Sequential drug release liposome hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of pharmaceutical chemicals, and discloses sequential drug release liposome hydrogel and a preparation method and application thereof, and the preparation method of the liposome hydrogel comprises the following steps: step 1, preparation of drug-loaded positive charge liposome; step 2, preparing phenylboronic acid modified polysaccharide; and step 3, carrying out vortex mixing on the drug-loaded positive charge liposome solution, the phenylboronic acid modified polysaccharide hydrogel precursor powder and the spermidine solution to obtain the liposome hydrogel. According to the sequential drug release liposome hydrogel as well as the preparation method and the application thereof, the liposome hydrogel is prepared by taking high-molecular hyaluronic acid and phenylboronic acid as main raw materials, and the liposome hydrogel has microenvironment responsiveness and time-space characteristics of drug release, and can be used for treating osteoarthritis in an environment with active oxygen increase, so that the drug release effect is good, and the drug release effect is good. The functions of early-stage anti-inflammation and continuous cartilage regeneration are achieved by sequentially releasing double drugs, synovitis can be effectively relieved, cartilage regeneration can be promoted, and the compound can be used for preparing drugs for treating osteoarthritis.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemical engineering, and in particular to a sequential drug-releasing liposome hydrogel and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is a common joint degenerative disease with the main clinical symptoms of joint pain. As the disease progresses, it can lead to severe joint deformities. At present, the treatment of osteoarthritis is mainly symptomatic, such as non-steroidal anti-inflammatory drugs to control pain and inflammation, and injection of chondroitin sulfate to supplement cartilage matrix and reduce wear. Injectable hydrogels have unique properties that have attracted widespread attention and are ideal biomaterials for the treatment of OA. The natural polysaccharides that make up the hydrogel have good biosafety, biocompatibility, and biodegradability. More importantly, some polysaccharides have anti-inflammatory properties, such as hyaluronic acid and chitosan. At the same time, injectable hydrogels can be directly injected into the affected area without the need for surgery, minimizing surgical trauma and improving patient comfort.

[0003] However, traditional hydrogels have encountered some application challenges. For example, the current consensus in research and clinical treatment is that joint pain and inflammation can be quickly relieved in a short period of time, while cartilage lesions require long-term treatment. In addition, the microenvironment of osteoarthritis is complex, with the characteristics of "multiple sites and multiple targets". The inflammatory environment of the joints (especially synovitis) will promote the continuous dissolution of cartilage collagen. Traditional hydrogels only focus on the role of cartilage tissue repair and cannot accurately coordinate various biological events in the pathogenesis of OA. Summary of the invention

[0004] The purpose of the present invention is to provide a liposome hydrogel with sequential drug release, and a preparation method and application thereof. The liposome hydrogel is prepared with high molecular weight hyaluronic acid and phenylboronic acid as main raw materials. The liposome hydrogel has microenvironment responsiveness and spatiotemporal characteristics of drug release. In an environment of increased reactive oxygen species in osteoarthritis, the liposome hydrogel can achieve the functions of early anti-inflammatory and sustained cartilage regeneration by sequentially releasing two drugs, which can effectively reduce synovial inflammation, promote cartilage regeneration, and meet the needs of osteoarthritis treatment.

[0005] To achieve the above object, the present invention provides a method for preparing a liposome hydrogel for sequential drug release, comprising the following steps: Step 1, preparation of drug-loaded positively charged liposomes: Dissolving lecithin, cholesterol, octadecylamine and loaded drugs in an organic solvent, fully mixing, and removing the solvent by rotary evaporation to obtain a fat-soluble film; subjecting the fat-soluble film to hydration treatment and ultrasonic treatment respectively to obtain a drug-loaded positively charged liposome solution; Step 2, preparation of phenylboronic acid modified polysaccharide: Phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HoBt are added to the polysaccharide solution, fully mixed under weak acid conditions, dialyzed, and freeze-dried to obtain a phenylboronic acid-modified polysaccharide hydrogel precursor powder; Step 3, preparation of liposome hydrogel: The drug-loaded positively charged liposome solution of step 1, the phenylboronic acid-modified polysaccharide hydrogel precursor powder of step 2 and the spermidine solution are vortex-mixed to obtain a liposome hydrogel.

[0006] Furthermore, in step 1, the ultrasonic treatment conditions are as follows: the power is 135 W and the time is 3 min; the organic solvent is selected from one or more of dichloromethane, chloroform, and ethanol.

[0007] Furthermore, the mass ratio of lecithin, cholesterol, octadecylamine and loaded drug is (100-300): (20-60): (5-20): (1-5).

[0008] Furthermore, the loaded drug in step 1 includes an anti-inflammatory and / or cartilage repair drug, and the anti-inflammatory and / or cartilage repair drug is one or more of disulfiram, diclofenac or diacerein.

[0009] Furthermore, in step 2, the reaction pH value is 5-6.

[0010] Furthermore, in step 2, the mass ratio of phenylboric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HoBt is phenylboric acid:EDC:HoBt=(10-50):(5-10):(5-10).

[0011] Furthermore, in step 2, the polysaccharide is selected from one or more of sodium alginate, hyaluronic acid, chitosan and gelatin.

[0012] Furthermore, in step 3, the mass ratio of the drug-loaded positively charged liposomes, the phenylboronic acid-modified polysaccharide hydrogel precursor solution and spermidine is 2:(10-100):10.

[0013] The present invention also provides a sequential drug-releasing liposome hydrogel, which is prepared according to the above-mentioned preparation method.

[0014] The present invention also provides an application of a sequential drug-releasing liposome hydrogel in the preparation of a drug for treating osteoarthritis.

[0015] The advantages and positive effects of the sequential drug release liposome hydrogel and its preparation method and application described in the present invention are: 1. The liposome hydrogel in the present invention is a biomaterial with a suitable particle size and good stability, injectability and safety.

[0016] 2. Aiming at the microenvironmental characteristics of osteoarthritis where reactive oxygen species are significantly elevated, the present invention provides a functional biomaterial that responds to and regulates the microenvironment of reactive oxygen species and a preparation method thereof. The liposome hydrogel of the present invention achieves a cascade effect of early anti-inflammatory and cartilage regeneration by sequentially releasing dual drugs, and has great application potential in the treatment of osteoarthritis.

[0017] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The characterization diagrams of liposomes in the embodiments of the present invention, wherein A is a particle size diagram, B is a potential diagram, and C is a transmission electron microscope diagram; Figure 2 The synthetic route diagram of phenylboronic acid grafted polysaccharide in the embodiment of the present invention and the verified H NMR spectrum diagram; Figure 3 This is a macroscopic inversion phenomenon diagram of the liposome hydrogel in an embodiment of the present invention; Figure 4 Graph showing the rheological properties of the liposome hydrogel in an embodiment of the present invention; Figure 5 This is a SEM image of the liposome hydrogel in the embodiment of the present invention; Figure 6 This is a macroscopic injection performance diagram of liposome hydrogel in an embodiment of the present invention; Figure 7 is the cumulative drug release rate of spermidine (SP) and disulfiram (DS) in the liposome hydrogel at different times in vitro in the embodiment of the present invention; Figure 8 The evaluation results of the in vitro cartilage repair ability of the liposome hydrogel in the embodiment of the present invention, wherein A is the staining result and B is the protein expression result; Fig. 9 The in vivo imaging diagram of the fluorescent dye of the liposome hydrogel in the embodiment of the present invention at different time in vivo; Fig.10 The results of in vivo biocompatibility evaluation in the embodiments of the present invention are HE images of major organs (heart, liver, spleen, lung, and kidney) stained with hematoxylin-eosin after the liposome hydrogel was injected into the body; Fig.11 The results of evaluating the in vivo cartilage repair ability of the liposome hydrogel in the embodiments of the present invention include articular cartilage staining images of hematoxylin-eosin (HE), safranin fast green (S / F), and toluidine blue (Tb); Fig.12 This is an OARSI scoring diagram of osteoarthritis pathological tissue in an embodiment of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0020] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following examples are usually measured in accordance with national standards. The experimental reagents without source in the following examples are all commercially available raw materials.

[0022] The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0023] Unless otherwise defined or described, all professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0024] In the present invention, EDC is the English abbreviation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and HoBt is the English abbreviation of 1-hydroxybenzotriazole.

[0025] Example 1 A method for preparing a liposome hydrogel for sequential drug release, the specific steps are as follows: 1) Preparation of drug-loaded positively charged liposomes: Phosphatidylcholine, cholesterol, octadecylamine and disulfiram were dissolved in dichloromethane, and then the mixed solution was subjected to rotary evaporation to remove the solvent to obtain a lipid-soluble film. Deionized water was then added to hydrate the lipid film on the wall of the round-bottom flask to form liposomes.

[0026] In order to further prepare single-layer liposomes with smaller particle size, after ultrasonic treatment, nano-scale positively charged liposomes with uniform particle size distribution were extruded through a liposome extruder. The ultrasonic power was 135W, and the time was 3min (start 3s, stop 3s). The optimized results are shown in Table 1, and the results show that the particle size of the liposomes after ultrasonic treatment is more uniform.

[0027] Table 1 Ultrasonic optimization results ;

[0028] 2) Preparation of phenylboronic acid modified polysaccharides: Hyaluronic acid was fully dissolved in deionized water, and then phenylboronic acid, EDC and HoBt were added to the hyaluronic acid solution, and dilute hydrochloric acid was added to adjust the pH value. The solution was mixed and stirred at room temperature. Finally, it was dialyzed with deionized water and freeze-dried. Among them, the adjusted pH value was 5.5; the stirring time was 48h; the dialysis and freeze-drying time was 72h; the optimized results are shown in Table 2, and the results show that phenylboronic acid can better react with hyaluronic acid under weak acid conditions.

[0029] Table 2 Reaction conditions optimization results ;

[0030] 3) Preparation of liposome hydrogel: The liposome solution was premixed with the polysaccharide hydrogel precursor solution modified with phenylboronic acid, and then the spermidine solution was quickly mixed, and the liquid flow was observed to prepare the liposome hydrogel. The blank hydrogel was recorded without the addition of liposome solution. The mass ratio of drug-loaded positively charged liposomes, phenylboronic acid-modified polysaccharide hydrogel precursor powder and spermidine was 2: (10-100): 10. The optimized results are shown in Table 3. The results show that when the mass ratio is 2: 50: 10, the gelation speed and injectability are better.

[0031] Table 3 Mass ratio optimization results ;

[0032] For subsequent therapeutic tests, drug-loaded positively charged liposomes and blank hydrogels without liposomes were used as control groups for control experiments.

[0033] Example 2 Morphology Characterization 1. The drug-loaded positively charged liposomes prepared in Example 1 were characterized and analyzed by dynamic light scattering (DLS) and transmission electron microscopy.

[0034] The results are as follows Figure 1 As shown, the particle size of the drug-loaded positively charged liposomes is 125 nm ( Figure 1 A), the potential is 41.35mV ( Figure 1 In B, the transmission electron microscopy image of the liposomes prepared in Example 1 shows a spherical vesicle structure ( Figure 1 Middle C).

[0035] The phenylboronic acid grafted hyaluronic acid HA-PBA was characterized by H NMR. Figure 2 As shown, it can be observed that relevant characteristic peaks of HA and PBA appear in the NMR of HA-PBA.

[0036] 2. Premix the prepared liposome solution with the polysaccharide hydrogel precursor solution modified with phenylboronic acid (corresponding to step 3 in Example 1), and then quickly mix with the spermidine solution. Figure 3 As shown, it can be observed that after the liposome hydrogel is formed, there is no liquid flow in the bottle, which proves the successful preparation of the liposome hydrogel.

[0037] 3. The morphology of the liposome hydrogel prepared in Example 1 was observed by scanning electron microscopy. Figure 4 , a scanning electron micrograph of the liposome hydrogel of Example 1. The unique pore structure of the hydrogel can be observed.

[0038] 4. The rheological properties of the liposome hydrogel prepared in Example 1 were evaluated by TA rheometer (DHR-2). The stiffness of the liposome hydrogel was evaluated by 1% constant strain and 0.1-100 Hz frequency oscillation test at 37°C. Figure 5 As shown, within a certain frequency range, the storage modulus (G') of the liposome hydrogel is higher than the loss modulus (G''), indicating that the hydrogel formation is stable.

[0039] 5. The liposome hydrogel prepared in Example 1 was sucked up with a syringe and then injected into different shapes through the syringe needle, such as Figure 6 As shown, liposomal hydrogels can be injected into different shapes, presenting an “OA” shape.

[0040] Example 3 Sequential drug release capability verification To further investigate the sequential drug release capability of the liposome hydrogel, the liposome hydrogel of Example 1 was first transferred to a dialysis bag, and then the dialysis bag was immersed in a 1 mM H2O2 solution and oscillated in an oscillator at 37°C. The release medium was collected at different time points for analysis. Figure 7 As shown, in the H2O2 environment, about 50% of SP was released from the hydrogel within the first 24 hours, and the SP was completely released on the 12th day. The release rate of DS was significantly lower, and the release was sustained within 24 days (68.3%), which proved that the liposome hydrogel prepared in Example 1 had the ability to release drugs in different time sequences.

[0041] Example 4 In vitro cell validation The efficacy of liposome hydrogel was further verified in in vitro cell experiments. A co-culture experiment of chondrocytes and macrophages was established to simulate the microenvironment of osteoarthritis. Figure 8As shown, after inflammatory macrophages and chondrocytes were co-incubated for 24 hours, the degradation solution of the 21-day hydrogel was added for treatment for 24 hours, and the chondrocytes were collected for analysis. The results showed that compared with the PBS group, the hydrogel without liposome loading and the liposome hydrogel had a certain effect on the treatment of cartilage degeneration (synthetic protein COL II was upregulated, degradation proteins MMP3 and MMP13 were downregulated, and glycosaminoglycan staining was significantly deepened), but the effect of liposome hydrogel treatment was more obvious, indicating that the liposome hydrogel has a synergistic therapeutic effect after sequential drug release.

[0042] Example 5 In vivo validation The sequential drug release capability of liposome hydrogel was further verified in vivo. Two different fluorescent dyes, Cy5 and Ir780, were used to replace the above-mentioned SP and DS drugs, and loaded into the liposome hydrogel prepared in Example 1, injected into rats with osteoarthritis model, and in vivo imaging was performed at different times. Fig. 9 As shown, the signals of the two fluorescent dyes can be stably detected within 24 hours, confirming that the hydrogel was successfully injected into the joint cavity. However, the fluorescence signal of the Cy5 dye quickly dropped to the lowest level within 14 days. The fluorescence signal of Ir780 was still observable on the 28th day, although it gradually decreased over time. The above data demonstrate the ability of the liposome hydrogel prepared in Example 1 to release drugs in different time sequences.

[0043] Example 6 1. After the liposome hydrogel prepared in Example 1 was injected into rats with osteoarthritis model, different tissues were collected. First, the heart, liver, spleen, lung, and kidney tissues were sliced ​​and observed by hematoxylin-eosin staining. Fig.10 As shown, the liposomes, blank hydrogel and liposome hydrogel prepared in Example 1 have no obvious toxicity to the major organs such as heart, liver, spleen, lung and kidney, and have good biocompatibility.

[0044] 2. Articular cartilage was collected for sectioning and the efficacy of osteoarthritis was evaluated by OARSI scoring. Cartilage degeneration is an important pathological manifestation of osteoarthritis. Fig.11 and Fig.12 As shown in the figure, after CIOA modeling, the cartilage in the Saline saline group showed wear and the matrix was significantly reduced, and the OARSI score was significantly increased. The liposomes prepared in Example 1, the blank hydrogel and the liposome hydrogel treatment groups all restored this phenomenon to a certain extent, but the OARSI score of the liposome hydrogel was the lowest, proving that the liposome hydrogel synergistically inhibited cartilage degeneration through sequential drug release, and is expected to be further used in the treatment of osteoarthritis.

[0045] The above results show that the liposome hydrogel with sequential release capability provided by the present invention has microenvironment responsiveness and spatiotemporal characteristics of drug release. In the environment of increased reactive oxygen species in osteoarthritis, the sequential release of dual drugs can achieve the functions of early anti-inflammatory and sustained cartilage regeneration. In vitro and in vivo studies further confirmed this feature. The liposome hydrogel provided by the present invention has great application potential in the multi-stage treatment of osteoarthritis.

[0046] Therefore, the present invention adopts the above-mentioned sequential drug-releasing liposome hydrogel and its preparation method and application, and uses high molecular weight hyaluronic acid and phenylboronic acid as main raw materials to prepare liposome hydrogel, which has microenvironment responsiveness and spatiotemporal characteristics of drug release. In the environment of increased reactive oxygen species in osteoarthritis, it can achieve the functions of early anti-inflammatory and continuous cartilage regeneration by sequentially releasing dual drugs, which can effectively reduce synovial inflammation, promote cartilage regeneration, and meet the needs of osteoarthritis treatment.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a liposome hydrogel for sequential drug release, characterized in that: The steps include: Step 1, preparation of drug-loaded positively charged liposomes: Dissolving lecithin, cholesterol, octadecylamine and loaded drugs in an organic solvent, fully mixing, and removing the solvent by rotary evaporation to obtain a fat-soluble film; subjecting the fat-soluble film to hydration treatment and ultrasonic treatment respectively to obtain a drug-loaded positively charged liposome solution; Step 2, preparation of phenylboronic acid modified polysaccharide: Phenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HoBt are added to the polysaccharide solution, fully mixed under weak acid conditions, dialyzed, and freeze-dried to obtain a phenylboronic acid-modified polysaccharide hydrogel precursor powder; Step 3, preparation of liposome hydrogel: The drug-loaded positively charged liposome solution of step 1, the phenylboronic acid-modified polysaccharide hydrogel precursor powder of step 2 and the spermidine solution are vortex-mixed to obtain a liposome hydrogel.

2. The preparation method according to claim 1, characterized in that: In step 1, the ultrasonic treatment conditions are as follows: the power is 135 W and the time is 3 min; the organic solvent is selected from one or more of dichloromethane, chloroform and ethanol.

3. The preparation method according to claim 1, characterized in that: In step 1, the mass ratio of lecithin, cholesterol, octadecylamine and loaded drug is (100-300): (20-60): (5-20): (1-5).

4. The preparation method according to claim 1, characterized in that: The loaded drug in step 1 includes an anti-inflammatory and / or cartilage repair drug, and the anti-inflammatory and / or cartilage repair drug is one or more of disulfiram, diclofenac or diacerein.

5. The preparation method according to claim 1, characterized in that: The reaction pH in step 2 is 5-6.

6. The preparation method according to claim 1, characterized in that: In step 2, the mass ratio of phenylboric acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 1-hydroxybenzotriazole HoBt is phenylboric acid:EDC:HoBt=(10-50):(5-10):(5-10).

7. The preparation method according to claim 1, characterized in that: In step 2, the polysaccharide is selected from one or more of sodium alginate, hyaluronic acid, chitosan and gelatin.

8. The preparation method according to claim 1, characterized in that: In step 3, the mass ratio of drug-loaded positively charged liposomes, phenylboronic acid-modified polysaccharide hydrogel precursor solution and spermidine is 2:(10-100):

10.

9. A liposome hydrogel for sequential drug release, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the sequential drug-releasing liposome hydrogel according to claim 9 in preparing a drug for treating osteoarthritis.

Images