CN104940138A - Preparation method of stimulation-sensitive hyaluronic acid in-situ gel - Google Patents

Abstract

The invention discloses a temperature-stimulus-sensitive hyaluronic acid in-situ gel and a preparation method thereof, the carrier of which is two biodegradable polymers of hyaluronic acid-poloxamer and chitosan-poloxamer Polymers, drugs are physically entrapped into the gel. The gel has the characteristics of a temperature-sensitive reverse gel. When the temperature is lower than body temperature, it exists in the form of a nanogel liquid and can be injected into the body through a syringe; when the temperature in the body reaches body temperature, it transforms from a nanosuspension It is a solid or semi-solid gel to maintain slow drug release. The gel of the invention can carry insoluble drugs, has good biocompatibility, and is safe and nontoxic.

Description

A preparation method of stimulation-sensitive hyaluronic acid in situ gel

technical field

The invention belongs to the technical field of medicine, and is specifically designed for temperature-stimulation-sensitive hyaluronic acid in-situ gel for local injection into a joint cavity and a preparation method thereof.

Background technique

Intra-articular injection can directly act on the target site of the drug, increase the local concentration, avoid the physiological transport barrier in the body, change the distribution mode of the drug, exert the drug effect in a small dose, and improve the gastrointestinal adverse reactions that are easy to occur when oral drugs are taken for a long time , so it is widely used in the treatment of arthritis. Patents CN100594029, CN101112378 and CN101940587B all disclose methods for treating arthritis by intra-articular injection. This preparation has many advantages: slow-release and long-acting; it can reduce the clearance rate of drugs in the joint cavity; it can reduce the irritation of large doses of drugs; it can avoid infection after repeated administration; Toxic and side effects etc. This type of preparation is injected once every few days or even several months, which significantly enhances the effectiveness and safety of the drug and improves patient compliance.

A temperature-sensitive in situ gel is a gel that undergoes a phase transition depending on temperature. It is a free-flowing liquid under storage conditions. After being injected into the human body, it can fill the tissue space, undergo phase transition rapidly, and form a semi-solid gel at the injection site to achieve local administration or delay drug release. It has the advantages of injectability, less trauma, convenient administration, controlled drug release, etc., and is suitable for local injection in vivo. Poloxamer is the most well-studied polymer material for preparing temperature-sensitive in situ gels. Most of the main matrix materials of thermosensitive gels reported in current patents and literature are Poloxamer 407 (trade name: poloxamer 407). ), such as CN1230108A, CN101185650A, CN1593386A, CN1377706A, CN100422268C, CN02109503.5, etc. Among them, the F127 aqueous solution with a concentration of 20%-30% has the property of reverse gelation when heated, that is, it is a free-flowing liquid at refrigerated temperature, and forms a clear gel at room temperature or body temperature. Although poloxamers have good biocompatibility, safety, and non-irritating allergies, they are costly and have a wide temperature regulation range. Patents such as CN101327183A and CN102125516A disclose the preparation method of chitosan temperature-sensitive in-situ gel medicament. temperature sensitivity.

Intra-articular injection of hyaluronic acid is a common method for the treatment of arthritis. At present, hyaluronic acid intra-articular injections commonly used in clinic include Hyalgan, Artz, NRD-101, Hylan G-F20, etc. Among them, Hylan G-F20 is a hyaluronic acid gel injection produced by Genzyme Company of the United States. Patents CN02822420.5, US2006003964 and CN101112381A all disclose methods for intra-articular administration of hyaluronic acid to treat arthritis. However, only a single hyaluronic acid ordinary gel injection still has certain defects, such as a short residence time in the body and is easily degraded by HA enzyme; it is difficult to use after forming a gel in vitro; it does not contain therapeutic drugs and has no effect Slower wait. Therefore, it is particularly important to modify the structure of hyaluronic acid to prepare drug-loaded sustained-release preparations. Literature and other studies have also reported that the combined treatment of hyaluronic acid and NSAIDs can rapidly improve the clinical symptoms of arthritis patients, and can significantly reduce the dosage of NSAIDs while maintaining good curative effect. The gel prepared by the hyaluronic acid-poloxamer and the chitosan-poloxamer in the present invention can not only better adjust the sensitive temperature, but also can increase the hydrophobic area of the gel and increase the drug loading capacity.

Contents of the invention

The purpose of the present invention is to provide a hyaluronic acid in situ gel preparation sensitive to temperature stimulation, so as to increase the drug loading, reduce adverse drug reactions, reduce the number of administrations, and exert the effect of synergistic treatment of arthritis. The gel of the present invention is formed by physical cross-linking of two biocompatible polymers loaded with drugs.

Another object of the present invention is to provide a method for preparing a temperature-sensitive in-situ gel loaded with drugs.

The above-mentioned gel is composed of high molecular polymers chemically synthesized hyaluronic acid-poloxamer and chitosan-poloxamer.

Wherein, the molecular weight of hyaluronic acid is 500,000-5 million daltons, preferably 700,000-2 million daltons; the deacetylation degree of chitosan is 70%-100%, preferably 80%-95%. The molecular weight of chitosan is 100,000-3 million, preferably 250,000-900,000; the poloxamer is Pluronic F127.

The weight ratio of hyaluronic acid-poloxamer and chitosan-poloxamer in the gel is between 10:1-1:10, preferably between 3:1-1:2.

The drugs in the gel include one or more combinations of celecoxib, diclofenac, meloxicam, etodolac, ketoprofen, nabumetone, indomethacin, and rofecoxib.

The drug in the above gel can be entrapped or dispersed in the polymer. The concentration and drug loading of the polymer are not limited unless the gelation behavior of the polymer is affected so that it cannot form a gel.

The above-mentioned gel is temperature-sensitive. When the temperature is lower than body temperature, it exists in the form of nano-suspension. When the temperature of the injection site rises to body temperature, the preparation changes from nano-suspension to gel, prolonging the release time of the drug.

The present invention is achieved through the following technical scheme, linking Pluronic F-127 to the main chain of hyaluronic acid and chitosan respectively to form an amphiphilic copolymer, and the polymer solution passes through the Self-assembly entrapped drug to prepare nanogel suspension. Then, the nanogel is injected into the body to form an in situ gel at body temperature.

Description of drawings

Figure 1 shows the toxic effect of the gel on MC3T3-E1 cells.

Figure 2 is the drug release curve of the gel at different temperatures.

Detailed ways

The present invention will be described in more detail through specific examples below, but the scope of the present invention is not limited to the following examples.

Example 1

Dissolve the activated poloxamer F127 and hyaluronic acid in deionized water, add copper sulfate and sodium ascorbate, and react at 40°C for 48 hours under nitrogen protection. After the reaction, dialyze in deionized water for 3 days, and freeze-dry to obtain hyaluronic acid-poloxamer polymer.

The polymer was suspended in PBS buffer (pH 7.4), and the probe was sonicated for 6 minutes (working power 90W, working pulse was working for 1s and pausing for 5s), and a blank nanogel suspension was obtained. Dissolve rofecoxib in DMF solution, dissolve the copolymer in formamide solution, mix after stirring overnight, dialyze with PBS buffer solution (pH 7.4) for 12h, centrifuge to take supernatant, filter and freeze-dry to obtain Drug-loaded thermosensitive nanogel suspension.

Example 2

Using 4-lutidine (DMAP) as a catalyst, poloxamer and succinic anhydride were dissolved in 1,4-dioxane, a small amount of triethylamine was added dropwise, and reacted at room temperature for 24 hours. Then, dissolve it and chitosan in the buffer solution of pH 4-5, add EDC/NHS after activation, and react with stirring at room temperature for 24 hours. After the reaction was completed, it was dialyzed in distilled water for three days, and freeze-dried to obtain a white chitosan-poloxamer polymer.

Example 3

Hyaluronic acid-poloxamer and chitosan-poloxamer polymers were dissolved in PBS buffer (pH 7.4), and after stirring overnight, ultrasonic cell pulverizer probe ultrasonic 6min (working power 90W, working pulse is working 1s pause 5s), prepare a blank nanogel suspension. Its toxicity to cells was determined (Figure 1).

Example 4

Celecoxib was dissolved in DMF solution, the two polymers were dissolved in formamide solution, stirred overnight and mixed, then dialyzed with PBS buffer (pH 7.4) for 12 hours, centrifuged to obtain supernatant, filtered and lyophilized, Redissolve to prepare drug-loaded thermosensitive nanogel suspension. The in vitro drug release curves at different temperatures are shown in Fig. 2 .

Claims (10)

1. thermal stimulus responsive type hyaluronic acid situ-gel and preparation method thereof, is characterized in that, this gel is formed by the mode of physical crosslinking by the biocompatibility of bag medicine carrying thing and the high molecular polymer of biodegradability.

2. gel as claimed in claim 1, high molecular polymer is wherein made up of the hyaluronic acid-poloxamer of chemosynthesis and chitosan-poloxamer.

3. gel according to claim 2, hyaluronic molecular weight is wherein 500,000-500 ten thousand dalton, preferably 700,000-200 ten thousand dalton.

4. gel according to claim 2, the deacetylation of chitosan is wherein 70%-100%, preferred 80%-95%.The molecular weight of chitosan is 100,000-300 ten thousand, preferably 250,000-90 ten thousand.

5. gel according to claim 2, poloxamer is wherein Pluronic F127.

6. gel according to claim 1, hyaluronic acid-poloxamer wherein and the weight ratio of chitosan-poloxamer between 10:1-1:10, between preferred 3:1-1:2.

7. gel according to claim 1, wherein medicine comprises the one or more combination of celecoxib, diclofenac, meloxicam, etodolac, ketoprofen, nabumetone, indomethacin, rofecoxib.

8. gel according to claim 1, its Chinese medicine can wrap and carry or be scattered in polymer.Concentration and the drug loading of polymer are unrestricted, unless had influence on the gelling behavior of polymer, make it not form gel.

9. gel according to claim 1, has responsive to temperature character, and when temperature is lower than body temperature, exist with nanosuspension form, in injection site, temperature is increased to body temperature, and preparation is transformed into gel by nanosuspension, the release time of prolong drug.

10. the preparation method of thermal stimulus responsive type hyaluronic acid situ-gel according to claim 1, wherein Pluronic F-127 is linked on hyaluronic acid and chitosan main chain respectively, form amphipathic copolymer, temperature is lower than under the condition of 30 DEG C, polymer solution, by self assembly bag medicine carrying thing, prepares nanogel suspension.Then, nanogel is expelled in body, under temperature, forms situ-gel.