CN115737574A

CN115737574A - Acid-responsive chitosan nano micelle freeze-dried powder and preparation method and application thereof

Info

Publication number: CN115737574A
Application number: CN202211661334.4A
Authority: CN
Inventors: 李昊欢; 徐傅能; 崔超; 谢海龙; 袁棚
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-12-23
Filing date: 2022-12-23
Publication date: 2023-03-07

Abstract

The invention provides chitosan nano micelle freeze-dried powder with acid response as well as a preparation method and application thereof, wherein the preparation method of the chitosan nano micelle freeze-dried powder comprises the following steps: reacting amine compound with acid anhydride compound, reacting the reaction product with chitosan under the action of catalyst to form chitosan nano micelle, and freeze-drying. The chitosan nano micelle freeze-dried powder has good pH responsiveness, so that the chitosan nano micelle freeze-dried powder can release medicine in a bacterial infection acidic microenvironment, and the accumulation of the medicine at an infected part is effectively improved; the photosensitizer can be loaded through the hydrophobic effect, so that the encapsulation efficiency of the photosensitizer is greatly improved, and the obtained nano preparation has a good escherichia coli inhibition effect as proved by in vitro experiments.

Description

Acid-responsive chitosan nano micelle freeze-dried powder and preparation method and application thereof

Technical Field

The invention relates to the technical field of micelle preparation, in particular to acid-responsive chitosan nano micelle freeze-dried powder and a preparation method and application thereof.

Background

Chitosan is a natural polymer polysaccharide, has good natural antibacterial effect, is safe and nontoxic, and is widely applied to the field of medicines. Hydroxyethyl chitosan (HECTS) is a chitosan derivative obtained by modifying chitosan through hydroxyethylation, and is widely applied in multiple fields of antibiosis, drug synthesis, high molecular material synthesis and the like.

The existing hydrogel is prepared by combining chitosan with an anti-inflammatory agent, an antibacterial agent and the like, so that the hydrogel has good antibacterial effect and tissue repair capability, but the hydrogel lacks the targeting property of an infected part. Similar to the tumor microenvironment, during bacterial infection, the microenvironment also presents a slightly acidic environment, because some relevant active factors secreted by the bacteria can reduce the PH of the surrounding environment through fermentation to produce acidic substances. Therefore, if an acid-responsive chitosan nano micelle can be prepared for antibacterial through an acidic environment of an infection site, more excellent antibacterial and bacteriostatic effects may be produced.

In addition, photodynamic therapy (PDT) has promising applications in combating bacterial infections as an emerging disease treatment. Phototherapy is usually performed by using some drugs that are changed under specific light irradiation, for example, a photosensitizer can perform a photochemical reaction under the light irradiation and generate active oxygen, thereby destroying active substances in the pellicle. It is not easy to make bacteria produce drug resistance, and has little harm to organism itself, thus it has been widely paid attention to in the antibacterial field.

The invention aims to carry out preliminary exploration research on the acid response chitosan nano micelle combined photodynamic therapy.

Disclosure of Invention

In view of the above, the invention provides an acid-responsive chitosan nano micelle freeze-dried powder, and a preparation method and application thereof, the nano micelle is connected with amine through acid anhydride by utilizing the advantages of good water solubility, biocompatibility and the like of chitosan, and can be self-assembled in water to form the nano micelle due to the hydrophilic chitosan part and the hydrophobic amine part.

The micelle can be used as a drug carrier and can encapsulate a photosensitizer in a hydrophobic core thereof. And the micelle can be developed into a photodynamic therapy carrier and widely applied to the field of therapy.

The technical scheme provided by the invention is as follows:

in a first aspect, the invention provides a preparation method of chitosan nano micelle freeze-dried powder with acid response, which comprises the following steps: reacting amine compound with acid anhydride compound, reacting the reaction product with chitosan under the action of catalyst to form chitosan nano micelle, and freeze-drying.

Further, the preparation method comprises the following steps:

step 1, dissolving octadecylamine in a first solvent, dissolving cis-aconitic anhydride in a second solvent, then adding a cis-aconitic anhydride solution into an octadecylamine solution under an oxygen-free condition for mixing reaction, and removing the solvent after the reaction is finished to obtain a crude cis-aconityl octadecamine (ASA);

and 2, dissolving the ASA crude product in a solvent III, adding catalysts 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NSH) for activation, adding a solution of hydroxyethyl chitosan dissolved in the solvent III, dialyzing and centrifuging after complete reaction, and taking supernatant for freeze-drying to obtain the chitosan nano micelle freeze-dried powder.

Further, the solvent one is at least one selected from chloroform, dichloromethane, isopropanol and methanol, and preferably chloroform.

Further, the second solvent is methanol, acetonitrile, dimethyl sulfoxide or tetrahydrofuran, preferably acetonitrile.

Further, the molar ratio of the octadecylamine to the cis-aconitic anhydride in the step 1 is 0.1-1: 1, preferably 0.52:1.

preferably, the mixing reaction in the step 1 is carried out at the temperature of-10 to 0 ℃ for 2h +/-10 min.

Further, the solvent III is DMSO.

Further, the final concentration of the crude ASA in the third solvent in the step 2 is 20 mg/mL.

Further, the molar ratio of the catalysts EDC and NSH to the crude ASA product in the step 2 is 1-2: 1 to 2:1, preferably 1.5:1.5:1.

further, the activation time of ASA in the step 2 is 20 +/-1 min.

Further, the mass ratio of the hydroxyethyl chitosan to the ASA crude product in the step 2 is 1 to 3:1.

in a second aspect, the invention provides an acid-responsive chitosan nano micelle freeze-dried powder prepared by the preparation method.

In a third aspect, the invention also provides an application of the acid-responsive chitosan nano micelle freeze-dried powder in preparation of an antibacterial drug.

In a fourth aspect, the invention also provides a preparation method of the antibacterial nano preparation, which comprises the step of taking the chitosan nano micelle freeze-dried powder with acid response as a carrier and encapsulating a photosensitizer to form the nano preparation.

Further, the preparation method of the nano preparation specifically comprises the following steps: respectively dissolving a photosensitizer and chitosan nano micelle freeze-dried powder in DMSO, mixing and stirring the two solutions at room temperature for 2 to 6h, carrying out ultrasonic treatment at the temperature of-10 to 0 ℃ for 10 to 60 min, dialyzing, and carrying out freeze drying to obtain the chitosan nano micelle freeze-dried powder.

Further, in the preparation method, the mass ratio of the photosensitizer to the chitosan nano micelle freeze-dried powder is 0.05 to 0.5:1, preferably 0.1 to 0.3.

Further, the photosensitizer is chlorin e6 (Ce 6).

Preferably, in the preparation method, the mixing and stirring time at room temperature is 3 to 5h.

Preferably, in the preparation method, the temperature is kept at minus 10 to 0 ℃ for 20 to 40min by ultrasonic sound.

In a fifth aspect, the invention provides an antibacterial nano preparation, which is obtained by adopting the preparation method.

The invention has the beneficial effects that:

(1) The chitosan nano micelle freeze-dried powder provided by the invention is easy to obtain in materials, has high safety, is non-toxic and environment-friendly, is biodegradable, and has good safety and biocompatibility.

(2) The chitosan nano micelle freeze-dried powder is prepared from octadecylamine, cis-aconitic anhydride and chitosan, has good pH responsiveness, can release a drug in a bacterial infection acidic microenvironment, and effectively improves the accumulation of the drug at an infected part.

(3) The acid-response chitosan nano micelle freeze-dried powder can load photosensitizer through hydrophobic effect, so that the encapsulation efficiency of the photosensitizer is greatly improved, and the obtained nano preparation has good escherichia coli bacteriostasis effect as proved by in vitro experiments.

Drawings

FIG. 1 is a nuclear magnetic resonance spectrum of a crude ASA product obtained in Experimental example 1 of the present invention.

FIG. 2 is a nuclear magnetic resonance spectrum of chitosan nano micelle freeze-dried powder obtained in example 1 of the present invention.

FIG. 3 is a graph showing the cumulative release rate of Ce6 at pH 6.0 and pH7.4 of the antibacterial nano-formulation ASA-CC of example 7 of the present invention.

FIG. 4 is a graph showing the in vitro antibacterial effects of PBS and ASA-CC in example 8 of the present invention, wherein (a) is PBS group and (b) is ASA-CC group.

Detailed Description

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1

The embodiment provides a preparation method of chitosan nano micelle freeze-dried powder with acid response, which comprises the following steps:

(1) 0.14 g (0.52 mmol) of octadecylamine was weighed into a dry 50 mL three-necked flask, 6 mL of chloroform was added to dissolve it completely, and the solution was placed in an ice bath at 0 ℃ under nitrogen. 0.16 g (1.03 mmol) of cis-aconitic anhydride is weighed and dissolved in 3 mL of acetonitrile solution, the solution is added into a constant pressure dropping funnel and slowly dripped into a trichloromethane solution of octadecylamine, and the mixture is stirred for 2h at the temperature of 0 ℃. And removing the reaction solvent by rotary evaporation to obtain crude ASA.

(2) Weighing 10 mg of ASA obtained in the step (1) and dissolving the ASA in DMSO to form a 20 mg/mL solution, adding a mixed solution of catalysts EDC and NHS (molar ratio EDC: NHS: ASA = 1.5.

In this embodiment, the chloroform in step (1) may be replaced by dichloromethane, isopropanol, or methanol; acetonitrile can be replaced by methanol, dimethyl sulfoxide or tetrahydrofuran.

In the embodiment, the molar ratio of octadecylamine to cis-aconitic anhydride is controlled to be 0.1-1: 1 ASA was obtained but in a molar ratio of 0.52:1 has relatively few nuclear magnetic impurities. In addition, the reaction temperature of octadecylamine and cis-aconitic anhydride is controlled to be-10 to 0 ℃, and the reaction time is preferably about 2 hours.

In this example, the molar ratio of the catalysts EDC, NSH and ASA crude product is 1 to 2:1 to 2:1, preferably 1.5:1.5:1.

example 2

(1) 0.14 g (0.52 mmol) of octadecylamine was weighed into a dry 50 mL three-necked flask, 6 mL of chloroform was added to dissolve completely, and the solution was placed in an ice bath at 0 ℃ under nitrogen. 0.08 g (0.52 mmol) of cis-aconitic anhydride is weighed and dissolved in 3 mL of acetonitrile solution, the solution is added into a constant pressure dropping funnel and slowly dropped into a trichloromethane solution of octadecylamine, and the mixture is stirred for 2h under the condition of heat preservation at 0 ℃. And removing the reaction solvent by rotary evaporation to obtain crude ASA.

(2) Weighing 10 mg of ASA obtained in the step (1) and dissolving in DMSO to form a 20 mg/mL solution, adding a mixed solution of catalyst EDC and NHS and dissolving in DMSO (molar ratio EDC: NHS: ASA =1.

Example 3

(1) 0.14 g (0.52 mmol) of octadecylamine was weighed into a dry 50 mL three-necked flask, 6 mL of chloroform was added to dissolve completely, and the solution was placed in an ice bath at 0 ℃ under nitrogen. 0.81 g (5.2 mmol) of cis-aconitic anhydride is weighed and dissolved in 3 mL of acetonitrile solution, the solution is added into a constant pressure dropping funnel and slowly dropped into a trichloromethane solution of octadecylamine, and the mixture is stirred for 2h under the condition of heat preservation at 0 ℃. And removing the reaction solvent by rotary evaporation to obtain crude ASA.

(2) Weighing 10 mg of ASA obtained in the step (1) and dissolving the ASA in DMSO to form a 20 mg/mL solution, adding a solution of catalyst EDC and NHS mixed in DMSO (molar ratio EDC: NHS: ASA = 2.

Example 4

The embodiment provides an antibacterial nano preparation, which is a nano preparation taking the acid-responsive chitosan nano micelle freeze-dried powder prepared in the embodiment 1 as a carrier and encapsulating a photosensitizer, and the specific preparation process is as follows:

7 mg of chitosan nano micelle lyophilized powder prepared in example 1 was dissolved in 2.33 mL of DMSO. Weighing 0.77 mg of chlorin e6 (Ce 6), dissolving in 0.194 mL of DMSO, slowly dropwise adding into the chitosan nano micelle solution in the dark, magnetically stirring at room temperature for reaction for 4h, carrying out ultrasonic treatment in an ice bath for 30 min, transferring the solution into a dialysis bag, dialyzing with 50% ethanol for 24h, replacing deionized water for dialysis for 24h, and carrying out freeze drying to obtain the nano preparation ASA-CC.

Experimental example 5

In this example, the nuclear magnetic resonance hydrogen spectroscopy is used to confirm the structure of ASA and chitosan nano micelle lyophilized powder (ASA-shell), which is as follows:

crude ASA prepared in example 1 and ASA-shell were dissolved in deuterated DMSO for NMR analysis, see FIGS. 1 and 2.

FIG. 1 shows the result of hydrogen spectrum of ASA. It can be seen that the characteristic peaks of octadecylamine appeared at 0.86ppm (CH 3-), 0.86ppm (-CH 2-). A characteristic peak (= CH-) appears at 6.72ppm of cis-aconitic anhydride, which indicates that cis-aconitic anhydride is coupled to octadecylamine, so that ASA is successfully prepared.

FIG. 2 shows the result of the hydrogen spectrum of ASA-shell. It can be seen that, based on the nuclear magnetic results of ASA, the characteristic peak of hydroxyethyl Chitosan (CTS) appears at 4.37-4.84ppm, indicating that CTS is coupled to ASA, thereby successfully preparing ASA-shell.

Experimental example 6

This example characterizes the physical and chemical properties of ASA, ASA-shell, ASA-CC as follows:

the method of example 1 is adopted to prepare crude ASA, 4 mg is taken and added with 2 mL of deionized water to prepare a solution with the concentration of 2 mg/mL, the solution is subjected to ultrasonic treatment to be completely dissolved, the solution is filtered by 0.45 μm and 0.22 μm microporous filter membranes in sequence, 1mL of the filtered solution is added into a sample cell, and then the sample cell is placed into a laser particle size analyzer for particle size measurement.

The ASA-shell is prepared by the method of example 1, 4 mg is precisely weighed, 2 mL of deionized water is added to prepare a solution with the concentration of 2 mg/mL, the solution is completely dissolved by ultrasonic treatment, the solution is sequentially filtered by 0.45 μm and 0.22 μm microporous filter membranes, 1mL of the filtered solution is added into a sample cell, and then the sample cell is placed into a laser particle size analyzer for particle size measurement.

The method comprises the steps of preparing ASA-CC by the method of example 1, precisely weighing 4 mg, adding 2 mL of deionized water to prepare a solution with the concentration of 2 mg/mL, carrying out ultrasonic treatment to completely dissolve the solution, filtering the solution by 0.45 mu m and 0.22 mu m microporous filter membranes in sequence, adding 1mL of the filtered solution into a sample cell, and then placing the sample cell into a laser particle size analyzer to carry out particle size measurement.

ASA-CC 4 mg obtained in example 4 was precisely weighed, deionized water was added to prepare a solution with a concentration of 2 mg/mL, stirred at 30 ℃ for 30 min, filtered through a 0.22 μm microporous membrane, diluted to an appropriate concentration with a mobile phase, and the Ce6 content was measured by HPLC.

The results are shown in Table 1. The particle sizes of ASA, ASA-shell and ASA-CC are 171.2 nm, 234.7 nm and 238.3 nm respectively, which shows that the chitosan is successfully connected, and the average particle size of micelle is not influenced by the entrapment of Ce 6; the drug loading of Ce6 was 13.3%.

TABLE 1 physicochemical characterization of the micelles

Nano-particle	Size (nm)	Drug loading
			ASA	171.2 nm	---
ASA-shell	234.7 nm	---
			ASA-CC	238.3 nm	13.3%

Experimental example 7

This example uses dialysis to study the release behavior of the drug from the micelles, and the in vitro release test was performed using ASA-CC obtained as described in example 4, as follows:

the nano-preparation ASA-CC obtained in example 4 was weighed out to prepare a nanoparticle solution with a final concentration of 1 mg/mL, and 1.0 mL of the solution was taken out in a dialysis bag with a molecular weight cut-off of 3500 Da. 50 mL of phosphate buffer (pH 7.4, 6.5, respectively, containing 0.2% Tween 80) was used as a release medium, and the medium was placed in a constant temperature shaking table at 37 ℃ and a shaking speed of 100 rpm. At regular intervals, 2 mL of the sample was taken and immediately replenished with the same volume of release medium. The cumulative amount of released Ce6 was measured. The content of the released drug was measured by HPLC method, and the cumulative release rate of the drug was calculated according to the formula, and the result is shown in fig. 3. Under the condition of pH 6.0, the 24h cumulative release rate of Ce6 is about 80 percent and is far higher than that of a group with pH7.4, and the ASA-CC has acid response effect.

Example 8

In vitro antibacterial test

10 mg of ASA-CC obtained by the method of example 4 was dissolved in 5 mL of PBS to obtain a 2 mg/mL ASA-CC solution, which was then filtered through a 0.22 μm sterile filter, 100 μ L ASA-CC solution (n = 3) was added to a 96-well plate, and the E.coli concentration was adjusted to 1X 10 ⁶ Adding 100 μ L of CFU bacteria solution into each well, mixing well, and placing each well in a 660 nm laser at 0.5W/cm ² Irradiating for 5 min, placing into a bacteria incubator, and culturing for 12 h. In the control group, ASA-CC solution was replaced with an equal volume of PBS solution (n = 3). After 12h, 20. Mu.L of the suspension was pipetted into each well for plating, and the colony growth was observed. FIG. 4 shows the colony growth results of one of the ASA-CC groups and the control group, which indicates that the ASA-CC nano-preparation has a good Escherichia coli inhibition effect compared with the control group (PBS group).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A preparation method of chitosan nano micelle freeze-dried powder with acid response is characterized by comprising the following steps: the method comprises the following steps: reacting amine compound with acid anhydride compound, reacting the reaction product with chitosan under the action of catalyst to form chitosan nano micelle, and freeze-drying.

2. The method for preparing the chitosan nano micelle freeze-dried powder with acid response according to claim 1, which is characterized in that: the preparation method comprises the following steps:

step 1, dissolving octadecylamine in a first solvent, dissolving cis-aconitic anhydride in a second solvent, then adding a cis-aconitic anhydride solution into an octadecylamine solution under an oxygen-free condition for mixing reaction, and removing the solvent after the reaction is finished to obtain a crude cis-aconityl octadecamine;

and 2, dissolving the crude cis-aconityl octadecanamide in a third solvent, adding a catalyst 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride) and N-hydroxysuccinimide for activation, adding a solution of hydroxyethyl chitosan dissolved in the third solvent, dialyzing and centrifuging after complete reaction, and taking supernatant for freeze-drying to obtain the chitosan nano micelle freeze-dried powder.

3. The method for preparing acid-responsive chitosan nano micelle lyophilized powder according to claim 2, wherein: in the step 1, the molar ratio of octadecylamine to cis-aconitic anhydride is 0.1-1: 1.

4. the method for preparing the chitosan nano micelle freeze-dried powder with acid response according to claim 2, which is characterized in that: in the step 1, the mixing reaction temperature is-10 to 0 ℃, and the time is 2h +/-10 min.

5. The method for preparing acid-responsive chitosan nano micelle lyophilized powder according to claim 2, wherein: the molar ratio of the catalysts EDC and NSH to the ASA crude product in the step 2 is 1 to 2:1 to 2:1.

6. the method for preparing acid-responsive chitosan nano micelle lyophilized powder according to claim 2, wherein: in the step 2, the mass ratio of the hydroxyethyl chitosan to the ASA crude product is 1 to 3:1.

7. an acid-responsive chitosan nano micelle freeze-dried powder is characterized in that: is prepared by the preparation method of any one of claims 1 to 6.

8. The use of chitosan nano micelle lyophilized powder of claim 7 in the preparation of antibacterial drugs.

9. A preparation method of an antibacterial nano preparation is characterized by comprising the following steps: comprises the steps of taking the chitosan nano micelle freeze-dried powder as a carrier and encapsulating a photosensitizer to form a nano preparation.

10. An antibacterial nano-formulation obtained by the preparation method of claim 9.