CN107583049B

CN107583049B - Preparation method of injectable hydrogel with photo-thermal property

Info

Publication number: CN107583049B
Application number: CN201710820795.4A
Authority: CN
Inventors: 施冬健; 曾金凤; 杨雯迪; 李小杰; 陈明清
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2017-10-30
Filing date: 2017-10-30
Publication date: 2021-01-01
Anticipated expiration: 2037-10-30
Also published as: CN107583049A

Abstract

A preparation method of injectable hydrogel with photo-thermal performance, belonging to the field of functional materials. The invention provides a method for treating malignant tumor for multiple times by injecting medicine once based on the phenomenon that the malignant tumor needs to be treated by taking medicine for multiple times. Firstly, preparing dopamine modified sodium alginate biological macromolecules (Alg-DA); regulating the gelation temperature of the temperature-sensitive hydrogel (CGP) of Chitosan (CS) and beta-sodium glycerophosphate (beta-GP) to be at the physiological temperature of a human body, so that the hydrogel has the characteristic of being sol at normal temperature and being gel after reaching the human body; mixing Alg-DA and CGP to prepare temperature-sensitive hydrogel with injectability, and doping gold nanorods (Au-PDA NRs) coated with pre-prepared polydopamine in the hydrogel to obtain injectable hydrogel with photo-thermal property (CGP/Alg-DA @ Au-PDA). The gel can fix Au NRs at a tumor part, and can realize multiple photo-thermal conversion under laser irradiation, thereby realizing the purpose of treating tumors by utilizing high temperature. The prepared injectable hydrogel with photo-thermal property has potential application value in the field of biomedical science.

Description

Preparation method of injectable hydrogel with photo-thermal property

Technical Field

An injectable hydrogel with photo-thermal performance belongs to the technical field of functional materials.

Background

The medicine is used for treating malignant tumors, and is transported to a lesion part through a body circulation system, so that the purpose of treatment is achieved. However, the drug delivery system has a series of problems such as non-specific distribution in vivo and possibility of being rapidly cleared in vitro. The hydrogel as a local drug delivery system can be fixed at the focus position to continuously and effectively release the drug, so that the loading amount and the retention time of the drug can be selected according to the deterioration degree of the disease, and the problems can be effectively solved. Due to the invasiveness and drug resistance of malignant tumors, repeated administration is needed in the treatment process, so that the treatment efficiency is limited and great pain is brought to patients. Photothermal therapy (PTT) based on nanomaterials is a minimally invasive tumor therapy technology that has rapidly developed in recent years. After the photo-thermal agent is accumulated at a tumor part, the temperature of a lesion position is raised by utilizing heat generated by a photo-thermal conversion effect under the excitation of incident light, and the effect of killing tumor cells is realized after a certain temperature is reached. Moreover, the device can irradiate the same part for multiple times, thereby achieving the purpose of multiple treatments by one-time injection. Currently, the photo-thermal conversion agents which are researched more include gold nanoparticles, graphene oxide, carbon nanotubes, organic dyes and the like. Among them, gold nanorods (Au NRs) have the advantages of stable chemical properties, excellent optical properties, good biocompatibility, easy surface modification, etc., and are widely used in the field of tumor photothermal therapy. The photosensitizer Au NRs is fixedly embedded in hydrogel containing a large number of pore channel structures, and after the photosensitizer Au NRs is injected into a body, the photosensitizer Au NRs stays in a tumor part for a long time, so that multiple times of photothermal treatment can be performed according to the tumor type and the deterioration degree.

Temperature sensitive hydrogels that are capable of responding to changes in ambient temperature have attracted interest to researchers in order to make hydrogels directly injectable. Temperature sensitive gels can undergo a sol-gel transition with a change in temperature. The gelation temperature of part of temperature-sensitive gel materials is close to the physiological temperature of human bodies, namely the temperature-sensitive gel materials are sol at normal temperature and have injectability, but the temperature-sensitive gel materials are transformed into gel after being injected into the bodies, so the temperature-sensitive gel materials can be used as drug carriers for local injection therapy and have good application prospect in biomedicine. Among them, the temperature-sensitive hydrogel based on chitosan/beta-sodium glycerophosphate (CS/beta-GP, CGP) is widely concerned in the fields of drug delivery, cell scaffolds and the like. However, researches show that the CGP temperature-sensitive hydrogel has poor mechanical properties and is fragile and difficult to maintain the complete form in a living body. In order to improve the defects, researchers mix the hydrogel with other biomacromolecules such as hydroxyethyl cellulose, gelatin, polyvinyl alcohol, sodium alginate and the like to obtain the composite temperature-sensitive hydrogel, and the mechanical strength of the hydrogel can be enhanced to a certain extent. But the medicine still has no photothermal/photodynamic effect and cannot meet the effects of one-time injection and multiple treatments.

The CGP temperature-sensitive hydrogel is used as a main raw material and is compounded with dopamine-modified sodium alginate; meanwhile, the modified gold nanorods with the proper length-diameter ratio are prepared, so that the composite gel has a photo-thermal conversion effect, proper treatment time can be selected according to different tumor types and different deterioration degrees, side effects of multiple drug injections and multiple drug cycles on normal tissues are avoided, and the composite gel has a wide application prospect in the biomedical fields of tumor photo-thermal treatment and the like.

Disclosure of Invention

The invention aims to prepare the gold nanorod doped chitosan/sodium alginate injectable hydrogel with photo-thermal performance.

The technical scheme of the invention is as follows: the characteristic that the gelation temperature of the temperature-sensitive hydrogel of Chitosan (CS) and beta-sodium glycerophosphate (beta-GP) can be controlled at the physiological temperature of a human body is utilized; mixing pre-prepared dopamine modified alginic acid (Alg-DA) with CS and beta-GP to prepare temperature-sensitive hydrogel (CGP/Alg-DA) with injectability, doping gold nanorods (Au-PDA NRs) wrapped by polydopamine in the hydrogel, and uniformly and stably dispersing the Au-PDA NRs in the gel by utilizing the adhesion between dopamine on the surface of the Au NRs and the Alg-DA to obtain the injectable hydrogel (CGP/Alg-DA @ Au-PDA) with photo-thermal performance.

The dopamine is grafted and modified with sodium alginate through amidation reaction, the molar ratio of the dopamine to the sodium alginate is controlled to be 1: 1-1: 5, the grafting rate of the dopamine can be controlled to be 9% -50%, and the modified dopamine is expressed as Alg-DA, and the structural formula of the modified dopamine is as follows.

The length of the gold nanorod is controlled to be 60 +/-2 nm, the width is controlled to be 16 +/-4 nm, the length-diameter ratio is controlled to be 4.0, and the LSPR characteristic absorption peak is at 830 nm. By means of the oxidative self-polymerization characteristic of dopamine in an alkaline environment, a uniform polydopamine layer is wrapped around the gold nanorods, and the thickness of the polydopamine layer is controlled to be 5.0-9.0 nm.

The invention has the beneficial effects that: the surface of the gold nanorod is wrapped with the poly dopamine layer, and the gold nanorod can be uniformly and stably dispersed in the gel through the interaction between dopamine by utilizing the adhesion effect between the poly dopamine layer on the surface of the gold nanorod and the dopamine on the molecular chain of the sodium alginate. The gelation transition temperature of the CGP temperature-sensitive hydrogel can be controlled near the physiological temperature of a human body, the CGP temperature-sensitive hydrogel has injectability, the photothermal conversion material can be effectively fixed at a focus position, and the gel temperature can be raised to 64 +/-2.2 ℃ after laser irradiation; after multiple times of irradiation, the temperature of the gel can be raised to 60 +/-2.5 ℃ for multiple times; the time of photothermal therapy can thus be selected according to the type of tumor and the degree of deterioration.

Drawings

FIG. 1 NMR spectrum of Alg-DA

FIG. 2(a) Transmission Electron micrograph of Au NRs and (b) Au-PDA NRs

FIG. 3 shows that the CGP/Alg-DA @ Au-PDA temperature-sensitive composite hydrogel is irradiated by laser (808nm, 1.50W/cm)²) Temperature Change Pattern after 10 times

Detailed Description

Example 1 preparation of dopamine graft-modified sodium alginate (Alg-DA)

Sodium alginate (2g) was dissolved in 100mL of PBS buffer (0.2mol/L, pH 5.5), EDC (3.88g) and NHS (4.68g) were added in this order after complete dissolution, stirred at room temperature for 45min, then 1.9g of dopamine hydrochloride (DA · HCl) was added in a molar ratio of 1:1, nitrogen was passed through a double-line tube, vacuum was applied, and the flask was evacuated to remove air to prevent oxidation of DA. Stirring and reacting for 24h at normal temperature, precipitating with a large amount of ethanol to obtain light brown solid after reaction, washing with ethanol and centrifuging for three times, dissolving the precipitate in deionized water, dialyzing for three days, and freeze drying to obtain dopamine grafted modified sodium alginate labeled as Alg-DA₁。

Note that the amount of dopamine hydrochloride (DA) added in example 1 was changed to 5.8g and 9.6g (i.e., n)_Alg/n_DA1:3 and 1:5) respectively, Alg-DA with different degrees of substitution can be obtained, and respectively counted as Alg-DA₃And Alg-DA₅。

Example 2 preparation of gold nanorods (Au-PDA NRs) coated with Polydopamine

Preparing gold nanorods: HAuCl₄·3H₂O (0.25mL, 0.01mol/L) was added to a CTAB (9.75mL, 0.1mol/L) solution,slowly stirring, and adding freshly prepared, frozen stock NaBH₄The solution (0.6mL, 0.01mol/L) is rapidly added into the mixed solution, stirred for reaction for 2min, and placed at normal temperature for at least 2h to obtain a seed solution. Adding HAuCl₄·3H₂O(0.5mL，0.01mol/L)，Ag NO₃(0.1mL, 0.01mol/L) was mixed well with CTAB (10mL, 0.1mol/L), ascorbic acid (0.08mL, 0.1mol/L) was added and the solution changed from yellow to colorless. And then adding 0.2mL of HCl (1.0mol/L) to adjust the pH of the system to 1-2, finally adding 0.024mL of seed solution, slowly stirring for 10s, standing at room temperature for at least 6h, and centrifugally washing with ultrapure water for three times to obtain a gold nanorod (Au NRs) solution with the concentration of 0.2 nmol/L.

Polyethylene glycol-modified gold nanorods (Au-PEG NRs): 10mL of the prepared Au NRs solution was centrifuged at 7200r/min for 10min, and the lower precipitate was dispersed to 5mL of SH-PEG-CH₃In an aqueous solution (2mg/mL, Mw 2000), Au NRs were dispersed sufficiently with ultrasonic oscillation for 3min, and then stirred at room temperature for 16h, and centrifuged and washed three times with ultrapure water to obtain an Au-PEG NRs solution.

Polydopamine encapsulated Au-PEG NRs (Au-PDA NRs): preparing dopamine-Tris-HCl buffer solution (0.1mg/mL, pH 8.5) with dopamine concentration, centrifuging the prepared Au-PEG NRs solution, dispersing the Au-PEG NRs solution into 5mL of dopamine-Tris-HCl buffer solution, ultrasonically oscillating for 3min, and stirring at room temperature for reaction for 30min to enable dopamine to be wrapped on the surface of Au-PEG NRs in a self-polymerization manner. Centrifuging and washing the polydopamine-coated Au-PEG NRs solution for 3 times to obtain Au-PDA_0.1NRs。

It should be noted that, by changing the dopamine-Tris-HCl buffer solution to 0.25mg/mL and 0.5mg/mL in example 2, Au-PEG NRs with different polydopamine wrapping thicknesses can be obtained, which are respectively marked as Au-PDA_0.25NRs and Au-PDA_0.5NRs。

Example 3 preparation of gold nanorod-doped chitosan/dopamine-modified sodium alginate thermo-sensitive hydrogel (CGP/Alg-DA @ Au-PDA)

Gold nanorods (Au-PDA) coated with the prepared polydopamine_0.25NRs) is centrifugally washed, and is directly dispersed into Alg-DA solution, ultrasonic treatment is carried out for 3min, so that the composite gold nanorods can be uniformly distributedDispersed in Alg-DA solution, counted as Alg-DA @ Au-PDA_0.25. Accurately transferring 3mL of CS solution, adding 0.5mL of beta-GP solution at 4 ℃, uniformly stirring, and adding a certain amount of Alg-DA @ Au-PDA_0.25The solution is stirred and reacted for 30min in ice-water bath. Placing in a constant temperature water bath at 37 deg.C, mixing the solutions to form a crosslinked composite gel, recording the time of gel formation, and calculating the formed gel as CGP/Alg-DA @ Au-PDA_0.25And (4) compounding the gel.

Claims

1. A preparation method of injectable hydrogel with photo-thermal performance is characterized in that dopamine modified sodium alginate is compounded with chitosan and beta-sodium glycerophosphate to prepare chitosan-sodium glycerophosphate/alginic acid-dopamine gel; and doping gold nanorods wrapped by polydopamine in the gel, and uniformly and stably dispersing the gold nanorods in the gel by utilizing the interaction between the dopamine to prepare the injectable temperature-sensitive hydrogel with photo-thermal performance.

2. The preparation method of claim 1, wherein the molar ratio of sodium alginate to dopamine in the dopamine-grafted modified sodium alginate is 1: 1-1: 5, the grafting ratio of dopamine is controlled to be 9% -50%, and the structural formula of the dopamine-modified sodium alginate is as follows:

can be expressed as: alginic acid-dopamine, abbreviated as: Alg-DA.

3. The preparation method of claim 1, wherein in the composite gel of alginic acid-dopamine, chitosan and sodium beta-glycerophosphate, the mass ratio of chitosan to sodium beta-glycerophosphate is firstly regulated to 1: 5-1: 10, so that the gelation transition temperature of the gel of chitosan and sodium beta-glycerophosphate is controlled at 37 ℃; and then adding alginic acid-dopamine with different masses according to the mass ratio of chitosan to alginic acid-dopamine of 10: 0-5: 5, so that the gelation time of the composite gel at 37 ℃ is controlled within 30min, wherein the composite gel is represented as: chitosan-sodium glycerophosphate/alginic acid-dopamine, abbreviated CGP/Alg-DA.

4. The method according to claim 1, wherein the gold nanorods coated with polydopamine are prepared by first preparing gold nanorods with an absorption peak at 830nm, expressed as AuNRs; modifying the gold nanorods by using sulfhydryl polyethylene glycol so as to improve the dispersion stability and biocompatibility of the gold nanorods in water; and finally, placing the gold nanorods in an alkaline buffer solution of dopamine, and depositing polydopamine on the surfaces of the gold nanorods by utilizing the self-polymerization reaction of the dopamine in an alkaline environment to obtain the polydopamine-coated gold nanorods, which are expressed as Au-PDANRs.

5. The method according to claim 4, wherein the concentration of the alkaline buffer solution of dopamine is controlled to be 0.1-0.5 mg/mL, the pH is 8.5, and the thickness of polydopamine deposited on the AuNRs surface is controlled to be 5.0-9.0 nm.

6. The preparation method of claim 1, wherein the injectable hydrogel with photo-thermal properties is prepared by mixing and doping gold nanorod solution wrapped with polydopamine with concentration of 0.4nmol/L and chitosan-sodium glycerophosphate/alginic acid-dopamine, and the hydrogel is expressed as: chitosan-sodium glycerophosphate/alginic acid-dopamine @ gold nanorods, abbreviated as: CGP/Alg-DA @ Au-PDA.

7. The preparation method according to claim 6, wherein the dopamine on the surface of the gold nanorods and the dopamine in alginic acid-dopamine interact with each other, so that the gold nanorods coated with dopamine can be uniformly dispersed in the gel.

8. The method according to claim 1, wherein the injectable hydrogel is prepared so that the gold nanorods can be fixed on a tumor site and have photothermal conversion ability; after the gel is irradiated by laser, the temperature can be raised to 64 +/-2.2 ℃; after multiple times of irradiation, the temperature of the gel can be raised to 60 +/-2.5 ℃ for multiple times, and the purposes of one-time injection and multiple photo-thermal treatment can be realized.