CN112006978A - Targeted chitosan nano-silver gel and preparation method and application thereof - Google Patents
Targeted chitosan nano-silver gel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a targeted chitosan nano-silver gel and a preparation method and application thereof, belonging to the technical field of biological medicines. The method utilizes chitosan and imidazole-4, 5-dicarboxylic acid dimethyl ester to prepare imidazole cross-linked chitosan, and adopts hyaluronic acid to coat and endow the chitosan with targeting property; the cross-linked gel, silver nitrate and sodium borohydride are adopted to synthesize the target nano-silver gel by 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 and the like, has obvious inhibition effect on tumor cells such as Hela and the like, has potential inhibition effect on bacteria, viruses and the like, and has wide application prospect.
Description
Technical Field
The invention relates to a targeted chitosan nano-silver gel and a preparation method and application thereof, in particular to an antibacterial and cancer-suppressing material with a surface coated with hyaluronic acid and imidazole crosslinked chitosan nano-silver gel, and a preparation method and application thereof, belonging to the technical field of biological medicines.
Background
The nanogel has the advantages of high stability, good biocompatibility, large drug-loading rate and the like, and can prevent drug degradation and clearing of a circulating system. Various molecules such as deoxyribonucleic acid, ribonucleic acid, protein and inorganic materials are loaded in the nanogel structure, so that the residence time of the medicine in systemic circulation and the biocompatibility of the medicine are improved, and the nanogel can be used as a novel delivery carrier.
The metal-based medicines such as nano silver and the like have unique structure and function, and can inhibit cell division of bacteria or fungi, promote wound healing and inhibit proliferation of cancer cells by acting on targets such as mitochondria and the like. However, the application of nano silver in the field of biomedicine still has some problems, such as poor stability, low cellular uptake, high biotoxicity, low targeting property and the like. Researches show that the carrier loaded with the nano-silver can obviously improve the biocompatibility of the nano-silver, improve the bioavailability of the nano-silver and obtain better treatment effect.
Chitosan (CS) is a derivative of chitosan deacetylated, consisting of β -1, 4-linked glucosamine and a small amount of N-acetylglucosamine, and has the advantages of reactive functional groups, biocompatibility, biodegradability, non-toxicity, high charge density, etc., and is widely used in the field of medicine. The composite nanogel formed by the chitosan and the nano silver can improve the bacteriostatic performance of the gel. The method comprises the steps of dispersing silver nitrate in chitosan double-network gel by the department of pigment and recording, and the like, and obtaining the nano-silver composite gel through ultraviolet irradiation, thereby further prolonging the inhibition time of escherichia coli.
Hyaluronic Acid (HA) is a linear glycosaminoglycan composed of alternately repeated N-acetylglucosamine and glucuronobiose, is a major part of the extracellular matrix, and HAs a CD44 receptor targeting function; the CD44 receptor is over-expressed on the surface of most fungi and cancer cells, and the targeting performance of drug delivery can be remarkably enhanced by performing surface modification on the drug delivery nanogel by using HA.
Imidazolyl metal complexes are widely used in clinical treatment of various diseases. On one hand, the imidazole ring can interact with various metal ions to form the imidazole-based metal medicament through the actions of coordination bonds, hydrogen bonds, pi-pi stacking and the like, 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, the metal may protect the imidazole ligand from enzymatic degradation before reaching the target. Imidazolyl can also enhance cellular uptake through surface charge reversal in the tumor microenvironment (pH 6.8), and its strong proton sponge effect can facilitate endosome escape.
The preparation method adopts bifunctional molecules containing imidazole to crosslink chitosan, complexes metal ions such as silver and the like, constructs metal-based drugs such as nano silver and the like through in-situ reduction, and coats hyaluronic acid to enhance the circulation stability, cell targeting and intracellular endosome escape performance. Therefore, the chitosan nano silver gel antibacterial and cancer-inhibiting material is prepared by combining the biocompatibility and targeting property of the chitosan and hyaluronic acid and the biological function of imidazole, and is expected to realize safe and efficient metal-based drug delivery. According to the research results of the current literature, no report exists on the targeted chitosan nano gel material and the application thereof in the antibacterial and cancer-inhibiting fields.
Disclosure of Invention
The invention aims to overcome the defects of the existing nano silver reagent and provides a material containing imidazole crosslinked chitosan, an in-situ complexing reduced 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 targeted chitosan nano-silver gel, and particularly relates to the preparation method of the targeted chitosan nano-silver gel, which 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 nano-silver 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) preparing formyl chitosan: adding 1.0-5.0 g of chitosan into NaAc/HAc buffer solution with the pH value of 4.5, adding 0.1-0.5 g of sodium periodate under the protection of nitrogen, stirring and reacting for a period of time under an ice bath, and adding ethylene glycol 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 2-50 mL of methanol, adding 1-4 mL of 60-80% hydrazine hydrate under a reflux condition, refluxing for a period of time, washing with methanol, centrifuging, and drying in vacuum to obtain imidazole-4, 5-dicarboxylic acid dihydrazide;
(3) preparation of imidazole hydrazine crosslinked chitosan nanogel: dripping 0.01-0.1 mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide/dimethyl sulfoxide solution into 0.1-0.5 mg/mL formyl chitosan aqueous solution, heating and refluxing for a period of time, dialyzing with deionized water, and freeze-drying to obtain imidazole cross-linked chitosan gel;
(4) preparing targeted chitosan nano silver gel: adding 10-30 mL of deionized water into 5-20 mg of imidazole crosslinked chitosan gel, performing ultrasonic dissolution in a water bath, dropwise adding 1-5 mL of 1.0-2.0 mg/mL silver nitrate solution under an ultrasonic condition, performing ultrasonic treatment for 1-10 min, adjusting the pH value to 8-9 with 0.1-1M of 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 in a volume ratio of 1: 1-3: 1 to the imidazole crosslinked 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, the stirring reaction in the step (1) is carried out for 24-48 h under ice bath; the addition amount of the ethylene glycol is 1-10 mL.
Further, in the technical scheme, the reflux temperature in the step (2) is 50-80 ℃; the reflux time is 1-5 h.
Further, in the technical scheme, the reflux temperature in the step (3) is 50-80 ℃; the reflux time is 4-12 h.
The method according to claim 1, wherein the temperature of the water bath in the step (4) is 40 to 70 ℃.
Further, in the above technical solution, the molecular weight Mw of the hyaluronic acid is 10 to 100 kDa.
Further, in the above technical solution, the molecular weight Mw of the chitosan is 1 to 100 kDa.
The invention also provides a targeted chitosan nano silver gel which is prepared according to the preparation method and has a structure of imidazole crosslinked chitosan and in-situ complexing reduced nano silver and a hyaluronic acid targeted structure.
The invention also provides application of the targeted 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 bifunctional imidazole crosslinked chitosan material is adopted to reduce nano silver in situ in a limited domain, so that the nano silver gel with good biocompatibility and high stability is obtained, the seepage of high-toxicity silver ions is effectively controlled, and meanwhile, the material is endowed with good pH buffering capacity, so that the nano silver gel is easier to escape from cell lysosomes to play a role in treatment. The material can be used in the fields of antibiosis and cancer inhibition, and avoids 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 and the like, has obvious inhibition effect on tumor cells such as Hela and the like, has potential inhibition effect on bacteria, viruses and the like, and has wide application prospect
Drawings
FIG. 1 is a diagram of imidazole-4, 5-dicarboxylic acid dihydrazide prepared in example 11HNMR spectrogram, in the figure, a is imidazole-4, 5-dicarboxylic acid dimethyl ester, and b is imidazole-4, 5-dicarboxylic acid dihydrazide.
FIG. 2 shows the imidazole-crosslinked chitosan CFCS and the targeted imidazole-crosslinked chitosan prepared in example 2
FTIR spectrum of HCFC S.
Figure 3 is TEM pictures of the targeted imidazole crosslinked chitosan HCFCS prepared in example 2 and the targeted nanosilver gel HCFCS @ Ag prepared in example 3.
Figure 4 is the XRD result of the targeted nanosilver gel HCFCS @ Ag prepared in example 3.
FIG. 5 shows the results of the cell uptake assay of the targeted chitosan nanogel HCFCS prepared in example 2.
FIG. 6 shows the evaluation results of the antibacterial properties of the targeting chitosan nanogel HCFCS prepared in example 2 and the targeting nanosilver gel HCFCS @ Ag prepared in example 3; in the figure, A is candida albicans and B
Is staphylococcus aureus, 1 is solvent and 2 is. The target chitosan nano gel (HCFS), 3 is target nano silver gel (HCFS @ Ag), and 4 is nano silver.
FIG. 7 shows the results of the evaluation of the inhibitory effect of Hela tumor cell activity on the target chitosan nanogel HCFCS prepared in example 2 and the target nanosilver gel HCFCS @ Ag prepared in example 3.
Detailed Description
The present invention will be described in detail below with reference to specific examples and the accompanying drawings. It is to be understood that the specific embodiments and figures described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Placing 0.5g of imidazole-4, 5-dicarboxylic acid dimethyl ester into a 100mL three-neck round-bottom flask, adding 10mL of methanol, heating and refluxing at 70 ℃, adding 2mL of 80% hydrazine hydrate and refluxing for 3h after the imidazole-4, 5-dicarboxylic acid dimethyl ester is completely dissolved, washing with methanol, centrifuging twice, and drying the obtained sample in vacuum to obtain the product imidazole-4, 5-dicarboxylic acid dihydrazide. As shown in figure 1Process for preparing imidazole-4, 5-dicarboxylic acid dihydrazide1HNMR spectrogram. Imidazole-4, 5-dicarboxylic acid dimethyl ester (fig. 1 a): 3.8(CH3), 7.9(CH), 13.5 (NH). Imidazole-4, 5-dicarboxylic acid dihydrazide (fig. 1 b): and (3) after hydrazinolysis, the proton resonance peak of methoxyl 3.8 disappears, new resonance peaks of 9.7 and 12.0 are assigned as a hydrazide group NH peak, and 4.7 is assigned as a hydrazide group NH2 peak, which indicates that hydrazinolysis is successful, and a target product is obtained.
Example 2
1.7760g of chitosan (MW 5KDa) was weighed into a 250mL three-necked round bottom flask, dissolved in 50mL NaAc/HAc buffer solution with 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product.
Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dripping 1% hyaluronic acid aqueous solution (HA) with a volume ratio of 1:1 with imidazole crosslinked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and lyophilizing to obtain target chitosan nano gel (HCFC). FTIR spectra of imidazole-crosslinked chitosan gel (CFCS) and targeted chitosan nanogel (HCFC) prepared in this example are shown in FIG. 2, wherein the wave number is 1640cm-1、1519cm-1The peak was assigned as the-CO-NH-vibration absorption of the amide.
Example 3
Weighing 15mg formyl chitosan (MW & lt 5kDa) in 30mL deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution at the same time until the formyl chitosan is completely dissolved, dropwise adding 2mL 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonic treatment for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to be 8-9 by using sodium hydroxide solution, dropwise adding 0.5mL 10% sodium borohydride solution, dropwise adding 1% hyaluronic acid solution in a volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS), continuously ultrasonically treating for 20min, dialyzing (MWCO & lt 2000kDa) product liquid, and freeze-drying to obtain the target nano silver gel (HCFS @ Ag). FIG. 3 shows TEM images of the targeted chitosan nanogel (HCFS) prepared in example 2 and the targeted nanosilver gel (HCFS @ Ag) prepared in this example. The particle size of the targeting gel is distributed in the range of 50-70 nm; the particle size of the targeted nano-silver gel is 50-80 nm; the grain diameter of the target nano gel embedded nano silver is about 10 nm. Fig. 4 shows the XRD pattern of the targeting nano silver gel (HCFCS @ Ag) prepared in this example. The target nano silver gel in the spectrogram has nano silver crystal diffraction peaks at 2 theta of 38.5 degrees, 44.5 degrees, 64.8 degrees and 77.5 degrees, the diffraction peaks are obvious and have no impurity peaks, and the nano silver in the prepared nano silver gel has face-centered three-dimensional structures of (111), (200), (220) and (311).
Example 4
1.7760g of chitosan (MW 1KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water (MWCO is 500Da), freezing the dialyzate, and freeze-drying to obtain the product of imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nano gel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 5
1.7760g of chitosan (MW 10KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 1:1 to imidazole cross-linked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nano gel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 6
1.7760g of chitosan (MW 5KDa) was weighed into a 250mL three-necked round bottom flask, dissolved in 50mL NaAc/HAc buffer solution with 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nano gel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 7
1.7760g of chitosan (MW 1KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nano gel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 8
1.7760g of chitosan (MW 10KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 3:1 to imidazole cross-linked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nano gel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 9
1.7760g of chitosan (MW 3KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 1:1 of imidazole crosslinked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nanogel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 10
1.7760g of chitosan (MW 3KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 3:1 of imidazole crosslinked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nanogel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 11
1.7760g of chitosan (MW 20KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 1:1 of imidazole crosslinked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nanogel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 12
1.7760g of chitosan (MW 20KDa) was weighed, placed in a 250mL three-necked round bottom flask, 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 and are filled with nitrogen, 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. And taking out the product liquid, pouring the product liquid into a culture dish, freezing the culture dish in a refrigerator at the temperature of 20 ℃ below zero, taking out the solidified sample, and freeze-drying the solidified sample in a freeze dryer to obtain the formyl chitosan product. Weighing 0.05g of formyl chitosan into a single-neck flask, adding 20mL of deionized water for dissolving, dissolving 0.0055g of imidazole-4, 5-dicarboxylic acid dihydrazide by using 10mL of dimethyl sulfoxide, dropwise adding the imidazole-4, 5-dicarboxylic acid dihydrazide solution into the single-neck flask containing the formyl chitosan solution, heating and refluxing for 6h at 70 ℃, taking out a product liquid after the reaction is finished, dialyzing for two days by using the deionized water, freezing the dialyzate, and freeze-drying to obtain the imidazole cross-linked chitosan gel (CFCS). Dropwise adding 1% hyaluronic acid aqueous solution with volume ratio of 3:1 of imidazole crosslinked chitosan gel (CFCS) under ultrasonic condition, continuing ultrasonic treatment, dialyzing the obtained resultant, and freeze-drying to obtain the target chitosan nanogel (HCFC S). The structure is verified to be correct by FTIR spectrum.
Example 13
Weighing 15mg formyl chitosan (MW & lt 5kDa) in 30mL deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution at the same time until the formyl chitosan is completely dissolved, ultrasonically treating for 20min, dropwise adding 2mL 1.7mg/mL silver nitrate solution into the reaction solution, continuously ultrasonically treating for 10min, adjusting the pH value to be 8-9 by using sodium hydroxide solution, dropwise adding 0.5mL 10% sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 1:1, continuously ultrasonically treating for 20min, dialyzing the product solution (MWCO & lt 2kDa), and freeze-drying to obtain the target nano-silver gel (HCFCS @ Ag).
Example 14
The formyl chitosan used in this example was prepared as in example 2, except that it was prepared using chitosan of MW ═ 1kDa as the starting material.
Weighing 15mg of formyl chitosan (MW ═ 1kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to be 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, then dropwise adding 1% hyaluronic acid solution with the volume ratio of 1:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFCS @ Ag).
Example 15
The formyl chitosan used in this example was prepared as in example 2, except that it was prepared using chitosan of MW 10kDa as the starting material.
Weighing 15mg of formyl chitosan (MW 10kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 1:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFC S @ Ag).
Example 16
Weighing 15mg of formyl chitosan (MW & lt 5kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, ultrasonically treating for 20min, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 3:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFCS @ Ag).
Example 17
Weighing 15mg of formyl chitosan (MW ═ 1kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to be 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, then dropwise adding 1% hyaluronic acid solution with the volume ratio of 3:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFCS @ Ag).
Example 18
Weighing 15mg of formyl chitosan (MW 10kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 3:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFC S @ Ag).
Example 19
The formyl chitosan used in this example was prepared as in example 2, except that it was prepared using chitosan of MW ═ 3kDa as the starting material.
Weighing 15mg of formyl chitosan (MW & lt 3kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, then dropwise adding 1% hyaluronic acid solution with the volume ratio of 1:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFCS @ Ag).
Example 20
Weighing 15mg of formyl chitosan (MW & lt 3kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, then dropwise adding 1% hyaluronic acid solution with the volume ratio of 3:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFCS @ Ag).
Example 21
The formyl chitosan used in this example was prepared as in example 2, except that it was prepared using chitosan with MW ═ 20kDa as the starting material.
Weighing 15mg of formyl chitosan (MW 20kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 1:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFC S @ Ag).
Example 22
Weighing 15mg of formyl chitosan (MW 20kDa) in 30mL of deionized water, heating and ultrasonically treating in a water bath at 70 ℃, slowly dropwise adding 5mL of 0.6mg/mL imidazole-4, 5-dicarboxylic acid diformylhydrazine solution after completely dissolving, dropwise adding 2mL of 1.7mg/mL silver nitrate solution into the reaction solution after ultrasonically treating for 20min, continuously ultrasonically treating for 10min, adjusting the pH value to 8-9 by using sodium hydroxide solution, dropwise adding sodium borohydride solution, dropwise adding 1% hyaluronic acid solution with the volume ratio of 3:1, continuously ultrasonically treating for 20min, dialyzing and freeze-drying the product liquid, and obtaining the target nano silver gel (HCFC S @ Ag).
Application example 1 evaluation of cellular uptake
The cell uptake performance of imidazole crosslinked chitosan gel (CFCS), targeted chitosan nanogel (HCFS) and targeted nano silver gel (HCFS @ Ag) was evaluated by Hela cervical cancer cells (Shanghai cell bank of Chinese academy of sciences). Preparing cells cultured until the confluency is more than 80% into cell suspension, plating in a 24-well plate until the cell confluency reaches 80%, adding 10 μ L of FITC labeled gel material into each well (the gel material and FITC are stirred and react for 24h at 40 ℃ in a molar ratio of 3:1 to construct FITC labeled gel material), sucking out the culture medium after culturing for 4h, adding 0.04% Trypan Blue (Trypan), washing 3 times with 200 μ L PBS, adding 200 μ L0.25% trypsin into each well for digestion, discarding the trypsin after 1min, adding 500 μ L of high-sugar DMEM (containing serum and double antibody) into each well, preparing the cells into cell suspension, and detecting by a flow cytometer. The results are shown in fig. 5, and the results show that the cellular uptake of the targeted nano-silver gel reaches 70%, which is significantly higher than that of the imidazole crosslinked nano-gel.
Application example 2 evaluation of antibacterial Properties
Respectively inoculating staphylococcus aureus and candida albicans on LB solid culture medium and SCDLP solid culture medium for resuscitation, and culturing at 37 ℃ for 1 day and at 28 ℃ for 2 days. Selecting bacterial colony with inoculating loop, preparing bacterial suspension in LB liquid culture medium and SCDLP liquid culture medium, and counting with cell counting plate to obtain cell suspension with concentration of 104cfu/mL～105cfu/mL. Respectively inoculating 3mL of bacterial suspension into 50mL of LB liquid culture medium and 50mL of SCDLP liquid culture medium, pouring a flat plate, adding an Oxford cup, respectively adding 100 mu L of PBS control, nano-silver, targeted chitosan nano-gel (HCFS) and targeted nano-silver gel (HCFS @ Ag) into the Oxford cup, respectively culturing in an incubator at 37 ℃ and 28 ℃, and observing the size of a bacteriostatic circle after a certain time. The results are shown in FIG. 6. The targeted nano-silver gel has a larger inhibition zone on candida albicans than nano-silver, which shows that the targeted nano-silver gel has higher inhibition performance. The targeted nano-silver gel has higher inhibition effect on the growth of candida albicans than staphylococcus aureus, and due to the overexpression of a CD44 receptor on the cell membrane surface of the candida albicans, the uptake of the targeted nano-silver gel is increased.
Application example 3 evaluation of cancer inhibitory Effect
Hela cells in logarithmic growth phase are digested with 0.25% pancreatin and added into DMEM high-sugar medium (containing serum and double antibody) to prepare cell suspension. After counting with a cell counting plate, cells were seeded in a 96-well plate (5X 10)3One/well) to a final volume of 100. mu.L/well. The well plate was then placed at 37 ℃ with 5% CO2The cells are cultured in the cell culture box for 18-24 hours, so that the cell density reaches about 80%. Removing the culture medium, respectively diluting the nano-silver, the target chitosan nano-gel (HCFS) and the target nano-silver gel (HCFS @ Ag) to 100 mu L with the culture medium, so that the final concentration of the drug substance is 15, 30 and 45 mu g/mL, arranging 3 multiple holes for each concentration, and respectively adding the multiple holes into each hole. After the sample addition, the well plate was continuously placed at 37 ℃ in 5% CO2The cell culture box is used for culturing for 24 hours. And (3) taking out the 96-well plate, placing the 96-well plate in a superclean workbench, adding 10 mu L of MTT into each well, shaking up the well by gentle shaking, and placing the well in an incubator to continue incubation for 4 hours. And detecting the OD value by using a multifunctional microplate reader, wherein the detection wavelength is 570 nm. The results are shown in FIG. 7. The targeted 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 nano-silver (30-45 mug/mL), and the targeted nano-silver gel has high-efficiency tumor inhibition performance. Statistical analysis shows that the results have statistical significance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A preparation method of 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 coated by hyaluronic acid, and the targeted chitosan nano-silver gel is obtained.
2. The method of claim 1, comprising the steps of:
(1) preparing formyl chitosan: adding 1.0-5.0 g of chitosan into NaAc/HAc buffer solution with the pH value of 4.0-5.0, adding 0.1-0.5 g of sodium periodate under the protection of nitrogen, stirring and reacting for a period of time under an ice bath, and adding ethylene glycol 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 a reflux condition, refluxing for a period of time, washing with methanol, centrifuging, and drying in vacuum to obtain imidazole-4, 5-dicarboxylic acid dimethyl hydrazide;
(3) preparation of imidazole hydrazine crosslinked chitosan nanogel: dripping 0.01-0.1 mg/mL of imidazole-4, 5-dicarboxylic acid dihydrazide/dimethyl sulfoxide solution into 0.1-0.5 mg/mL formyl chitosan aqueous solution, heating and refluxing for a period of time, dialyzing with deionized water, and freeze-drying to obtain imidazole cross-linked chitosan gel;
(4) preparing targeted chitosan nano silver gel: adding deionized water into 5-20 mg of imidazole crosslinked chitosan gel, performing ultrasonic dissolution in a water bath, dropwise adding 1-5 mL of 1.0-2.0 mg/mL silver nitrate solution under the ultrasonic condition, performing ultrasonic treatment for 1-10 min, adjusting the pH value to 8-9 with 0.1-1M sodium hydroxide solution, dropwise adding 0.1-1 mg/mL sodium borohydride solution, continuing performing ultrasonic treatment for 30-120 min, dropwise adding 0.1-1 mg/mL hyaluronic acid solution in a volume ratio of 1: 1-3: 1 to the imidazole crosslinked chitosan gel, continuing ultrasonic treatment for 10-30 min, dialyzing, and freeze-drying to obtain the targeted chitosan nano-silver gel.
3. The preparation method according to claim 1, wherein the stirring reaction in the step (1) is carried out for 24-48 h under ice bath; the addition amount of the ethylene glycol is 1-10 mL.
4. The method according to claim 1, wherein the reflux temperature in step (2) is 50 to 80 ℃; the reflux time is 1-5 h.
5. The method according to claim 1, wherein the reflux temperature in step (3) is 50 to 80 ℃; the reflux time is 4-12 h.
6. The method according to claim 1, wherein the temperature of the water bath in the step (4) is 40 to 70 ℃.
7. The method according to claim 1, wherein the molecular weight Mw of the hyaluronic acid is 10-100 kDa.
8. The method according to claim 1, wherein the chitosan has a molecular weight Mw of 1 to 100 kDa.
9. A targeted chitosan nano silver gel, which is prepared according to the preparation method of any one of claims 1 to 8, and has a structure of imidazole crosslinked chitosan and in-situ complexing reduced nano silver and a hyaluronic acid targeted structure coated on the outer layer.
10. The use of a targeted chitosan nanosilver gel according to claim 9, characterized by the use in the preparation of a fungal, bacterial, viral or tumor cell inhibitor.
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