Bio-functional micelles self-assembled from a folate-conjugated block copolymer for targeted intracellular delivery of anticancer drugs

In this study, a block copolymer, poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-co-2-aminoethyl methacrylate)-b-poly(10-undecenoic acid) (P(NIPAAm-co-DMAAm-co-AMA)-b-PUA) was synthesized, and folic acid was conjugated to the hydrophilic block through the amine group in AMA. This polymer was self-assembled into micelles, which exhibited pH-induced temperature sensitivity. They were smaller in size, and possessed a better-defined core-shell structure as well as more stable hydrophobic core than the random copolymer P(NIPAAm-co-DMAAm-co-UA), and provided a shell with folate molecules. An anti-cancer drug, doxorubicin (DOX) was encapsulated into the micelles. The mean diameter of the blank and DOX-loaded micelles was less than 100 nm. DOX release was pH-dependent, being faster at low pH (endosomes/lysosomes). Therefore, DOX was readily released from the micelles into the nucleus after being taken up. More importantly, IC50 of DOX-loaded micelles with folate against folate receptor-expressing 4T1 and KB cells was much lower than that of the DOX-loaded micelles without folate (3.8 vs. 7.6 mg/L for 4T1 cells and 1.2 vs. 3.0mg/L for KB cells). In vivo experiments conducted in a 4T1 mouse breast cancer model demonstrated that DOX-loaded micelles had a longer blood circulation time than free DOX (t(1/2): 30 min and 140 min, respectively). In addition, the micelles delivered an increased amount of DOX to the tumor when compared to free DOX. These bio-functional micelles may make a promising carrier to transport anticancer drugs specifically to tumor cells and release the drug molecules inside the cells to the cytosols for improved chemotherapy.     

Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors

The aim of this study was to exploit the possibility of combination of active targeting function of folic acid by folate receptor-mediated endocytosis and overcoming multidrug resistance (MDR) by Pluronic block copolymers to promote drug delivery to MDR tumor following intravenous administration with paclitaxel (PTX) as model drug. Folic acid functionalized Pluronic P123/F127 mixed micelles encapsulating PTX (FPF-PTX) was firstly developed and tested in vitro and in vivo, while PTX-loaded Pluronic P123/F127 mixed micelles (PF-PTX) and Taxol were used as control. FPF-PTX was about 20 nm in diameter with spherical shape and high encapsulation efficiency. Cellular uptake of FPF-PTX was found to be higher than that of PF-PTX due to the folate receptor-mediated endocytosis effect. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies also revealed that FPF-PTX was more potent than those of PF-PTX and Taxol. In vivo pharmacokinetic study in rats showed that the polymeric micelles significantly enhanced the bioavailability of PTX (～3 fold) than Taxol. Moreover, in BALB/c mice bearing KBv MDR tumor xenografts, stronger antitumor efficacy was shown in FPF-PTX group, with good correlation between in vitro and in vivo. In conclusion, folate-conjugated Pluronic micelles could be a potential vehicle for delivering hydrophobic chemotherapeutic drugs to MDR tumors.     

Thermoresponsive nanostructured polycarbonate block copolymers as biodegradable therapeutic delivery carriers

Water-soluble, thermoresponsive block copolymers based on a biodegradable platform were synthesized by the ring opening polymerization of cyclic carbonate monomers functionalized with hydrophilic and hydrophobic groups for application as nanocarriers in medicine. The approach based on cyclic carbonate monomers derived from 2,2-bis(methylol)propionic acid (bis-MPA) allowed a simple and versatile route to functional monomers capable of undergoing ring opening polymerization (ROP). The resulting polymers possessed the predicted molecular weights based on the molar ratio between monomers to initiators and the narrow molecular weight distributions. Transmittance measurement for aqueous polymer solutions provided an evidence for temperature-responsiveness with lower critical solution temperature (LCST) in the range of 36?°C-60?°C, depending on the molecular weight of hydrophilic poly(ethylene glycol) (PEG) chains, compositions of copolymers, molar ratios of hydrophilic to hydrophobic monomers in the corona, and the hydrophobic core. This study showed synthetic advancement toward the design and preparation of biodegradable thermoresponsive polymers with extremely low CMC values for injectable drug delivery systems. TRC350-10,30,60, which possessed an LCST of 36?°C in PBS, was identified as a useful model polymer. Paclitaxel, an anti-cancer drug, was loaded into the micelles efficiently, giving rise to nano-sized particles with a narrow size distribution. Paclitaxel release from the micelles was faster, and cellular uptake of the micelles was higher at the body temperature (i.e. 37?°C) as compared to a temperature below the LCST. While the polymer was not cytotoxic, paclitaxel-loaded micelles killed HepG2 human liver carcinoma cells more efficiently at the body temperature as compared to free paclitaxel and paclitaxel-loaded micelles at the temperature below the LCST. These micelles are ideally suited to deliver anti-cancer drugs to tumor tissues through local injection.     

Paclitaxel-conjugated PEG and arginine-grafted bioreducible poly (disulfide amine) micelles for co-delivery of drug and gene

We developed a paclitaxel-conjugated polymeric micelle, ABP-PEG(3.5k)-Paclitaxel (APP) consisting of poly (ethylene glycol) (PEG) and arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP) for the co-delivery of gene and drug. The APP polymer self-assembled into cationic polymeric micelles with a critical micelle concentration (CMC) value of approximately 0.062?mg/mL, which was determined from measurements of the UV absorption of pyrene. The micelles have an average size of about 3?nm and a zeta potential of about?+14?mV. Due to the positive surface charge, APP micelles formed polyplexes with plasmid DNA approximately 200?nm in diameter. The luciferase gene and mouse interleukin-12 (IL-12) gene was used to monitor gene delivery potency. APP polyplexes showed increased gene delivery efficiency and cellular uptake with higher anticancer potency than paclitaxel alone. These results demonstrate that an APP micelle-based delivery system is well suitable for the co-delivery of gene and drug.     

Preparation and characterization of methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric nanospheres for tumor-specific folate-mediated targeting of anticancer drugs

Biodegradable methoxy poly(ethylene glycol)/poly(-caprolactone) (MPEG/PCL) amphiphilic block copolymer nanospheres coupled to folic acid have been designed to target a folate-binding protein that is overexpressed on the surface of many tumoral cells. For this purpose, hydroxy groups terminated on the MPEG/PCL copolymer were converted into primary amino groups, which were used to conjugate with the carboxylic group of folic acid. Nanospheres were prepared by the formation of micelles of the copolymer with or without the anticancer agent paclitaxel. Folate-mediated MPEG/PCL nanospheres were compared with hydroxyl- and amino-terminated nanospheres in terms of their size, surface characteristics, and drug-loading efficiency. Regardless of the type of terminal group, the MPEG/PCL nanospheres showed a narrow size distribution with an average diameter <80 nm without paclitaxel, and an average diameter of 115 nm when loaded with the drug. The results from zeta potential and X-ray photoelectron spectroscopy measurements revealed that the folate molecules were partially exposed, and were expressed on the surface of the nanospheres allowing folate receptor recognition. In in vitro, cytotoxicity tests, the nanospheres loaded with paclitaxel showed a higher cell viability than in cases where paclitaxel was absent. Thus, folate-mediated nanospheres composed of MPEG and PCL are potentially new drug carriers for tumor cell-selective targeting treatments.     

Self-assembled star-shaped chlorin-core poly(epsilon-caprolactone)-poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies

Amphiphilic 4-armed star-shaped chlorin-core diblock copolymers based on methoxy poly(ethylene glycol) (mPEG) and poly(varepsilon-caprolactone) (PCL) were synthesized and characterized in this study. The synthesized photosensitizer-centered amphiphilic star block copolymer that forms assembled micelle-like structures can be used in a photodynamic therapy (PDT)-functionalized drug delivery system. Moreover, the hydrophobic chemotherapeutic agent, paclitaxel, can be trapped in the hydrophobic inner core of micelles. In our results, the star-polymer-formed micelle exhibited efficient singlet oxygen generation, whereas the hydrophobic photosensitizer failed due to aggregation in aqueous solution. The chlorin-core micelle without paclitaxel loading exhibited obvious phototoxicity in MCF-7 breast cancer cells with 7J/cm2 or 14J/cm2 light irradiation at a chlorin concentration of 125microg/ml. After paclitaxel loading, the size of micelle increased from 71.4nm to 103.2nm. Surprisingly, these micelles were found to improve the cytotoxicity of paclitaxel significantly in MCF-7 cells after irradiation through a synergistic effect evaluated by median effect analysis. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer.     

Tumor targeting by pH-sensitive,biodegradable, cross-linked N-(2-hydroxypropyl) methacrylamide copolymer micelles

Increasing the molecular weight of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers by using micellar structures could result in more pronounced enhanced permeability and retention effect, thus increase the tumor accumulation of drug. However, most micellar formulations are relatively unstable and release their drug non-specifically. To improve on these disadvantages, we developed a micellar drug delivery system based on self-assembly of HPMA copolymers. Amphiphilic conjugates were synthesized by conjugating the hydrophobic drug doxorubicin and hydrophobic β-sitosterol to the hydrophilic HPMA polymer backbone via pH-sensitive hydrazone linkages. This linkage is quite stable at physiological pH but hydrolyzes easily at acidic pH. After conjugates self-assembly into micelles, HPMA copolymer side chains were cross-linked through the hydrazone linkages to ensure micelle stability in the blood. Using this approach, cross-linked micelles were obtained with molecular weight of 1030 KD and diameter of 10–20 nm. These micelles remained stable with undetectable doxorubicin release at pH 7.4 or mouse plasma, whereas collapsed quickly with 80% of the drug released at pH 5 which corresponds to the pH of lyso/endosome compartments of tumor cells. Both cross-linked and non-cross-linked micelles displayed similar in vitro anti-tumor activity as linear copolymer conjugates in Hep G2 and A549 cancer cell lines with internalization mechanism by caveolin, clathrin, and giant macropinocytosis. In vivo studies in an H22 mouse xenograft model of hepatocarcinoma showed the tumor accumulation (1633 μCi/L*h) and anti-tumor rate (71.8%) of cross-linked micelles were significantly higher than non-cross-linked ones (698 μCi/L*h, 64.3%). Neither type of micelle showed significant toxicity in heart, lung, liver, spleen or kidney. These results suggest that cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability show excellent potential as carriers of anti-cancer drugs.     

Self-assembled pH-responsive MPEG-b-(PLA-co-PAE) block copolymer micelles for anticancer drug delivery

A series of amphiphilic pH-responsive poly (ethylene glycol) methyl ether-b-(poly lactic acid-co-poly (β-amino esters)) (MPEG-b-(PLA-co-PAE)) block copolymers with different PLA/PAE ratios were designed and synthesized via a Michael-type step polymerization. The molecular structures of the copolymers were confirmed with (1)H NMR and gel permeation chromatography (GPC). These amphiphilic copolymers were shown to self-assemble into core/shell micelles in aqueous solution at low concentrations, and their critical micelle concentrations (CMC) in water were 1.2-9.5 mg/L. The pH-responsive PAE segment was insoluble at pH 7.4, but it became positively charged and soluble via protonation of amino groups at pH lower than 6.5. The average particle size and zeta potential of micelles increased from 180 nm and 15 mV to 220 nm and 40 mV, respectively, when the pH decreased from 7.4 to 5.0. Doxorubicin (DOX) was loaded into the core of these micelles with a high drug loading of 18%. The in vitro DOX release from the micelles was significantly accelerated when solution pH decreased from 7.4 to 5.0. DOX release in the first 10 h appeared to follow Fickian diffusion mechanism. Toxicity test showed that the copolymers had low toxicity whereas the DOX-loaded micelles remained high cytotoxicity for HepG2 cells. The results indicate the pH-sensitive MPEG-b-(PLA-co-PAE) micelle may be a potential hydrophobic drug delivery carrier for cancer targeting therapy with sustained release.     

The synthesis and co-micellization of PCL-P(HEMA/HEMA-LA) and PCL-P(HEMA/HEMA-FA) as shell cross-linked drug carriers with target/redox properties

In order to obtain target/redox shell cross-linked micelles (TCM), copolymers poly(ε-caprolactone)-poly(2-hydroxyethyl methacrylate/methacrylate-alpha lipoic acid) and poly(ε-caprolactone)-poly(2-hydroxyethyl methacrylate/methacrylate-folate, PCL-P(HEMA/HEMA-LA) and PCL-P(HEMA/HEMA-FA) were designed and synthesized. The copolymers PCL-P(HEMA/HEMA-LA) could form reduction-sensitive cross-linked micelles (CM) by using a catalytic amount of DTT. The micelles maintained high stability against dilution but were destroyed in 10?mM dithiothreitol (DTT). The drug loaded content (DLC) of CM was 8.9%, which was almost twice as much as non-cross-linked micelle (NCM). In vitro drug release at pH 7.4 showed that the cumulative release rate of CM in 36?h was less than 30%, while it was about 50% for NCM. When PCL-P(HEMA/HEMA-LA) and PCL-P(HEMA/HEMA-FA) (FA 1%, 3% and 5%) formed target/redox micelles, IC50 of TCM with FA 3% was the lowest (1.4?µg/mL) to Hela cells with excessive expression folate receptors. The cell uptake of TCM by Hela cells is higher than target non-cross-linked micelles (TNCM), while there was not much difference between both micelles uptaken by A549 cells, which are lack of folate receptors. Therefore, the drug carriers of TCM have potential to be explored as shell cross-linked target/redox drug carriers to the cancer cells on the surface with excessive folate receptors.     

A one-pot synthesis of oleic acid end-capped temperature- and pH-sensitive amphiphilic polymers

A one-pot synthesis of an amphiphilic oleic acid-end capped random poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (2) is reported. In aqueous media, the solubility of 2 was temperature- and pH-sensitive. Both the lower critical solution temperature and critical micelle concentration (CMC) of 2 were pH-dependent. The LCST of 2 was around 35.2 degrees C in acidic buffer solutions (pH=2.00-5.00), and it was increased significantly to around 38.4 degrees C in neutral and alkaline buffer solutions (pH 7.00-11.00). Polymer 2 exhibited a phase transition pH around 6.7, below which the polymer became significantly less water-soluble. The CMC of 2 was 40 mg/l in pH 2.0 buffer solution, and it was increased markedly to 60-67 mg/l in pH 7.0 and pH 11.0 buffer solutions. Micelle solutions of 2 in different pH conditions were prepared by a membrane-dialysis method. In aqueous solution, dynamic light scattering studies revealed that the size of micelles was 50-90 nm with the particle size being larger in acidic solutions. In solid state, transmission electron microscope studies showed that micelles were roughly spherical, their sizes were 25-90 nm and it decreased with the increase of solution pH. The pH-sensitivity of 2 was triggered by the -COOH in the hydrophobic segment. The temperature- and pH-sensitivity of the novel polymeric micelles would make an interesting drug delivery system.     

Gold nanoparticles with a monolayer of doxorubicin-conjugated amphiphilic block copolymer for tumor-targeted drug delivery

Gold (Au) nanoparticles (NPs) stabilized with a monolayer of folate-conjugated poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol) copolymer (Au-P(LA-DOX)-b-PEG-OH/FA) was synthesized as a tumor-targeted drug delivery carrier. The Au-P(LA-DOX)-b-PEG-OH/FA NPs consist of an Au core, a hydrophobic poly(l-aspartate-doxorubicin) (P(LA-DOX)) inner shell, and a hydrophilic poly(ethylene glycol) and folate-conjugated poly(ethylene glycol) outer shell (PEG-OH/FA). The anticancer drug, doxorubicin (DOX), was covalently conjugated onto the hydrophobic inner shell by acid-cleavable hydrazone linkage. The DOX loading level was determined to be 17 wt%. The Au-P(LA-DOX)-b-PEG-OH/FA NPs formed stable unimolecular micelles in aqueous solution. The size of the Au-P(LA-DOX)-b-PEG-OH/FA micelles were determined as 24–52 and 10–25 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The conjugated DOX was released from the Au-P(LA-DOX)-b-PEG-OH/FA micelles much more rapidly at pH 5.3 and 6.6 than at pH 7.4, which is a desirable characteristic for tumor-targeted drug delivery. Cellular uptake of the Au-P(LA-DOX)-b-PEG-OH/FA micelles facilitated by the folate-receptor-mediated endocytosis process was higher than that of the micelles without folate. This was consistent with the higher cytotoxicity observed with the Au-P(LA-DOX)-b-PEG-OH/FA micelles against the 4T1 mouse mammary carcinoma cell line. These results suggest that Au-P(LA-DOX)-b-PEG-OH/FA NPs could be used as a carrier with pH-triggered drug releasing properties for tumor-targeted drug delivery.     

Doxorubicin-loaded,charge reversible,folate modified HPMA copolymer conjugates for active cancer cell targeting

Although folate exhibits many advantages over other targeting ligands, it has one major defect: poor water solubility. Once it was conjugated to hydrophilic drug carrier such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, the hydrophobic folate may be buried inside the random polymer coil and not exposed to be accessible to its receptor on the cell surface, thus losing its active targeting ability. To address this folate dilemma, the positive charge was introduced in the present study. The obtained cationic folate-functionalized HPMA copolymers exhibited a synergistic enhancing effect on cellular uptake by folate receptor (FR) positive Hela cells via electrostatic absorptive endocytosis and folate receptor-mediated endocytosis, with the involvement of multiple internalization pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and energy-dependent endocytosis. As demonstrated in binding efficiency study, the FR antibody bound to 71.2% of tested cells in the competition with neutral folate modified HPMA copolymers, while the FR antibody-bounded cells decreased to only 34.0% in competition with cationic folate modified HPMA copolymers, indicating that the positively charge could probably amplify the binding efficiency of folate to its receptor due to close proximity of the conjugates to the cell surface by the electronic adhesion. In addition, the cell uptake study on FR negative A549 cells also confirmed the specific role of folate as targeting ligand. Then, to avoid non-specific binding by positive charge in the circulation, the charge shielding/deshielding approach was further employed. With selective hydrolysis of the charge shielding groups 2,3-dimethylmaleic anhydride (DMA) at tumor extracellular pH 6.8, the conjugates underwent a quick charge-reversible process with more than 80% DMA cleavage within 2 h and endocytosed into the endo/lysosomes much more rapidly than at physiological pH 7.4. And then the drug release was triggered by the cleavage of hydrazone spacer at another level of pH 5 in endo/lysosomal compartment. Furthermore, the anticancer activity results showed that Dox-loaded, charge-switchable, folate modified HPMA copolymer conjugates could indeed lead to enhanced cytotoxicity, stronger apoptosis and greater tumor spheroid inhibition towards Hela cells, indicating the great potential feasibility of this multiple responsive drug delivery system.     

The potential of pH-responsive PEG-hyperbranched polyacylhydrazone micelles for cancer therapy

pH-responsive hyperbranched polymers have attracted much attention due to their unique properties for tumor-targeted drug delivery. In this study, we describe a pH-responsive drug carrier, poly (ethylene glycol) (PEG)-hyperbranched polyacylhydrazone (HPAH), which can form nanoscale micelles to be used as anti cancer drug carriers with pH-controlled drug release. The molecular structure of PEG-HPAH was confirmed by nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). The drug-loaded micelles with a diameter of approximately 190 nm, were prepared using a dialysis method against PBS with a pH of 8.0. The drug-loaded micelles showed the desired pH-dependent drug release properties. The drug release levels were low at neutral and alkaline pH, but increased significantly with a decrease in the pH of the medium. Intracellular uptake results indicated that the PEG-HPAH-drug micelles could efficiently deliver chemotherapeutic drugs into the cells. In addition, it was found that the subcellular localization of the drug-loaded micelles was different from that of free drugs, in which the drug-loaded micelles were mainly in the cytoplasm. The docetaxel (DTX)-loaded PEG-HPAH micelles presented a high cytotoxic activity against tumor cells in vitro. When combined with the administration of glucose, the PEG-HPAH-DTX micelles exhibited a superior anti-tumor efficacy and a lower systemic toxicity in vivo. The biodistribution profile showed increased accumulated drug levels in tumor tissue and plasma in micelles treated group. The results indicate that the nanoscale PEG-HPAH-DTX micelles may serve as a selective tumor-targeting drug delivery system.     

Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel -polymer micelles to overcome multi-drug resistance

Multidrug resistance (MDR) in tumor cells is a significant obstacle for successful cancer chemotherapy. Overexpression of drug efflux transporters such as P-glycoprotein (P-gp) is a key factor contributing to the development of tumor drug resistance. Verapamil (VRP), a P-gp inhibitor, has been reported to be able to reverse completely the resistance caused by P-gp. For optimal synergy, the drug and inhibitor combination may need to be temporally colocalized in the tumor cells. Herein, we investigated the effectiveness of simultaneous and targeted delivery of anticancer drug, paclitaxel (PTX), along with VRP, using DOMC-FA micelles to overcome tumor drug resistance. The floate-functionalized dual agent loaded micelles resulted in the similar cytotoxicity to PTX-loaded micelles/free VRP combination and co-administration of two single-agent loaded micelles, which was higher than that of PTX-loaded micelles. Enhanced therapeutic efficacy of dual agent micelles could be ascribe to increased accumulation of PTX in drug-resistant tumor cells. We suggest that the synergistic effect of folate receptor-mediated internalization and VRP-mediated overcoming MDR could be beneficial in treatment of MDR solid tumors by targeting delivery of micellar PTX into tumor cells. As a result, the difunctional micelle systems is a very promising approach to overcome tumor drug resistance.     

Biamphiphilic triblock copolymer micelles as a multifunctional platform for anticancer drug delivery

Novel micelles from biamphiphilic triblock copolymer poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(acrylic acid) (PEG-b-PCL-b-PAA) as new multifunctional nanocarriers to delivery anticancer drugs were evaluated. The well-defined triblock copolymers prepared by controlled polymerizations self-assembled into micelles in aqueous solution with a hydrodynamic radius of 13 nm as obtained by dynamic light scattering (DLS) and a low critical micellization concentration of 2.9 × 10(-4) g/L. The hydrophobic PCL cores of micelles were applied to load hydrophobic drug doxorubicin and the functional PAA subcoronas clung to the micellar core were used to carry cisplatin through covalent interaction. The results indicated that two anticancer drugs had been loaded by different mechanism either separately or simultaneously. Drug loading content and efficiency as well as release profiles were evaluated. Furthermore, internalization and cytotoxicity of the anticancer nanoparticles against human bladder carcinoma EJ cells were studied. The biamphiphilic triblock copolymer micelles provided not only biocompatibility and biodegradability, but also abilities for loading single and dual anticancer drugs, indicating that this was a useful multifunctional platform for anticancer drug delivery.     

Folate-functionalized polymeric micelles for tumor targeted delivery of a potent multidrug-resistance modulator FG020326

To overcome multidrug resistance (MDR) existing in tumor chemotherapy, polymeric micelles encoded with folic acid on the micelle surface were prepared with the encapsulation of a potent MDR modulator, FG020326. The micelles were fabricated from diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable poly(epsilon-caprolactone) (PCL) with folate attached to the distal ends of PEG chains. The folate-conjugated copolymers, folate-PEG-PCL, were synthesized by multistep chemical reactions. First, allyl-terminated copolymer (allyl-PEG-PCL) was synthesized through a ring-opening polymerization of epsilon-caprolactone in bulk employing monoallyl-PEG as a macroinitiator. Second, the allyl terminal groups of copolymers were converted into primary amino groups by a radical addition reaction, followed by conjugation of the carboxylic group of folic acid. In vitro studies at 37 degrees C demonstrated that FG020326 release from micelles at pH 5.0 was faster than that at pH 7.4. Cytotoxicity studies with MTT assays indicated that folate-functionalized and FG020326-loaded micelles resensitized the cells approximately five times more than their folate-free counterparts (p < 0.01) in human KB(v200) cells treated with vincristine (VCR). The in vitro Rhodamine 123 efflux experiment using MDR KB(v200) cells revealed that when cells were pretreated with folate-attached and FG020326-loaded micelles, the P-glycoprotein (P-gp) drug efflux function was significantly inhibited.     

Amphiphilic multi-arm-block copolymer conjugated with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery

Folate-conjugated unimolecular micelles based on amphiphilic hyperbranched block copolymer, Boltorn^® H40-poly(l-aspartate-doxorubicin)-b-poly(ethylene glycol)/FA-conjugated poly(ethylene glycol) (H40-P(LA-DOX)-b-PEG-OH/FA), were synthesized as a carrier for tumor-targeted drug delivery. The anticancer drug DOX was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms by pH-sensitive hydrazone linkage. The size of the unimolecular micelles was determined as 17–36 and 10–20 nm by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The release profiles of the DOX from the H40-P(LA-DOX)-b-PEG-OH/FA micelles showed a strong dependence on the environmental pH values. The DOX release rate increased in the acidic medium due to the acid-cleavable hydrazone linkage between the DOX and micelles. Cellular uptake of the H40-P(LA-DOX)-b-PEG-OH/FA micelles was found to be higher than that of the H40-P(LA-DOX)-b-PEG-OH micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. Degradation studies showed that the H40-P(LA-DOX)-b-PEG-OH/FA copolymer hydrolytically degraded into polymer fragments within six weeks. These results suggest that H40-P(LA-DOX)-b-PEG-OH/FA micelles could be a promising nanocarrier with excellent in vivo stability for targeting the drugs to cancer cells and releasing the drug molecules inside the cells by sensing the acidic environment of the endosomal compartments.     

Addition of TPGS to folate-conjugated polymer micelles for selective tumor targeting

Folate-conjugated polymer micelles poly(D,L-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) was fabricated to encapsulate anticancer drug doxorubicin for targeting delivery to cancer cells with overexpression of folate receptors. To increase therapeutic effect, D-alpha-tocopheryl polyethylene glycol succinate (TPGS) was added during the micelles preparation. The physicochemical study showed that the mixed micelles of PLGA-PEG-FOL and TPGS formed a homogeneous population. The addition of TPGS did not result in much variation in the micellar size, surface charge, and drug encapsulation efficiency. The cellular uptake study showed that mixed micelles with TPGS had higher cellular uptake compared with the ones without TPGS to drug-resistant cancer cells. These mixed micelles also selectively increased the cytotoxicity of drug on cancer cells but exhibited minimal cytotoxic enhancement on normal fibroblasts. Furthermore, the accumulation of rhodamine study showed that the mixed micelles with TPGS increased the cellular uptake of drugs on Caco-2 cells. This indicates that TPGS in the mixed micelles may act as P-glycoprotein inhibitor to reduce drug efflux. This new formulation with TPGS may have dual functions of folate-mediated targeting and multidrug resistance inhibition and can be promising in improving the therapeutic efficacy of polymer micellar targeting delivery system.     

Stealth properties of poly(ethylene oxide)-based triblock copolymer micelles: a prerequisite for a pH-triggered targeting system

Evaluation of the biocompatibility of pH-triggered targeting micelles was performed with the goal of studying the effect of a poly(ethylene oxide) (PEO) coating on micelle stealth properties. Upon protonation under acidic conditions, pH-sensitive poly(2-vinylpyridine) (P2VP) blocks were stretched, exhibiting positive charges at the periphery of the micelles as well as being a model targeting unit. The polymer micelles were based on two different macromolecular architectures, an ABC miktoarm star terpolymer and an ABC linear triblock copolymer, which combined three different polymer blocks, i.e. hydrophobic poly(ε-caprolactone), PEO and P2VP. Neutral polymer micelles were formed at physiological pH. These systems were tested for their ability to avoid macrophage uptake, their complement activation and their pharmacological behavior after systemic injection in mice, as a function of their conformation (neutral or protonated). After protonation, complement activation and macrophage uptake were up to twofold higher than for neutral systems. By contrast, when P2VP blocks and the targeting unit were buried by the PEO shell at physiological pH, micelle stealth properties were improved, allowing their future systemic injection with an expected long circulation in blood. Smart systems responsive to pH were thus developed which therefore hold great promise for targeted drug delivery to an acidic tumoral environment.     

Self-assembled, fluorescent polymeric micelles of a graft copolymer containing carbazole for thermo-controlled drug delivery in vitro

A novel thermosensitive amphiphilic graft copolymer PNIPAAm-g-PCbzEA appending carbazole group was successfully designed and synthesized by the free radical copolymerization of N-isopropylacrylamide with hydrophobic precursor polymers of vinyl-functionalized poly(2-(N-carbazolyl)ethyl acrylate) (PCbzEA) in DMF. The PNIPAAm-g-PCbzEA copolymer was characterized by FTIR, (1)H NMR, GPC analysis, UV-vis spectroscopy and fluorescence spectroscopy. The TEM observation shows that the graft copolymer may self-assemble into polymeric micelles exhibiting a nanospheric morphology within a narrow size range of 30-60 nm in aqueous solution. From the (1)H NMR and FTIR analysis, the polymer micelles are composed of hydrophobic PCbzEA segments as the cores and the hydrophilic PNIPAAm segements as outer shells. The resulting micelles exhibited the temperature sensitivity with a lower critical solution temperature (LCST) of 31.5 degrees C and a critical micelle concentration (CMC) of 12.9 mg/L in water. In the study of drug release, an "on-off" drug release profile was found in response to stepwise temperature changes between 20 and 40 degrees C. The cytotoxicity assays for vero cells shows good biocompatibility of the graft copolymer in vitro.