CN105520906B - A kind of temperature sensitive self-healing hydrogel and preparation method thereof being loaded with doxorubicin hydrochloride - Google Patents

Abstract

The invention relates to a thermosensitive self-healing hydrogel loaded with doxorubicin hydrochloride and a preparation method thereof, which comprises chitosan, sodium β-glycerophosphate, and polyethylene glycol with benzaldehyde groups at both ends . The present invention utilizes the amino groups in the chitosan structure to react with the benzaldehyde groups at both ends of polyethylene glycol to form an imine bond with self-healing properties, which is combined with the chitosan/β-sodium glycerophosphate thermosensitive gel, through physical packaging Combined with doxorubicin hydrochloride loaded therein, a new temperature-sensitive self-healing hydrogel drug delivery system was prepared. The drug delivery system is liquid at room temperature, and can gel rapidly at body temperature after injection into the tumor site to form a drug depot. Compared with traditional in-situ gels, the temperature-sensitive self-healing gel has stronger mechanical Intensity, and under the action of external force or tissue damage, it can achieve rapid self-repair, thereby avoiding sudden release of drugs, further delaying drug release, in order to prolong the residence time of drugs in tumor sites, enhance drug efficacy, and reduce side effects.

Description

A temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride and its preparation method

technical field

The invention relates to a temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride and a preparation method thereof, belonging to the field of pharmaceutical preparations.

Background technique

At present, the main methods of tumor treatment are surgical resection, radiotherapy and chemotherapy. In the process of treating malignant tumors, the first consideration of most patients is to remove as much tumor tissue as possible through surgery, but the indications for surgery are limited to early-stage tumors. Adjuvant preoperative radiotherapy or chemotherapy is needed, and some patients also need postoperative radiotherapy and chemotherapy for consolidative treatment. However, during the course of radiotherapy and chemotherapy, the residual tumor cells may cause tumor recurrence or metastasis due to the administration of systemic chemotherapy or local radiotherapy. Therefore, the administration method of intratumoral injection can achieve relative improvement of the anticancer activity of antitumor drugs and effectively prevent recurrence and metastasis of cancer. This way of administration can reduce the drug concentration in the system, increase the drug concentration at the tumor site, and improve the anti-tumor effect on the basis of reducing side effects.

In-situ gel, also known as in-body gel, is in liquid state at room temperature. After being injected into the medication site, affected by the physiological conditions of the body or other environmental factors, it undergoes a phase transition to form a semi-solid gel state preparation. At present, the most researched is the chitosan/β-glycerophosphate (β-glycerophosphate) temperature-sensitive in-situ gel system. The mixture of chitosan and β-glycerophosphate is liquid at room temperature. , undergoes a phase transition into a semi-solid gel. Chitosan thermosensitive gel has the advantages of non-toxicity, good biocompatibility and biodegradability. After the local injection of the gel system, a drug reservoir can be formed at the site of application, which is beneficial to the slow release of the drug, prolongs the time for the drug to remain in the injection site, and enhances the efficacy of the drug. However, the existing thermosensitive gel drug delivery system for intratumoral injection still has problems such as insufficient strength and sudden drug release.

Self-healing hydrogels, invented by bioengineers at the University of California, San Diego, can allow hydrogel material scaffolds or drug delivery systems implanted in the human body to heal automatically after repeated trauma. It has been reported in the literature that a hydrogel with self-healing ability has been prepared by using chitosan as the main raw material through simple material synthesis. The current self-healing gel is mainly in solid form, which can only be applied through surgical implantation, and the operation is complicated. The present invention synthesizes a polyethylene glycol (diabenzaldehyde-terminated telechelic polyethylene glycol, DF-PEG) with benzaldehyde groups at both ends, and utilizes the amino groups in the chitosan structure to carry out esterification with the benzaldehyde groups at both ends of the polyethylene glycol. Chemical reaction to form imine bonds with self-healing properties, combined with traditional chitosan/β-sodium glycerophosphate thermosensitive gel to prepare a new type of injectable thermosensitive self-healing hydrogel system. When the system is used for treatment, it can avoid trauma, is easier to operate, and is easier to be accepted by patients; at the same time, it improves the disadvantages of weak strength and easy burst release of traditional thermosensitive gels.

Doxorubicin Hydrochloride (DOX), also known as doxorubicin, its mechanism of action is mainly that DOX molecules intercalate into DNA and inhibit the synthesis of nucleic acids. , strong activity and good curative effect, etc., but the serious side effects of doxorubicin hydrochloride greatly limit its application in chemotherapy. The present invention cleverly combines self-healing properties with a temperature-sensitive gel system, uses doxorubicin hydrochloride as a drug model, loads doxorubicin hydrochloride in the gel solution through simple physical mixing, and directly injects it into the tumor site without passing through Surgical implantation is simple and convenient, and the broken hydrogel during the injection process can quickly realize self-healing, "fix" the drug at the expected position, and then release it slowly to avoid sudden release of the drug and achieve the purpose of treatment.

Contents of the invention

The purpose of the present invention is to provide a practical, convenient, stable and reliable doxorubicin hydrochloride temperature-sensitive self-healing hydrogel for treating tumors and its preparation method, aiming to overcome the weak mechanical strength and drug breakthrough of traditional temperature-sensitive gels. Insufficient release.

To achieve the above object, the present invention provides the following technical solutions:

A temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride and its preparation method, characterized in that the gel is composed of chitosan, β-sodium glycerophosphate and DF-PEG, and loaded with doxorubicin hydrochloride through physical mixing white.

The doxorubicin hydrochloride temperature-sensitive self-healing hydrogel intratumoral injection preparation provided by the invention, wherein DF-PEG is synthesized by esterification reaction of PEG ₂₀₀₀ and p-formylbenzoic acid, and the dosage is 1.0-2.0 mg/mL.

The doxorubicin hydrochloride temperature-sensitive self-healing hydrogel intratumoral injection preparation provided by the invention, wherein the chitosan solution is chitosan glacial acetic acid aqueous solution, and the dosage is 10.0-15.0 mg/mL.

In the doxorubicin hydrochloride temperature-sensitive self-healing hydrogel intratumoral injection preparation provided by the invention, the dosage of β-glycerophosphate sodium is 10.0-20.0 mg/mL.

The preparation method of the doxorubicin hydrochloride thermosensitive self-healing hydrogel intratumoral injection preparation mainly includes the following steps:

(1) Weigh a certain amount of PEG ₂₀₀₀ and p-formylbenzoic acid in 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1Ethyl3(3dimethylaminopropyl)carbodiie hydrochlide, EDC .HCL) and 4-dimethylaminopyridine (4-dimethylaminopyridine, DMAP) under reaction conditions for 18 hours, the product was precipitated with ether and then suction filtered, and the operation was repeated three times to obtain a white solid; dialyzed with a dialysis bag (MWCO: 8000-14000) Then get DF-PEG.

(2) Dissolving the DF-PEG obtained in step (1) in deionized water, ultrasonication, and preparing an aqueous solution of DF-PEG with a certain concentration.

(3) A certain amount of chitosan was weighed, dissolved in glacial acetic acid solution, prepared into a certain concentration of chitosan in glacial acetic acid aqueous solution, and stored at 4° C. for later use.

(4) Weighing a certain amount of sodium β-glycerophosphate and dissolving it in deionized water to prepare a certain concentration of sodium β-glycerophosphate aqueous solution.

(5) Weigh a certain amount of doxorubicin hydrochloride in the chitosan solution, stir to dissolve it completely, add DF-PEG solution dropwise to the chitosan solution, stir, and add β-PEG dropwise under ice bath conditions. Sodium glycerophosphate solution, continue to stir to make it evenly mixed, and obtain the preparation of the present invention.

The preparation of the present invention is a liquid at room temperature, and after being injected into the tumor site, it can quickly gel to form a semi-solid gel at a physiological temperature, and slowly release the drug as a drug storage. Compared with the traditional temperature-sensitive in-situ gel, The temperature-sensitive self-healing gel has strong mechanical strength, and under the action of external force or tissue damage, the self-healing gel can realize rapid self-healing, thereby avoiding drug burst release and further delaying drug release. The present invention is expected to be used as a new type of intratumoral injection drug delivery system, which achieves the purpose of treatment through slow drug release, is easy to operate, reduces drug toxicity, and solves the shortcomings of traditional intravenous and implant drug delivery methods.

Description of drawings

Accompanying drawing 1: (a) infrared spectrum and (b) nuclear magnetic spectrum of the DF-PEG obtained in specific example 1.

Accompanying drawing 2: The in vitro gelation diagram of the temperature-sensitive self-healing hydrogel preparation obtained in specific example 1.

Accompanying drawing 3: The investigation of the self-healing performance of the temperature-sensitive self-healing hydrogel obtained in specific example 1.

Accompanying drawing 4: The in vitro drug release curve of the gel preparation obtained in specific example 1, wherein DOX-CG represents the chitosan/β-sodium glycerophosphate thermosensitive gel loaded with doxorubicin hydrochloride, and DOX-CGD represents the loaded doxorubicin hydrochloride Chitosan/β-sodium glycerophosphate/DF-PEG thermosensitive self-healing hydrogel.

Detailed ways

The present invention will be further explained and illustrated below in conjunction with specific examples. It should be understood that the given examples are only illustrative and do not limit the scope of the present invention in any way.

Specific embodiment 1

A certain amount of PEG ₂₀₀₀ , p-formylbenzoic acid and DMAP was weighed, dissolved in 50 mL of dichloromethane, an appropriate amount of EDC.HCL was added, stirred at room temperature for 18 hours, and a white solid was obtained by suction filtration. The solid obtained from the reaction was redissolved in dichloromethane, and precipitated with glacial ether, and the operation was repeated three times. The resulting solid was dialyzed in a dialysis bag for two days, and DF-PEG was obtained after freeze-drying. NMR and IR spectra are shown in Figure 1.

Dissolve chitosan (0.04g) in 2mL of glacial acetic acid solution (0.1M), add doxorubicin hydrochloride powder to dissolve, add DF-PEG solution drop by drop under stirring condition, add β -Sodium glycerophosphate solution, continue to stir to make it evenly mixed, and obtain the temperature-sensitive self-healing hydrogel preparation of doxorubicin hydrochloride. The preparation is in a liquid state at room temperature, has good needle penetration, and can gel in 3 minutes in a water bath at 37°C. See Figure 2 for the in vitro gelation test.

Specific embodiment 2

The self-healing performance of the temperature-sensitive self-healing hydrogel obtained in Example 1 of the present invention was investigated, see Figure 3.

Preliminary investigation of self-healing ability: the prepared thermosensitive self-healing hydrogel was cut in half from the middle, and one half was stained with methylene blue. Put the cut gels back together to see if the two gels can fully self-heal and return to the original intact gel. Repeat the operation to see if the gel can achieve repeated self-healing.

Further investigation of self-healing ability: take appropriate amount of chitosan/β-sodium glycerophosphate (CS/β-GP) solution and chitosan/β-sodium glycerophosphate/DF-PEG (CS/β-GP/DF) respectively -PEG) solution in a 2.5mL syringe, placed in a 37±1°C water bath until completely gelled. Take out the syringe, and use a 0.5mm needle to completely inject the gel into the vial until the gel system is completely destroyed. Stand still for a period of time, observe the gel form in the vial.

Specific embodiment 3

The thermosensitive self-healing hydrogel obtained in Example 1 of the present invention was subjected to an in vitro drug release behavior test, and the release curve is shown in Figure 4.

Take 1 mL of CS/β-GP solution loaded with doxorubicin hydrochloride and 3 parts of CS/β-GP/DF-PEG solution in a dialysis bag, seal it, and let it gel completely at 37±1°C for a period of time , add 100mL pH 6.8 phosphate buffer solution, and shake at 100rpm constant temperature. Sampling at the set time point, each sampling is 2ml, at the same time add the same amount of fresh phosphate buffer solution to maintain the effect of the sink, the sampling time is 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, Samples were then taken every two days. The sample taken out was filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was taken for injection, and the amount of doxorubicin hydrochloride was measured at a wavelength of 490 nm, and the cumulative release amount was calculated.

Claims (3)

1. A temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride is characterized in that: polyethylene glycol with benzaldehyde groups modified at both ends by chitosan, β-sodium glycerophosphate, and by physical mixing The loading consisted of doxorubicin hydrochloride. 2. The temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride according to claim 1, characterized in that: the polyethylene glycol modified with benzaldehyde groups at both ends is made of PEG2000 and p-formylbenzene Formic acid is synthesized by esterification. 3. The preparation method of the temperature-sensitive self-healing hydrogel loaded with doxorubicin hydrochloride as claimed in claim 1, characterized in that: comprising the following steps: Step 1, PEG2000 and p-formylbenzoic acid were reacted for 18h under the conditions of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine, and the product was diethyl ether Suction filtration after precipitation, repeat the operation three times, dialyze the obtained white solid with a dialysis bag to obtain polyethylene glycol modified with benzaldehyde groups at both ends; Step 2, dissolving polyethylene glycol modified with benzaldehyde groups at both ends in deionized water, and ultrasonicating to obtain an aqueous solution of polyethylene glycol modified with benzaldehyde groups at both ends, with a concentration of 1.0-2.0 mg/mL; Step 3, dissolving chitosan in glacial acetic acid aqueous solution to obtain chitosan glacial acetic acid aqueous solution with a concentration of 10.0-15.0 mg/mL, and storing at 4°C for later use; Step 4, dissolving sodium β-glycerophosphate in deionized water to obtain an aqueous solution of sodium β-glycerophosphate with a concentration of 10.0-20.0 mg/mL; Step 5, dissolving doxorubicin hydrochloride in glacial acetic acid aqueous solution of chitosan, adding dropwise polyethylene glycol aqueous solution modified with benzaldehyde groups at both ends, stirring, adding dropwise β-sodium glycerophosphate aqueous solution under ice bath conditions, Continue to stir to make it evenly mixed.