Self-assembled poly(butadiene)-b-poly(ethylene oxide) polymersomes as paclitaxel carriers

In this work, self-assembled poly(butadiene)-b-poly(ethylene oxide) (PB-PEO) polymersomes (polymer vesicles) and worm micelles were evaluated as paclitaxel carriers. Paclitaxel was successfully incorporated into PB-PEO polymersomes and worm micelles. The loading capacity of paclitaxel inside PB-PEO colloids ranged from 6.7% to 13.7% w/w, depending on the morphology of copolymer colloids and the molecular weight of diblock copolymer. Paclitaxel loaded OB4 (PB219-PEO121) polymersome formulations were colloidally stable for 4 months at 4 degrees C and exhibited slow steady release of paclitaxel over a 5 week period at 37 degrees C. Evaluation of the in vitro cytotoxicity of paclitaxel-polymersome formulations showed that the ability of paclitaxel-loaded polymersomes to inhibit proliferation of MCF-7 human breast cancer cells was less compared to paclitaxel alone. By increasing the concentration of paclitaxel in polymersomes from 0.02 to 0.2 mug/mL, paclitaxel-polymersome formulations showed comparable activity in inhibiting the growth of MCF-7 cells. Taken together, these results demonstrate that paclitaxel-polymersomes have desirable restrained release profile and exhibit long-term stability.     

Hydrotropic polymeric micelles for enhanced paclitaxel solubility: in vitro and in vivo characterization

The purpose of this investigation was to characterize the in vitro stability and in vivo disposition of paclitaxel in rats after solubilization of paclitaxel into hydrotropic polymeric micelles. The amphiphilic block copolymers consisted of a micellar shell-forming poly(ethylene glycol) (PEG) block and a core-forming poly(2-(4-vinylbenzyloxy)-N,N-diethylnicotinamide) (P(VBODENA)) block. N,N-Diethylnicotinamide (DENA) in the micellar inner core resulted in effective paclitaxel solubilization and stabilization. Solubilization of paclitaxel using polymeric micelles of poly(ethylene glycol)-b-P(D,L-lactide) (PEG-b-PLA) served as a control for the stability study. Up to 37.4 wt % paclitaxel could be loaded in PEG-b-P(VBODENA) micelles, whereas the maximum loading amount for PEG-b-PLA micelles was 27.6 wt %. Thermal analysis showed that paclitaxel in the polymeric micelles existed in the molecularly dispersed amorphous state even at loadings over 30 wt %. Paclitaxel-loaded hydrotropic polymeric micelles retained their stability in water for weeks, whereas paclitaxel-loaded PEG-b-PLA micelles precipitated in a few days. Hydrotropic polymer micelles were more effective than PEG-PLA micelle formulations in inhibiting the proliferation of human cancer cells. Paclitaxel in hydrotropic polymer micelles was administered orally (3.8 mg/kg), intravenously (2.5 mg/kg), or via the portal vein (2.5 mg/kg) to rats. The oral bioavailability was 12.4% of the intravenous administration. Our data suggest that polymeric micelles with a hydrotropic structure are superior as a carrier of paclitaxel due to a high solubilizing capacity combined with long-term stability, which has not been accomplished by other existing polymeric micelle systems.     

Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide)

End-functionalized poly(N-isopropylacrylamide) (PNIPA) was synthesized by living free radical polymerization and conventional free radical polymerization and was used to prepare graft copolymers with poly(ethylenimine) (PEI). The copolymers exhibited lower critical solution temperature (LCST) behavior between 30 and 32 degrees C and formed complexes with plasmid DNA. The LCST of the copolymers in the DNA complexes increased slightly to approximately 34-35 degrees C. Cytotoxicity of the copolymers was evaluated by measuring lactate dehydrogenase (LDH) release from cells. The copolymers exhibited temperature-dependent toxicity, with higher levels of LDH release observed at temperatures above the LCST. Cellular uptake and transfection activity of the DNA complexes with the PEI-g-PNIPA copolymers were lower than those of the control PEI/DNA complexes at temperature below the LCST but increased to the PEI/DNA levels at temperatures above the LCST.     

Thermally controlled release of anticancer drug from self-assembled γ-substituted amphiphilic poly(ε-caprolactone) micellar nanoparticles

A thermo-responsive poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone}-b-poly(γ-octyloxy-ε-caprolactone) (PMEEECL-b-POCTCL) diblock copolymer was synthesized by ring-opening polymerization using tin octanoate (Sn(Oct)(2)) catalyst and a fluorescent dansyl initiator. The PMEEECL-b-POCTCL had a lower critical solution temperature (LCST) of 38 °C, and it was employed to prepare thermally responsive micelles. Nile Red and Doxorubicin (DOX) were loaded into the micelles, and the micellar stability and drug carrying ability were investigated. The size and the morphology of the cargo-loaded micelles were determined by DLS, AFM, and TEM. The Nile-Red-loaded polymeric micelles were found to be stable in the presence of both fetal bovine serum and bovine serum albumin over a 72 h period and displayed thermo-responsive in vitro drug release. The blank micelles showed a low cytotoxicity. As comparison, the micelles loaded with DOX showed a much higher in vitro cytotoxicity against MCF-7 human breast cancer cell line when the incubation temperature was elevated above the LCST. Confocal laser scanning microscopy was used to study the cellular uptake and showed that the DOX-loaded micelles were internalized into the cells via an endocytosis pathway.     

Novel stimuli-responsive micelle self-assembled from Y-shaped P(UA-Y-NIPAAm) copolymer for drug delivery

A new amphiphilic Y-shaped copolymer, comprised of hydrophobic poly(undecylenic acid) (PUA) and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm), was designed and synthesized. A cytotoxicity study revealed that P(UA-Y-NIPAAm) copolymers did not exhibit apparent inhibition impact on the proliferation of cells when the concentration of the copolymer was below 1000 mg/L. Characterization demonstrated that the P(UA-Y-NIPAAm) copolymer is thermosensitive with a lower critical solution temperature (LCST) of 31 degrees C. In water, the P(UA-Y-NIPAAm) copolymer would self-assemble into micelles with a critical micelle concentration (CMC) of 20 mg/L. Self-assembled P(UA-Y-NIPAAm) micelles exhibited a nanospherical morphology of 40 to approximately 80 nm in size. The controlled drug release behavior of the P(UA-Y-NIPAAm) micelles was further investigated, and self-assembled micelles exhibited improved properties in controlled drug release.     

载紫杉醇共聚物胶束的合成与表征

目的:制备一种包封率、载药量高的紫杉醇载体材料。方法:开环聚合法一步合成了两种两亲性共聚物PTL1和PTL2,以核磁和凝胶渗透色谱进行了产物的表征,以固体分散-超声法制备紫杉醇胶束,考察了胶束的载药量、包封率。结果:核磁和凝胶渗透色谱的结果显示得到了目标产物,所制备得到的载紫杉醇胶束包封率可以达到90%以上,载药量为9.5%以上。结论:实验结果表明我们所合成的PTL1和PTL2是好的紫杉醇载体材料。     

载紫杉醇共聚物胶束的合成与表征

目的：制备一种包封率、载药量高的紫杉醇载体材料。方法：开环聚合法一步合成了两种两亲性共聚物PTL1和PTL2,以核磁和凝胶渗透色谱进行了产物的表征,以固体分散一超声法制备紫杉醇胶束,考察了胶束的载药量、包封率。结果：核磁和凝胶渗透色谱的结果显示得到了目标产物,所制备得到的载紫杉醇胶束包封率可以达到90％以上,载药量为9．5％以上。结论：实验结果表明我们所合成的PTL1和PTL2是好的紫杉醇栽体材料。     

Micelles self-assembled from thermoresponsive 2-hydroxy-3-butoxypropyl starches for drug delivery

A new class of thermoresponsive polymers, 2-hydroxy-3-butoxypropyl starches (HBPS), was synthesized by changing the hydrophobic-hydrophilic balance of starch using butyl glycidyl ether as hydrophobic reagent. The lower critical solution temperatures (LCSTs) of HBPS can be adjusted by varying the molar substitution (MS) of hydrophobic groups in the range of 4.5-32.5 °C. In water, HBPS can self-assemble into micelles below the LCST, and the micelles are deformed and aggregate into more polar and larger objects above the LCST. The drug loading HBPS micelles showed thermoresponsive controlled release, namely, the drug release is accelerated dramatically above the LCST.     

TAT Peptide-Conjugated Magnetic PLA-PEG Nanocapsules for the Targeted Delivery of Paclitaxel: <Emphasis Type="Italic">In Vitro</Emphasis> and Cell Studies

Paclitaxel (PTX) and organophilic iron oxide nanocrystals of 7 nm average size were co-encapsulated in the oily core of poly(lactide)-poly(ethyleneglycol) (PLA-PEG) nanocapsules in order to develop magnetically responsive nanocarriers of PTX. The nanocapsules were prepared by a solvent displacement technique and exhibited satisfactory drug and iron oxide loading efficiency, high colloidal stability, and sustained drug release properties. Drug release also proved responsive to an alternating magnetic field. Magnetophoresis experiments showed that the magnetic responsiveness of the nanocapsules depended on their SPION content. The PTX-loaded nanocapsules exhibited comparable to free PTX cytotoxicity against the A549 lung cancer cell line at 24 h of incubation but higher cytotoxicity than free drug at 48 h of incubation. The conjugation of a cysteine-modified TAT peptide (HCys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH₂) on the surface of the nanocapsules resulted to highly increased uptake of nanocapsules by cancer cells, as well as to profound improvement of their cytotoxicity against the cancer cells. The results obtained justify further investigation of the prospects of these multifunctional PLA-PEG nanocapsules as a targeted delivery system of paclitaxel.     

Combined Delivery of Paclitaxel and Tanespimycin via Micellar Nanocarriers: Pharmacokinetics,Efficacy and Metabolomic Analysis

Background

Despite the promising anticancer efficacy observed in preclinical studies, paclitaxel and tanespimycin (17-AAG) combination therapy has yielded meager responses in a phase I clinical trial. One serious problem associated with paclitaxel/17-AAG combination therapy is the employment of large quantities of toxic organic surfactants and solvents for drug solubilization. The goal of this study was to evaluate a micellar formulation for the concurrent delivery of paclitaxel and 17-AAG in vivo.

Methodology/Principal Findings

Paclitaxel/17-AAG-loaded micelles were assessed in mice bearing human ovarian tumor xenografts. Compared with the free drugs at equivalent doses, intravenous administration of paclitaxel/17-AAG-loaded micelles led to 3.5- and 1.7-fold increase in the tumor concentrations of paclitaxel and 17-AAG, respectively, without significant altering drug levels in normal organs. The enhanced tumor accumulation of the micellar drugs was further confirmed by the whole-body near infrared imaging using indocyanine green-labeled micelles. Subsequently, the anticancer efficacy of paclitaxel/17-AAG-loaded micelles was examined in comparison with the free drugs (weekly 20 mg/kg paclitaxel, twice-weekly 37.5 mg/kg 17-AAG). We found that paclitaxel/17-AAG-loaded micelles caused near-complete arrest of tumor growth, whereas the free drug-treated tumors experienced rapid growth shortly after the 3-week treatment period ended. Furthermore, comparative metabolomic profiling by proton nuclear magnetic resonance revealed significant decrease in glucose, lactate and alanine with simultaneous increase in glutamine, glutamate, aspartate, choline, creatine and acetate levels in the tumors of mice treated with paclitaxel/17-AAG-loaded micelles.

Conclusions/Significance

We have demonstrated in the current wok a safe and efficacious nano-sized formulation for the combined delivery of paclitaxel and 17-AAG, and uncovered unique metabolomic signatures in the tumor that correlate with the favorable therapeutic response to paclitaxel/17-AAG combination therapy.     

长循环紫杉醇纳米脂质体的合成及其活性评估

目的：研制甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺（mPEG2000-DSPE）修饰的长循环紫杉醇纳米脂质体（PEG-PTX-LP）,减少市售紫杉醇制剂的不良反应并增强疗效。方法：采用薄膜超声分散法制备PEG-PTX-LP,采用激光散射粒度分析仪和透射电镜观察其物理性状,超滤法检测药物包封率,透析法检测药物缓释能力,通过细胞摄取试验观察人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells,HUVEC）、A549肺癌细胞对PEG-PTX-LP的摄取能力。结果：透射电镜显示长循环紫杉醇纳米脂质体呈圆形囊泡样结构,粒径检测其平均粒径为99.1 nm,制备后第2、7、14、21、30天的紫杉醇包封率均大于99%,在血清中的缓释能力优于泰素溶液,HUVEC、A549细胞对PEG-PTX-LP中紫杉醇的摄取量明显高于泰素溶液（Taxol）。结论：采用mPEG2000-DSPE修饰的PEG-PTX-LP具有更高的稳定性和缓释能力,对肿瘤细胞和血管内皮细胞有一定的特异性,是一种更有效的紫杉醇新剂型。     

Intratumoral Delivery of Paclitaxel in Solid Tumor from Biodegradable Hyaluronan Nanoparticle Formulations

In the current study, novel paclitaxel-loaded cross-linked hyaluronan nanoparticles were engineered for the local delivery of paclitaxel as a prototype drug for cancer therapy. The nanoparticles were prepared using a desolvation method with polymer cross-linking. In vitro cytotoxicity studies demonstrated that less than 75% of the MDA-MB-231 and ZR-75-1 breast cancer cells were viable after 2-day exposure to paclitaxel-loaded hyaluronan nanoparticles or free paclitaxel, regardless of the dose. These results suggest that hyaluronan nanoparticles maintain the pharmacological activity of paclitaxel and efficiently deliver it to the cells. Furthermore, in vivo administration of the drug-loaded nanoparticles via direct intratumoral injection to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor in female rats was studied. The paclitaxel-loaded nanoparticles treated group showed effective inhibition of tumor growth in all treated rats. Interestingly, there was one case of complete remission of tumor nodule and two cases of persistent reduction of tumor size that was observed on subsequent days. In the case of free paclitaxel-treated group, the mean tumor volume increased almost linearly (R ² = 0.93) with time to a size that was 4.9-fold larger than the baseline volume at 57 days post-drug administration. Intratumoral administration of paclitaxel-loaded hyaluronan nanoparticles could be a promising treatment modality for solid mammary tumors.     

Methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) copolymer micelles for formulation of hydrophobic drugs

Six amphiphilic diblock copolymers based on methoxy poly(ethylene glycol) (MePEG) and poly(delta-valerolactone) (PVL) with varying hydrophilic and hydrophobic block lengths were synthesized via a metal-free cationic polymerization method. MePEG-b-PVL copolymers were synthesized using MePEG with Mn = 2000 or Mn = 5000 as the macroinitiator. 1H NMR and GPC analyses confirmed the synthesis of diblock copolymers with relatively narrow molecular weight distributions (Mn/Mw = 1.05-1.14). DSC analysis revealed that the melting temperatures (Tm) of the copolymers (47-58 degrees C) approach the Tm of MePEG as the PVL content is decreased. MePEG-b-PVL copolymer aggregates loaded with the hydrophobic anti-cancer drug paclitaxel were found to have effective mean diameters ranging from 31 to 970 nm depending on the composition of the copolymers. A MePEG-b-PVL copolymer of a specific composition was found to form drug-loaded micelles of 31 nm in diameter with a narrow size distribution and improve the apparent aqueous solubility of paclitaxel by more than 9000-fold. The biological activity of paclitaxel formulated in the MePEG-b-PVL micelles was confirmed in human MCF-7 breast and A2780 ovarian cancer cells. Furthermore, the biocompatibility of the copolymers was established in CHO-K1 fibroblast cells using a cell viability assay. The in vitro hydrolytic and enzymatic degradation of the micelles was also evaluated over a period of one month. The present study indicates that the MePEG-b-PVL copolymers are suitable biomaterials for hydrophobic drug formulation and delivery.     

Oil-encapsulating PEO-PPO-PEO/PEG shell cross-linked nanocapsules for target-specific delivery of paclitaxel

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.     

姜黄素共聚物胶束的制备与体外细胞毒评价

目的:制备一种姜黄素共聚物胶束以提高姜黄素的水溶性及其抗肿瘤活性。方法:采用乳化溶剂挥发法制备了载姜黄素的共聚物胶束(Cur/PTL1胶束),对其粒径、载药量、包封率和体外药物释放行为进行了考察;并采用MTT法考察了PTL1空白胶束和Cur/PTL1胶束的体外细胞毒作用。结果:制备了粒径在40 nm左右的载姜黄素共聚物胶束,载药量为9.78±0.29%,包封率为97.24±2.68%。体外药物释放实验表明,游离姜黄素在24 h内的药物累积释放率达到90%以上,而Cur/PTL1胶束在24 h内药物累积释放率为23.8%,能够持续释放14天,14天内累积释放率为85.9%,具有一定的缓释能力。MTT实验结果表明,当PTL1空白胶束浓度达到1 mg/mL时,细胞的存活率仍在90%以上;Cur/PTL1胶束组IC50为4.73±0.23μg/mL,游离姜黄素组IC50为6.42±0.35μg/mL。结论:实验结果表明,Cur/PTL1胶束可以作为一种有前景的纳米药物输送系统。     

Core-cross-linked polymeric micelles as paclitaxel carriers

Cross-linkable di- and triblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized. These amphiphilic copolymers self-assembled into nanoscale micelles capable of encapsulating hydrophobic paclitaxel in their hydrophobic cores in aqueous solutions. To further enhance their thermodynamic stability, the micelles were cross-linked by radical polymerization of the double bonds introduced into the PCL blocks. Reaction conditions were found to significantly affect both the cross-linking efficiency and the micelle size. The encapsulation of paclitaxel into the micelles was confirmed by the proton nuclear magnetic resonance (1H NMR) spectroscopy. Encouragingly, paclitaxel-loading efficiency of micelles was enhanced significantly upon micelle core-cross-linking. Both the micelle size and the drug loading efficiency increased markedly with increasing the PCL block lengths, no matter if the micelles were core-cross-linked or not. However, paclitaxel-loading did not obviously affect the micelle size or size distribution. The cross-linked micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents. The efficient cellular uptake of paclitaxel loaded in the nanomicelles was demonstrated by confocal laser scanning microscopy (CLSM) imaging. This new biodegradable nanoscale carrier system merits further investigations for parenteral drug delivery.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Novel fast degradable thermosensitive polymeric micelles based on PEG-block-poly(N-(2-hydroxyethyl)methacrylamide-oligolactates)

The aim of this study was to design a thermosensitive polymeric micelle system with a relatively fast degradation time of around 1 day. These micelles are of interest for the (targeted) delivery of biologically active molecules. Therefore, N-(2-hydroxyethyl)methacrylamide-oligolactates (HEMAm-Lac(n)()) were synthesized and used as building blocks for biodegradable (block co) polymers. p(HEMAm-Lac(2)) is a thermosensitive polymer with a cloud point (CP) of 22 degrees C which could be lowered by copolymerization with HEMAm-Lac(4). The block copolymer PEG-b-((80%HEMAm-Lac(2))-(20%HEMAm-Lac(4))) self-assembled into compact spherical micelles with an average size of 80 nm above the CP of the thermosensitive block (6 degrees C). Under physiological conditions (pH 7.4; 37 degrees C), the micelles started to swell after 4 h and were fully destabilized within 8 h due to hydrolysis of the lactate side chains. Rapidly degrading thermosensitive polymeric micelles based on PEG-b-((80%HEMAm-Lac(2))-(20%HEMAm-Lac(4))) have attractive features as a (targeted) drug carrier system for therapeutic applications.     

Paclitaxel and vinorelbine cause synergistic increases in apoptosis but not in microtubular disruption in human lung adenocarcinoma cells (A-549)

Concurrent administration of paclitaxel and vinorelbine results in cytotoxicity in vivo and in vitro in a number of tumor cell lines, yet the mechanisms of enhanced cell killing are undefined. In studies here, we show that low concentrations (1 nM) of paclitaxel and vinorelbine in combination result in enhanced cell killing by apoptosis (P<0.05) in the human lung adenocarcinoma cell line, A-549. In contrast, necrotic cell death and formation of multinucleated cells, which were significantly increased by paclitaxel (P<0.05) alone, but not vinorelbine, were not increased synergistically by both drugs. Paclitaxel also caused microtubular disruption which was not observed with vinorelbine. These data provide further rationale for the combined use of paclitaxel and vinorelbine in clinical trials, and suggest that the cooperative effects of drugs on apoptosis are not mediated through similar disruptional effects on microtubules.     

Enhancement of paclitaxel production by temperature shift in suspension culture of Taxus chinensis

Effect of temperature shift during culture period on cell growth and paclitaxel was investigated to optimize paclitaxel production in suspension culture of Taxus chinensis. Cell growth showed the optimum at 24 degrees C while paclitaxel synthesis showed the maximum at 29 degrees C. To minimize the inhibitory effect of higher temperature on cell growth, temperature was shifted after a certain period of culture time at 24 degrees C. Paclitaxel synthesis in plant cell culture increased dramatically during day 14 to day 21 regardless of treatment, reaching the maximum production of 137.5 mg paclitaxel/L. When the temperature was maintained at 29 degrees C after day 21, the specific productivity of paclitaxel was sustained for prolonged period of 42 days. The possible relationship between temperature and paclitaxel synthetic pathway was also suggested.