CN113082002B - PH sensitive microsphere carrying sodium hyaluronate gold nanocluster and preparation method - Google Patents
PH sensitive microsphere carrying sodium hyaluronate gold nanocluster and preparation method Download PDFInfo
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Abstract
The invention discloses a pH sensitive microsphere carrying sodium hyaluronate gold nanoclusters and a preparation method thereof. The microsphere preparation is mainly characterized in that a biodegradable high polymer material with pH sensitivity is used as a framework, and a gold nanocluster loaded with sodium hyaluronate is wrapped inside the microsphere. The nano cluster has active targeting property by modifying a targeting group targeting on inflammatory macrophages in the bone joint cavity, the novel preparation not only can achieve the aim of slowly releasing the medicament, but also has the performance of accurately targeting treatment on inflammation, can effectively solve the problems of frequent injection administration, low bioavailability and the like in osteoarthritis treatment, and has stronger research significance.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a pH sensitive microsphere carrying sodium hyaluronate gold nanoclusters and a preparation method thereof.
Technical Field
Osteoarthritis is the leading cause of chronic joint pain worldwide, often occurring in the knee, and is called knee osteoarthritis. With the aging population and the increase of obesity, the number of affected people is continuously increased, and serious burden is caused to families and society of patients. Knee osteoarthritis is a chronic progressive synovial joint pain disease, is characterized by cartilage degradation, subchondral bone remodeling, osteophyte formation and synovial inflammation, and is clinically manifested as chronic joint activity pain, stiffness, and severe joint deformity and even paralysis.
Sodium hyaluronate is a mucopolysaccharide widely present in human connective tissues and synovial fluids, provides a main means for retaining aggrecan on the cartilage matrix or cell surface thereof, increases the viscoelasticity of synovial fluid, and has analgesic, anti-inflammatory and anti-nociceptive effects. Sodium hyaluronate is widely used in clinic as a joint mucus supplement, can relieve pain of patients and improve joint functions. However, with the increasing clinical application, the problems are increased. For example, sodium hyaluronate (sodium hyaluronate injection) is still injected into joint cavities for effective treatment of osteoarthritis at present, the medicine can rapidly enter body circulation after being injected, the retention time in the joint cavities is short, and therefore frequent injection is needed, so that local swelling and pain after injection even cause infection. Although various sustained-release preparations have been widely studied, for example, microsphere preparations are new pharmaceutical preparations developed in recent years, in which a drug is encapsulated in a matrix material and solidified to form solid beads. After injection administration, the medicine can be slowly released along with the degradation and erosion of the high molecular material so as to achieve the purpose of slow release. However, most of such sustained-release preparations are mainly made of synthetic polymer materials, and although these polymer materials are biodegradable, their degradation products are generally acidic, which inevitably leads to local low pH, irritates tissues, and has the risk of causing inflammation again.
The polyketal is a novel biodegradable high molecular material, and a ketal structure exists in a polymer skeleton, and the structure is easy to hydrolyze and break when encountering free hydrogen ions, so that the material has the pH sensitive characteristic; meanwhile, the polyketal degradation product is acetone, does not generate an acidic environment, has the advantages of good biocompatibility and the like, and is a good carrier material. Therefore, the novel sustained-release preparation for bone joint cavity injection, which can delay the release of the drug, is prepared by utilizing the advantages of the polyketal material, and has great potential and application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel sustained-release preparation which can treat osteoarthritis in a targeted manner, solve the problems of sudden release and frequent administration times of the medicament and finally achieve the aim of slowly releasing the medicament. Specifically, the invention provides a pH sensitive microsphere carrying sodium hyaluronate gold nanoclusters and a preparation method thereof.
The invention provides the following technical scheme:
the preparation method of the pH sensitive microsphere carrying the sodium hyaluronate gold nanocluster comprises the following steps:
(1) Preparation of folic acid modified gold nano-cluster
Dissolving Polyethyleneimine (PEI) and Folic Acid (FA) in dimethyl sulfoxide solution to prepare a folic acid solution and a polyethyleneimine solution respectively, then dropwise adding the folic acid solution into the polyethyleneimine solution under the stirring condition of 200-500rpm, stirring and reacting at room temperature for 24 hours, and then dissolving sodium hyaluronate in the solution and slowly stirring for later use;
slowly dripping the standby solution into a nano-gold solution with the concentration of 0.01-5mg/mL under the stirring condition of 100-400 rpm under the water bath condition of 60 ℃, reacting for 1h, and performing ultrafiltration concentration to obtain a gold nano-cluster suspension carrying sodium hyaluronate and folic acid modification;
wherein the mass ratio of the folic acid to the sodium hyaluronate to the polyethyleneimine to the nanogold is (5-50): (40-1000): (500-2000): (10-300);
(2) Preparation of nanocluster-loaded microspheres
Dropwise adding the gold nanocluster suspension loaded with the sodium hyaluronate and folic acid modification prepared in the step (1) into an organic solvent of polyketal or a derivative thereof under the stirring condition to form W/O type colostrum, dispersing the prepared colostrum into a polyvinyl alcohol aqueous solution with the mass concentration of 0.5-3% at 3000-8000rpm to form W/O/W type multiple emulsion, and stirring the obtained multiple emulsion for 1-3h at 100-400rmp to obtain solidified microspheres;
and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the pH sensitive microspheres carrying the sodium hyaluronate gold nanoclusters.
Preferably, the polyketal or the derivative thereof contains a ketal unit structure, contains two oxygen atoms and has a weight average molecular weight of 4000 to 20000.
Preferably, the concentration of the nano gold solution is 0.05-2mg/mL.
Preferably, the mass concentration of the polyketal or the derivative thereof in the oil phase is 5-40%. Further preferably, the mass concentration of the polyketal or the derivative thereof in the oil phase is 15% to 25%.
Preferably, the organic solvent is selected from one or more of chloroform, dichloromethane, ethyl acetate, methanol, diethyl ether and acetonitrile.
Preferably, the organic solvent is selected from dichloromethane.
Preferably, the volume ratio of the colostrum to the aqueous solution of the polyvinyl alcohol is 1:50-500.
Preferably, the polyvinyl alcohol is replaced by one or more of polyvinylpyrrolidone, sodium di-ethylhexyl succinate sulfonate, polyethylene glycol, span, tween, poloxamer and ethyl cellulose.
The invention provides another technical scheme:
the pH sensitive microsphere of the sodium hyaluronate-loaded gold nanocluster prepared by the method.
The pH sensitive microsphere of the sodium hyaluronate gold nano cluster prepared by the method is mainly applied to treatment of osteoarthritis and related diseases.
Different from the technical route of realizing the slow release effect by the diffusion of an effective preparation in the prior art, the invention provides another slow release route of carrying out targeted slow release by charge adsorption on a pH-sensitive slow release microsphere preparation. The pH sensitive microsphere of the sodium hyaluronate gold nanocluster prepared by the invention is combined with polyethylene imine with positive electricity through the surface of the nanogold, so that the nanogold is positively charged, and is mutually attracted with sodium hyaluronate which is a therapeutic drug with negative electricity through charge attraction to form a nanocluster with a slow release effect, and the nanocluster is wrapped in the microsphere, and the sodium hyaluronate can be released from the microsphere only by getting rid of the constraint of the nanocluster and the double barrier of the microsphere, so that the aim of reducing the burst release of the drug from the microsphere can be achieved, the problem of frequent injection of a common preparation can be solved, and the aim of slow release can be achieved. Moreover, after the novel preparation is injected into the articular cavity, because the inflammation part is obvious in acidity, the pH sensitive microspheres are degraded at the part in advance by induction, then the embedded gold nanoclusters are exposed, and the proinflammatory cytokines with positive charges competitively attract sodium hyaluronate to promote the sodium hyaluronate to fall off from the gold nanoclusters so as to release the medicine, thereby realizing the purpose of gradual release.
Advantageous effects
The pH sensitive microsphere preparation carrying the sodium hyaluronate gold nanoclusters, which is prepared by the invention, takes a biodegradable high molecular material with pH sensitivity as a framework, the gold nanoclusters carrying the sodium hyaluronate are wrapped inside the microspheres, and the nanoclusters have active targeting property by modifying a targeting group targeting inflammatory macrophages in a bone joint cavity. Therefore, the pH sensitive microsphere carrying the sodium hyaluronate gold nanoclusters prepared by the invention has great advantages in the aspect of treating arthritis by injecting in joint cavities.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a scanning electron micrograph of sodium hyaluronate-loaded gold nanocluster pH sensitive microspheres in example 1 of the present invention;
fig. 2a is an in vitro release curve of sodium hyaluronate-loaded gold nanocluster pH sensitive microspheres in example 1 of the present invention;
fig. 2b is an in vitro release curve of the sodium hyaluronate-loaded gold nanocluster pH sensitive microspheres in example 1 of the present invention;
FIG. 3 is an in vitro release profile of sodium hyaluronate loaded gold nanoclusters of comparative example 1 of the present invention;
FIG. 4 is an in vitro release profile of sodium hyaluronate-loaded polyketal microspheres of comparative example 2 of the present invention;
fig. 5 is an in vitro release profile of the sodium hyaluronate-gold nanoclustered polylactic acid-glycolic acid copolymer microspheres of comparative example 3 according to the present invention.
Detailed Description
The present invention will now be described in more detail, wherein preferred embodiments of the invention are shown, it being understood that one skilled in the art could modify the invention herein described while still achieving the beneficial results of the present invention. Accordingly, the following description should be construed as broadly as possible to those skilled in the art and not as limiting the invention.
Example 1
Preparation of pH sensitive microspheres of sodium hyaluronate gold nanocluster
Respectively dissolving 500mg of polyethyleneimine in 5ml of dimethyl sulfoxide solution, dissolving 20mg of folic acid in 1ml of dimethyl sulfoxide, dropwise adding the folic acid solution into the polyethyleneimine solution under the magnetic stirring condition of 200rpm, and reacting for 24 hours at room temperature under the stirring condition; weighing 100mg of sodium hyaluronate, dissolving in the solution, and slowly stirring for later use. Slowly dripping the obtained solution into 20mL of nano gold solution with the mass concentration of 1mg/mL and the water bath heat preservation at the temperature of 60 ℃ under the magnetic stirring condition of 400 rpm, reacting for 1 hour, and performing ultrafiltration concentration to obtain gold nano cluster suspension carrying sodium hyaluronate and folic acid modification;
0.2ml of nanocluster suspension is dripped into 1ml of dichloromethane containing 10% of polyketal or derivatives thereof by mass concentration under the stirring condition of 300rpm to form W/O type colostrum, the prepared colostrum is dispersed into 0.5% of polyvinyl alcohol aqueous solution by mass concentration at 5000rpm to form W/O/W type multiple emulsion, and the obtained multiple emulsion is stirred for 3 hours at 400rmp to obtain solidified microspheres; and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the nanocluster microspheres. The scanning electron microscope of the sodium hyaluronate-loaded gold nanocluster microsphere is shown in figure 1, and the prepared microsphere is smooth and round in surface and uniform in particle size.
In vitro release tests are carried out on the sodium hyaluronate-loaded gold nanocluster microspheres in the embodiment, and the tests show that the sodium hyaluronate-loaded gold nanocluster microspheres prepared by the invention have pH responsiveness in an acidic environment, overcome the problem of burst release and have an obvious slow release effect, as shown in fig. 2 a.
Example 2
Preparation of pH sensitive microsphere of sodium hyaluronate gold nano cluster
Respectively dissolving 1000mg of polyethyleneimine in 5ml of dimethyl sulfoxide solution, dissolving 30mg of folic acid in 1ml of dimethyl sulfoxide, dropwise adding the folic acid solution into the polyethyleneimine solution under the magnetic stirring condition of 200rpm, and reacting for 24 hours at room temperature under the stirring condition; 300mg of sodium hyaluronate is weighed and dissolved in the solution, and is slowly stirred for standby. Slowly dripping the obtained solution into 30mL of nano-gold solution with the mass concentration of 3mg/mL and the heat preservation in a water bath at the temperature of 60 ℃ under the magnetic stirring condition of 300rpm, reacting for 1h, and performing ultrafiltration and concentration to obtain a gold nano-cluster suspension carrying the modification of the sodium hyaluronate and the folic acid;
0.5ml of nano-cluster suspension is dripped into 2ml of 20 mass percent polyketal or methylene dichloride of derivatives thereof under the stirring condition of 300rpm to form W/O type colostrum, the prepared colostrum is dispersed into 1 mass percent polyvinyl alcohol aqueous solution at 6000rpm to form W/O/W type multiple emulsion, and the obtained multiple emulsion is stirred for 3 hours at 400rmp to obtain solidified microspheres; and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the nanocluster microspheres.
In vitro release tests are performed on the sodium hyaluronate-loaded gold nanocluster microspheres in the example, as shown in fig. 2b, the test results are similar to the slow release effect of example 1, but all show that the prepared sodium hyaluronate-loaded gold nanocluster microspheres have pH responsiveness and an obvious slow release effect in an acidic environment.
The examples 1 and 2 show that the pH sensitive microspheres of the sodium hyaluronate-loaded gold nanocluster prepared by the invention have an obvious slow release effect. According to the invention, the surface of the nanogold is combined with the polyethyleneimine with positive charge, so that the nanogold is positively charged, and the therapeutic drug sodium hyaluronate with negative charge is mutually attracted through charge attraction to form the nanocluster with a slow release effect. Furthermore, because the inflammation part is obvious in acidity, the pH sensitive microspheres are degraded at the part in advance by induction, then the embedded gold nanoclusters are exposed, and the positively charged proinflammatory cytokines can competitively attract sodium hyaluronate to promote the sodium hyaluronate to fall off from the gold nanoclusters, so that the aim of gradually releasing the medicine is fulfilled; moreover, the polyketal degradation product is neutral acetone, and does not secondarily stimulate inflammation and peripheral tissues.
In addition, since folate receptor is expressed to a high degree on osteoarthritis activated macrophages, sodium hyaluronate is a natural polysaccharide that binds specifically to the CD44 receptor, and CD44 is an adhesion receptor that is widely distributed on epithelial cells and activated lymphocytes, CD44 expression on leukocytes and activated macrophages increases rapidly when exposed to various inflammatory stimuli. Therefore, sodium hyaluronate and folic acid are synergistically targeted to macrophages to improve the therapeutic effect.
The technical route of obtaining the pH sensitive microspheres loaded with sodium hyaluronate gold nanoclusters of the present invention is further illustrated by comparative experiments.
Comparative example 1
Preparation of sodium hyaluronate-loaded nanoclusters
Respectively dissolving 500mg of polyethyleneimine in 5ml of dimethyl sulfoxide solution, dissolving 20mg of folic acid in 1ml of dimethyl sulfoxide, dropwise adding the folic acid solution into the polyethyleneimine solution under the magnetic stirring condition of 200rpm, and reacting for 24 hours at room temperature under the stirring condition; weighing 100mg of sodium hyaluronate, dissolving in the solution, and slowly stirring for later use. Slowly and dropwise adding the obtained solution into 20mL of nano gold solution with the mass concentration of 1mg/mL and the water bath temperature of 60 ℃ under the magnetic stirring condition of 400 rpm, reacting for 1h, performing ultrafiltration concentration to obtain gold nano cluster suspension carrying sodium hyaluronate and folic acid modification, and performing freeze drying to obtain the product. The nanocluster in vitro release profile loaded with sodium hyaluronate is shown in figure 3.
Comparative example 2
Preparation of sodium hyaluronate-loaded polyketal microspheres
Weighing 20mg of sodium hyaluronate, dissolving the sodium hyaluronate in 0.5ml of aqueous solution, dropwise adding the sodium hyaluronate into 2ml of dichloromethane containing 20% of polyketal in mass concentration under the shearing condition of 6000rpm to form W/O type colostrum, dispersing the prepared colostrum at 8000rpm into aqueous solution of polyvinyl alcohol with 1% of mass concentration to form W/O/W type multiple emulsion, and stirring the obtained multiple emulsion for 3 hours at 400rmp to obtain cured microspheres; and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the polyketal microspheres. The in vitro release curve of the sodium hyaluronate loaded polyketal microspheres is shown in figure 4.
Comparative example 3
Preparation of sodium hyaluronate-loaded nanocluster polylactic acid-glycolic acid copolymer microspheres
Respectively dissolving 1000mg of polyethyleneimine in 5ml of dimethyl sulfoxide solution, dissolving 30mg of folic acid in 1ml of dimethyl sulfoxide, dropwise adding the folic acid solution into the polyethyleneimine solution under the magnetic stirring condition of 200rpm, and reacting for 24 hours at room temperature under the stirring condition; 300mg of sodium hyaluronate is weighed and dissolved in the solution, and is slowly stirred for standby. Slowly dripping the obtained solution into 30mL of nano-gold solution with the mass concentration of 3mg/mL and the heat preservation in a water bath at the temperature of 60 ℃ under the magnetic stirring condition of 300rpm, reacting for 1h, and performing ultrafiltration and concentration to obtain a gold nano-cluster suspension carrying the modification of the sodium hyaluronate and the folic acid;
0.5ml of nanocluster suspension is dropwise added into 2ml of dichloromethane containing PLGA with the mass concentration of 20% under the stirring condition to form W/O type colostrum, the prepared colostrum is dispersed into 1% polyvinyl alcohol aqueous solution with the mass concentration of 7000rpm to form W/O/W type multiple emulsion, and the obtained multiple emulsion is stirred for 3 hours at 400rmp to obtain solidified microspheres; and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the nanocluster microspheres. The in vitro release profile of PLGA microspheres loaded with sodium hyaluronate nanoclusters is shown in fig. 5.
Comparison of the in vitro release profiles of example 1 with comparative examples 1, 2 shows that: the nano-cluster microspheres loaded with sodium hyaluronate have pH responsiveness in an acidic environment and release faster than those in a neutral release medium; in addition, sodium hyaluronate needs to get rid of the restraint of nano-clusters and the double-layer barrier of microspheres to be released from the microspheres, so that the burst release phenomenon is better solved; the nano-cluster and the polyketal microspheres are single in release, and the slow release effect is not as obvious as that of the nano-cluster and the polyketal microspheres. The nano-cluster microspheres prepared by the invention can effectively meet the requirement that the common sustained-release preparation needs frequent injection to achieve the purpose of slow release.
Also, a comparison of the in vitro release profiles of example 1 and comparative example 3 shows that: although both have slow release effects, the microsphere material adopting the polyketal or the derivative thereof has obvious pH sensitive characteristic, and the drug release can be accelerated because the inflammation part is obvious in acidity, and the neutral acetone does not stimulate the part again because the product is neutral. Although the PLGA microspheres of the nanoclusters are released slowly, as the PLGA degradation product is lactic acid, the inflammatory part can be re-stimulated and the disease condition is even aggravated, so that the pH sensitive microspheres carrying the sodium hyaluronate gold nanoclusters have obvious advantages, and the PLGA is not adopted in the invention.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The above-described embodiments of the invention are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and not by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (8)
1. The preparation method of the pH sensitive microsphere carrying the sodium hyaluronate gold nanocluster is characterized by comprising the following steps:
(1) Preparation of folic acid modified gold nano-cluster
Dissolving Polyethyleneimine (PEI) and Folic Acid (FA) in dimethyl sulfoxide solution to prepare a folic acid solution and a polyethyleneimine solution respectively, then dropwise adding the folic acid solution into the polyethyleneimine solution under the stirring condition of 200-500rpm, stirring and reacting at room temperature for 24 hours, and then dissolving sodium hyaluronate in the solution and slowly stirring for later use;
slowly dripping the standby solution into a nano-gold solution with the concentration of 0.01-5mg/mL under the stirring condition of 100-400 rpm under the water bath condition of 60 ℃, reacting for 1h, and performing ultrafiltration concentration to obtain a gold nano-cluster suspension carrying sodium hyaluronate and folic acid modification;
wherein the mass ratio of folic acid, sodium hyaluronate, polyethyleneimine and nanogold is 5-50:40-1000:500-2000:10-300;
(2) Preparation of nanocluster-loaded microspheres
Dropwise adding the gold nanocluster suspension loaded with the sodium hyaluronate and folic acid modification prepared in the step (1) into an organic solvent of polyketal or a derivative thereof under the stirring condition to form W/O type colostrum, dispersing the prepared colostrum into a polyvinyl alcohol aqueous solution with the mass concentration of 0.5-3% at 3000-8000rpm to form W/O/W type multiple emulsion, and stirring the obtained multiple emulsion for 1-3h at 100-400rmp to obtain solidified microspheres;
and centrifuging, washing and freeze-drying the obtained solidified microspheres to obtain the pH sensitive microspheres carrying the sodium hyaluronate gold nanoclusters.
2. The method according to claim 1, wherein the polyketal or a derivative thereof has a ketal unit structure and a weight average molecular weight of 4000 to 20000.
3. The method according to claim 1, wherein the concentration of the nanogold solution is 0.05 to 2mg/mL.
4. The method according to claim 1, wherein the organic solvent is selected from chloroform, dichloromethane, ethyl acetate, methanol, diethyl ether, and acetonitrile.
5. The method according to claim 4, wherein the organic solvent is selected from methylene chloride.
6. The method according to claim 1, wherein the volume ratio of colostrum to aqueous solution of polyvinyl alcohol is 1:50-500.
7. The method according to claim 6, wherein the polyvinyl alcohol is replaced with one or more of polyvinylpyrrolidone, sodium 2-ethylhexyl succinate, polyethylene glycol, span, tween, poloxamer and ethylcellulose.
8. The pH sensitive microsphere of sodium hyaluronate-loaded gold nanocluster prepared by the method of any one of claims 1 to 7.
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