CN112006978B - Targeting chitosan nano silver gel and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a targeting chitosan nano silver gel and a preparation method and application thereof, belonging to the technical field of biological medicine. The method utilizes chitosan and imidazole-4, 5-dicarboxylic acid dimethyl ester to prepare imidazole cross-linked chitosan, and adopts hyaluronic acid coating to endow the chitosan with targeting; the cross-linked gel, silver nitrate and sodium borohydride are adopted to synthesize the targeting nano silver gel through in-situ reduction. The nano silver prepared by the invention has good dispersibility and stability, has obvious inhibition effect on fungi such as candida albicans, has obvious inhibition effect on tumor cells such as Hela, has potential inhibition effect on bacteria, viruses and the like, and has wide application prospect.
Description
Technical Field
The invention relates to a targeting chitosan nano silver gel and a preparation method and application thereof, in particular to a hyaluronic acid and imidazole cross-linked chitosan nano silver gel antibacterial and cancer inhibiting material coated on the surface and a preparation method and application thereof, belonging to the technical field of biological medicine.
Background
The nano gel has the advantages of high stability, good biocompatibility, large drug loading capacity and the like, and can prevent drug degradation and clearance of a circulatory system. Various molecules such as deoxyribonucleic acid, ribonucleic acid, protein and inorganic materials are loaded in the nano gel structure, so that the retention time of the medicine in the systemic circulation and the biocompatibility of the medicine are improved, and the nano gel can be used as a novel delivery carrier.
The nano silver and other metal-based medicines have unique structure and function, and can inhibit bacterial or fungal cell division, promote wound healing and inhibit cancer cell proliferation by acting on targets such as mitochondria. However, the nano silver has some problems in the application in the biomedical field, such as poor stability, low cell uptake, high biotoxicity, low targeting and the like. Researches show that the nano silver is loaded by adopting the carrier, so that the biocompatibility of the nano silver can be obviously improved, the bioavailability of the nano silver is improved, and a better treatment effect is obtained.
Chitosan (CS) is a deacetylated chitin derivative composed of beta-1, 4-linked glucosamine and a small amount of N-acetylglucosamine, and has the advantages of reactive functional group, biocompatibility, biodegradability, non-toxicity, high charge density and the like, and is widely applied to the field of medicine. The composite nano gel formed by the chitosan and the nano silver can improve the antibacterial performance of the gel. Yan Luke and the like are used for dispersing silver nitrate in the chitosan double-network gel, and the nano silver composite gel is obtained through ultraviolet irradiation, so that the inhibition time of escherichia coli can be further prolonged.
Hyaluronic Acid (HA) is a linear mucopolysaccharide consisting of alternately repeated N-acetylglucosamine and glucuronic acid disaccharide, is a major part constituting the extracellular matrix, and HAs a CD44 receptor targeting function; the CD44 receptor is over-expressed on the surfaces of most fungi and cancer cells, and the targeting performance of drug delivery can be remarkably enhanced by adopting HA to carry out surface modification on the drug delivery nanogel.
Imidazolyl metal complexes are widely used in the clinical treatment of a variety of diseases. On one hand, the imidazole ring can interact with various metal ions to form imidazole-based metal medicaments through coordination bonds, hydrogen bonds, pi-pi stacking and other actions, so that the metal can obtain better hydrophilic/hydrophobic ratio, the inherent toxicity and drug resistance of the metal are reduced, and the biocompatibility and bioavailability of the metal are improved. On the other hand, metals may protect the imidazole ligands from enzymatic degradation prior to reaching the target. Imidazolyl groups can also enhance cellular uptake by surface charge reversal in tumor microenvironment (ph=6.8), and their strong proton sponge effect can promote endosomal escape.
The imidazole-containing difunctional molecule is adopted to crosslink chitosan, silver and other metal ions are complexed and reduced in situ to construct nano silver and other metal-based drugs, and hyaluronic acid is coated, so that the circulation stability, cell targeting and intracellular endosome escape performance of the nano silver and other metal-based drugs are enhanced. Therefore, the biological compatibility and targeting of chitosan and hyaluronic acid, combined with the biological function of imidazole, are hopeful to realize safe and efficient metal-based drug delivery, and the targeted chitosan nano silver gel antibacterial and cancer-inhibiting material is prepared. As proved by the research results of the current literature, no targeting chitosan nanogel material has been reported and has application in the fields of antibiosis and cancer inhibition.
Disclosure of Invention
The invention aims to overcome the defects of the existing nano silver reagent and provide a material containing imidazole cross-linked chitosan, an in-situ complexing reduction nano silver structure and a hyaluronic acid targeting structure and a preparation method thereof.
The invention aims to provide a novel CD44 targeted chitosan nano silver gel antibacterial and cancer-inhibiting material, and a preparation method and application thereof.
The invention provides a preparation method of a targeted chitosan nano silver gel, which specifically comprises the steps of loading nano silver on the surface of imidazole crosslinked chitosan gel after preparing the imidazole crosslinked chitosan gel, and coating the imidazole crosslinked chitosan gel with hyaluronic acid to obtain the targeted chitosan nano silver gel.
Further, in the above technical scheme, the preparation method of the targeted chitosan nano silver gel comprises the following steps:
(1) Preparation of formyl chitosan: 1.0-5.0 g chitosan is added into NaAc/HAc buffer solution with pH=4.5, 0.1-0.5 g sodium periodate is added under the protection of nitrogen, and after stirring reaction for a period of time in ice bath, ethylene glycol is added to stop the reaction; dialyzing, and freeze-drying to obtain formyl chitosan;
(2) Preparation of imidazole-4, 5-dicarboxylic acid dihydrazide: adding 2-50 mL of methanol into 0.1-0.5 g of imidazole-4, 5-dicarboxylic acid dimethyl ester, adding 1-4 mL of 60% -80% hydrazine hydrate under reflux condition, refluxing for a period of time, washing with methanol, centrifuging, and vacuum drying to obtain imidazole-4, 5-dicarboxylic acid diformyl hydrazine;
(3) Preparation of imidazole hydrazine crosslinked chitosan nanogel: dropwise adding 0.01-0.1 mg/mL imidazole-4, 5-dicarboxylic acid diformyl hydrazine/dimethyl sulfoxide solution into 0.1-0.5 mg/mL formyl chitosan water solution, heating and refluxing for a period of time, dialyzing with deionized water, and freeze-drying to obtain imidazole crosslinked chitosan gel;
(4) Preparing targeted chitosan nano silver gel: adding 10-30 mL of deionized water into 5-20 mg of imidazole cross-linked chitosan gel, carrying out ultrasonic dissolution in a water bath, dropwise adding 1-5 mL of 1.0-2.0 mg/mL of silver nitrate solution under an ultrasonic condition, regulating the pH value to 8-9 by using 0.1-1M sodium hydroxide solution after ultrasonic treatment for 1-10 min, dropwise adding 0.1-1 mg/mL of sodium borohydride solution, continuing ultrasonic treatment for 30-120 min, dropwise adding 0.1-1 mg/mL of hyaluronic acid solution with the volume ratio of 1:1-3:1 of imidazole cross-linked chitosan gel, continuing ultrasonic treatment for 10-30 min, dialyzing and freeze-drying to obtain the targeted chitosan nano silver gel.
Further, in the technical scheme, stirring and reacting for 24-48 hours under the ice bath in the step (1); the addition amount of the ethylene glycol is 1-10 mL.
Further, in the above technical scheme, the temperature of the reflux in the step (2) is 50-80 ℃; the reflux time is 1-5 h.
Further, in the above technical scheme, the temperature of the reflux in the step (3) is 50-80 ℃; the reflux time is 4-12 h.
In the preparation method, the temperature of the water bath in the step (4) is 40-70 ℃.
Further, in the above technical scheme, the molecular weight Mw of the hyaluronic acid is=10 to 100kDa.
Further, in the above technical scheme, the molecular weight Mw of the chitosan is=1 to 100kDa.
The invention also provides a targeting chitosan nano silver gel, which is prepared according to the preparation method, and has a structure of imidazole cross-linked chitosan and in-situ complexing reduction nano silver and a hyaluronic acid targeting structure.
The invention also provides application of the targeting chitosan nano silver gel in preparation of fungi, bacteria, viruses or tumor cell inhibitors.
Compared with the prior art, the invention has the following advantages:
the nano silver gel with good biocompatibility and high stability is obtained by adopting the bifunctional imidazole cross-linked chitosan material and reducing nano silver in situ in a limited area, so that exudation of high-toxicity silver ions is effectively controlled, and meanwhile, the material is endowed with good pH buffering capacity, so that the material can escape from a cell lysosome more easily to play a therapeutic role. The material can be used in the fields of antibiosis and cancer inhibition, and can avoid the drug resistance of conventional drugs.
The nano silver prepared by the invention has good dispersibility and stability, has obvious inhibition effect on fungi such as candida albicans, has obvious inhibition effect on tumor cells such as Hela, has potential inhibition effect on bacteria, viruses and the like, and has wide application prospect
Drawings
FIG. 1 shows the imidazole-4, 5-dicarboxylic acid dihydrazide prepared in example 1 1 HNMR spectra, wherein a is imidazole-4, 5-dicarboxylic acid dimethyl ester and b is imidazole-4, 5-dicarboxylic acid diformylhydrazine.
FIG. 2 is a schematic diagram of imidazole cross-linked chitosan CFCS and targeted imidazole cross-linked chitosan prepared in example 2
FTIR spectra of HCFCS.
Fig. 3 is a TEM photograph of the targeted imidazole cross-linked chitosan HCFCS prepared in example 2 and the targeted nano silver gel hcfcs@ag prepared in example 3.
Fig. 4 shows XRD results of the targeted nano silver gel hcfcs@ag prepared in example 3.
FIG. 5 shows the results of cell uptake assays of the targeted chitosan nanogel HCFCs prepared in example 2.
FIG. 6 shows the results of evaluation of the antibacterial properties of the targeted chitosan nanogel HCFCs prepared in example 2 and the targeted nanogel HCFCs@Ag prepared in example 3; in the figure, A is candida albicans, B
Staphylococcus aureus, 1 is solvent, 2 is solvent. Targeting chitosan nanogel (HCFCS), 3 is targeting nano silver gel (HCFCS@Ag), and 4 is nano silver.
FIG. 7 shows the evaluation results of the inhibition effect of the HCFCs targeting chitosan nanogel prepared in example 2 and the HCFCs@Ag Hela tumor cells targeting nanogel prepared in example 3.
Detailed Description
The present invention will be described in detail with reference to the following specific examples and the accompanying drawings. It is to be understood that the specific embodiments and figures described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
Example 1
Placing 0.5g of imidazole-4, 5-dicarboxylic acid dimethyl ester into a 100mL three-necked round bottom flask, adding 10mL of methanol, heating and refluxing at 70 ℃, adding 2mL of 80% hydrazine hydrate for refluxing for 3h after the imidazole-4, 5-dicarboxylic acid dimethyl ester is completely dissolved, washing with methanol, centrifuging for two times, and vacuum drying the obtained sample to obtain the product imidazole-4, 5-dicarboxylic acid diformylhydrazine. Shown in FIG. 1 as imidazole-4, 5-dicarboxylic acid dihydrazide 1 HNMR spectra. Imidazole-4, 5-dicarboxylic acid dimethyl ester (fig. 1 a): δ3.8 (CH 3), δ7.9 (CH), δ13.5 (NH). Imidazole-4, 5-dicarboxylic acid dihydrazide (fig. 1 b): after hydrazinolysis, the proton formants of methoxy delta 3.8 disappear, and new formants of delta 9.7 and delta 12.0 belong to acylThe hydrazine group NH peak, delta 4.7 is attributed to the hydrazide group NH2 peak, which shows that the hydrazinolysis is successful, and the target product is obtained.
Example 2
1.7760g of chitosan (MW=5 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan.
0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution (HA) with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 1:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). FTIR spectra of imidazole crosslinked chitosan gel (CFCS) and targeting chitosan nanogel (HCFCS) prepared in this example are shown in FIG. 2, wherein wave number is 1640cm -1 、1519cm -1 The absorption peak of-CO-NH-vibration belonging to amide.
Example 3
15mg of formyl chitosan (MW=5 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, 0.5mL of 10% sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with the volume ratio of 1:1 with imidazole cross-linked chitosan gel (CFCS) is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed (MWCO=2000 kDa) and freeze-dried to obtain the targeted nano silver gel (HCFCS@Ag). TEM images of the targeted chitosan nanogel (HCFCS) prepared in example 2 and the targeted nano silver gel (HCFCS@Ag) prepared in this example are shown in FIG. 3. The grain size of the targeting gel is distributed at 50-70 nm; the grain size of the targeting nano silver gel is distributed at 50-80 nm; the particle size of the targeting nano gel embedded nano silver is about 10 nm. The XRD pattern of the targeted nano silver gel (HCFCS@Ag) prepared in this example is shown in FIG. 4. The targeted nano silver gel in the spectrogram has nano silver crystal diffraction peaks at 38.5 degrees, 44.5 degrees, 64.8 degrees and 77.5 degrees, the diffraction peaks are obvious, and no impurity peak exists, so that the nano silver in the prepared nano silver gel has a face center three-dimensional structure of (111), (200), (220) and (311).
Example 4
1.7760g of chitosan (MW=1 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, after two days of deionized water dialysis (MWCO=500 Da), dialyzate is frozen and freeze-dried, and the imidazole crosslinked chitosan gel (CFCS) is obtained. Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 1:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 5
1.7760g of chitosan (MW=10kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 1:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 6
1.7760g of chitosan (MW=5 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 3:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 7
1.7760g of chitosan (MW=1 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 3:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 8
1.7760g of chitosan (MW=10kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) of 3:1 under the ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 9
1.7760g of chitosan (MW=3 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of 1:1 with imidazole cross-linked chitosan gel (CFCS) under the ultrasonic condition, continuing to carry out ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 10
1.7760g of chitosan (MW=3 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) to 3:1 under the ultrasonic condition, continuing to carry out ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 11
1.7760g of chitosan (MW=20 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of 1:1 with imidazole cross-linked chitosan gel (CFCS) under the ultrasonic condition, continuing to carry out ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 12
1.7760g of chitosan (MW=20 kDa) was weighed into a 250mL three-necked round bottom flask and dissolved in 50mL NaAc/HAc buffer solution at pH=4.5 and sonicated for 30min to dissolve. 0.2246g of sodium periodate is weighed and dissolved in 20mL of NaAc/HAc buffer solution with pH=4.5, the two solutions are respectively placed in an ice bath, nitrogen is filled, the two solutions are respectively degassed for 20min and then mixed, and after stirring for 24h under the ice bath, 10mL of ethylene glycol is added to stop the reaction. Taking out the product liquid, pouring the product liquid into a culture dish, putting the culture dish into a refrigerator for freezing at the temperature of minus 20 ℃, taking out the solidified sample, and putting the solidified sample into a freeze dryer for freeze drying to obtain the product formyl chitosan. 0.05g of formyl chitosan is weighed into a single-neck flask, 20mL of deionized water is added for dissolution, 10mL of dimethyl sulfoxide is used for dissolution of 0.0055g of imidazole-4, 5-dicarboxylic acid diformylhydrazine, imidazole-4, 5-dicarboxylic acid diformylhydrazine solution is dropwise added into the single-neck flask filled with the formyl chitosan solution, heating reflux is carried out at 70 ℃ for 6 hours, product liquid is taken out after the reaction is finished, deionized water is dialyzed for two days, and dialyzate is frozen and freeze-dried to obtain the product imidazole crosslinked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with the volume ratio of imidazole cross-linked chitosan gel (CFCS) to 3:1 under the ultrasonic condition, continuing to carry out ultrasonic treatment, dialyzing the obtained resultant solution, and freeze-drying to obtain the targeted chitosan nanogel (HCFCS). The structural correctness was verified by FTIR spectroscopy.
Example 13
15mg of formyl chitosan (MW=5 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, 0.5mL of 10% sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 1:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed (MWCO=2 kDa) and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 14
The preparation of formylcellulose used in this example was as in example 2, except that chitosan with mw=1 kDa was used as starting material.
15mg of formyl chitosan (MW=1 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 1:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 15
The preparation of formylcellulose used in this example was as in example 2, except that chitosan with mw=10 kDa was used as starting material.
15mg of formyl chitosan (MW=10kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 1:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 16
15mg of formyl chitosan (MW=5 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 3:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 17
15mg of formyl chitosan (MW=1 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 3:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 18
15mg of formyl chitosan (MW=10kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 3:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 19
The preparation of formylcellulose used in this example was as in example 2, except that chitosan with mw=3 kDa was used as starting material.
15mg of formyl chitosan (MW=3 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 1:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 20
15mg of formyl chitosan (MW=3 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 3:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried to obtain targeted nano silver gel (HCFCS@Ag).
Example 21
The preparation of formylcellulose used in this example was as in example 2, except that chitosan with mw=20 kDa was used as starting material.
15mg of formyl chitosan (MW=20 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 1:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried, thus obtaining targeted nano silver gel (HCFCS@Ag).
Example 22
15mg of formyl chitosan (MW=20 kDa) is weighed in 30mL of deionized water, ultrasound is heated in a water bath at 70 ℃ until the formyl chitosan is completely dissolved, 5mL of 0.6mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide solution is slowly added dropwise, after 20min of ultrasound, 2mL of 1.7mg/mL of silver nitrate solution is added dropwise into the reaction solution, ultrasound is continued for 10min, pH value is regulated to 8-9 by sodium hydroxide solution, sodium borohydride solution is added dropwise, 1% hyaluronic acid solution with volume ratio of 3:1 is added dropwise, ultrasound is continued for 20min, and the product solution is dialyzed and freeze-dried, thus obtaining targeted nano silver gel (HCFCS@Ag).
Application example 1 evaluation of cell uptake
Cell uptake properties of imidazole cross-linked chitosan gel (CFCS), targeted chitosan nanogel (HCFCS) and targeted nanosilver gel (hcfcs@ag) were evaluated using Hela cervical cancer cells (Shanghai cell bank, national academy of sciences). Cells cultured to a confluence of >80% were prepared as a cell suspension, plated in 24 well plates, 10. Mu.L of FITC-labeled gel material (gel material and FITC were reacted at a molar ratio of 3:1 under stirring at 40℃for 24 hours) was added to each well after the confluence of 80%, the medium was aspirated after 4 hours of culture, 0.04% Trypan Blue (Trypan Blue) was added, washed 3 times with 200. Mu.L of PBS, digested with 200. Mu.L of 0.25% trypsin for 1min, and 500. Mu.L of high-sugar DMEM (serum-containing, double antibody-containing) was added to each well to prepare a cell suspension, which was examined by flow cytometer. The results are shown in fig. 5, and indicate that the cell uptake of the targeted nano-silver gel reaches 70% and is significantly higher than that of the imidazole crosslinked nano-gel.
Application example 2 evaluation of antibacterial Properties
Staphylococcus aureus and candida albicans are respectively inoculated on an LB solid culture medium and an SCDLP solid culture medium for resuscitation, and are cultured for 1 day at 37 ℃ and 2 days at 28 ℃. Bacterial suspension is prepared by picking colonies in LB liquid medium and SCDLP liquid medium respectively by an inoculating loop, and the bacterial suspension is counted by a cell counting plate to make the concentration of the prepared cell suspension be 10 4 cfu/mL～10 5 cfu/mL. Taking 3mL of bacterial suspension, respectively inoculating the bacterial suspension into 50mL of LB liquid medium and 50mL of SCDLP liquid medium, pouring the bacterial suspension into a flat plate, adding oxford cups, respectively adding 100 mu L of PBS control, nano silver, targeted chitosan nano gel (HCFCS) and targeted nano silver gel (HCFCS@Ag) into the oxford cups, respectively placing the oxford cups in a culture box at 37 ℃ and 28 ℃ for culture, and observing the size of a bacteriostasis zone after a certain period of time. The results are shown in FIG. 6. The antibacterial circle of the targeted nano silver gel on candida albicans is larger than that of nano silver, which shows that the antibacterial performance of the targeted nano silver gel is higher. The inhibition effect of the targeting nano silver gel on the growth of candida albicans is higher than that of staphylococcus aureus, and is attributed to the overexpression of CD44 receptors on the surface of candida albicans cell membranesThe intake of the targeted nano silver gel is increased.
Application example 3 evaluation of cancer inhibitory Effect
HeLa cells in logarithmic growth phase were taken, digested with 0.25% pancreatin, and then added with DMEM high sugar medium (containing serum and diabody) respectively to prepare cell suspensions. After counting with a cell counting plate, cells were seeded in 96-well plates (5X 10) 3 And/or hole) to a final volume of 100. Mu.L/hole. The well plate was then placed at 37℃with 5% CO 2 The cells are cultured in a cell culture box for 18 to 24 hours, so that the cell density reaches about 80 percent. And removing the culture medium, diluting the nano silver, the targeted chitosan nano gel (HCFCS) and the targeted nano silver gel (HCFCS@Ag) to 100 mu L by using the culture medium respectively, so that the final concentration of the medicine mass is 15, 30 and 45 mu g/mL, setting 3 compound holes for each concentration, and adding the compound holes into each hole respectively. After the sample addition, the well plate is continuously placed at 37 ℃ and 5 percent CO 2 Is cultured in a cell culture incubator for 24 hours. The 96-well plate is taken out and placed in an ultra-clean workbench, 10 mu L of MTT is added into each well, and after shaking evenly with light shaking, the well plate is placed in an incubator for continuous incubation for 4 hours. The OD value is detected by a multifunctional enzyme-labeled instrument, and the detection wavelength is 570nm. The results are shown in FIG. 7. The targeting nanogel has no obvious cell proliferation inhibition effect, which indicates good biological safety. When the concentration of the targeted nano silver is 45 mug/mL (the silver loading rate is 5.94 mug/mL), the proliferation inhibition effect on cells is equivalent to that of the nano silver (30-45 mug/mL), which indicates that the targeted nano silver gel has high-efficiency tumor inhibition performance. Statistical analysis shows that the result has statistical significance.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (6)
1. The preparation method of the targeted chitosan nano silver gel is characterized in that after imidazole cross-linked chitosan gel is prepared, nano silver is loaded on the surface of the imidazole cross-linked chitosan gel, and the targeted chitosan nano silver gel is obtained by coating the surface of the imidazole cross-linked chitosan gel with hyaluronic acid; the method specifically comprises the following steps:
(1) Preparation of formyl chitosan: 1.0-5.0 g chitosan is added into NaAc/HAc buffer solution with pH value of 4.0-5.0, 0.1-0.5 g sodium periodate is added under the protection of nitrogen, and after stirring reaction for a period of time in ice bath, ethylene glycol is added to stop the reaction; dialyzing, and freeze-drying to obtain formyl chitosan;
(2) Preparation of imidazole-4, 5-dicarboxylic acid dihydrazide: adding 0.1-0.5 g of imidazole-4, 5-dicarboxylic acid dimethyl ester into methanol, adding 1-4 mL of 60% -80% hydrazine hydrate under reflux condition, refluxing for a period of time, washing with methanol, centrifuging, and vacuum drying to obtain imidazole-4, 5-dicarboxylic acid diformyl hydrazine;
(3) Preparation of imidazole hydrazine crosslinked chitosan nanogel: dropwise adding 0.01-0.1 mg/mL imidazole-4, 5-dicarboxylic acid diformyl hydrazine/dimethyl sulfoxide solution into 0.1-0.5 mg/mL formyl chitosan water solution, heating and refluxing for a period of time, dialyzing with deionized water, and freeze-drying to obtain imidazole crosslinked chitosan gel;
(4) Preparing targeted chitosan nano silver gel: adding deionized water into 5-20 mg of imidazole cross-linked chitosan gel, performing ultrasonic dissolution in a water bath, dropwise adding 1-5 mL of 1.0-2.0 mg/mL of silver nitrate solution under an ultrasonic condition, performing ultrasonic treatment for 1-10 min, adjusting the pH value to 8-9 by using 0.1-1M sodium hydroxide solution, dropwise adding 0.1-1 mg/mL of sodium borohydride solution, continuing ultrasonic treatment for 30-120 min, dropwise adding 0.1-1 mg/mL of hyaluronic acid solution with the volume ratio of 1:1-3:1 of imidazole cross-linked chitosan gel, continuing ultrasonic treatment for 10-30 min, performing dialysis and freeze-drying to obtain targeted chitosan nano silver gel;
wherein, stirring and reacting for 24-48 h under the ice bath in the step (1); the addition amount of the ethylene glycol is 1-10 mL;
wherein the temperature of the reflux in the step (2) is 50-80 ℃; the reflux time is 1-5 h;
wherein the temperature of the reflux in the step (3) is 50-80 ℃; the reflux time is 4-12 h.
2. The method according to claim 1, wherein the temperature of the water bath in step (4) is 40 to 70 ℃.
3. The method according to claim 1, wherein the hyaluronic acid has a molecular weight mw=10 to 100kDa.
4. The method according to claim 1, wherein the molecular weight Mw of the chitosan is comprised between 1 and 100kDa.
5. The targeted chitosan nano silver gel is characterized by being prepared by the preparation method according to any one of claims 1-4, and has a structure of imidazole cross-linked chitosan and in-situ complexing reduction nano silver and a hyaluronic acid targeted structure coated on an outer layer.
6. The use of the targeted chitosan nanosilver gel of claim 5, wherein the use in the preparation of a fungal, bacterial, viral or tumor cell inhibitor.
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