Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.     

pH-敏感甲硝唑壳聚糖-聚乙烯吡咯烷酮水凝胶的研究

 目的 制备pH-敏感的壳聚糖-聚乙烯吡咯烷酮（PVP）水凝胶，作为甲硝唑治疗胃溃疡控释给药系统的载体。方法 用戊二醛交联适宜比例混合的壳聚糖、PVP混合物，负载甲硝唑或不负载甲硝唑，制备载药水凝胶及空白水凝胶，再冷冻干燥形成半渗透聚合网状物Semi-IPN。用扫描电镜观察性状结构，用测角仪测定其辛烷接触角，用电子天平测其在不同pH介质中的膨胀率，用红外测定了其特征峰，同时考察了载药水凝胶体外释药特性，初步药理实验判断其对实验性胃溃疡的疗效。结果 水凝胶具多孔性，且可很好地负载甲硝唑。辛烷接触角为(143.8±1.02)°，显示本品有较大的亲水性。膨胀率随pH的降低而增大，红外图谱说明水凝胶在酸性介质中膨胀时有-NH₂质子化。甲硝唑在酸性介质中的释放也随pH降低而增大，在pH 1.0介质中，3 h达最大释放量87%。初步药理实验表明，与对照组及甲硝唑片治疗组相比，该甲硝唑水凝胶对胃溃疡具有较优的治疗作用（P分别<0.01及P<0.05）。结论 甲硝唑水凝胶具有pH-敏感膨胀性，可改善甲硝唑对实验性胃溃疡的治疗作用，可作为治疗胃溃疡的新型胃部控释系统研究开发。     

海藻酸钙凝胶微球中模型药物的pH值依赖性释放

目的:考察海藻酸钙凝胶微球中模型药物的pH值依赖性释放.方法:以硝苯地平为模型药,采用滴制法制备了含药微球;考察了含药微球在不同pH值介质中的释放特性.结果:含药微球在水及pH 1.0的介质中几乎不溶胀,12 h累积释放百分率为23.1%和23.4%;在pH6.8的介质中微球溶胀至完全溶蚀,且呈现缓慢释放的趋势,12 h药物的累积释放百分率为92.5%;换介质的释放中,0～2 h微球几乎不溶胀,2 h累积释放8.4%,介质pH改变后微球很快崩解,3 h累积释放82.4%.结论:海藻酸钙凝胶微球中硝苯地平的释放具有pH值依赖性,在pH 6.8的介质中缓释.     

阿霉素化学键接聚合物纳米颗粒的合成及pH敏感释放

合成了前驱体三-(2-氨乙基)-N,N',N"-三硫辛酸氨,该前驱体经三丁基膦(TBUP)还原后得到末端带有6个巯基的小分子单体(C-6SH)并共价偶联腙键修饰的阿霉素-(6-马来酰亚胺基己酰)腙(MAL-DOX)。通过巯基与丙烯酸酯的点击化学反应,以二甲基丙烯酸乙二醇酯(EGDMA)为交联剂,连续投料制备了具有内核交联、表面聚乙二醇(PEG)化与pH敏感药物释放特性的聚合物纳米颗粒。采用动态光散射粒径分析(DLS)和透射电镜(TEM)对纳米颗粒的尺寸和形貌进行表征,结果表明该纳米颗粒呈球形,平均尺寸为42 nm。体外药物释放表明:通过腙键连接阿霉素(DOX)的聚合物纳米颗粒具有良好的pH响应性释放能力,即在酸性条件下(pH=5.5)的释放速率比生理pH环境下要明显加快。     

Design and synthesis of pH-sensitive polymeric micelles for oral delivery of poorly water-soluble drugs

pH-sensitive polymer poly (polylactide-co-methacrylic acid)–b-poly (acrylic acid) was synthesized using atom transfer radical polymerization and ring-opening polymerization and characterized by gel permeation chromatography and ¹H NMR. The polymers can self-assemble to form micelles in aqueous medium, which respond rapidly to pH change within the gastrointestinal relevant pH range. Critical micelle concentrations and pH response behavior of the polymeric micelle were investigated. Water-insoluble drug nifedipine was loaded and the drug-loading content can be controlled by tuning the composition of the polymers. The in vitro release studies indicate pH sensitivity enabled rapid drug release at the environment of simulated intestinal fluid (pH 7.36), the cumulative released amount of NFD reached more than 80% within 24 h, while only 35% in the simulated gastric fluid (pH 1.35). All the results showed that the pH-sensitive P(PLAMA-co-MAA)–b-PAA micelle may be a prospective candidate as oral drug delivery carrier for hydrophobic drugs with controlled release behavior.     

pH-sensitive carbonate apatite nanoparticles as DNA vaccine carriers enhance humoral and cellular immunity

To demonstrate the potential of pH-sensitive carbonate apatite (CO₃Ap) nanoparticles as DNA vaccine carriers to enhance vaccination efficacy, we examined the humoral and cellular immune responses of C57BL/6 mice immunized with the plasmid expression vector pCI-neo encoding the full-length soluble ovalbumin (OVA) (pCI-neo-sOVA), pCI-neo-sOVA/CO₃Ap complexes, or pCI-neo/CO₃Ap complexes as a control. Mice immunized with a low dose of pCI-neo-sOVA-loaded CO₃Ap (10 μg) produced ex vivo splenocyte proliferation after stimulation with CD8 T-cell but not CD4 T-cell epitopes and a delayed-type-hypersensitivity reaction more efficiently than mice in the other groups. Furthermore, mice receiving this immunization generated the same levels of OVA-specific antibodies and interferon (IFN)-γ secretion after CD8 T-cell and CD4 T-cell epitope challenges as those in mice treated with 100 μg of free pCI-neo-sOVA, whereas mice injected with a high dose of pCI-neo-sOVA-loaded CO₃Ap (100 μg) or with control plasmids produced negligible levels of OVA-specific antibodies or IFN-γ. Therefore, our results showed that 10 μg of pCI-neo-sOVA delivered by CO₃Ap strongly elicited humoral and cellular immune responses. This study is the first to demonstrate the promising potential of CO₃Ap nanoparticles for DNA vaccine delivery.     

Inhibition of hypoxia-induced proliferation of pulmonary arterial smooth muscle cells by a mTOR siRNA-loaded cyclodextrin nanovector

The proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH), an intractable disease, for which pharmacotherapy is limited and only slight improvement in survival outcomes have achieved over the past few decades. RNA interference provides a highly promising strategy to the treatment of this chronic lung disease, while efficient delivery of small interfering RNA (siRNA) remains a key challenge for the development of clinically acceptable siRNA therapeutics. With the aim to construct useful nanomedicines, the mammalian target of rapamycin (mTOR) siRNA was loaded into hybrid nanoparticles based on low molecular weight (Mw) polyethylenimine (PEI) and a pH-responsive cyclodextrin material (Ac-aCD) or poly(lactic-co-glycolic acid) (PLGA). This hybrid nanoplatform gave rise to desirable siRNA loading, and the payload release could be modulated by the hydrolysis characteristics of carrier materials. Fluorescence observation and flow cytometry quantification suggested that both Ac-aCD and PLGA nanovectors (NVs) may enter PASMCs under either normoxia or hypoxia conditions as well as in the presence of serum, with uptake and transfection efficiency significantly higher than those of cationic vectors such as PEI with Mw of 25 kDa (PEI25k) and Lipofectamine 2000 (Lipo 2k). Hybrid Ac-aCD or PLGA NV containing siRNA remarkably inhibited proliferation and activated apoptosis of hypoxic PASMCs, largely resulting from effective suppression of mTOR signaling as evidenced by significantly lowered expression of mTOR mRNA and phosphorylated protein. Moreover, these hybrid nanomedicines were more effective than commonly used cationic vectors like PEI25k and Lipo 2k, with respect to cell growth inhibition, apoptosis activation, and expression attenuation of mTOR mRNA and protein. Therefore, mTOR siRNA nanomedicines based on hybrid Ac-aCD or PLGA NV may be promising therapeutics for diseases related to hypoxic abnormal growth of PASMCs.     

The anti-tumor efficacy of curcumin when delivered by size/charge-changing multistage polymeric micelles based on amphiphilic poly(β-amino ester) derivates

Modifying positive surface charge and reducing bulk size of nanoparticles has been proven beneficial to cancer cellular delivery, but meanwhile results in fast clearance and unspecific distribution in body after intravenous injection. How to balance these problems is still a challenge to construct an ideal nano-scaled drug delivery system in cancer treatment. Herein, we developed a multistage drug delivery system to enhance anticancer efficacy of curcumin (CUR), which could intelligently alter its size and surface charge after long-circulation and extravasation from leaky blood vessels at tumor sites. This micellar system was constructed by amphiphilic and pH-sensitive methoxy poly(ethylene glycol)-poly(lactide)-poly(β-amino ester) (MPEG-PLA-PAE) copolymers. As compared with MPEG-PLA micelles, MPEG-PLA-PAE micelles displayed several advantageous characteristics for drug delivery and treatment. We found that CUR-loaded MPEG-PLA-PAE micelles remained stable in murine plasma at 37 °C even with high drug loading. More interestingly, when the media pH decreased from 7.4 to 5.5, the micelles shrank from 171.0 nm to 22.6 nm and their surface charge increased to 24.8 mV meanwhile, which resulted in the significantly improved cell uptake of CUR by human breast cancer MCF-7 cells. Using indocyanine green (ICG) as a fluorescence probe, it was observed that MPEG-PLA-PAE micelles experienced longer circulation than MPEG-PLA micelles followed by accumulation at tumors with stronger fluorescence intensity. Consequently, MPEG-PLA-PAE micelles achieved enhanced cancer growth inhibition of 65.6% in vivo. All these findings demonstrated the potential of size/charge–changing MPEG-PLA-PAE micelles as a promising drug delivery system for tumor-targeted therapy.     

Potentiation of pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy

Cationic lipid-incorporated liposomes modified with pH-sensitive polymers were prepared by introducing 3, 5-didodecyloxybenzamidine as a cationic lipid to egg yolk phosphatidylcholine liposomes modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG) as a pH-sensitive polymer. These liposomes were stable at neutral pH, but were destabilized below pH 6.0 because MGlu-HPG changed its characteristics from hydrophilic to hydrophobic in response to the pH decrease. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and association of liposomes with murine dendritic cell (DC) lines. Cationic lipid-incorporated liposomes delivered entrapped ovalbumin (OVA) molecules not only to cytosol but also to endosome/lysosome. Treatment with cationic lipid-incorporated liposomes induced up-regulation of antigen presentation-involved molecules on DCs, the promotion of cytokine production, and antigen presentation via both major histocompatibility complex (MHC) class I and II molecules. Especially, antigen presentation via MHC class II was promoted by cationic lipid inclusion, which might correspond to efficient endosome/lysosome delivery of OVA. Subcutaneous administration of OVA-loaded cationic lipid-incorporated liposomes induced antigen-specific antibody production in serum and Th1-dominant immune responses in the spleen. Furthermore, administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor more significantly reduced the tumor volume than liposomes without cationic lipids. Therefore, cationic lipid inclusion into pH-sensitive polymer-modified liposomes, which can achieve both efficient antigen intracellular delivery and activation of antigen presenting cell, is an effective approach to develop antigen carriers for efficient cancer immunotherapy.