CN109734937B - Preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel - Google Patents
Preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel Download PDFInfo
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Abstract
The invention relates to a preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel, belonging to the technical field of biomedical materials. Firstly, preparing an OHPPMC solution with the mass percentage concentration of 1-3 wt%; then preparing an HA-ADA solution with the mass percentage concentration of 1-3 wt%; uniformly mixing the OHPMC solution and the HA-ADA solution in equal volume to obtain an OHPMC-HA mixed solution; and finally, adding the VA @ PLGA-CS-HA microspheres into the OHPPMC-HA mixed solution, and uniformly mixing to obtain the OHPPMC-HA microsphere. The hydrogel disclosed by the invention has good rheological property, degradation property, excellent swelling property and antibacterial property, and good biological safety, and is suitable for being used as a bone defect repair material. In addition, the method has controllable conditions, simple and effective whole process and good clinical application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel.
Background
A hydrogel is a polymer that swells in water and retains a large amount of water without dissolving. Hydrogel polymers have the property of absorbing a large amount of water, which is distributed throughout the polymer network and extends to a greater extent to the crosslinked macromolecular chains, giving the overall material a fluid character, which is very similar to body tissue filled with large amounts of aqueous fluids, with soft, moist surfaces and a greatly reduced irritation to tissue. Injectable hydrogels refer to a class of hydrogels that have some fluidity and that can be applied by injection. The injectable hydrogel has temperature sensitivity, and can be converted into biodegradable semisolid gel at body temperature after the embedded drug is injected into human body. In addition, defects or depressions in the body cavity can also be filled with injectable hydrogels. In recent years, some novel hydrogels have attracted attention and studied extensively in drug delivery applications based on their advantages of excellent pharmacokinetics, easily controllable mechanical properties, easy injection, high drug entrapment rate, and site-specific drug release.
The microsphere is a drug delivery system which wraps drug molecules in a high molecular polymer to realize long-time stable release of the drug, and has attracted extensive attention due to the characteristic of sustained and controlled release. Currently, the most widely effective antibiotic delivery system material for clinical applications is Polymethylmethacrylate (PMMA), and the commonly used antibiotic-loaded materials include gentamicin, tobramycin, vancomycin, and the like. The degradable inorganic and organic carrier materials reported at present, which have been used for sustained release of local antibiotics, are Hydroxyapatite (HAP), Calcium Phosphate Cement (CPC), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), Polycaprolactone (PCL), chitosan, collagen, alginate, etc. Chitosan (Chitosan, CS) has been demonstrated to have many excellent properties, such as: biological adhesion, no toxicity, good tissue compatibility, biodegradability, wide source and the like. Sodium Hyaluronate (HAS) is a natural biomolecule widely present in skin and other tissues, is a main component constituting connective tissues such as human intercellular substance, joint synovial fluid, and exists in a nearly pure form in joint cavities. It can promote cell proliferation and differentiation, eliminate oxygen free radical, promote wound healing, regulate osmotic pressure, lubricate, promote cell repair and improve joint. Hydroxypropyl methylcellulose (HPMC) is an important cellulose derivative, belongs to a non-ionic water-soluble polymer, is odorless, tasteless and nontoxic, and is widely used for oral matrix controlled release and sustained release preparations as a release retarding material for regulating the release of medicaments.
The invention prepares the antibacterial injectable hydrogel VA @ PLGA-CS-HA/OHPPMC-HA by integrating the advantages of HPMC, PLGA, hyaluronic acid, chitosan, vancomycin and other materials.
Disclosure of Invention
Aiming at the background technology, the invention aims to provide a preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel.
In order to achieve the above purpose of the present invention, the technical solution adopted by the present invention is as follows:
a preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel comprises the following steps:
(1) dissolving oxidized hydroxypropyl carboxymethyl cellulose (OHPMC) in distilled water to prepare an OHPMC solution with the mass percentage concentration of 1-3 wt%; dissolving aminated hyaluronic acid (HA-ADA) in distilled water to prepare an HA-ADA solution with the mass percentage concentration of 1-3 wt%; then, the OHPMC solution and the HA-ADA solution are mixed uniformly in equal volume to obtain an OHPMC-HA uniform mixed solution;
(2) and (2) adding VA @ PLGA-CS-HA microspheres into the OHPPMC-HA uniform mixed solution prepared in the step (1) according to a proportion, and uniformly mixing to form gel, thus preparing the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel.
Further, in the above technical scheme, the oxidized hydroxypropyl carboxymethyl cellulose in step (1) is prepared by oxidizing and modifying hydroxypropyl carboxymethyl cellulose (HPMC) with sodium periodate, and the oxidation modification method specifically comprises the following steps:
preparing a hydroxypropyl carboxymethyl cellulose aqueous solution with the mass percentage concentration of 8-12%, and then preparing the hydroxypropyl carboxymethyl cellulose aqueous solution according to the volume ratio VHydroxypropyl carboxymethyl cellulose solution：VSodium periodate solution5: adding a sodium periodate solution according to the proportion of 1, reacting for 24 hours in a dark place, adding ethylene glycol which is equal to the sodium periodate solution in volume into a reaction system, continuously stirring for 30-45 min, dialyzing for 2-3 d with distilled water, and freeze-drying to obtain the sodium periodate.
Preferably, in the above technical solution, the mass percentage concentration of the hydroxypropyl carboxymethyl cellulose aqueous solution is 10%, and the mass concentration of the sodium periodate solution is 100 mg/mL.
Further, in the above technical scheme, the aminated hyaluronic acid in step (1) is prepared by amination and modification of hyaluronic acid with azodicarbonamide (ADA), and the amination and modification method specifically comprises the following steps:
preparing a hyaluronic acid solution with the mass percentage concentration of 1-3%, and then mixing the hyaluronic acid solution with the mass percentage concentration of 1: 1, adding adipic acid dihydrazide, adjusting the pH value of the reaction system to 4.7-4.8 after complete dissolution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, continuously adjusting the pH value of the reaction system to 4.7-4.8 after uniform mixing, finally reacting for 12h at room temperature, dialyzing for 2-3 d with distilled water, and freeze-drying to obtain the product.
Preferably, in the above technical solution, the molar ratio of the hyaluronic acid to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1: 2.
further, in the technical scheme, the dosage ratio of the VA @ PLGA-CS-HA microspheres to the OHPPMC-HA hydrogel in the step (2) is 0.1-0.3 parts by mass: 1 part by volume, wherein: the mass part and the volume part are as follows: ml is used as a reference.
Further, in the above technical solution, the concentration of the OHPMC solution in the step (1) is preferably 2 wt%; the HA-ADA solution is preferably 2 wt% in mass percentage concentration.
Further, according to the technical scheme, the gelling time in the step (2) is preferably 3-7 min.
Further, in the technical scheme, the VA @ PLGA-CS-HA microspheres in the step (2) are prepared by the following method, and the specific steps are as follows:
(a) dissolving polylactic acid-polyglycolic acid copolymer (PLGA) in dichloromethane, and preparing a PLGA solution with the mass percentage concentration of 5-15 wt%; dissolving Chitosan (CS) in glacial acetic acid to prepare a chitosan solution with the mass percentage concentration of 0.5-1.5 wt% for later use; dissolving sodium Hyaluronate (HAS) in distilled water to prepare a sodium hyaluronate solution with the mass percentage concentration of 1-3 wt% for later use; dissolving vancomycin hydrochloride (VA) in distilled water to prepare a vancomycin solution with the mass percent concentration of 25-100 mg/ml; dissolving polyvinyl alcohol (PVA) in distilled water to prepare a chitosan solution with the mass percentage concentration of 0.3-3 wt% for later use;
(b) uniformly mixing the PLGA solution and the VA solution in the step (a) according to the proportion, and then carrying out ultrasonic dispersion to form W1an/O emulsion, wherein: the volume ratio of the PLGA solution to the VA solution is 2-10: 1;
(c) uniformly mixing the chitosan solution obtained in the step (a) with a PVA (polyvinyl alcohol) aqueous solution according to a proportion to obtain a CS/PVA solution; wherein: the volume ratio of the chitosan solution to the PVA aqueous solution is 0.5-2: 1;
(d) proportionally mixing the CS/PVA solution of step (c) with the W of step (b)1Mixing the emulsion and dispersing the mixture by ultrasonic to obtain stable W1/O/W2An emulsion, wherein: the CS/PVA solution and W1The volume ratio of the/O emulsion is 2-6: 1;
(e) taking a proper amount of the sodium hyaluronate solution obtained in the step (a), adding a PVA solution to prepare a HAS/PVA solution, wherein: the volume ratio of the sodium hyaluronate solution to the chitosan solution adopted in the step (c) is 1: 1, the HAS/PVA solution and W1/O/W2The volume ratio of the emulsion is 4-6: 1;
(f) subjecting W obtained in step (d) to1/O/W2And (e) mixing the emulsion with the HAS/PVA solution obtained in the step (e), stirring at room temperature for 0.5-2 h, heating to 45 ℃, continuously stirring for 20-40 min, adding a proper amount of sodium tripolyphosphate aqueous solution, stirring for 1-2 h, standing for 8-12 h, centrifuging, washing, collecting precipitates, and freeze-drying to obtain the VA @ PLGA-CS-HA microspheres.
Preferably, in the above technical solution, the concentration of the PLGA solution in step (a) is preferably 10 wt%; the mass percentage concentration of the chitosan solution is preferably 1 wt%; the mass percent concentration of the sodium hyaluronate solution is preferably 1.5 wt%.
Preferably, in the above technical solution, the volume ratio of the PLGA solution to the VA solution in step (b) is preferably 5: 1.
preferably, in the above technical solution, the volume ratio of the chitosan solution to the PVA aqueous solution in step (c) is preferably 1: 1, the PVA aqueous solution preferably has a mass percent concentration of 3 wt%.
Preferably, the above technical solution, the CS/PVA solution of step (d) and W1The volume ratio of the/O emulsion is preferably 4: 1.
preferably, the step (e) of mixing the HAS/PVA solution with W is performed according to the above technical scheme1/O/W2The volume ratio of the emulsion is 5: 1, the concentration of the added PVA solution is preferably 0.4 wt%.
Preferably, in the above technical solution, the mass percentage concentration of the aqueous solution of sodium tripolyphosphate in the step (f) is preferably 5 wt%, and the volume ratio of the aqueous solution of sodium tripolyphosphate to the chitosan solution adopted in the step (c) is preferably 1: 1.
compared with the prior art, the preparation method of the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel HAs the following beneficial effects:
(1) the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel prepared by the invention HAs good mechanical property, rheological property, degradation property, excellent swelling property and antibacterial property, can inhibit the growth of bacteria for 56 days, and is suitable for being used as a bone defect repair material.
(2) The raw materials adopted by the method have good biological safety, the safety risk of clinical application is greatly reduced, the preparation method does not relate to organic reagents or organic chemical reactions, the reaction system is mild, the conditions are controllable, the problem of residues of toxic cross-linking agents or auxiliary agents and the like is solved, the whole process is simple and effective, and the method has good clinical application prospect.
Drawings
FIG. 1 is a graph showing the results of rheological tests on OHPMC-HA hydrogels prepared in examples 1 to 9;
FIG. 2 is a graph showing a comparison of swelling kinetics curves of xerogels of OHPMC-HA hydrogels prepared in examples 1 to 9 in PBS buffer solution;
FIG. 3 is a VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel prepared in examples 10-12;
FIG. 4 is a graph comparing the swelling kinetics curves of the VA @ PLGA-CS-HA/OHPPMC-HA hydrogels prepared in examples 10 to 12 in PBS buffer solution;
FIG. 5 is a comparison graph of the results of the antibacterial property tests of the VA @ PLGA-CS-HA/OHPPMC-HA hydrogel prepared in examples 10 to 12;
wherein: in fig. 1 and 2, Group1 represents example 1, Group2 represents example 2, and so on, and Group9 represents example 9.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. The present invention is implemented on the premise of the technology of the present invention, and the detailed embodiments and specific procedures are given to illustrate the inventive aspects of the present invention, but the scope of the present invention is not limited to the following embodiments.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". Accordingly, unless expressly indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Example 1
The preparation method of the OHPMC-HA injectable hydrogel of the present example comprises the following steps:
(1) preparation of oxidized hydroxypropyl carboxymethyl cellulose (OHPPMC)
Dissolving hydroxypropyl carboxymethyl cellulose in distilled water to prepare 10 percent hydroxypropyl carboxymethyl cellulose solution according to the mass percentage concentration, and then preparing the solution according to the volume ratio VHydroxypropyl carboxymethyl cellulose solution：VSodium periodate solution5: 1 adding 100mg/mL sodium periodate solution, reacting for 24h in a dark place, adding ethylene glycol with the same volume as the sodium periodate solution, continuing stirring for 30-45 min, dialyzing with distilled water for 2-3 d (molecular weight cut-off: 500), and freeze-drying at 50 ℃.
(2) Preparation of aminated hyaluronic acid (HA-ADA)
Dissolving sodium hyaluronate in distilled water to prepare a hyaluronic acid solution with the mass percentage concentration of 2%, and then mixing the hyaluronic acid solution with the mass percentage concentration of 1: 1, adding adipic acid dihydrazide, after the two are completely dissolved, adjusting the pH of a reaction system to 4.7-4.8 by using 0.1M HCl, and then mixing the two solutions according to a molar ratio of 1: 2, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, adjusting the pH value to 4.7-4.8, reacting for 12 hours at room temperature, dialyzing for 2-3 d (molecular weight cut-off: 500) with distilled water, and freeze-drying at 50 ℃ to obtain the product.
(3) Preparation of oxidized hydroxypropyl carboxymethyl cellulose-aminated hyaluronic acid hydrogel (OHPMC-HA)
Dissolving the oxidized hydroxypropyl carboxymethyl cellulose prepared in the step (1) in distilled water to prepare an oxidized hydroxypropyl carboxymethyl cellulose solution with the mass percentage concentration of 1%; dissolving the aminated hyaluronic acid prepared in the step (2) in distilled water to prepare an aminated hyaluronic acid solution with the mass percentage concentration of 1%; mixing the two solutions in equal volume, whirling for 10s, and gelling.
Example 2
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 1 percent, and the mass percent concentration of the aminated hyaluronic acid solution is 1.5 percent.
Example 3
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 1 percent, and the mass percent concentration of the aminated hyaluronic acid solution is 2 percent.
Example 4
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 1.5%, and the mass percent concentration of the aminated hyaluronic acid solution is 1%.
Example 5
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 1.5%, and the mass percent concentration of the aminated hyaluronic acid solution is 1.5%.
Example 6
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 1.5%, and the mass percent concentration of the aminated hyaluronic acid solution is 2%.
Example 7
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 2%, and the mass percent concentration of the aminated hyaluronic acid solution is 1%.
Example 8
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 2%, and the mass percent concentration of the aminated hyaluronic acid solution is 1.5%.
Example 9
The preparation method of the OHPMC-HA injectable hydrogel of this example is substantially the same as that of example 1, except that: in the step (3), the mass percent concentration of the oxidized hydroxypropyl carboxymethyl cellulose solution is 2%, and the mass percent concentration of the aminated hyaluronic acid solution is 2%.
Analytical testing was as follows:
1. gel formation time test for OHPPMC-HA injectable hydrogels
The mixture obtained by vortex mixing in the examples 1 to 9 was respectively sucked by a syringe and injected into a glass vial. The mixture was observed by pouring the vial, the gel was obtained when the solution no longer flowed, the time was recorded and each set was repeated 3 times, and the gel time for each example is shown in table 1.
TABLE 1 comparative gel formation time tables for OHPPMC-HA hydrogels of examples 1-9
As can be seen from Table 1, the gel forming time of the injectable hydrogel is most suitable for 3-7 min (too fast gel forming time is not favorable for the dispersion of the subsequently added VA @ PLGA-CS-HA microspheres, and too long gel forming time can cause the later added VA @ PLGA-CS-HA microspheres to deposit). The gel forming time of all the groups except 3 groups of the example 1, the example 2 and the example 3 in the 9 groups of samples meets the requirement of 3-7 min.
2. Rheological Performance testing of OHPPMC-HA injectable hydrogels
The rheology of the OHPMC-HA hydrogel prepared in examples 1 to 9 was analyzed using a rheometer. The prepared OHPMC-HA hydrogel (20.0mm diameter and 1.0mm thickness) of different composition ratios was placed for 1h and then placed on the lower plate of a 37 ℃ rheometer. The angular frequency is swept from 1 to 150rad/s at 1% strain. The results of rheological testing of the OHPMC-HA hydrogel of each example are shown in figure 1.
The basic mechanical properties of the hydrogel of the system at 37 ℃ are analyzed by a rotational rheometer. The strength of the hydrogel can be judged by the size of the storage modulus G ', and it can be seen that the storage modulus G' of the 9 groups of samples is the largest, while the storage modulus G 'and the loss modulus G' of the 3 groups of samples are relatively close, so that the 3 groups of hydrogels can be judged to be in a viscous state, have poor mechanical properties and do not meet the requirements of being injectable hydrogels.
3. Swelling performance testing of OHPPMC-HA injectable hydrogels
The mass of the gel sample after freeze drying (5 mm. times.5 mm) was accurately weighed as Wd, and then placed in a 15m L centrifuge tube, soaked with 10mL of PBS buffer (pH 7.4), and finally placed in a constant temperature shaker at 37 ℃ and continuously shaken at 150 r/min. Taking out samples at regular intervals, carefully wiping the surface moisture of the samples with filter paper, and measuring the mass of the samples at this time to be recorded as Ws, wherein the swelling ratio of the gel can be calculated by the following formula: q ═ Ws/Wd (3 replicates per sample were used in a panel).
The swelling kinetics of the hydrogels were obtained by soaking the dried gels after freeze-drying of each set of hydrogels in 37 ℃ PBS buffer (pH 7.4) to swelling equilibrium, with different time intervals chosen to monitor the change in gel weight after water absorption. It can be seen from FIG. 2 that the hydrogels of examples 4-9 all reached swelling equilibrium rapidly, and reached equilibrium approximately at 120min, with the swelling ratio of example 9 being a minimum of 3.66. The hydrogels of examples 1-3 showed substantial collapse after 120min due to too high water absorption, which is consistent with the results of the rheological test.
From the above 3 test results, it can be seen that the hydrogel in example 9 HAs a suitable gel forming time, a better mechanical strength and a smaller swelling ratio, and the hydrogel in group9 HAs better comprehensive performance, so that in the preparation of the following examples 10 to 12VA @ PLGA-CS-HA/OHPMC-HA antibacterial hydrogel, the concentrations and ratios of OHPMC and HA-ADA are the same as the raw material ratios used for preparing the hydrogel in example 9.
Example 10
The preparation method of the VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel of this embodiment comprises the following steps:
(1) dissolving oxidized hydroxypropyl carboxymethyl cellulose (OHPMC) in distilled water to prepare an OHPMC solution with the mass percentage concentration of 2 wt%; dissolving aminated hyaluronic acid (HA-ADA) in distilled water to prepare an HA-ADA solution with the mass percentage concentration of 2 wt%; then, the OHPMC solution and the HA-ADA solution are mixed uniformly in equal volume to obtain an OHPMC-HA uniform mixed solution;
(2) according to the dosage ratio of VA @ PLGA-CS-HA microspheres to OHPPMC-HA hydrogel of 0.1 g: adding VA @ PLGA-CS-HA microspheres into the OHPPMC-HA uniform mixed solution prepared in the step (1) in a proportion of 1mL, and uniformly mixing to prepare the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel named VA @ PLGA-CS-HA/OHPPMC-HA 01;
wherein:
the VA @ PLGA-CS-HA microspheres prepared in the step (2) are prepared by the following method, and the method comprises the following steps:
(a) dissolving PLGA in dichloromethane to prepare PLGA solution with the mass percentage concentration of 10 wt% for later use; dissolving chitosan in glacial acetic acid with the mass percentage concentration of 2 wt% to prepare a chitosan solution with the mass percentage concentration of 1 wt% for later use; dissolving sodium hyaluronate in distilled water to prepare a sodium hyaluronate solution with the mass percentage concentration of 1.5 wt% for later use; dissolving vancomycin hydrochloride (VA) in distilled water to prepare a vancomycin solution with the mass percentage concentration of 50mg/ml for later use; dissolving polyvinyl alcohol (PVA) in distilled water to prepare a PVA solution with the mass percentage concentration of 3 wt% for later use; dissolving sodium tripolyphosphate in distilled water to prepare a sodium tripolyphosphate solution with the mass percentage concentration of 5 wt% for later use;
(b) in volume ratio VPLGA：VVA5: 1, mixing PLGA solution and VA solution, vortex mixing for 5min, and then carrying out ultrasonic treatment for 5min to form W1an/O emulsion;
(c) in volume ratio VCS：VPVA1: 1, uniformly mixing a chitosan solution and a PVA solution to obtain a CS/PVA solution;
(d) in volume ratio VCS/PVA：VW1/O4: 1, mixing the CS/PVA solution with W1Mixing the emulsion/O, carrying out vortex mixing for 5min, carrying out ultrasonic treatment for 5min, and repeating for 2-3 times to obtain stable W1/O/W2An emulsion;
(e) measuring a sodium hyaluronate solution with the volume equivalent to that of the chitosan solution used in the step (3), and then adding a PVA solution with the mass percentage concentration of 0.4 wt% to prepare a HAS/PVA solution, wherein the total volume of the HAS/PVA solution is W1/O/W 25 times the volume of the emulsion;
(f) w obtained in the step (4)1/O/W2And (e) mixing the emulsion with the HAS/PVA solution obtained in the step (e), mechanically stirring at room temperature at 800rpm for 1h, heating to 45 ℃ at 1000rpm, stirring for 30min, adding a sodium tripolyphosphate solution with the volume equivalent to that of the chitosan solution used in the step (3) at 800rpm, continuously stirring for 1.5h, standing overnight (12h), centrifuging at 8000rpm for 5min, washing with distilled water for dispersion, centrifuging at 8000rpm, collecting precipitates, and freeze-drying to obtain the VA @ PLGA/CS/HA-50 composite microspheres.
Example 11
The preparation method of the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel of the embodiment is basically the same as that of the embodiment 1, and the difference is only that: in this example, the dosage ratio of VA @ PLGA-CS-HA microspheres to OHPPMC-HA hydrogel in step (2) is 0.2 g: 1mL, designated VA @ PLGA-CS-HA/OHPPMC-HA 02.
Example 12
The preparation method of the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel of the embodiment is basically the same as that of the embodiment 1, and the difference is only that: in this example, the dosage ratio of VA @ PLGA-CS-HA microspheres to OHPPMC-HA hydrogel in step (2) is 0.3 g: 1mL, designated VA @ PLGA-CS-HA/OHPPMC-HA 03.
Performance testing and characterization
1. The gel forming time of the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel of the example 10-12 is tested, and the test results are shown in Table 2.
TABLE 2 comparison table of gelation time of VA @ PLGA-CS-HA/OHPPMC-HA hydrogels of examples 10-12
The gel forming time of the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel containing different VA @ PLGA-CS-HA microspheres is shown in Table 2, and it can be seen that the addition of the VA @ PLGA-CS-HA microspheres HAs a slight influence on the gel forming time of the hydrogel, and the gel forming time is increased with the increase of the amount of the microspheres contained in the hydrogel, but the overall influence is small.
2. Rheological property test of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel
The products VA @ PLGA-CS-HA/OHPPMC-HA 01, VA @ PLGA-CS-HA/OHPPMC-HA 02 and VA @ PLGA-CS-HA/OHPPMC-HA 03 prepared in the above examples 10 to 12 were subjected to rheological property tests, and the test results are shown in FIG. 3. As can be seen from FIG. 3, the addition of the microspheres can increase the mechanical properties of the hydrogel, and the storage modulus of the hydrogel is obviously improved with the increase of the addition amount of the microspheres.
3. Swelling performance test of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel
FIG. 4 is a graph comparing the swelling kinetics curves of the VA @ PLGA-CS-HA/OHPPMC-HA hydrogels prepared in examples 10 to 12 in PBS buffer solution; from FIG. 4, it can be seen that the addition of VA @ PLGA-CS-HA microspheres can slightly decrease the swelling ratio of the hydrogel, but generally HAs little effect on the swelling performance of the OHPPMC-HA hydrogel.
4. Antibacterial performance test of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel
The antibacterial performance test method comprises the following steps:
(1) preparing LB culture solution and normal saline, reviving staphylococcus aureus, and diluting the bacteria solution to a bacteria solution concentration of 10 by using normal saline5cfu/ml。
(2) Weighing 50mg of hydrogel sample, carrying out ultraviolet sterilization treatment, placing the hydrogel sample into a 50mL conical flask, adding 1mL of LB removing culture solution and 0.2mL of bacterial solution, and co-culturing the sample and the bacterial solution under the condition of shaking table 120rpm at 37 ℃ (co-culturing time points are respectively 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d and 56 d).
(3) And inoculating 100 mu L of mixed solution into a plate by adopting a culture medium pouring method, placing the plate in a bacterial incubator at 37 ℃ for 24 hours, and calculating the colony number. Calculating the bacteriostasis rate according to the following formula:
Relative CFU％＝CFU(24h)/CFU(0h)×100％。
FIG. 5 shows the results of the tests on the antibacterial properties of the VA @ PLGA-CS-HA/OHPPMC-HA hydrogels containing different amounts of VA @ PLGA-CS-HA microspheres, which show that the bactericidal rate of all 3 groups of hydrogels reaches 90% at day 7, the bacteriostatic rate of all 3 groups at day 14 approaches 99%, and that the Relative CFU of all 3 groups slightly increases at the later stage of the test, but the Relative CFU of all 3 groups still effectively inhibits the growth of bacteria (the Relative CFU of all 3 groups is still less than 1% at day 56).
Claims (9)
1. A preparation method of VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel is characterized by comprising the following steps: the method comprises the following steps:
(1) dissolving oxidized hydroxypropyl carboxymethyl cellulose (OHPMC) in distilled water to prepare an OHPMC solution with the mass percentage concentration of 1-3 wt%; dissolving aminated hyaluronic acid HA-ADA in distilled water to prepare an HA-ADA solution with the mass percentage concentration of 1-3 wt%; then, the OHPMC solution and the HA-ADA solution are mixed uniformly in equal volume to obtain an OHPMC-HA uniform mixed solution;
(2) adding VA @ PLGA-CS-HA microspheres into the OHPPMC-HA uniform mixed solution prepared in the step (1) according to a proportion, and uniformly mixing to form gel to prepare the VA @ PLGA-CS-HA/OHPPMC-HA antibacterial injectable hydrogel;
wherein: the VA @ PLGA-CS-HA microspheres in the step (2) are prepared by the following method, and the specific steps are as follows:
(a) dissolving polylactic acid-polyglycolic acid copolymer (PLGA) in dichloromethane, and preparing a PLGA solution with the mass percentage concentration of 5-15 wt%; dissolving chitosan CS in glacial acetic acid to prepare a chitosan solution with the mass percentage concentration of 0.5-1.5 wt% for later use; dissolving sodium hyaluronate HAS in distilled water to prepare a sodium hyaluronate solution with the mass percentage concentration of 1-3 wt% for later use; dissolving vancomycin hydrochloride VA in distilled water to prepare a vancomycin solution with the mass percentage concentration of 25-100 mg/ml; dissolving polyvinyl alcohol (PVA) in distilled water to prepare a chitosan solution with the mass percentage concentration of 0.3-3 wt% for later use;
(b) uniformly mixing the PLGA solution and the VA solution in the step (a) according to the proportion, and then carrying out ultrasonic dispersion to form W1an/O emulsion, wherein: the volume ratio of the PLGA solution to the VA solution is 2-10: 1;
(c) uniformly mixing the chitosan solution obtained in the step (a) with a PVA (polyvinyl alcohol) aqueous solution according to a proportion to obtain a CS/PVA solution; wherein: the volume ratio of the chitosan solution to the PVA aqueous solution is 0.5-2: 1;
(d) proportionally mixing the CS/PVA solution of step (c) with the W of step (b)1Mixing the emulsion and dispersing the mixture by ultrasonic to obtain stable W1/O/W2An emulsion, wherein: the CS/PVA solution and W1The volume ratio of the/O emulsion is 2-6: 1;
(e) taking a proper amount of the sodium hyaluronate solution obtained in the step (a), adding a PVA solution to prepare a HAS/PVA solution, wherein: the volume ratio of the sodium hyaluronate solution to the chitosan solution adopted in the step (c) is 1: 1, the HAS/PVA solution and W1/O/W2Volume of emulsionThe ratio is 4-6: 1;
(f) subjecting W obtained in step (d) to1/O/W2And (e) mixing the emulsion with the HAS/PVA solution obtained in the step (e), stirring at room temperature for 0.5-2 h, heating to 45 ℃, continuously stirring for 20-40 min, adding a proper amount of sodium tripolyphosphate aqueous solution, stirring for 1-2 h, standing for 8-12 h, centrifuging, washing, collecting precipitates, and freeze-drying to obtain the VA @ PLGA-CS-HA microspheres.
2. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the oxidized hydroxypropyl carboxymethyl cellulose in the step (1) is prepared by oxidizing and modifying hydroxypropyl carboxymethyl cellulose (HPMC), and the oxidizing and modifying method comprises the following specific steps:
preparing a hydroxypropyl carboxymethyl cellulose aqueous solution with the mass percentage concentration of 8-12%, and then preparing the hydroxypropyl carboxymethyl cellulose aqueous solution according to the volume ratio VHydroxypropyl carboxymethyl cellulose solution：VSodium periodate solution5: adding a sodium periodate solution according to the proportion of 1, reacting for 24 hours in a dark place, adding ethylene glycol which is equal to the sodium periodate solution in volume into a reaction system, continuously stirring for 30-45 min, dialyzing for 2-3 d with distilled water, and freeze-drying to obtain the sodium periodate.
3. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the aminated hyaluronic acid in the step (1) is prepared by amination and modification of hyaluronic acid by using azodicarbonamide (ADA), and the amination and modification method comprises the following specific steps:
preparing a hyaluronic acid solution with the mass percentage concentration of 1-3%, and then mixing the hyaluronic acid solution with the mass percentage concentration of 1: 1, adding adipic acid dihydrazide, adjusting the pH value of the reaction system to 4.7-4.8 after complete dissolution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, continuously adjusting the pH value of the reaction system to 4.7-4.8 after uniform mixing, finally reacting for 12h under the condition of room temperature, dialyzing for 2-3 d with distilled water, and freeze-drying to obtain the product.
4. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the dosage ratio of the VA @ PLGA-CS-HA microspheres to the OHPPMC-HA hydrogel in the step (2) is 0.1-0.3 parts by mass: 1 part by volume, wherein: the mass part and the volume part are as follows: ml is used as a reference.
5. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the OHPPMC solution in the step (1) has the mass percentage concentration of 2 wt%; the mass percentage concentration of the HA-ADA solution is 2 wt%.
6. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: and (3) gelling time in the step (2) is 3-7 min.
7. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the PLGA solution of the step (a) has the mass percentage concentration of 10 wt%; the mass percentage concentration of the chitosan solution is 1 wt%; the mass percent concentration of the sodium hyaluronate solution is 1.5 wt%.
8. The method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: the volume ratio of the PLGA solution to the VA solution in the step (b) is 5: 1; the volume ratio of the chitosan solution to the PVA aqueous solution in the step (c) is 1: 1.
9. the method for preparing VA @ PLGA-CS-HA/OHPMC-HA antibacterial injectable hydrogel according to claim 1, wherein: step (e) mixing the HAS/PVA solution with W1/O/W2The volume ratio of the emulsion is 5: 1.
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