Poly(ethylene glycol)-modified Nanocarriers for Tumor-targeted and Intracellular Delivery

The success of anti-cancer therapies largely depends on the ability of the therapeutics to reach their designated cellular and intracellular target sites, while minimizing accumulation and action at non-specific sites. Surface modification of nanoparticulate carriers with poly(ethylene glycol) (PEG)/poly(ethylene oxide) (PEO) has emerged as a strategy to enhance solubility of hydrophobic drugs, prolong circulation time, minimize non-specific uptake, and allow for specific tumor-targeting through the enhanced permeability and retention effect. Furthermore, PEG/PEO modification has emerged as a platform for incorporation of active targeting ligands, thereby providing the drug and gene carriers with specific tumor-targeting properties through a flexible tether. This review focuses on the recent developments surrounding such PEG/PEO-surface modification of polymeric nanocarriers to promote tumor-targeting capabilities, thereby enhancing efficacy of anti-cancer therapeutic strategies.     

Poly(ethylene glycol)-Modified Proteins: Implications for Poly(lactide-co-glycolide)-Based Microsphere Delivery

The reduced injection frequency and more nearly constant serum concentrations afforded by sustained release devices have been exploited for the chronic delivery of several therapeutic peptides via poly(lactide-co-glycolide) (PLG) microspheres. The clinical success of these formulations has motivated the exploration of similar depot systems for chronic protein delivery; however, this application has not been fully realized in practice. Problems with the delivery of unmodified proteins in PLG depot systems include high initial “burst” release and irreversible adsorption of protein to the biodegradable polymer. Further, protein activity may be lost due to the damaging effects of protein-interface and protein-surface interactions that occur during both microsphere formation and release. Several techniques are discussed in this review that may improve the performance of PLG depot delivery systems for proteins. One promising approach is the covalent attachment of poly(ethylene glycol) (PEG) to the protein prior to encapsulation in the PLG microspheres. The combination of the extended circulation time of PEGylated proteins and the shielding and potential stabilizing effects of the attached PEG may lead to improved release kinetics from PLG microsphere system and more complete release of the active conjugate.     

Surface Modification of Poly(lactide-co-glycolide) Nanospheres by Biodegradable Poly(lactide)-Poly(ethylene glycol) Copolymers

The modification of surface properties of biodegradable poly(lactide-co-glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethlene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da, respectively) were studied. The results reveal the formation of a PLA: PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems. A similar biodistribution pattern of PLA:PEG 3:4 to PEG 2:5 coated particles may indicate that poly(ethylene glycol) chains in the range of 2000 to 5000 can produce a comparable effect on in vivo behaviour.     

Controlled Delivery Systems for Proteins Based on Poly(Lactic/Glycolic Acid) Microspheres

This paper describes an investigation of the use of poly(lactic/glycolic acid) polymers for long-term delivery of high molecular weight, water-soluble proteins. Poly(lactic/glycolic acid) (PLGA) microspheres, containing (fluorescein isothiocyanate)-labeled bovine serum albumin and (fluorescein isothiocyanate)-labeled horseradish peroxidase, were prepared by a modified solvent evaporation method using a double emulsion. The microspheres were spherical with diameters of 55–95 µm and encapsulated more than 90% of the protein. The preparation method was gentle and maintained enzyme activity and protein solubility. Stability studies showed that the encapsulation of an enzyme inside PLGA microspheres can protect them from activity loss. When not placed inside PLGA microspheres, (fluorescein isothiocyanate)-labeled horseradish peroxidase lost 80% of its activity in solution at 37°C in a few days, whereas inside the PLGA microspheres it retained more than 55% of its activity after 21 days of incubation at 37°C. In vitro release studies revealed that different release profiles (i.e., near-constant or biphasic) and release rates can be achieved by simply modifying factors in the preparation procedure such as mixing rate and volume of inner water and organic phases. Degradation studies by scanning electron microscopy and gel-permeation chromatography suggested that the mechanism responsible for protein release is mainly through matrix erosion.     

Synthesis of Novel Biodegradable Methoxy Poly(ethylene glycol)-Zein Micelles for Effective Delivery of Curcumin

Novel biodegradable micelles were synthesized by conjugating methoxy poly(ethylene glycol) (mPEG) to zein, a biodegradable hydrophobic plant protein. The mPEG-zein micelles were in the size range of 95-125 nm with a low CMC (5.5 × 10(-2) g/L). The micelles were nonimmunogenic and were stable upon dilution with buffer as well as 10% serum. Curcumin, an anticancer agent with multiple delivery challenges, was encapsulated in mPEG-zein micelles. The micelles significantly enhanced the aqueous solubility (by 1000-2000-fold) and stability (by 6-fold) of curcumin. PEG-zein micelles sustained the release of curcumin up to 24 h in vitro. Curcumin-loaded mPEG-zein micelles showed significantly higher cell uptake than free curcumin in drug-resistant NCI/ADR-RES cancer cells in vitro. Micellar curcumin formulation was more potent than free curcumin in NCI/ADR-RES cancer cells, as evidenced from the 3-fold reduction in IC(50) value of curcumin. Overall, this study for the first time reports a natural protein core based polymeric micelle and demonstrates its application for the delivery of hydrophobic anticancer drugs such as curcumin.     

聚(ε-己内酯) -聚乙二醇-聚(ε-己内酯)两亲三嵌段共聚物蛋白大分子药物微球的研究

目的:研究以聚(ε-己内酯) -聚乙二醇-聚(ε-己内酯) (PCE)制备蛋白大分子药物微球的方法及其与成品理化性质和释放动力学的关系。方法:采用复乳溶剂挥发法制备牛血清白蛋白(BSA) PCE微球,以扫描电镜观察微球的表面形态,以Mi-croBCA法测定微球载药量和包封率,以累积释放量考察微球体外释药特性。结果:微球外形圆整、表面光滑。不同分子量PCE微球载药量和包封率相近,但体外释药特性显著不同,释放机制为扩散-降解,其中PCE4000因扩散作用释出的蛋白量明显低于其它分子量所制微球。微球体外释药规律符合扩散-溶蚀(Q=k1t1/2+k2t+k3t2+k4t3)(r=0.997)方程。结论:以PCE制备蛋白大分子药物微球具有良好的缓释效果,突释小,释放完全。     

Improving Protein Delivery from Microparticles Using Blends of Poly(DL lactide co-glycolide) and Poly(ethylene oxide)-poly(propylene oxide) Copolymers

Purpose. Microparticles containing ovalbumin as a model for protein drugs were formulated from blends of poly(DL lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide) copolymers (Pluronic). The objectives were to achieve uniform release characteristics and improved protein delivery capacity. Methods. The water- in oil -in oil emulsion/solvent extraction technique was used for microparticle production. Results. A protein loading level of over 40% (w/w) was attained in microparticles having a mean diameter of approximately 5 µm. Linear protein release profiles over 25 days in vitro were exhibited by certain blend formulations incorporating hydrophilic Pluronic F127. The release profile tended to plateau after 10 days when the more hydrophobic Pluronic L121 copolymer was used to prepare microparticles. A delivery capacity of 3 µg OVA/mg particles/ day was achieved by formulation of microparticles using a 1:2 blend of PLG:Pluronic F127. Conclusions. The w/o/o formulation approach in combination with PLG:Pluronic blends shows potential for improving the delivery of therapeutic proteins and peptides from microparticulate systems. Novel vaccine formulations are also feasible by incorporation of Pluronic L121 in the microparticles as a co-adjuvant.     

双氨基聚乙二醇的制备及分离纯化

聚乙二醇2000经氯代、叠氮化、氢化还原3步反应制得双氨基聚乙二醇（1）粗品,总收率57%.采用阳离子交换树脂,以不同浓度的乙酸盐缓冲液（pH 5.7）梯度洗脱,得到的1纯品采用IR、^1HNMR和HPLC等方法进行检测,并用高效分子排阻色谱法测定相对分子量.     

Novel Biodegradable Polylactide/poly(ethylene glycol) Micelles Prepared by Direct Dissolution Method for Controlled Delivery of Anticancer Drugs

Purpose  

The aim of this study is to develop novel polylactide/poly(ethylene glycol) (PLA/PEG) micelles as carrier of hydrophobic drug (paclitaxel) by direct dissolution method without using any organic solvents. The in vitro and in vivo release properties were studied in comparison with micelles prepared by dialysis.     

Poly(ethylene glycol)-mesalazine conjugate for colon specific delivery

Chronic inflammatory bowel diseases (IBDs) are still waiting for improved and innovative therapeutic treatments, which can overcome the limits of the current approaches. Since IBDs affect mainly the lower tract of the intestine, a localized therapy in the colon tract will avoid most of the problems caused by systemic or poor selective therapies. Particularly promising are the advance drug delivery systems that can reach specific colon delivery, thus guaranteeing active agent release only at the site of action. This approach can meet two aims at the same time, first of all the drug will not affect healthy tissue and second a lower drug dose may be used because all the administered active agent will reach the target. To obtain a specific colon delivery we exploited the azoreductase enzymes, selectively present only in colon, by inserting an azo linker between a selected drug and a macromolecular carrier. The drug employed is mesalazine, a well know and used agent against IBDs. Poly(ethylene glycol) (PEG), of different molecular weights and structures, was used as carrier. Three different conjugates were synthesized and characterized, and the most promising one, with highest drug loading thanks to the use of diamino PEG of 4 kDa, was further investigated in vitro on mouse colonic epithelial cells (CMT-9) and in vivo on model mice with induced colitis. The data presented here demonstrate that PEG conjugation of mesalazine prevents drug release and absorption in upper intestine, after oral administration of the conjugates, and that the azo linker ensures a good drug release in the colon tract. The results in vivo take into consideration mice bodyweight gain, tissue histology and interleukin-2 beta as an index of inflammation. These parameters, all together, demonstrated the conjugate effectiveness against the controls.     

热敏性三嵌段聚酯-聚乙二醇共聚物的特性与应用

三嵌段聚酯-聚乙二醇共聚物具有热敏性、生物相容性、生物降解性、增溶性及缓释特性,是一种良好的注射用缓释制剂载体。本文综述了此类共聚物的特性及其在药物传递系统中的应用。     

Stabilization and Controlled Release of Bovine Serum Albumin Encapsulated in Poly(D,L-lactide) and Poly(ethylene glycol) Microsphere Blends

Purpose. The acidic microclimate in poly(D, L-lactide-co-glycolide) 50/50 microspheres has been previously demonstrated by our group as the primary instability source of encapsulated bovine serum albumin (BSA). The objectives of this study were to stabilize the encapsulated model protein, BSA, and to achieve continuous protein release by using a blend of: slowly degrading poly(D, L-lactide) (PLA), to reduce the production of acidic species during BSA release; and pore-forming poly(ethylene glycol) (PEG), to increase diffusion of BSA and polymer degradation products out of the polymer. Methods. Microspheres were formulated from blends of PLA (Mw 145,000) and PEG (Mw 10,000 or 35,000) by using an anhydrous oil-in-oil emulsion and solvent extraction (O/O) method. The polymer blend composition and phase miscibility were examined by FT-IR and DSC, respectively. Microsphere surface morphology, water uptake, and BSA release kinetics were also investigated. The stability of BSA encapsulated in microspheres was examined by losses in protein solubility, SDS-PAGE, IEF, CD, and fluorescence spectroscopy. Results. PEG was successfully incorporated in PLA microspheres and shown to possess partial miscibility with PLA. A protein loading level of 5% (w/w) was attained in PLA/PEG microspheres with a mean diameter of approximately 100 m. When PEG content was less than 20% in the blend, incomplete release of BSA was observed with the formation of insoluble, and primarily non-covalent aggregates. When 20%-30% PEG was incorporated in the blend formulation, in vitro continuous protein release over 29 days was exhibited. Unreleased BSA in these formulations was water-soluble and structurally intact. Conclusions. Stabilization and controlled relaease of BSA from PLA/PEG microspheres was achieved due to low acid and high water content in the blend formulation.     

氧化还原调控的甲氧基聚乙二醇-二硫键-聚乙烯亚胺非病毒基因载体的研制

合成了氧化还原调控的甲氧基聚乙二醇-二硫键-聚乙烯亚胺[methoxy poly (ethylene glycol)-SS-polyethylenimine,mPEG-SS-PEI].采用薄层色谱验证了该聚合物中二硫键的存在.琼脂糖凝胶电泳结果显示,mPEG-SS-PEI-pEGFP复合物在含10％胎牛血清的DMEM细胞培养液中稳定.透射电镜观察表明,不含二硫键的mPEG-PEI及mPEG-SS-PEI 与pEGFP复合物的粒径均约为200 nm.体外转染试验中,荧光显微镜定性及流式细胞定量结果均表明,与mPEG-PEIpEGFP相比,mPEG-SS-PEI-pEGFP能显著提高对U87细胞的转染效率.原因是mPEG-SS-PEI-pEGFP复合物进入细胞后能在较高浓度谷胱甘肽的还原下脱去PEG,从而恢复PEI的质子泵效应.并且,聚合物中加入二硫键未显著增加PEG化PEI的体外细胞毒性.     

Composite Poly(vinyl alcohol) Beads for Controlled Drug Delivery

A new method of preparing composite poly(vinyl alcohol) (PVA) beads with a double-layer structure has been developed, which involves a stepwise saponification of suspension polymerized poly (vinyl acetate) (PVAc) beads and subsequent stepwise cross-linking of the PVA core and shell with glu-taraldehyde. This process results in PVA beads with thin, highly cross-linked outer shells and lightly cross-linked inner cores of different degrees of cross-linking. In addition to structural characterization of the polymer based on equilibrium swelling measurements, the kinetics of water swelling and drug release from these beads were studied at 37°C using acetaminophen and proxyphylline as model drugs. The results show that the outer shell functions as a rate-controlling membrane upon increasing its cross-linking ratio, X, above 0.47. This aspect is reflected in the observed diffusional time lags and constant-rate regions during swelling and drug release. Based on observed time lags, the diffusion coefficient of water through the outer PVA shell with a high cross-linking ratio of X = 0.5 is estimated to be at least six times higher than that of acetaminophen and proxyphylline. In addition, drug diffusion coefficients in the lightly cross-linked PVA core appear to be at least 10 times larger than that in the highly cross-linked outer shell. At lower shell cross-linking ratios (X < 0.4), the diffusional time lags appear to be absent and the diffusion profiles are apparently first-order (Fickian) in nature.     

Preparation of Biodegradable Polycaprolactone/Poly (ethylene glycol)/Polycaprolactone (PCEC) Nanoparticles

Biodegradable polyetherester copolymer (PCL/PEG/PCL, PCEC) was synthesized by ring-opening polymerization of ε-caprolactone initiated by poly(ethylene glycol) (PEG). The PCEC nanoparticles were prepared by solvent diffusion method or w/o/w double emulsion method. The obtained particles' morphology was observed on scanning electron microscopy, and the particle size distribution was determined using Malvern laser particle sizer. Bovine serum albumin was used as the model water-soluble protein drug, which was successfully encapsulated in PCEC nanoparticles, the drug release behavior was studied in detail. The hydrolytic degradation behavior of the PCEC nanoparticles was also studied.     

聚乙二醇-聚左旋赖氨酸共聚物生物降解性的研究

目的:研究非病毒基因载体聚左旋赖氨酸(PLL)及其聚乙二醇(PEG)修饰共聚物的生物降解性。方法:将合成的不同分子量的PLL及PEG-PLL共聚物,采用体外酶解试验,用乌氏黏度计测定不同分子量的PLL及其共聚物在不同浓度胰蛋白酶作用下的增比黏度(ηsp)并考察其变化以评价聚合物结构与其生物降解性之间的关系。结果:所合成的PEG-PLL共聚物在胰蛋白酶作用下能有效地催化其生物降解,酶解反应速度与酶浓度成线性关系(R2=0.9775)。另随着PLL分子量的增加,酶解速度降低;而随着PEG含量的增加,酶解速度加快。结论:PLL的分子量及用于修饰的PEG的量对PEG-PLL共聚物生物降解性具有一定的影响。     

Promising Inhibitors of Poly(ethylene glycol) Oxidation in Aqueous Solutions

Pharmaceutical Chemistry Journal -     

Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery

Poly(ethylene glycol) (PEG)-modified thiolated gelatin (PEG-SHGel) anoparticles were developed as a long-circulating passively targeted delivery system that responds to intracellular glutathione concentrations to enhance DNA delivery and transfection. Reporter plasmid expressing enhanced green fluorescent protein (EGFP-N1) was encapsulated in the nanoparticles. DNA-containing gelatin (Gel) and thiolated gelatin (SHGel) nanoparticles were found to have a size range of 220 to 250 nm, whereas surface modification with PEG resulted in particles with a slightly larger size range of 310 to 350 nm. PEG modification was confirmed by electron spectroscopy for chemical analysis (ESCA), where an increase in the ether peak intensities of the C1s spectra corresponds to the surface presence of ethylene oxide residues. In addition, the PEG-SHGel nanoparticles released encapsulate plasmid DNA in response to varying concentrations of glutathione (up to 5.0 mM GSH in phosphate-buffered saline, or PBS). The stability of the encapsulated DNA was confirmed by agarose gel electrophoresis. Finally, from the qualitative and quantitative results of in vitro transfection studies in murine fibroblast cells (NIH3T3), PEG-Gel and PEG-SHGel nanoparticles afforded the highest transfection efficiency of the reporter plasmid. The results of these studies show that PEG-modified thiolated gelatin nanoparticles could serve as a very efficient nanoparticulate vector for systemic DNA delivery to solid tumors where the cells are known to have significantly higher intracellular GSH concentrations.     

姜黄素聚乙二醇-聚己内酯纳米粒的制备及制剂学性质分析

目的制备具有肝脏靶向性的姜黄素聚乙二醇-聚己内酯纳米粒。方法采用乳化溶剂挥发法,通过正交实验筛选最佳处方工艺,并按处方工艺制备姜黄素聚乙二醇-聚己内酯纳米粒。结果当选择聚乙二醇-聚己内酯100 mg、泊洛沙姆188浓度1.5%、乙酸乙酯与二氯甲烷体积比2∶1、有机相与内水相体积比为1∶4时,制备的姜黄素聚乙二醇-聚己内酯纳米粒粒径为(102.84±2.68)nm、多分散系数为0.105±0.010、包封率为84.36%±1.19%、载药量为4.70%±0.27%。结论本方法成功制备了姜黄素聚乙二醇-聚己内酯纳米粒,为下一步的药动学及体外抗肿瘤活性研究奠定基础。     

Poly(ethylene glycol) conjugated drugs and prodrugs: a comprehensive review

Low molecular weight Poly(ethylene glycol) (PEG) (< 20,000)-drug conjugates, prepared over a 20-year period, have been scrutinized and their properties and efficacy reviewed. No commercial products have thus far been reported for these types of compounds. However, during the past 5 years a renaissance in the field of PEG-(anticancer) drug conjugates has taken place, initiated by the use of higher molecular weight PEGs (> 20,000), especially 40,000, which is estimated to have a plasma circulating half-life of approximately 8-9 h. This recent resuscitation of small organic molecule delivery by high molecular weight PEG conjugates was founded on meaningful in vivo testing using established tumor models and has led to a clinical candidate. Recent applications of high molecular weight PEG prodrug strategies to amino-containing drugs are also detailed.