WO2023077699A1 - 一种pH响应型水凝胶生物载体及应用 - Google Patents
一种pH响应型水凝胶生物载体及应用 Download PDFInfo
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- WO2023077699A1 WO2023077699A1 PCT/CN2022/079518 CN2022079518W WO2023077699A1 WO 2023077699 A1 WO2023077699 A1 WO 2023077699A1 CN 2022079518 W CN2022079518 W CN 2022079518W WO 2023077699 A1 WO2023077699 A1 WO 2023077699A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F299/00—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/46—Polymerisation initiated by wave energy or particle radiation
- C08F2/48—Polymerisation initiated by wave energy or particle radiation by ultraviolet or visible light
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
Definitions
- the invention discloses a hydrogel biological carrier, which belongs to the technical field of pharmacy.
- Platelet-derived growth factor is a cationic glycoprotein that is heat-resistant, acid-resistant, and easily hydrolyzed by proteases. It can be secreted by a variety of cells, such as: platelets, fibroblasts, macrophages, etc.
- PDGF has four monomers PDGF-A, PDGF-B, PDGF-C and PDGF-D, and these monomers are connected by disulfide bonds to form a dimer form.
- PDGF-BB the dimer form of PDGF-B, acts as a mitosis promoter, which can stimulate the proliferation and migration of fibroblasts and smooth muscle cells, and promote the production of macrophages and the secretion of growth factors. It is more effective in wound repair, tissue regeneration, bone and tooth regeneration, and joint repair. Especially in wound repair, it is the only growth factor approved by the US Food and Drug Administration (FDA) for the treatment of diabetic ulcers.
- FDA US Food and Drug Administration
- Wound repair and tissue regeneration diseases require growth factors to maintain a certain concentration in the diseased area for a long time, but PDGF-BB has a short half-life ( ⁇ 2min) and is easily degraded by proteases, and there are a lot of proteases in chronic wound beds, which makes PDGF-BB more effective. Almost deactivated.
- multiple high-dose administrations are used to maintain the drug concentration, but this has the risk of tumorigenesis. Therefore, there is a need in this field for a carrier, which can not only improve the stability of PDGF-BB, but also maintain a certain drug concentration for a long time, so as to ensure the safety and effectiveness of PDGF medication.
- the object of the present invention is to provide a hydrogel biological carrier, which can release the biological activity of PDGF-BB controllably, improve its stability in drug effect, The biosafety of drug use can also be ensured through the degradation of the carrier.
- the present invention comprehensively considers the biocompatibility of the methacrylic anhydride hyaluronic acid hydrogel and the characteristics of being able to protect the slow release of growth factors, and introduces PDGF-BB into the methacrylic anhydride hyaluronic acid hydrogel network
- a pH-responsive hydrogel biocarrier combined with growth factors was prepared, aiming to improve the biocompatibility, pH responsiveness, cell sheet desorption and protection, and slow release of growth factors of traditional hydrogels.
- the present invention firstly provides a pH-responsive hydrogel biocarrier, the pH-responsive hydrogel biocarrier is composed of methacrylated hyaluronic acid in the presence of a photoinitiator and a cross-linking agent. , prepared by irradiating with 365nm ultraviolet laser to initiate cross-linking reaction.
- the photoinitiator is I2959
- the crosslinking agent is the first crosslinking agent GDMA and the second crosslinking agent AI102.
- the methacrylated hyaluronic acid is synthesized by esterification of sodium hyaluronate and methacrylic anhydride in a molar ratio of 1:30.
- the molar ratio of the first crosslinking agent GDMA to the second crosslinking agent AI102 is (1-4):(0-2).
- the present invention provides a method for preparing the above-mentioned pH-responsive hydrogel biocarrier, the method comprising making methacrylated hyaluronic acid in a photoinitiator, a first cross-linking agent GDMA and a second cross-linking agent In the presence of agent AI102, the cross-linking reaction was initiated by 365nm ultraviolet laser irradiation.
- the present invention provides the above-mentioned pH-responsive hydrogel biocarrier loaded with therapeutic protein, and the pH-responsive hydrogel biocarrier loaded with therapeutic protein is loaded by hydrogel biological
- the carrier is lyophilized, and the therapeutic protein is loaded in a pH-responsive hydrogel biological carrier by a dry soaking method.
- the therapeutic protein is platelet-derived growth factor.
- the platelet-derived growth factor is in the form of a BB dimer.
- the present invention provides a method for preparing the above-mentioned pH-responsive hydrogel biocarrier loaded with therapeutic proteins, the method comprising lyophilizing the hydrogel biocarrier, and loading the therapeutic protein with a dry soaking method Steps for fabrication within pH-responsive hydrogel biocarriers.
- the present invention provides the application of the above-mentioned pH-responsive hydrogel biocarrier loaded with therapeutic proteins in the preparation of drugs for promoting wound repair and tissue regeneration.
- the invention adopts the method of in-situ free radical polymerization to synthesize the pH-responsive hydrogel biocarrier, and uses the dry soaking method to load the therapeutic protein in the pH-responsive hydrogel biocarrier, the reaction conditions are mild, and the PDGF-BB The biological activity of PDGF-BB will not be affected; the PDGF-BB is encapsulated in the hydrogel to avoid exposure to the enzyme environment and improve stability. The release of PDGF-BB is achieved through the degradation of the hydrogel.
- the present invention selects two cross-linking agents that respond to the degradation of the alkaline pH value: the first cross-linking agent GDMA and the second cross-linking agent AI102, and the two cross-linking agents
- the degradation of crosslinkers is based on their own ester bond groups, but these two crosslinkers degrade at different rates under alkaline conditions.
- the pH-responsive hydrogel biological carrier provided by the present invention can control the compatibility ratio according to the different types of wounds, that is, the pH value of the wound site, so as to control the degradation rate of the gel to meet the application requirements.
- the degradation rates of the two cross-linking agents are different at 8.0, the second cross-linking agent is more water-soluble than the first cross-linking agent, and the ester The number of bonds is relatively large, so the degradation rate is faster than that of the first cross-linking agent.
- the ratio of the two for example, when the ratio of GDMA:AI102 is 1:2, the release rate is the slowest, which provides control of the ratio between the two cross-linking agents to achieve different rates of degradation of the hydrogel.
- the appropriate cross-linker ratio can be selected for the treatment of different diseases and wound types (see Figure 1).
- Figure 1 Schematic diagram of the preparation of pH-responsive hydrogels with controlled release of PDGF-BB.
- Figure 3 The physical picture of pH-responsive hydrogel with controlled release of PDGF-BB.
- Figure 4 Compressive performance test of pH-responsive hydrogels with different cross-linking agent ratios.
- Figure 5 Scanning electron micrographs of pH-responsive hydrogels with different cross-linking agent ratios.
- Figure 6 Swelling ratio diagram of pH-responsive hydrogels with different cross-linking agent ratios.
- Figure 7 The cumulative release rate of PDGF-BB from pH-responsive hydrogels with different cross-linking agent ratios.
- Figure 8 pH-responsive hydrogel cytotoxicity assay.
- Figure 9 Statistical diagram of the migration effect of pH-responsive hydrogels on 3T3 cells.
- Fig. 11 Observation diagram of full-thickness skin wound healing in mice with different treatment methods.
- Figure 1 shows a schematic diagram of the preparation of PDGF-BB pH-responsive hydrogel, where A is methacrylic anhydride, B is sodium hyaluronate, 1 is the first crosslinking agent GDMA, and 2 is the second crosslinking agent AI102 .
- A is methacrylic anhydride
- B is sodium hyaluronate
- 1 is the first crosslinking agent GDMA
- 2 is the second crosslinking agent AI102 .
- the preparation steps are described in detail below.
- the methacrylate derivatives of hyaluronic acid are synthesized by esterifying sodium hyaluronate (HA) and methacrylic anhydride (MA), and the specific steps include the following.
- m-HA Dissolve m-HA in deuterated water as a solvent with a concentration of 6 mg/ml, and carry out NMR characterization.
- the NMR image of m-HA is shown in Figure 2. From the 1 H-NMR spectrum, it can be seen that at ⁇ 4.79ppm is the characteristic peak of D2O ; ⁇ 3-4ppm is the characteristic peak of proton hydrogen in the HA ring structure; ⁇ 1.86ppm is the characteristic peak of H on the methyl group in HA Peak; ⁇ 2.03ppm is the characteristic peak of methyl H on side chain N-acetylglucosamine.
- the two new peaks of m--HA at ⁇ 5.68ppm and ⁇ 6.13ppm are characteristic peaks of olefin protons in methacrylic anhydride, which can indicate that methacrylic anhydride has been successfully grafted to HA in the molecular structure.
- GDMA glycol dimethacrylate, the first crosslinking agent
- AI102 polylactic acid-polyethylene glycol-polylactic acid acrylate, the second crosslinking agent
- the methacrylated hyaluronic acid and the second crosslinking agent AI102 were prepared into an aqueous solution; the first crosslinking agent GDMA and the initiator I2959 were respectively added into DMSO to prepare a solution.
- the molar ratio of the first crosslinking agent GDMA: the second crosslinking agent AI102 is (1-4): (0-2).
- Figure 3 is a physical picture of the pH-responsive hydrogel with controlled release of PDGF-BB.
- the test results of the compressive properties of pH-responsive hydrogels with different cross-linking agent ratios are shown in Fig. 4 .
- PDGF-BB (purchased from the Military Medical Research Institute) was loaded into the pH-responsive hydrogel biocarrier using the dry soaking method, that is, after the hydrogel was completely freeze-dried, it was soaked in the PDGF-BB solution, Make PDGF-BB fully loaded on the hydrogel. Upon completion of the reaction a hydrogel comprising PDGF-BB was formed.
- the hydrogel is sampled with a puncher to make a cylinder with a diameter of 15mm and a height of 8mm, then the hydrogel is freeze-dried for 3 days, and then the freeze-dried hydrogel is soaked in 5ml Concentration of 50ng/ml containing PDGF-BB solution for 48h, to ensure that the hydrogel is fully absorbed, then washed with PBS 3 times, the PDGF-BB not bound to the hydrogel was washed away, and the growth factor-binding Hydrogel biocarriers.
- Example 2 Characterization of m-HA hydrogel loaded with platelet-derived growth factor.
- ELISA enzyme-linked immunoassay
- Figure 7 shows the cumulative release rate of PDGF-BB from pH-responsive hydrogels with different cross-linking agent ratios.
- the GDMA:AI102 ratio is 1:0, the release rate is the fastest.
- the ratio of the two is 1:2, the release rate is the slowest.
- Cytotoxicity test Take the prepared hydrogel without PDGF-BB and the hydrogel with PDGF-BB and put them into a 24-well plate. After continuing to incubate for 24 h in the incubator, the culture medium was removed and passed through a 0.22 ⁇ m filter membrane. Inoculate 1 ⁇ 10 4 cells per well and incubate overnight at 37°C, 5% CO 2 . After adding the above extract solution, continue to incubate for 24 h, and then use the CCK-8 kit to verify whether the hydrogel has cytotoxicity. The ratio of the first cross-linking agent: the second cross-linking agent of the hydrogel used is 1:2.
- Plate laying take a 6-hole plate, and use a marker to draw 5 horizontal lines through the holes on the back of the plate. Dilute the cells to 5 ⁇ 10 4 cells/ml and inoculate 2 ml in each well. Incubate at 37°C, 5% CO 2 , so that the bottom of the well plate is covered with a monolayer.
- Figure 9 is a statistical diagram of the migration of pH-responsive hydrogels on 3T3 cells
- Figure 10 is an observation diagram of the migration of pH-responsive hydrogels on 3T3 cells.
- the ratio of the first cross-linking agent: the second cross-linking agent of the hydrogel used is 1:2.
- 3T3 cells migrated continuously to the middle part, and the area of the scratch was significantly reduced.
- the wound area was quantified by ImageJ, and it was found that the cell migration rate of the unwrapped PDGF-BB hydrogel reached 37% within 36 h, and the cell migration rate of the PDGF-BB-wrapped hydrogel reached 42% within 36 h, while The cell migration rate in the control group was only about 10%. This proves that the PDGF-BB-encapsulated hydrogel can promote the migration of 3T3 cells, which may have a promoting effect on wound healing.
- mice were divided into three groups: hydrogel without PDGF-BB and hydrogel with PDGF-BB.
- the ratio of the first cross-linking agent: the second cross-linking agent of the hydrogel used is 1:2.
- Wound closure rate% (S 0 -S N )/S 0 ⁇ 100 , where S 0 is the initial wound area, and S N is the wound area at day N.
- Fig. 11 is a diagram showing the healing of full-thickness skin wounds in mice with different treatment methods. It can be seen from Figure 11 that the wounds of diabetic mice were gradually shrinking, but the wound healing of the PDGF-BB group wrapped in hydrogel was more significant than that of the other two groups.
- Figure 12 shows the change of mouse wound area over time. At 7 days, the average wound areas of the three groups were 83.4%, 72.8% and 65.0%, respectively; at 14 days, the average wound areas of the three groups became 54.9, 25.1% and 7.8%, compared with the other two groups, the wound area of the hydrogel-wrapped PDGF-BB group decreased the most, so it can be seen that the hydrogel-wrapped PDGF-BB group is beneficial to wound healing.
- the invention provides a pH-responsive hydrogel biocarrier, which is easy for industrial production and has industrial applicability.
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Abstract
本发明公开了一种pH响应型水凝胶生物载体，所述pH响应型水凝胶生物载体由透明质酸钠与甲基丙烯酸酐在光引发剂存在的条件下，用365nm紫外激光照射，透明质酸钠侧链上的羟基通过光致交联经过酯化作用聚合而成透明质酸的甲基丙烯酸酯衍生物。本发明还公开了一种装载有治疗性蛋白PDGF-BB的pH响应型水凝胶生物载体。本发明采用原位自由基聚合的方法合成，反应条件温和，对PDGF-BB的生物活性不会产生影响；把PDGF-BB包裹在水凝胶内，避免暴露在酶环境中，提高了PDGF-BB生物活性的稳定性。
Description
本发明公开一种水凝胶生物载体，属于制药学技术领域。
血小板衍生生长因子（Platelet-derived growth factor, PDGF）是一种耐热、耐酸以及易被蛋白酶水解的阳离子糖蛋白，可由多种细胞分泌，如：血小板、成纤维细胞、巨噬细胞等等。PDGF有四种单体PDGF-A、PDGF-B、PDGF-C和PDGF-D，这些单体通过二硫键连接形成二聚体形式。其中，PDGF-B 的二聚体形式PDGF-BB作为一种有丝分裂促进剂，可以刺激成纤维细胞和平滑肌细胞增殖和迁移、促进巨噬细胞产生和分泌生长因子。在伤口修复、组织再生骨骼和牙齿再生以及关节修复方面的作用更为显著。尤其在伤口修复方面，是美国食品药品监督管理局（FDA）唯一批准的用于糖尿病溃疡治疗的生长因子。
伤口修复和组织再生类疾病需要生长因子在患病部位长期维持一定浓度，但PDGF-BB半衰期短（＜2min），容易被蛋白酶降解，而像慢性伤口床中存在大量蛋白酶，使得PDGF-BB更容易失活。一般会采用多次大剂量给药的方式来维持药物浓度，但这样存在致瘤的风险。所以本领域需要一种载体，所述载体既能够提高PDGF-BB稳定性，又同时能够长期维持一定的药物浓度，以保证PDGF用药的安全性和有效性。
基于本领域存在的上述技术问题，本发明的目的就是提供一种水凝胶生物载体，该载体既能够通过可控性释放PDGF-BB的生物活性，提高其在药效作用上的稳定性，还能够通过所述载体的降解保证药物使用的生物安全性。
本发明综合考虑了甲基丙烯酸酐化透明质酸水凝胶的生物相容性和能够保护生长因子缓慢释放的特点，通过将PDGF-BB引入到甲基丙烯酸酐化透明质酸水凝胶网络中，制备得到结合生长因子型pH响应型水凝胶生物载体，目的在于提高传统水凝胶的生物相容性、pH响应性、细胞片层脱附和保护并缓慢释放生长因子等特性。
基于上述目的，本发明首先提供了一种pH响应型水凝胶生物载体，所述pH响应型水凝胶生物载体由甲基丙烯酸化透明质酸在光引发剂、交联剂存在的条件下，用365nm紫外激光照射引发交联反应制备而成。
在一个优选的技术方案中，所述光引发剂为I2959，所述交联剂为第一交联剂GDMA和第二交联剂AI102。
在另一个优选的技术方案中，所述甲基丙烯酸化透明质酸是由透明质酸钠与甲基丙烯酸酐以摩尔比例为 1：30经过酯化作用合成。
在一个优选的技术方案中，所述第一交联剂GDMA与第二交联剂AI102的摩尔比为(1-4):(0-2)。
其次，本发明提供了一种上述的pH响应型水凝胶生物载体的制备方法，所述方法包括使甲基丙烯酸化透明质酸在光引发剂、第一交联剂GDMA和第二交联剂AI102存在的条件下，用365nm紫外激光照射引发交联反应。
第三，本发明提供了一种装载有治疗性蛋白的上述的pH响应型水凝胶生物载体，所述装载有治疗性蛋白的pH响应型水凝胶生物载体装载是通过将水凝胶生物载体冻干，以干态浸泡法将治疗性蛋白负载在pH响应型水凝胶生物载体内制备而成。
在一个优选的技术方案中，所述治疗性蛋白为血小板衍生生长因子。
在一个更为优选的技术方案中，所述血小板衍生生长因子为BB型二聚体形式。
第四，本发明提供了上述的载有治疗性蛋白的pH响应型水凝胶生物载体的制备方法，所述方法包括将水凝胶生物载体冻干，以干态浸泡法将治疗性蛋白负载在pH响应型水凝胶生物载体内制备的步骤。
最后，本发明提供了上述的载有治疗性蛋白的pH响应型水凝胶生物载体在制备促进伤口修复和组织再生类药物中的应用。
本发明采用原位自由基聚合的方法合成pH响应型水凝胶生物载体，并使用干态浸泡法将治疗性蛋白负载在pH响应型水凝胶生物载体内，反应条件温和，对PDGF-BB的生物活性不会产生影响；把PDGF-BB包裹在水凝胶内，避免暴露在酶环境中，提高稳定性。PDGF-BB的释放是通过水凝胶的降解实现的，本发明选用了响应碱性pH值降解的两种交联剂：第一交联剂GDMA和第二交联剂AI102，两种交联剂的降解基于自身酯键基团，但这两种交联剂在碱性条件下降解速率不同。本发明提供的pH响应型水凝胶生物载体可根据伤口类型的不同，即，伤口部位的pH值的不同，通过控制配伍比例，从而控制了凝胶降解速度，以适应应用要求。鉴于人体伤口部位的pH值是在8.0左右，所述的两种交联剂在8.0时，降解的速度是不同的，第二交联剂比第一交联剂水溶性好，且含有的酯键数量相对较多，所以在降解速率上比第一交联剂快。通过调整两者的比例，例如GDMA：AI102比例为1：2时，释放速率最慢，这样就提供控制两个交联剂之间的比例实现水凝胶不同速率的降解。可以针对不同疾病以及伤口类型的治疗选择合适的交联剂比例（见图1）。
图1：可控释放PDGF-BB pH响应性水凝胶制备的示意图。
图2：m-HA
1H-NMR谱图。
图3：可控释放PDGF-BB pH响应性水凝胶实物图。
图4：不同交联剂比例pH响应性水凝胶压缩性能测试。
图5：不同交联剂比例pH响应性水凝胶扫描电子显微镜图。
图6：不同交联剂比例pH响应性水凝胶溶胀率图。
图7：不同交联剂比例pH响应性水凝胶累计PDGF-BB释放速率图。
图8：pH响应性水凝胶细胞毒性实验。
图9：pH响应性水凝胶对于3T3细胞的迁移作用统计图。
图10. pH响应性水凝胶对于3T3细胞的迁移作用观察图。
图11.不同处理方式小鼠全层皮肤伤口愈合情况观察图。
图12.不同处理方式小鼠全层皮肤伤口面积随时间变化曲线。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1. pH响应型水凝胶生物载体的制备。
图1给出了PDGF-BB pH响应性水凝胶制备的示意图，其中A为甲基丙烯酸酐，B为透明质酸钠，1为第一交联剂GDMA，2为第二交联剂AI102。以下具体介绍制备步骤。
1. 甲基丙烯酸化透明质酸（m-HA）的制备。
使透明质酸钠（HA）与甲基丙烯酸酐（MA）经过酯化作用合成透明质酸的甲基丙烯酸酯衍生物， 具体步骤包括如下。
（1）称取2g透明质酸钠（HA）粉末，溶解于100mL去离子水中，在4℃冰箱中搅拌过夜，将粉末充分溶解。
（2）通过加入5mol/L氢氧化钠调节HA溶液的pH，使反应的pH保持在8到9之间，随后在HA溶液中缓慢滴加1.6mL的甲基丙烯酸酐并在4℃连续搅拌反应24小时。
（3）随后，将m-HA沉淀在过量丙酮中，再用乙醇洗涤，然后溶解在50ml去离子水中。
（4）采用截留分子量为14000Da的透析袋用去离子水透析48小时，以去除未反应的甲基丙烯酸酐和其他副产物，获得纯化的m-HA，将其冻干备用。
以氘代水为溶剂溶解m-HA，浓度为6mg/ml，进行核磁表征，m-HA核磁图见图2。从
1H-NMR谱图可以看出，在δ4.79ppm处为D
2O的特征峰；δ3-4ppm为HA环结构中质子氢的特征峰；δ1.86ppm为HA中甲基上H的特征峰；δ2.03ppm为侧链N-乙酰葡糖胺上甲基H的特征峰。与HA核磁谱图相比，m--HA在δ5.68ppm和δ6.13ppm出现的两个新的峰为甲基丙烯酸酐中烯烃质子的特征峰，可以表明甲基丙烯酸酐成功接枝到了HA分子结构中。
2. pH响应型水凝胶生物载体的制备。
装载有血小板衍生生长因子的m-HA水凝胶的制备。
以m-HA为单体，GDMA（二甲基丙烯酸甘油酯，第一交联剂）和AI102（聚乳酸-聚乙二醇-聚乳酸丙烯酸酯，第二交联剂）为交联剂，然后加入引发剂I2959（2-羟基-4’-（2-羟乙氧基）-2-甲基苯丙酮），在紫外光365nm处引发反应，制备成pH响应型水凝胶生物载体，随后将其冷冻干燥。
具体步骤如下。
取甲基丙烯酸化透明质酸、第二交联剂AI102制备成水溶液；第一交联剂GDMA和引发剂I2959分别加入DMSO制备成溶液。
将m-HA、AI102水溶液、I2959溶液、GDMA溶液在反应管中混合均匀，在365nm紫外光下反应2min，形成pH响应型水凝胶生物载体。将其冻干后，将PDGF-BB负载到水凝胶上。
其中第一交联剂GDMA：第二交联剂AI102的摩尔比为(1-4):(0-2)。
（1） 溶液配制。
取冻干m-HA 20mg，加入去离子水1ml，制成20mg/ml的溶液。
取AI102 50mg，加入去离子水500μl，制成100mg/ml的溶液。
取GDMA 250mg，加入DMSO
500μl，制成500mg/ml的溶液。
取I2959 100mg，加入DMSO1ml，制成100mg/ml的溶液。
（2）制备不同交联剂比例的水凝胶。
表1水凝胶合成参数（交联剂比例为GDMA：AI102）。
将m-HA、AI102水溶液、I2959溶液、GDMA溶液在反应管中混合均匀，在365nm紫外光下反应2min，得到不同比例的水凝胶。
图3为可控释放PDGF-BB pH响应性水凝胶实物图。不同交联剂比例pH响应性水凝胶压缩性能测试结果如图4。随着交联剂量的增加，杨氏模量显著增加。杨氏模量越大，材料越不容易发生形变。相同的压缩应变下交联剂的量越多，水凝胶需要的应力就高，压缩模量就大。由结果可知，当GDMA：AI102=4：1时杨氏模量最大，说明受力时形变最小。
3. pH响应型水凝胶生物载体的PDGF-BB装载。
使用干态浸泡法将PDGF-BB（购自军事医学研究院）装载于pH响应型水凝胶生物载体之中，即，待水凝胶完全冻干，将其浸泡在PDGF-BB溶液中，使PDGF-BB充分负载到水凝胶上。反应完成形成包含PDGF-BB的水凝胶。
具体步骤如下：将水凝胶用打孔器打孔取样，制成直径为15mm，高度为8mm的圆柱体,随后将水凝胶冻干3天，之后将冻干的水凝胶浸泡于5ml浓度为50ng/ml的含有PDGF-BB溶液中48h，以确保水凝胶充分吸收，之后用PBS冲洗3次，将未结合到水凝胶中的PDGF-BB冲洗掉，制得结合生长因子的水凝胶生物载体。
实施例2. 装载有血小板衍生生长因子的m-HA水凝胶的表征。
1、水凝胶形貌表征。
将达到平衡溶胀的水凝胶样品置于-50℃的冻干机中冷冻干燥48h，所得样品在液氮中冷冻脆断后喷金制样，其截面形貌使用扫描电子显微镜（SEM）观察。图5为不同交联剂比例pH响应性水凝胶扫描电子显微镜图像。由此可见，交联剂比例越大，孔径越小，说明交联程度越高。当GDMA与AI102的摩尔比例为1：2时，交联程度最大。
2、水凝胶的溶胀率表征。
利用冷冻干燥后的水凝胶支架进行溶胀性能测试。将水凝胶样品用打孔器打孔，制成直径为1.5cm，高度为12mm的圆柱体，随后将样品冻干。利用分析天平称量并记录各组水凝胶的原始重量 W
0，然后将水凝胶置于10mL的 PBS 缓冲溶液中浸泡，观察水凝胶的溶胀变化，分别在 2 h，6 h，12 h，24 h，48 h 和 72 h 取样。用滤纸小心吸去水凝胶表面的水分，称量并记录水凝胶样品的重量 Wi，每种样品至少设置 3 个重复样，最后结果取平均值。水凝胶的溶胀率（W）可用以下公式计算：溶胀率(W)＝(Wi–W
0
)/W
0×100％。
如图6所示，当GDMA：AI102比例为1：0时，溶胀率最大。当两者比例为4：1时，溶胀率最小。水凝胶支架在刚开始 14 h
内，溶胀程度急剧上升，尤其是两种加入交联剂量少的水凝胶，主要是由于加入的交联剂少，交联度较低，形成的聚合物网络比较疏散，可以吸收大量的水。在浸泡 24 h
左右水凝胶支架达到了溶胀平衡。
3、不同交联剂比例pH响应性水凝胶释放动力学表征。
将负载PDGF-BB的不同交联剂比例的干燥HA-MA水凝胶放置于pH=8的PBS缓冲液中，37℃水浴锅中温育，进行药物控释研究。在特定时间（1、2、3、4、5、7、10天）取出200μL的释放液，同时补充200μL纯净的PBS缓冲液。将收集的释放液通过酶联免疫反应（ELISA）测定释放的PDGF-BB的含量。
不同交联剂比例pH响应性水凝胶累计PDGF-BB释放速率图见图7。当GDMA：AI102比例为1：0时，释放速率最快。当两者比例为1：2时，释放速率最慢。
4. pH响应性水凝胶生物相容性表征。
细胞毒性测试：取制备好的不含PDGF-BB的水凝胶和含PDGF-BB的水凝胶适量加入24孔板中，加入适量只含P/S的培养基（pH=8），在培养箱中继续孵育24 h后，取出培养基，过0.22 μm滤膜。每孔中接种1×10
4个细胞，37 ℃，5% CO
2条件下孵育过夜。把上述浸提液加入后继续孵育24 h，然后使用CCK-8试剂盒来验证水凝胶是否具有细胞毒性。所用到的水凝胶的第一交联剂：第二交联剂的比例为1：2。
pH响应性水凝胶细胞毒性实验结果见图8。包裹不同浓度PDGF-BB的水凝胶的生物活性都大于75%，所以认为制备的水凝胶生物相容性良好。
5. pH响应性水凝胶对于3T3细胞的迁移作用。
（1）无血清 DMEM 培养基(pH=8)配制：取 0.5 ml 双抗，然后加入 49.5 ml DMEM 高糖培养基。
（2）把直尺和记号笔放在超净台紫外杀菌 30
min，然后把 DMEM 完全培养基， 1×PBS 放到 37 ℃水浴锅温育 20 min，胰酶放在室温条件下温育。
（3）铺板：取 6 孔板，在板背部，用记号笔画 5 条过孔的横线。将细胞稀释成5×10
4个/ml，每孔接种 2 ml。37 ℃，5% CO
2条件下孵育，使孔板底部被单层铺满。
（4）为了消除完全培养基促进细胞增殖迁移对于结果的影响，加样前12 h，换成只含 P/S 的培养基。用10μL 枪头垂直于标记线方向划直线。移除旧培养基，用 1×PBS 冲洗 3 次以除去划落的细胞。
（5）用无血清 DMEM 培养基分别浸泡不含PDGF的水凝胶和包含PDGF-BB的水凝胶。取上述提取液 2 ml 分别加入到各个孔中。设置空白组（只含 P/S 的培养基）。加样后放入 5% CO2，37 ℃培养箱孵育。在同一个视野下分别拍摄 0、12、24、36、48 h的照片，使用 Image J 软件计算面积，进而计算出细胞迁移率。
图9为pH响应性水凝胶对于3T3细胞的迁移作用统计图；图10为 pH响应性水凝胶对于3T3细胞的迁移作用观察图。所用到的水凝胶的第一交联剂：第二交联剂的比例为1：2。与只含有水凝胶的对照组相比，使用未包裹的
PDGF-BB和水凝胶包裹的PDGF-BB作用的细胞，3T3细胞不断向中间部位迁移，划痕面积显著减小。通过 ImageJ 定量伤口面积，发现未包裹PDGF-BB 的水凝胶的细胞迁移率在36h 内能达到37%，包裹PDGF-BB的水凝胶细胞迁移率在36 h 时间内能达到42%，而对照组细胞迁移率仅为10%左右。这证明了包裹PDGF-BB的水凝胶能够促进 3T3 细胞迁移，可能对于伤口愈合具有促进作用。
6.小鼠全层皮肤伤口愈合实验。
将小鼠分为不含PDGF-BB的水凝胶和含PDGF-BB的水凝胶三组。所用到的水凝胶的第一交联剂：第二交联剂的比例为1：2。负载药物的水凝胶原位注射至伤口部位(pH=8)，随后覆盖非粘性无菌敷贴，并用3M敷贴固定。分别在不同时间点（0、3、7、10、14天）对小鼠伤口进行拍照，使用直尺作为参照。伤口面积使用Image
J软件计算。
创面闭合率%=（S
0-S
N）/S
0×100 ，其中，S
0为初始创面面积，S
N为N天时创面面积。
图11是不同处理方式小鼠全层皮肤伤口愈合情况图示。从图11中可以看到糖尿病小鼠的伤口都在逐渐缩小，但水凝胶包裹的PDGF-BB组与其他两组相比，伤口愈合更显著。图12的小鼠伤口面积随时间变化曲线显示，7 d时，3组的平均伤口面积分别为83.4%，72.8%和65.0%；14 d时，3组的平均伤口面积变为54.9，25.1%和7.8%，与其他两组相比，水凝胶包裹的PDGF-BB组伤口面积减小的最多，由此可以看出水凝胶包裹的PDGF-BB组有利于伤口愈合。
本发明提供了一种pH响应型水凝胶生物载体，所述pH响应型水凝胶生物载体易于工业化生产，具有工业实用性。
Claims (10)
- 一种pH响应型水凝胶生物载体，其特征在于，所述pH响应型水凝胶生物载体由甲基丙烯酸化透明质酸在光引发剂、交联剂存在的条件下，用365nm紫外激光照射引发交联反应制备而成。
- 根据权利要求1所述的pH响应型水凝胶生物载体，其特征在于，所述光引发剂为I2959，所述交联剂为第一交联剂GDMA和第二交联剂AI102。
- 根据权利要求1所述的pH响应型水凝胶生物载体，其特征在于，所述甲基丙烯酸化透明质酸是由透明质酸钠与甲基丙烯酸酐以摩尔比例为 1：30经过酯化作用合成。
- 根据权利要求2所述的pH响应型水凝胶生物载体，其特征在于，所述第一交联剂GDMA与第二交联剂AI102的摩尔比为(1-4):(0-2)。
- 权利要求1-3任一所述的pH响应型水凝胶生物载体的制备方法，其特征在于，所述方法包括使甲基丙烯酸化透明质酸在光引发剂、第一交联剂GDMA和第二交联剂AI102存在的条件下，用365nm紫外激光照射引发交联反应。
- 一种装载有治疗性蛋白的权利要求1-3任一所述的pH响应型水凝胶生物载体，其特征在于，所述装载有治疗性蛋白的pH响应型水凝胶生物载体装载是通过将水凝胶生物载体冻干，以干态浸泡法将治疗性蛋白负载在pH响应型水凝胶生物载体内制备而成。
- 根据权利要求6所述的装载有治疗性蛋白的pH响应型水凝胶生物载体，其特征在于，所述治疗性蛋白为血小板衍生生长因子。
- 根据权利要求7所述的装载有治疗性蛋白的pH响应型水凝胶生物载体，其特征在于，所述血小板衍生生长因子为BB型二聚体形式。
- 权利要求6-8任一所述的载有治疗性蛋白的pH响应型水凝胶生物载体的制备方法，其特征在于，所述方法包括将水凝胶生物载体冻干，以干态浸泡法将治疗性蛋白负载在pH响应型水凝胶生物载体内制备的步骤。
- 权利要求6-8任一所述的载有治疗性蛋白的pH响应型水凝胶生物载体在制备促进伤口修复和组织再生类药物中的应用。
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