注射用交联透明质酸钠凝胶的制备及其体外抗酶降解性的研究

目的筛选交联反应条件,制备强抗酶降解的注射用交联透明质酸钠(HA)凝胶(HA-凝胶),并建立其体外抗酶降解性的测定方法。方法用1,4-丁二醇二缩水甘油醚(BDDE)交联HA制备HA-凝胶,对其体外抗酶降解性的测定方法进行确证;采用正交试验考察6个因素对交联反应的影响,选择最佳反应条件。结果透明质酸酶(HAas)对HA-凝胶体外抗酶降解性测定方法无干扰,HAas在100~500 U/mL与产物显色后的吸光度呈线性关系(r=0.998 8)。正交试验极差分析和方差分析显示HA的起始反应浓度、BDDE与HA的比例对交联反应具有显著性影响,在氢氧化钠浓度1%、HA平均相对分子质量1.2×106、反应温度50℃、BDDE与HA的比例为1∶10(g/g)、反应时间4 h和HA浓度为15%的反应条件下,可得到抗酶降解性最好的HA-凝胶。结论选择适宜反应条件可以制备得到体外抗酶降解性强的HA-凝胶,为进一步开发注射美容和整形产品奠定了基础。     

交联固化时间对利多卡因水凝胶贴剂的影响

目的 研究交联固化时间对利多卡因水凝胶贴剂的初黏力、持黏力、剥离强度、体外释放度等的影响.方法 以初黏力、持黏力、剥离强度为黏性评价指标,采用桨碟法测定体外释放度,用计算相似因子f2值比较释放度,以水凝胶溶胀率和弹性模量E'分析其内部结构和成型机理.结果 初黏力、持黏力、剥离强度在交联固化前7d迅速降低,之后趋于稳定;利多卡因的体外释放度、水凝胶溶胀率和弹性模量E'随着交联固化时间的增加先增加再趋于稳定.结论 交联固化的初始7d为快速反应期,对初黏力、持黏力、剥离强度体外释放度的影响较大.交联固化7d之后,凝胶交联网络基本形成,水凝胶贴剂的质量相对稳定.     

阿霉素载药栓塞微球的制备和性能表征

目的：研究离子交换型阿霉素载药透明质酸栓塞微球（DHAMs）的制备方法及性能表征。方法：采用油包水（W/O）乳化交联法,以改性透明质酸（HA）为载药基质,通过离子交换原理制备载阿霉素的透明质酸栓塞微球。采用光学显微镜、扫描电子显微镜观察表面形态,考察粒径和溶胀率;采用恒温摇床模拟体内环境初步测定透明质酸微球降解率;采用紫外分光光度法测定DHAMs载药量、包封率和体外释放率;采用傅里叶变换红外光谱（FT-IR）和粉末X射线衍射法（PXRD）对DHAMs进行表征。结果：透明质酸微球形态圆整、表面光滑,平均粒径为（111.83 ± 12.21）μm,溶胀率为201.81%;微球56 d体外降解率为34.31%;微球载药量和包封率分别为（22.09% ± 0.05%）和（99.41% ± 0.21%）,且72 h累计释放量可达50%;FT-IR和PXRD分析显示,阿霉素在微球中以无定形形式存在。结论：阿霉素透明质酸微球有较好的载药性能,理化性能良好,具有动脉化疗栓塞的应用前景。     

胶原/氧化透明质酸复合水凝胶支架的制备与表征

目的制备复合水凝胶并初步考察其在软骨组织工程中的应用。方法用高碘酸钠对透明质酸进行改性,与中性的胶原反应,37℃下形成水凝胶。二甲氨基苯甲醛反应测透明质酸含量并用TNBS法测胶原与氧化透明质酸的交联度。软骨细胞包埋于水凝胶中培养并观察其生长状态。结果氧化透明质酸被有效固定于水凝胶中,胶原的氨基与氧化透明质酸的醛基反应形成西夫碱结构,二者交联度随氧化透明质酸含量的增加而提高。软骨细胞在水凝胶中均匀分布且活性良好,透明质酸的添加对细胞生长有一定促进作用。结论胶原/氧化透明质酸复合水凝胶仿生支架在软骨组织工程中有很大的应用前景。     

聚乙二醇制备的透明质酸衍生物的生物降解性

目的对聚乙二醇(PEG)交联制备的透明质酸(HA)衍生物薄膜的含水量及其体内和体外生物降解特性进行初步研究。方法25℃利用5 d时间制备200μm厚的HA衍生物薄膜,将其干燥后制备成10 mm×10 mm规格的试样,浸泡24 h后计算其含水量。将上述得到的HA衍生物薄膜浸入磷酸盐缓冲液(PBS)和植入到白鼠背部皮下组织,测试其体外和体内降解特性。结果当PEG浓度大于2.5%,pH 8.0时,经交联制备的HA衍生物水凝胶含水量可低于60%(w/w);当pH 8.0,PEG浓度为3.8%时HA衍生物薄膜在37℃浸泡在PBS中至少10 d无任何重量改变;将其植入白鼠皮下组织1周后,约有75%的交联薄膜仍存留在白鼠皮下组织中。结论可以利用PEG交联制备含水量较低、生物降解性小的HA衍生物凝胶材料。     

复合基质水凝胶微针的制备及性能研究

该研究以聚乙烯醇(PVA)和甲基乙烯基醚/马来酸共聚物(PVM/MA)或甲基乙烯基醚/马来酸酐共聚物(PMVE/MAH)作为复合基质材料，采用热交联法制备水凝胶微针，考察热交联温度、时间以及复合材料种类对水凝胶微针形态、溶胀率的影响，筛选并优化热交联工艺条件。PVM/MA和PVA(1∶1)在80℃/2 h、95℃/0.5 h和110℃/0.5 h以及PMVE/MAH和PVA(1∶1)在80℃/2 h、95℃/1 h和110℃/0.5 h条件下制备的水凝胶微针均显示出良好的溶胀率(>200%)、凝胶组分百分比(>80%)、疗效和安全性。对制备的水凝胶微针进行XRD和DSC分析，结果显示，与物理混合物相比，以PMVE/MAH和PVA(1∶1)制备的水凝胶微针的XRD图谱中2θ为19.45°处PVA衍射峰的消失，玻璃化转变温度(Tg)从约150℃降至约58℃，说明聚合物发生了酯化反应，成功交联。该研究为PVA和PVM/MA或PMVE/MAH复合基质水凝胶微针的透皮给药应用提供了参考。     

氨基葡萄糖-Cu(Ⅱ)配合物清除过氧化氢的研究

目的：研究氨基葡萄糖与Cu^2 形成的配合物对过氧化氢分解的催化性能及影响因素。方法：利用D-氨基萄萄糖盐酸盐在水溶液中与Cu^2 形成配合物；加入到一定浓度的H2O2中，在不同的条件下放置，于不同的时间测定H2O2的分解率。结果：在30℃及pH6．5条件下，氨基葡萄糖-Cu(Ⅱ)配合物作用12h，可使90％以上的H2O2降解,24h后分解几乎完全。结论：氨基葡萄糖-Cu(Ⅱ)配合物对H2O2分解具有明显的催化作用。     

用作盲肠位点药物传载的交联凝胶的体外降解

目的　考察用作结肠位点药物传载体的偶氮苯交联凝胶的降解机制及影响因素。方法　凝胶经大鼠结肠菌培养,在不同时间间隔内取出用蒸馏水洗净,加15%氢氧化钠加热水解后,用紫外光度法测定其降解率。结果　这类凝胶能被盲肠内的厌氧菌降解,其降解率与凝胶的溶胀程度有关,凝胶的溶胀程度越大,其降解率越高。凝胶的降解率与溶胀程度间的关系主要取决于网络孔密度和孔尺寸。影响网络孔密度和孔尺寸的因素包括网络上疏水基的长度、交联程度及组成等,通过改变这些因素可以达到调节网络孔密度和孔尺寸,从而控制其降解率和药物释放的目的。结论　电子载体对凝胶降解率的影响研究,不仅证实盲肠菌对芳香族偶氮化合物的还原发生在细胞体外,而且亦显示偶氮还原酶存在于细胞体内,电子载体的功能是穿梭于细胞内的偶氮还原酶与细胞外的偶氮基团之间起传递电子的作用。本实验结果对开发结肠位点的控释传载体有重要的指导意义。     

克林霉素磷酸酯聚氨酯水凝胶栓剂的制备及体外释放

目的：制备一种基于聚氨酯水凝胶的克林霉素磷酸酯缓释栓剂,并考察栓剂的体外释放及其影响因素。方法：以聚乙二醇6000、二环己基甲烷二异氰酸酯及1,2,6-己三醇为原料,采用一步法合成聚氨酯水凝胶材料,并通过傅里叶变换红外光谱及溶胀率对其进行初步表征。将聚氨酯水凝胶制成特定形状的空白栓剂,载入克林霉素磷酸酯后考察所得载药栓剂的体外释放行为,并探讨聚氨酯的化学组成、栓剂厚度、栓剂载药量及释放介质pH对载药栓剂体外释放的影响。通过Ritger—Peppas方程对释放数据进行拟合,探讨栓剂的释放机制。结果：合成了4批不同化学组成的聚氨酯水凝胶材料,其结构经FTIR确证,溶胀率则随交联率的增加而逐渐降低。制备的栓剂中克林霉素磷酸酯占目标载药量的103．0％（RSD=1．8％,／1,=10）。体外释放结果显示聚氨酯的化学组成及栓剂载药量对释放基本无影响;释放介质pH的升高及栓剂厚度的增加会减缓药物释放。释放数据分析结果表明克林霉素磷酸酯的释放由溶胀及扩散双重机制控制。结论：基于聚氨酯水凝胶的克林霉素磷酸酯栓剂制备方法简单、可控,体外释放完全,具有良好的应用前景。     

白藜芦醇改性壳聚糖水凝胶的制备及体外释放研究

目的研究聚乙二醇化的壳聚糖对白藜芦醇体外释放的影响及载药水凝胶对氧化应激状态下人视网膜色素上皮细胞的保护作用。方法通过有机合成、化学交联等方法制备聚乙二醇-壳聚糖水凝胶,以白藜芦醇为模型药物,采用透析袋法研究其释放机制,对释放曲线进行拟合分析;建立H2O2诱导的ARPE-19细胞的氧化应激模型,利用试剂盒测定乳酸脱氢酶释放率及丙二酸含量。结果与白藜芦醇对照组相比,载药水凝胶的体外释放度明显提高(前者为24%,后者为82%); H2O2能够增加乳酸脱氢酶释放率和丙二酸含量,应用白藜芦醇后降低了以上效应,呈现剂量依赖性;相较于同浓度白藜芦醇对照组,载药水凝胶的抑制作用更好。结论聚乙二醇-壳聚糖水凝胶可提高白藜芦醇的体外释放率、抗氧化保护作用更明显。     

Effect of Peptide Loading and Surfactant Concentration on the Characteristics of Physically Crosslinked Hydrogel

The purpose of this study was to investigate the effect of varying drug load and concentration of a surfactant (sodium lauryl sulfate [SLS]) on the release characteristics of a model peptide (bovine serum albumin [BSA]), and study the net effects of the swelling properties of the hydrogel matrix [poly(vinyl alcohol) (PVA)]. The PVA hydrogel was prepared by a freeze-thaw process in the absence of a chemical crosslinking agent. The effect of protein loading on drug release was examined at three levels (0.65, 1.3, and 2%), whereas the effect of SLS was studied at four levels (0, 0.07, 0.13, and 0.26%). The baseline time for reaching equilibrium swelling was 48 hr for the hydrogel containing 0.65% BSA, and the equilibrium swelling time decreased significantly as the protein load was increased to 2%. The net effect of increased BSA concentrations resulted in faster BSA dissolution from the hydrogel matrix. The equilibrium-swelling ratio decreased from 21 to 10% when SLS was added to the PVA solution, which resulted in a reduction in the extent of equilibrium swelling; however, the time to reach equilibrium swelling was increased. The investigation provided a mechanistic basis toward the development of a hydrogel formulation by altering the concentration of two fundamental components, i.e., drug and surfactant, within the delivery system.     

Effect of peptide loading and surfactant concentration on the characteristics of physically crosslinked hydrogel

The purpose of this study was to investigate the effect of varying drug load and concentration of a surfactant (sodium lauryl sulfate [SLS]) on the release characteristics of a model peptide (bovine serum albumin [BSA]), and study the net effects of the swelling properties of the hydrogel matrix [poly(vinyl alcohol) (PVA)]. The PVA hydrogel was prepared by a freeze-thaw process in the absence of a chemical crosslinking agent. The effect of protein loading on drug release was examined at three levels (0.65, 1.3, and 2%), whereas the effect of SLS was studied at four levels (0, 0.07, 0.13, and 0.26%). The baseline time for reaching equilibrium swelling was 48 hr for the hydrogel containing 0.65% BSA, and the equilibrium swelling time decreased significantly as the protein load was increased to 2%. The net effect of increased BSA concentrations resulted in faster BSA dissolution from the hydrogel matrix. The equilibrium-swelling ratio decreased from 21 to 10% when SLS was added to the PVA solution, which resulted in a reduction in the extent of equilibrium swelling; however, the time to reach equilibrium swelling was increased. The investigation provided a mechanistic basis toward the development of a hydrogel formulation by altering the concentration of two fundamental components, i.e., drug and surfactant, within the delivery system.     

Mechanism of acetaminophen-stimulated NADPH oxidation catalyzed by the peroxidase-H2O2 system.

The oxidation of NADPH catalyzed by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) is markedly increased by the presence of acetaminophen in a concentration-dependent manner. The oxidation follows pseudo-first order kinetics with respect to acetaminophen concentration. The product of the oxidation is enzymatically active NADP+. The stoichiometry of the reaction shows that 1.4 mol of NADPH are oxidized per mole of H2O2 added, and the addition of superoxide dismutase to the reaction mixture increases the ratio of NADPH oxidized:H2O2 consumed, which suggests formation of superoxide as a product. Monitoring cytochrome c reduction in the presence and absence of superoxide dismutase further suggests formation of superoxide. These results indicate that the HRP-H2O2 system oxidizes acetaminophen to the phenoxyl radical, N-acetyl-p-benzosemiquinone imime, which undergoes a rapid electron transfer reaction with NADPH. The NADP thus formed reacts with molecular oxygen to produce superoxide.     

Influence of β-cyclodextrin concentration and polyacrylic acid molecular weight on swelling and release characteristics of metoclopramide-containing hydrogels

We evaluated the effects of polyacrylic acid (PAA) molecular weight and crosslinking agent (β-cyclodextrin) concentration on swelling and drug (metoclopramide) release characteristics of PAA hydrogels. Both factors, and the interactions between them, were found to have significant effects on both hydrogel swelling and drug release. In particular, increased β-cyclodextrin concentrations led to reduced swelling and reduced drug release efficiency.     

Design and evaluation of buccoadhesive metoclopramide hydrogels composed of poly(acrylic acid) crosslinked with sucrose

We evaluated the effects of polyacrylic acid (PAA) molecular weight and crosslinking agent (sucrose) concentration on swelling and drug (metoclopramide) release characteristics of PAA (Carbopol) hydrogels. Both factors, and the interactions between them, were found to have significant effects on both hydrogel swelling and drug release. In particular, increased sucrose concentration led to reduced swelling and reduced drug release efficiency.     

透明质酸酶催化透明质酸水解的最适反应条件

目的确定透明质酸酶(HAase)催化透明质酸(HA)水解的最适条件。方法HAase不同条件下催化HA水解,反应结束后利用高效凝胶渗透色谱(HPGPC)法测量反应产物的相对分子质量及其分布系数。结果HAase催化HA水解受温度、pH值、酶浓度、底物浓度、反应时间等因素的影响。结论HAase催化HA水解的最适反应条件是底物浓度为10 g/L,酶浓度为150 000 U/L,pH为5.0,反应温度为50℃。     

An electron spin resonance study of the activation of benzidine by peroxidases

The oxidation of benzidine, a carcinogenic aromatic amine, by H2O2 is catalyzed by horseradish peroxidase or lactoperoxidase. The resulting cation free radical is moderately stable at pH 5.0, and was identified by electron spin resonance spectroscopy. Two-electron oxidation yields the benzidine di-imine. This species reacts with phenol or catechol derivatives to give colored adducts. Monoacetylbenzidine is a relatively poor peroxidase substrate, and the biological implications of this difference are discussed.     

Inactivation of cholinesterase induced by chlorpromazine cation radicals

To clarify the mechanism of the side effect of chlorpromazine, we examined the inactivation of cholinesterase induced by chlorpromazine. Cholinesterase was inactivated and its activity was lost in rat serum during interaction of chlorpromazine with horseradish peroxidase and H2O2. When chlorpromazine was oxidized by horseradish peroxidase and H2O2, the reaction solution colored pink and the visible absorption spectrum was consistent with the absorption spectrum of the chlorpromazine cation radical (CPZ*+). Adding cholinesterase immediately decreased the pink color of CPZ*+, indicating that CPZ*+ directly attacked cholinesterase to cause loss of the enzyme activity. Tryptophan residues in cholinesterase sharply decreased during the interaction of cholinesterase with horseradish peroxidase and H2O2. Presumably, loss of tryptophan residues changed the conformation of the cholinesterase protein and then the activity of the enzyme was lost. Other phenothiazine derivatives, including promethazine, triflupromazine, trifluoperazine, trimeprazine, thioridazine and perphenazine, also inactivated cholinesterase during the oxidation by horseradish peroxidase and H2O2. These results suggest that phenothiazine cation radicals participate in toxicological signs caused by the drugs.     

Structural and biological investigation of chitosan/hyaluronic acid with silanized-hydroxypropyl methylcellulose as an injectable reinforced interpenetrating network hydrogel for cartilage tissue engineering

Cartilage damage continues to pose a threat to humans, but no treatment is currently available to fully restore cartilage function. In this study, a new class of composite hydrogels derived from water-soluble chitosan (CS)/hyaluronic acid (HA) and silanized-hydroxypropyl methylcellulose (Si-HPMC) (CS/HA/Si-HPMC) has been synthesized and tested as injectable hydrogels for cartilage tissue engineering when combined without the addition of a chemical crosslinking agent. Mechanical studies of CS/HA and CS/HA/Si-HPMC hydrogels showed that as Si-HPMC content increased, swelling rate and rheological properties were higher, compressive strength decreased and degradation was faster. Our results demonstrate that the CS and HA-based hydrogel scaffolds, especially the ones with 3.0% (w/v) Si-HPMC and 2.5/4.0% (w/v) CS/HA, have suitable physical performance and bioactive properties, thus provide a potential opportunity to be used for cartilage tissue engineering. In vitro studies of CS/HA and CS/HA/Si-HPMC hydrogels encapsulated in chondrocytes have shown that the proper amount of Si-HPMC increases the proliferation and deposition of the cartilage extracellular matrix. The regeneration rate of the CS/HA/Si-HPMC (3%) hydrogel reached about 79.5% at 21 days for long retention periods, indicating relatively good in vivo bone regeneration. These CS/HA/Si-HPMC hydrogels are promising candidates for tissue compatibility injectable scaffolds. The data provide proof of the principle that the resulting hydrogel has an excellent ability to repair joint cartilage using a tissue-engineered approach.

RESEARCH HIGHLIGHTS

• An injectable hydrogel based on CS/HA/Si-HPMC composites was developed.
• The CS/HA/Si-HPMC hydrogel displays the tunable rheological with mechanical properties.
• The CS/HA/Si-HPMC hydrogel is highly porous with high swelling and degradation ratio.
• Increasing concentration of Si-HPMC promote an organized network in CS/HA/Si-HPMC hydrogels.
• Injectable CS/HA/Si-HPMC hydrogels have a high potential for cartilage tissue engineering.