel has an efficient tumor effect inhibit performance. Statistical analysis showed that the result was statistically significant.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. a preparation method of targeting chitosan nano-silver gel, is characterized in that, after preparing imidazole cross-linked chitosan gel, on imidazole cross-linked chitosan gel surface load nano silver, and use hyaluronic acid Coating to obtain targeted chitosan nano-silver gel. 2. preparation method according to claim 1, is characterized in that, comprises the steps: (1) Preparation of formyl chitosan: 1.0-5.0 g of chitosan was added to NaAc/HAc buffer solution with pH of 4.0-5.0, and 0.1-0.5 g of sodium periodate was added under nitrogen protection. After stirring and reacting for a period of time, ethylene glycol was added to stop the reaction; dialysis and freeze-drying were performed to obtain formyl chitosan; (2) Preparation of imidazole-4,5-dicarboxylic acid dicarboxyhydrazide: 0.1-0.5 g of imidazole-4,5-dicarboxylate dimethyl hydrazide was added to methanol, and under reflux conditions, 1-4 mL of 60%-80 % hydrazine hydrate was refluxed for a period of time, washed with methanol, centrifuged, and dried in vacuum to obtain imidazole-4,5-dicarboxylic acid dicarboxyhydrazide; (3) Preparation of imidazolium hydrazine cross-linked chitosan nanogels: add 0.01-0.1 mg/mL imidazole-4,5-dicarboxylic acid dicarboxylic acid dropwise to 0.1-0.5 mg/mL formyl chitosan aqueous solution Formic hydrazide/dimethyl sulfoxide solution, heated to reflux for a period of time, dialyzed with deionized water, freeze-dried to obtain imidazole cross-linked chitosan gel; (4) Preparation of targeted chitosan nano-silver gel: To 5-20 mg of imidazole cross-linked chitosan gel, add deionized water, dissolve by ultrasonic in a water bath, and add 1-5 mL of 1.0 ～2.0mg/mL silver nitrate solution, after sonicating for 1～10min, adjust the pH to 8～9 with 0.1～1M sodium hydroxide solution, add 0.1～1mg/mL sodium borohydride solution dropwise, continue to sonicate for 30～120min, then drip Add 0.1-1 mg/mL hyaluronic acid solution with a volume ratio of 1:1-3:1 cross-linked chitosan gel with imidazole, continue to ultrasonicate for 10-30 min, dialyze, freeze-dry, and obtain targeted chitosan nano-silver coagulation glue. 3 . The preparation method according to claim 1 , wherein the reaction is stirred under ice bath for 24-48 h in the step (1); the amount of ethylene glycol added is 1-10 mL. 4 . 4 . The preparation method according to claim 1 , wherein the reflux temperature in step (2) is 50-80° C.; the reflux time is 1-5 h. 5 . 5 . The preparation method according to claim 1 , wherein the reflux temperature in step (3) is 50-80° C.; the reflux time is 4-12 h. 6 . 6 . The preparation method according to claim 1 , wherein the temperature of the water bath in step (4) is 40-70° C. 7 . 7 . The preparation method according to claim 1 , wherein the molecular weight of the hyaluronic acid is Mw=10-100 kDa. 8 . 8 . The preparation method according to claim 1 , wherein the molecular weight of the chitosan is Mw=1˜100 kDa. 9 . 9. A targeted chitosan nano-silver gel, characterized in that it is prepared according to the preparation method described in any one of claims 1 to 8, and has imidazole cross-linked chitosan and in-situ complex reduction nano-gels. The structure of silver and the targeting structure of hyaluronic acid coated on the outer layer. 10 . The application of the targeted chitosan nano-silver gel according to claim 9 , wherein the application is in the preparation of fungi, bacteria, viruses or tumor cell inhibitors. 11 .

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