Co-delivery of docetaxel and doxorubicin using biodegradable PEG-PLA micelles for treatment of breast cancer with synergistic anti-tumour effects

Poly(ethylene glycol)-poly(lactic acid) copolymer, prepared by ring opening polymerization, was used as a single platform to co-deliver both hydrophilic doxorubicin and hydrophobic docetaxel (DTX) in a simulated physiological environment. The average size of the negatively charged drug loaded polymeric micelles were found to be 293 nm. The drug loading (%) and encapsulation efficiency (%) were calculated to be 1.21 and 59.0, respectively. The in vitro cytotoxicity test using MCF7 breast cancer cells was conducted using 1 × 10⁴ cells in 10% FBS and 1% antibiotic, and the absorbance of formazan was evaluated at 570 nm. Cell growth inhibition by MTT assay showed viability of 33% of the MCF7 cells after treatment with drug-loaded micelles for 48 h. Controlled release of drugs from the polymeric micelles indicated a burst release effect initially; whereas, 98% of drug could be released at pH 7.4 within a time period of 96 h. Time period for drug release shorten to 48 h only in simulated mild acidic pH (5.4) condition. The in vitro drug release study from micelles indicated synergistic cytotoxicity effect in human metastatic breast cancer MCF7 cell.     

Hyper-branched multifunctional carbon nanotubes carrier for targeted liver cancer therapy

The systemic toxicity of anticancer drugs regularly restricts the use of conventional chemotherapy to treat cancer. In this study, the limitations overcome by profitably fabricating a multifunctional nanocarrier system to carry the anticancer drug into the specific location of the cancer cells. The polyethylene glycol (PEG) was functionalized in the carboxylated multiwalled carbon nanotubes (MWCNT-COOH) through an esterification reaction (MWCNT-PEG). The targeting ligand of folic acid (FA) was covalently bonded with hyperbranched poly-L-lysine (HBPLL) using adipic acid (AA) as a cross-linking agent. Doxorubicin (DOX), an anticancer drug, was effectively loaded on MWCNT-PEG-AA-HBPLL-FA carrier loading, and in-vitro drug release was investigated by UV–Vis spectrophotometer. The chemical functionalization, morphological properties, crystalline nature, surface charge, and thermal stability of the synthesized materials were studied by FT-IR, FE-SEM, HR-TEM, DLS, and TGA techniques. In-vitro cytotoxicity and anticancer properties of DOX-loaded nanocarrier were studied in human liver cancer (HepG2) cells and human embryonic kidney (HEK293) cells. The activities of caspases (caspase ?3, ?8 & ?9) were analyzed using luminometry. The intrinsic apoptosis pathway proteins (Bcl-2 & BAX) were determined by western blot and RT-PCR analysis. The synthesized DOX-loaded nanocarriers exhibited increased cytotoxicity and apoptosis in liver HepG2 cells. The results suggest that the DOX-loaded nanocarrier possesses strong anticancer properties and could be an applicable and potential drug carrier for liver cancer chemotherapy.     

嵌段共聚物聚(异丙基丙烯酰胺)-b-聚(双丙酮丙烯酰胺)制备及其对叶酸的载负和释放

以N-异丙基丙烯酰胺(NIPAm)和双丙酮丙烯酰胺(DAAM)为原料,采用可逆加成 断裂链转移(RAFT)可控聚合反应法合成了两亲性两嵌段共聚物 聚(异丙基丙烯酰胺)-b-聚(双丙酮丙烯酰胺)(PNIPAm-b-PDAAM),用红外光谱(FT-IR)、核磁共振(1H NMR)和凝胶渗透色谱(GPC)对其结构和组成进行了表征。 这种共聚物在水溶液中能够自组装成稳定的聚合物胶束,通过荧光探针测得其低临界胶束浓度(CMC)约为7.0 mg/L。 采用扫描电子显微镜(SEM)和动态激光光散射(DLS)测得,PNIPAm-b-PDAAM在水溶液中自组装成核壳结构的球形胶束,SEM测得其直径约150 nm,且分散性良好。 以其聚合物胶束为载体、叶酸(FA)为模型药物,模拟人体生理环境进行药物体外释放。 结果表明,叶酸的负载量及负载率分别为25%和74%。 在人体温度37℃、pH值分别为4.0、6.86、9.18磷酸缓冲溶液(PBS)中,FA在20 h内的释放均比25 ℃快,释放速率随pH值增加而增大,最大累积释放率分别为31%、67%和72%。     

聚甘油酸-g-胆固醇胶束的制备及药物释放性能研究

以甘油酸为单体,通过本体缩聚制备了水溶性生物降解高分子聚甘油酸,利用聚甘油酸侧基上的羟基固定生物相容性好的疏水性分子胆固醇,通过亲疏水作用自组装形成胶束.以形成的胶束作为载体负载抗肿瘤药物阿霉素,研究了药物的体外释放行为.将肝癌细胞HepG2与载药胶束共培养研究其体外抗肿瘤效果.研究结果表明,聚甘油酸-g-胆固醇共聚物...     

Self-assembled micelles of novel graft amphiphilic copolymers for drug controlled release

In this study, with the aim of designing an ideal anticancer drug carrier, we synthesized novel amphiphilic graft copolymers, P(Glu-alt-PEG)-graft-PCLA, based on poly(ethylene glycol) (PEG) segments and glutamic acid (Glu) units as the hydrophilic main chain, and poly(?-caprolactone-co-lactide) (PCLA) as hydrophobic branches. The chemical structure of the copolymers was characterized by (1)H MNR and FT-IR. The self-assembly of the copolymers to form micelles was studied by TEM, DLS and fluorescence spectroscopy. In vitro doxorubicin controlled release studies demonstrated that these graft copolymer micelles had high drug loading capacity and good controlled released properties, demonstrating their potential as a novel anticancer drug carrier. The drug loaded graft copolymer micelles exhibited efficient inhibition of HeLa cells in in vitro studies.     

Preparation of poly(glutamic acid) shielding micelles self-assembled from polylysine-b-polyphenylalanine for gene and drug codelivery

A novel amphiphilic cationic block copolymer polylysine-b-polyphenylalanine(PLL-b-PPhe) was synthesized and self-assembled into micelles in aqueous solution,then shielded with poly(glutamic acid)(marked as PG/PLL-b-PPhe) to codeliver gene and drug for combination cancer therapy.Here,doxorubicin(DOX) was selected to be loaded into PLL-b-PPhe micelles and the drug loading efficiency was 8.0%.The drug release studies revealed that the PLL-b-PPhe micelles were pH sensitive and the released DOX could reach to 53.0%,65.0%,72.0% at pH 7.4,6.8 and 5.0,respectively.In order to reduce positive charge and cytotoxicity of PLL-b-PPhe micelles,PG was used as shelding,simultaneously condensed with Bcl2 siRNA to form gene carrier system.Compared with PEI,PG/PLL-b-PPhe had excellent gene transfection efficiency,especially when the molar ratio of PLL to PPhe was 30:60 and the mixed mass ratio of PLL-b-PPhe to gene was 5:1.More importantly,DOX and Bcl2 siRNA gene codelivery system displayed remarkable cytotoxicity against B16 F10 cells.Confocal laser scanning microscopy(CLSM) and flow cytometry were used to characterize endocytosis of the codelivery system,and confirmed that both DOX and Bcl2 siRNA had been endocytosed into B16 F10 cells.The above results indicated that gene and drug codelivery was a promising strategy in future cancer therapy.     

Synthesis and characterization of a biodegradable amphiphilic copolymer based on branched poly(ϵ‐caprolactone) and poly(ethylene glycol)

A novel biodegradable amphiphilic copolymer with hydrophobic poly(ε‐caprolactone) branches containing cholic acid moiety and a hydrophilic poly(ethylene glycol) chain was synthesized. The copolymer was characterized by FTIR, ¹H NMR, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), polarizing light microscopy (PLM), and wide‐angle X‐ray diffraction (WAXD) analysis. The amphiphilic copolymer could self‐assemble into micelles in an aqueous solution. The critical micelle concentration of the amphiphilic copolymer was determined by fluorescence spectroscopy. A nanoparticle drug delivery system with a regularly spherical shape was prepared with high encapsulation efficiency. The in vitro drug release from the drug‐loaded polymeric nanoparticles was investigated. Because of the branched structure of the hydrophobic part of the copolymer and the relatively fast degradation rate of the copolymer, an improved release behavior was observed. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5256–5265, 2007     

in vitro biological evaluation of folate-functionalized block copolymer micelles for selective anti-cancer drug delivery

The main objective of this study was to evaluate the ability of folic acid-functionalized diblock copolymer micelles to improve the delivery and uptake of two poorly water-soluble anti-tumor drugs, tamoxifen and paclitaxel, to cancer cells through folate receptor targeting. The diblock copolymer used in this study comprised a hydrophilic poly[2-(methacryloyloxy)ethyl phosphorylcholine] (MPC) block, carrying at the chain end the folate targeting moiety, and a pH-sensitive hydrophobic poly[2-(diisopropylamino)ethyl methacrylate] (DPA) block (FA-MPC-DPA). The drug-loading capacities of tamoxifen- and paclitaxel-loaded micelles were determined by high performance liquid chromatography and the micelle dimensions were determined by dynamic light scattering and transmission electron microscopy. Cell viability studies were carried out on human chronic myelogenous leukaemia (K-562) and colon carcinoma cell lines (Caco-2) in order to demonstrate that drug-loaded FA-MPC-DPA micelles exhibited higher cytotoxicities toward cancer cells than unfunctionalized MPC-DPA micelles. Uptake studies confirmed that folate-conjugated micelles led to increased drug uptake within cancer cells, demonstrating the expected selectivity toward these tumor cells.     

Comparative study of paclitaxel physically encapsulated in and chemically conjugated with PEG‐PLA

Paclitaxel‐loaded poly(ethylene glycol)‐b‐poly(l ‐lactide (LA)) (PEG‐PLA) micelles were prepared by two methods. One is physical encapsulation of paclitaxel in micelles composed of a PEG‐PLA block copolymer and the other is based on a PEG‐PLA–paclitaxel conjugate, abbreviated as “conjugate micelles”. Their physicochemical characteristics, e.g. critical micelle concentration (CMC), morphology, and micelle size distribution were then evaluated by means of fluorescence spectroscopy, scanning electron microscopy (SEM), and dynamic light scattering (DLS). The results show that the CMC of PEG‐PLA–paclitaxel and PEG‐PLA are 6.31 × 10^?4 and 1.78 × 10^?3 g L^?1, respectively. Both micelles assume a spherical shape with comparable diameters and have unimodal size distribution. Moreover, invitro drug delivery behavior was studied by high performance liquid chromatography (HPLC). The antitumor activity of the paclitaxel‐loaded micelles against human liver cancer H7402 cells was evaluated by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method. The conjugate micelles show a lower burst release during the initial stage and higher accumulative release amount of paclitaxel after a period of time while the encapsulated ones behave in the opposite way. Both the paclitaxel‐loaded micelles showed comparable anticancer efficacy with the free drug. Copyright © 2008 John Wiley & Sons, Ltd.     

Thermosensitive Micelles Based on Folate‐Conjugated Poly(N‐vinylcaprolactam)‐block‐Poly(ethylene glycol) for Tumor‐Targeted Drug Delivery

Thermosensitive PNVCL‐b‐PEG block copolymer coupled with folic acid was prepared as an anti‐cancer drug carrier. This polymer self‐assembled into stable micelles in aqueous solutions at above 33 °C. At 37 °C, the release profile of PNVCL‐b‐PEG‐FA micelles showed a slower and more controlled release of the entrapped 5‐FU than that at 25 °C. The blank and 5‐FU‐loaded PNVCL‐b‐PEG‐FA micelles did not induce remarkable cytotoxicity against the EA.hy 926 human endothelial cell line; however, 5‐FU‐loaded PNVCL‐b‐PEG‐FA micelles showed a cytotoxicity effect against 4T1 mouse mammary carcinoma cells due to the availability of loaded anti‐cancer drugs delivered to the inside of the cancer cells by the folate‐receptor‐mediated endocytosis process.

     

Comparison of drug delivery properties of PEG-<Emphasis Type="Italic">b</Emphasis>-pdhpc micelles with different compositions

An anti-tumor drug doxorubicin was encapsulated in micelles of poly（ethylene glycol）-b-poly（2,2-dihydroxyl-methyl propylene carbonate）（PEG-b-PDHPC） diblock copolymers.The morphology of both blank micelles and drug loaded micelles was characterized by TEM.The in vitro drug release profiles of micelles were investigated.The cytotoxicity of the micelles was evaluated by incubating with Hela tumor cells and 3T3 fibroblasts.The drug loaded micelles were co-cultured with HepG2 cells to evaluate the in vitro anti-tumor efficacies.The results showed that the mean sizes of both micelles with different copolymer compositions increased after being loaded with drugs.The drug release rate of PEG₄₅-b-PDHPC₃₄ micelles was faster than that of mPEG₁₁₄-b-PDHPC₂₆,micelles.Both of the two block copolymers were non-toxic.The confocal laser scanning microscopy and flow cytometry results showed that both the drug loaded micelles could be internalized efficiently in HepG2 cells.The PEG₄₅-b-PDHPC₃₄ micelles exhibited higher anti-tumor activity comparing to mPEG₁₁₄-b-PDHPC₂₆ micelles.     

Effect of pH and content of reduction-sensitive copolymer on the guest exchange kinetics of micelles

The micelles formed by reduction-sensitive amphiphilic copolymer have emerged as promising drug nanocarriers due to the controlled drug release and effective anticancer activity triggered by the reducing stimulation. However, the effect of pH on the stability and guest exchange of the micelles formed by reduction-sensitive copolymer have not been systemically investigated. Herein, the micelles formed by a reduction-sensitive copolymer poly(ε-caprolactone)-b-poly[oligo(ethylene glycol) methyl ether methacrylate] (PCL–SS–POEGA) with a single disulfide group at different pH values loaded with dyes 3,3′-dioctadecyloxacarbocyanine perchlorate/1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiO/DiI) were prepared through the precipitation-dialysis method. In addition, mixed micelles formed by different contents of reduction-sensitive and reduction-insensitive copolymers encapsulated with DiO/DiI at pH 7.5 were also prepared by the similar approach. The effects of pH and the content of reduction-sensitive copolymer on guest exchange of these micelles were studied by the fluorescence resonance energy transfer method. Results show that the pH value in the environment has great influence on the guest exchange rate of reduction-sensitive micelles in the presence of 10 mM dithiothreitol (DTT) and slight effect on that in the absence of DTT. Under a reducing environment, the guest exchange rate of the micelles containing various contents of disulfide-linked copolymer increases with the increasing content of PCL–SS–POEGA. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 1636–1644     

The synthesis of biodegradable graft copolymer cellulose-graft-poly(L-lactide) and the study of its controlled drug release

According to the concept of green chemistry and sustainable development, a new biodegradable copolymer comprised of hydrophobic poly(l-lactide) (PLLA) segments and hydrophilic cellulose segment (cellulose-g-PLLA) was designed and synthesized. The structure of the copolymer was characterized by (1)H NMR, FT-IR, (13)C NMR, DSC and WAXD. The cytotoxicity study shows that cellulose-g-PLLA exhibits good biocompatibility. The copolymer can self-assemble into micelles in water with the hydrophobic PLLA segments at the cores of micelles and the hydrophilic cellulose segments as the outer shells. Transmission electron microscopy (TEM) shows that the micelles exhibit nanospheric morphology within a size range of 30-80nm. The drug loaded micelles formed by the copolymer in aqueous media show sustained drug release which indicates their potential applicability in drug carrier.     

基于“分子胶”的新型两亲性嵌段共聚物β-CD-PLA的合成及其胶束化

以二重氢键为引导,二硫键连接疏水性聚乳酸(PLA)和亲水性β-环糊精(β-CD)合成了嵌段共聚物β-CD-PLA。采用1 H-NMR和GPC对嵌段共聚物β-CD-PLA的结构进行了表征,以芘作为荧光分子探针对嵌段共聚物β-CD-PLA自组装胶束的性质进行了表征,采用动态光散射纳米粒度仪(DLS)对自组装胶束的粒径进行了测试。结果表明:在二重氢键的引导作用力和碘的氧化作用下,中间体脱去保护基形成双二硫键,形成目标嵌段共聚物β-CD-PLA,该嵌段共聚物能够在水中自组装形成纳米胶束,临界胶束浓度(CMC)为0.089mg/mL,在稀溶液中具有良好的稳定性,自组装形成空白胶束的粒径为31nm,阿霉素盐酸盐(DOX)载药胶束的粒径为42nm。     

Effect of the Folate Ligand Density on the Targeting Property of Folated‐Conjugated Polymeric Nanoparticles

Targeted drug delivery systems have attracted increasing attention due to their ability for delivering anticancer drugs selectively to tumor cells. Folic acid (FA)‐conjugated targeted block copolymers, FA‐Pluronic‐polycaprolactone (FA‐Pluronic‐PCL) are synthesized in this study. The anticancer drug paclitaxel (PTX) is loaded in FA‐Pluronic‐PCL nanoparticles by nanoprecipitation method. The in vitro release of PTX from FA‐Pluronic‐PCL nanoparticles shows slow and sustained release behaviors. The effect of FA ligand density of FA‐Pluronic‐PCL nanoparticles on their targeting properties is examined by both cytotoxicity and fluorescence methods. It is shown that FA‐Pluronic‐PCL nanoparticles indicated better targeting ability than non‐targeted PCL‐Pluronic‐PCL nanoparticles. Furthermore, FA‐F127‐PCL nanoparticle with 10% FA molar content has more effective antitumor activity and higher cellular uptake than those with 50% and 91% FA molar content. These results prove that FA‐F127‐PCL nanoparticle with 10% FA molar content can be a better candidate as the drug carrier in targeted drug delivery systems.     

Gold-labeled block copolymer micelles reveal gold aggregates at multiple subcellular sites

There is increasing interest in the usefulness of block copolymer micelles as drug delivery vehicles. However, their subcellular distribution has not been explored extensively, mostly because of the lack of adequately labeled block copolymers. In a previous study, we showed that fluorescently labeled block copolymer micelles entered living cells and co-localized with cytoplasmic organelles selectively labeled with fluorescent dyes. The details of the observed co-localizations were, however, limited by the resolution of the fluorescence approach, which is ca. 500 nm. Using transmission electron microscopy (TEM), we established time- and concentration-dependent subcellular distributions of gold-labeled micelles within human embryonic kidney (HEK 293) cells and human lung carcinoma (A549) cells. Gold particles were incorporated into poly(4-vinylpyridine)-block-poly(ethylene oxide) (P4VP21-b-PEO45) micelles. Data from dynamic light scattering (DLS) and TEM analyses revealed that the sizes of the gold particles ranged from 4 to 8 nm. The cells survived up to 24 h in the presence of low gold-labeled micelle concentrations (0.73 microg/mL), but cell death occurred at higher concentrations (i.e., kidney cells are more susceptible than lung cells). Over 24 h periods of equivalent exposure, lung cells internalized significantly more gold-incorporated micelles than kidney cells. Although micelles were added to the cell culture media as dispersed colloidal particles, the presence of serum in these media caused aggregation. These aggregates occurred mainly close to the cell plasma membrane at early times (5-10 min); however, at later times (24 h) aggregated particles were seen inside endosomes and lysozomes. Thus, gold-incorporated (labeled) micelles can serve as a valuable extension of the fluorescence approach to visualizing the localization of micelles in subcellular compartments, improving the resolution by at least 20-fold.     

Microemulsions for intravesical delivery of gemcitabine

The objective of this work was to develop a safe and effective delivery vehicle for topical treatment of gemcitabine. The physicochemical properties, drug release rate, drug level in plasma and bladder, and histological changes of tissue after drug administration were investigated. The electrical conductivity, mean size, and viscosity of drug-loaded microemulsions were 0.8-102.0 μS/cm, 116.8-322.5 nm, and 42.9-105.0 cps×103, respectively. Gemcitabine loaded microemulsions showed a slower and sustained release. After intravesical administration of aqueous control and microemulsions treated, the drug concentrations in plasma were 15.11 μg/ml and 2.81-12.82 μg/ml, respectively, and the accumulation in bladder were 18.27 μg and 9.12-64.16 μg, respectively. Microemulsions slightly decreased the systemic absorption and significantly enhanced the accumulation in bladder tissue. Moreover, the preliminary toxicity studies revealed no overt adverse histological changes or tissue irritation by the microemulsion application. Therefore, the microemulsions were suggested to be a promising drug carrier for intravesical chemotherapy.     

两亲性嵌段共聚物mPEG-b-PCL/FA的合成及其药物释放性能

以苯甲醇为引发剂,在辛酸亚锡催化下引发ε-环己内酯(ε-CL )开环聚合,合成了聚己内酯(PCL).分别利用端甲氧基聚乙二醇(mPEG)、PCL及叶酸(FA)与三氯三嚎中不同的氯反应,得到两亲性嵌段共聚物mPEG-b-PCL/FA;采用傅立叶变换红外光谱仪和核磁共振谱仪等表征了mPEG-b-PCL/FA的组成和结构;采...     

Novel hepatoma‐targeting micelles based on chemoenzymatic synthesis and self‐assembly of galactose‐functionalized ribavirin‐containing amphiphilic random copolymer

Hepatoma‐targeting micelles were successfully prepared by self‐assembly of galactose‐functionalized ribavirin‐containing amphiphilic random copolymer as novel drug delivery vehicles. The ribavirin‐containing random copolymer with galactose as the targeting ligand was facilely synthesized by combining enzymatic transesterification with radical polymerization and fully characterized by FTIR, NMR, and GPC. The formation of micelle‐type aggregates from the random copolymer was verified by UV–vis and fluorescence spectroscopy using pyrene as the guest molecule. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) experiments revealed that the micelles were well dispersed as spherical nanoparticles in water, whose hydrodynamic diameter was 217 ± 19 nm. Their biological recognition to fluorescein‐labeled peanut agglutinin investigated by confocal laser scanning microscopy (CLSM) proved the existence of hydrophilic galactose targeting moieties on the surface of micelles. Cell cytotoxicity tests and the inhibition experiment of galactose performed by MTT assay showed that the micelles had evident targeting function to hepG2 cells and the galactose moieties on the surface of micelles mediated cellar uptake of micelles. In vitro release studies indicated that ribavirin could be slowly released from the copolymer with pseudo zero‐order kinetics. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2734–2744, 2008     

Block copolymer micelles conjugated with anti‐EGFR antibody for targeted delivery of anticancer drug

Antiepidermal growth factor receptor antibody (anti‐EGFR antibody) was conjugated with the block copolymer micelle based on poly(ethylene glycol) (PEG) and poly(ε‐caprolactone) (PCL) for active targeting to EGFR overexpressing cancer cells. Doxorubicin (DOX) was encapsulated in the core of the block copolymer (MePEG‐b‐PCL) micelle (DOX‐micelle). The mean diameters of the DOX‐micelle and the anti‐EGFR‐PEG‐b‐PCL copolymer micelles loaded with DOX (DOX‐anti‐EGFR‐micelle) were about 25 and 31 nm, respectively. The RKO human colorectal cancer cells expressing moderate degree of EGFR were incubated with free DOX, DOX‐micelle, or DOX‐anti‐EGFR‐micelle to study the distribution of DOX in the cells. When cells were incubated with free DOX, moderate degree of DOX fluorescence was observed in the nuclei. In the cells treated with DOX‐micelle, the DOX fluorescence intensity in the cytoplasm was much greater than that in the nuclei. On the other hand, the nuclei of the cells treated with DOX‐anti‐EGFR‐micelle exhibited DOX fluorescence intensity similar to that in the cytoplasm. The cytotoxicity of DOX‐anti‐EGFR‐micelle to induce apoptosis in RKO cells was significantly greater than that of free DOX or DOX‐micelle. These results demonstrated that the presence of anti‐EGFR antibody on the DOX‐micelle surface (DOX‐anti‐EGFR‐micelle) increased the internalization of the DOX‐micelle and nuclear accumulation of DOX, and enhanced the DOX‐induced cell death. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7321–7331, 2008