pH-sensitive pluronic micelles combined with oxidative stress amplification for enhancing multidrug resistance breast cancer therapy.

Multidrug resistance (MDR) is one of the major obstacles to clinical cancer chemotherapy. Herein, we designed new pH-sensitive pluronic micelles with the synergistic effects of oxidative therapy and MDR reversal. Pluronic (P123) was modified with α-tocopheryl succinate (α-TOS) via an acid-labile ortho ester (OE) linkage to give a pH-sensitive copolymer (POT). Self-assembled POT micelles exhibited desirable size (~80 nm), excellent anti-dilution ability, high drug loading (~85%), acid-triggered degradation and drug release behaviours. In vitro cell experiments verified that POT micelles could significantly reverse MDR through suppressing the function of drug effluxs mediated by P123 and induce more reactive oxygen species (ROS) generation mediated by α-TOS, resulting in enhanced cytotoxicity and apoptosis in MDR cells. In vivo studies further revealed that DOX-loaded POT micelles (POT-DOX) possessed the highest drug accumulation (3.03% ID/g at 24 h) and the strongest tumour growth inhibition (TGI 83.48%). Pathological analysis also indicated that POT-DOX could induce more apoptosis or necrosis at the site of tumour without distinct damage to normal tissues. Overall, these smart POT micelles have great potential as promising nano-carriers for MDR reversal and cancer treatment.

Redox-responsive F127-folate/F127-disulfide bond-d-α-tocopheryl polyethylene glycol 1000 succinate/P123 mixed micelles loaded with paclitaxel for the reversal of multidrug resistance in tumors.

INTRODUCTION: The development of nanodrug carriers utilizing tumor microenvironment has become a hotspot in reversing multidrug resistance (MDR). MATERIALS AND METHODS: This study synthesized a redox-sensitive copolymer, Pluronic F127-disulfide bond-d-α-tocopheryl polyethylene glycol 1000 succinate (FSST), through the connection of the reduction-sensitive disulfide bond between F127 and d-α-tocopheryl polyethylene glycol 1000 succinate. This polymer could induce the elevation of reactive oxygen species (ROS) levels, ultimately resulting in cytotoxicity. Moreover, the redox-responsive mixed micelles, F127-folate (FA)/FSST/P123 (FFSSTP), based on FSST, Pluronic F127-FA, and Pluronic P123, were prepared to load paclitaxel (PTX). RESULTS: The in vitro release study demonstrated that FFSSTP/PTX accelerated the PTX release through the breakage of disulfide bond in reductive environment. In cellular experiment, FFSSTP/PTX induced significant apoptosis in PTX-resistant MCF-7/PTX cells through inhibiting adenosine triphosphate (ATP)-binding cassette proteins from pumping out PTX by interfering with the mitochondrial function and ATP synthesis. Furthermore, FFSSTP/PTX induced apoptosis through elevating the intracellular levels of ROS. CONCLUSION: FFSSTP could become a potential carrier for the treatment of MDR tumors.

Use of mesoporous cellular foam (MCF) in preparation of polymeric microspheres for long acting injectable release formulations of paliperidone antipsychotic drug.

In this study, high surface area mesoporous silica foam with cellular pore morphology (MCF) was used for injectable delivery of paliperidone, an antipsychotic drug used in patients suffering from bipolar disorder. The aim was to enhance paliperidone solubility and simultaneously to prepare long active intractable microspheres. For this reason paliperidone was first loaded in MCF silica, and the whole system was further encapsulated into PLA and PLGA 75/25w/w copolymer in the form of microspheres. It was found that paliperidone, after its adsorption into MCF, was transformed in its amorphous state, thus leading to enhanced in vitro dissolution profile. Furthermore, incorporation of the drug-loaded MCF to polymeric microparticles (PLA and PLGA) prolonged the release time of paliperidone from 10 to 15days.

pH-responsive copolymers based on pluronic P123-poly(ß-amino ester): Synthesis, characterization and application of copolymer micelles.

A novel amphiphilic and pH-responsive copolymer, pluronie P123-poly(ß-amino ester) (P123-PAE), was firstly designed and synthesized using a Michael-type step polymerization. Nano-sized polymeric micelles based on P123-PAE block copolymer were prepared by self-assembly. Curcumin (Cur), a potential cancer therapy drug, was efficiently encapsulated into the P123-PAE micelles to enhance anticancer efficacy. The obtained Cur loaded P123-PAE micelles (Cur-P123-PAE) presented a spherical shape and high drug loading (18.4%). Interestingly, when the media pH decreased from 7.4 to 5.5, the particle size of the micelles shrank from 152.5nm to 122.1nm due to the protonation of PAE blocks, and the zeta potential of the P123-PAE micelles changed from weakly positive (1.5mV) to highly positive (9.0mV) over a pH range from 7.4 to 5.5. In vitro drug release studies demonstrated that the release rate of Cur was markedly influenced by pH. In vitro cytotoxicity tests showed that all the blank micelles were non-toxic. Cur-P123-PAE exhibited similar antitumor effect against MCF-7 and HepG2 cells compared to solubilized Cur solution. Using Coumarin-6 as a fluorescence probe, it was observed that Cur-P123-PAE micelles experienced longer circulation followed by accumulation at tumor tissues with stronger fluorescence intensity. The results of pharmacokinetics studies showed that the P123-PAE micelles could significantly prolong the retention time of Cur in vivo.

Effects of Bile Salt Sodium Glycodeoxycholate on the Self-Assembly of PEO-PPO-PEO Triblock Copolymer P123 in Aqueous Solution.

A comprehensive experimental study on the interaction between the PEO-PPO-PEO block copolymer P123 (EO20PO68EO20) and the anionic bile salt sodium glycodeoxycholate (NaGDC) in water has been performed. The work was aimed at investigating the suitability of using P123 as bile salt sequestrant beside the fundamental aspects of PEO-PPO-PEO block copolymer-bile salt interactions. Various experimental techniques including dynamic and static light scattering, small-angle X-ray scattering, and differential scanning calorimetry (DSC) were employed in combination with electrophoretic mobility measurements. The system was investigated at a constant P123 concentration of 1.74 mM and with varying bile salt concentrations up to approximately 250 mM NaGDC (or a molar ratio n(NaGDC)/n(P123) = 144). In the mixed P123-NaGDC solutions, the endothermic process related to the self-assembly of P123 was observed to gradually decrease in enthalpy and shift to higher temperatures upon progressive addition of NaGDC. To explain this effect, the formation of NaGDC micelles carrying partly dehydrated P123 unimers was proposed and translated into a stoichiometric model, which was able to fit the experimental DSC data. In the mixtures at low molar ratios, NaGDC monomers associated with the P123 micelle forming a charged "P123 micelle-NaGDC" complex with a dehydrated PPO core. These complexes disintegrated upon increasing NaGDC concentration to form small "NaGDC-P123" complexes visualized as bile salt micelles including one or a few P123 copolymer chains.

Reversal of P-glycoprotein-mediated multidrug resistance by CD44 antibody-targeted nanocomplexes for short hairpin RNA-encoding plasmid DNA delivery.

Multidrug resistance (MDR) remains one of the major reasons for the reductions in efficacy of many chemotherapeutic agents in cancer therapy. As a classical MDR phenotype of human malignancies, the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp) is an efflux protein with aberrant activity that has been linked to multidrug resistance in cancer. For the reversal of MDR by RNA interference (RNAi) technology, an U6-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed (abbreviated pDNA-iMDR1-shRNA). This study explored the feasibility of using Pluronic P123-conjugated polypropylenimine (PPI) dendrimer (P123-PPI) as a carrier for pDNA-iMDR1-shRNA to overcome tumor drug resistance in breast cancer cells. P123-PPI functionalized with anti-CD44 monoclonal antibody (CD44 receptor targeting ligand) (anti-CD44-P123-PPI) can efficiently condense pDNA into nanocomplexes to achieve efficient delivery of pDNA, tumor specificity and long circulation. The in vitro studies methodically evaluated the effect of P123-PPI and anti-CD44-P123-PPI on pDNA-iMDR1-shRNA delivery and P-gp downregulation. Our in vitro results indicated that the P123-PPI/pDNA and anti-CD44-P123-PPI/pDNA nanocomplexes with low cytotoxicity revealed higher transfection efficiency compared with the PPI/pDNA nanocomplexes and Lipofectamine™ 2000 in the presence of serum. The nanocomplexes loaded with pDNA-iMDR1-shRNA against P-gp could reverse MDR accompanied by the suppression of MDR1/P-gp expression at the mRNA and protein levels and improve the internalization and cytotoxicity of Adriamycin (ADR) in the MCF-7/ADR multidrug-resistant cell line. BALB/c nude mice bearing MCF-7/ADR tumor were utilized as a xenograft model to assess antitumor efficacy in vivo. The results demonstrated that the administration of anti-CD44-P123-PPI/pDNA-iMDR1-shRNA nanocomplexes combined with ADR could inhibit tumor growth more efficiently than ADR alone. The enhanced therapeutic efficacy of ADR may be correlated with increased accumulation of ADR in drug-resistant tumor cells. Consequently, these results suggested that the use of pDNA-iMDR1-shRNA-loaded nanocomplexes may be a promising gene delivery strategy to reverse MDR and improve the effectiveness of chemotherapy.

An interface-directed coassembly approach to synthesize uniform large-pore mesoporous silica spheres.

A facile and controllable interface-directed coassembly (IDCA) approach is developed for the first time to synthesize uniform discrete mesoporous silica particles with a large pore size (ca. 8 nm) by using 3-dimensional macroporous carbon (3DOMC) as the nanoreactor for the confined coassembly of template molecules and silica source. By controlling the amount of the precursor solution and using Pluronic templates with different compositions, we can synthesize mesoporous silica particles with diverse morphologies (spheres, hollow spheres, and hemispheres) and different mesostructure (e.g., 2-D hexagonal and 3D face centered cubic symmetry), high surface area of about 790 m(2)/g, and large pore volume (0.98 cm(3)/g). The particle size can be tunable from submicrometer to micrometer regimes by changing the macropore diameter of 3DOMC. Importantly, this synthesis concept can be extended to fabricate multifunctional mesoporous composite spheres with a magnetic core and a mesoporous silica shell, large saturated magnetization (23.5 emu/g), and high surface area (280 m(2)/g). With the use of the magnetic mesoporous silica spheres as a magnetically recyclable absorbent, a fast and efficient removal of microcystin from water is achieved, and they can be recycled for 10 times without a significant decrease of removal efficiency for microcystin.

Pluronic P85/poly(lactic acid) vesicles as novel carrier for oral insulin delivery.

Poly(lactic acid)-b-Pluronic-b-poly(lactic acid) (PLA-P85-PLA) vesicles were developed as novel carrier for oral insulin delivery. PLA-P85-PLA block copolymer was synthesized by ring opening polymerization of the monomer l-lactide using Pluronic copolymer P85 as the initiator. Insulin-loaded PLA-P85-PLA vesicles were prepared by dialysis method and the mean diameter of insulin-loaded PLA-P85-PLA vesicles was determined to be 178 nm. The cytotoxicity studies using human ovarian cancer cells OVCAR-3 indicate that PLA-P85-PLA block copolymer has good biocompatibility. Both in vitro and in vivo release behavior of insulin loaded in PLA-P85-PLA vesicles were studied. It was observed that insulin was released out gradually from PLA-P85-PLA vesicles and almost all insulin was released out 7.5h later. More importantly, for the oral administration of insulin-loaded PLA-P85-PLA vesicles at insulin doses of 200 IU/kg, the minimum blood glucose concentration was observed in the diabetic mice test after 2.5h, which was 15% of initial glucose level. Furthermore, the blood glucose concentration increased slowly to 31.8% of initial blood glucose concentration after 10.5h and was maintained at this level for at least an additional 14h (32.5% of initial blood glucose concentration at 24.5h). These results proved that PLA-P85-PLA vesicles could be promising polymeric carriers for oral insulin delivery application due to their sustained and enhanced hypoglycemic effect.

Mesoporous silicas synthesis and application for lignin peroxidase immobilization by covalent binding method.

Immobilization of enzymes on mesoporous silicas (MS) allows for good reusability. MS with two-dimensional hexagonal pores in diameter up to 14.13 nm were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene as a swelling agent in acetate buffer. The surface of MS was modified by the silanization reagents 3-aminopropyltriethoxysilane. Lignin peroxidase (LiP) was successfully immobilized on the modified MS through covalent binding method by four agents: glutaraldehyde, 1,4-phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide. Results showed that cyanotic chloride provided the best performance for LIP immobilization. The loaded protein concentration was 12.15 mg/g and the immobilized LiP activity was 812.9 U/L. Immobilized LiP had better pH stability. Acid Orange II was used to examine the reusability of immobilized LiP, showing more than 50% of the dye was decolorized at the fifth cycle.

Preparation and characterization of poly(pluronic-co-L-lactide) nanofibers for tissue engineering.

In this work, a new kind of biodegradable poly(pluronic-co-L-lactide) (Pluronic-PLLA copolymers) was successfully prepared by melt-polycondensation method from L-lactide, Pluronic and isophorone diisocyanate (IPDI). The obtained copolymers were characterized by (1)H NMR, FT-IR, X-ray, and TGA/DTA. Meanwhile, three-dimensional (3-D) porous scaffolds based on Pluronic-PLLA were prepared by the electrospinning method, the factors of concentration, flow rate and voltage that influence the formation of the Pluronic-PLLA nanofibers were studied and the structure of Pluronic-PLLA nanofibers were investigated by scanning electron microscopy (SEM). MTT results revealed that the Pluronic-PLLA scaffolds had good biocompatibility and nontoxicity. Morphological study using fluorescence micrographs and scanning electron microscopy showed that in vitro osteoblast cell culture demonstrated the electrospun Pluronic-PLLA composite scaffolds could provide a suitable environment for good cell attachment. These results suggested that such Pluronic-PLLA nanofibers membranes might have prospective applications in tissue engineering field.

Preparation and evaluation of folate-modified cationic pluronic micelles for poorly soluble anticancer drug.

The aim of this study was to construct novel targeting polymeric micelles. Folate-Poly (ethylenimine)-Pluronic copolymers were synthesized. A paclitaxel (PTX)-loaded mixed micelles consisting of Folate-Poly (ethylenimine)-Pluronic and Pluronic L121 copolymers have been developed. The mixed micelles showed nano-sized spherical morphology. The solubilization capacity of the mixed micelles was higher than Folate-Poly (ethylenimine)-Pluronic micelles because L121 has high solubilization capacity. MTT colorimetric test revealed that PTX in Folate-Poly (ethylenimine)-Pluronic micelles demonstrated the maximum anticancer activity. Pluronic-poly (ethylenimine) micelles and folate-modified Pluronic-poly(ethylenimine) micelles showed a marked increase of cellular accumulation compared with Pluronic P123 micelles. The biodistribution and retention of intravenously (i.v.) administered micelles to rats were determined. Folate-Poly (ethylenimine)-Pluronic micelles demonstrated enhanced pulmonary retention in rats after injection when compared to Pluronic P123 micelles.

Synthesis of poly(acrylic acid) (PAA) modified Pluronic P123 copolymers for pH-stimulated release of doxorubicin.

Pluronic P123 was chain-extended at their terminal groups using atom transfer radical polymerization to form poly(acrylic acid) (PAA) tails and obtain the PAA-b-P123-b-PAA (P123-PAA) copolymer. The incorporation of PAA had the effect of increasing the carrier's drug loading capacity of an anti-cancer drug, Doxorubicin (DOX), and also allowed for pH-controlled release of the drug. Drug release assays showed that up to 60% of DOX cargo could be retained in the DOX/P123-PAA complex for 3 days at normal physiological pH (7.4). This was then followed by a secondary burst release of DOX when the environment became more acidic (pH 5). Therefore, it was possible that the more acidic physiological environment of tumor sites could be used to trigger an accelerated release of DOX from the drug carriers. The material was demonstrated for potential application in the delivery of cationic drugs for cancer treatment.

Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery.

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.

Vesicles from Pluronic/poly(lactic acid) block copolymers as new carriers for oral insulin delivery.

The morphologies of poly(lactic acid)-b-Pluronic-b-poly(lactic acid) (PLA-F127-PLA) aggregates in aqueous solutions were reported previously to be vesicular nano-particles by our group. In the present study, we seek to investigate the feasibility of using PLA-F127-PLA vesicles as oral delivery carrier for insulin. Both in vitro and in vivo release behavior of insulin loaded in PLA-F127-PLA vesicles were studied. A biphasic release behavior was observed for the in vitro release of insulin from PLAF127-29 vesicles. More importantly, it was found in the diabetic mice tests that the blood glucose concentration of oral insulin-loaded PLAF127-29 vesicles decreased from 18.5 to 5.3 mmol/L within 4.5 h and the minimum blood glucose concentration (about 4.5 mmol/L) was achieved after about 5 h. Furthermore, the blood glucose concentration was maintained at this level for at least an additional 18.5 h. These results proved that PLA-F127-PLA vesicles could be promising polymeric carriers for oral insulin delivery application due to their prolonged hypoglycemic effect.

Novel adhesion prevention membrane based on a bioresorbable copoly(ester-ether) comprised of poly-L-lactide and Pluronic: in vitro and in vivo evaluations.

Block copolymers consisting of poly(L-lactide) (PLLA) and poly(oxyethylene-co-oxypropylene), with various compositions, were synthesized and characterized in vitro and in vivo for their application as postoperative adhesion prevention membranes. It was found that the flexibility and degradability of the cast films of the block copolymers grew with increasing Pluronic F68 [PN; poly(oxyethylene-co-oxypropylene] composition. The receding contact angle of the copolymer films against water became lower than that of the PLLA film, because the surface was predominantly covered with more hydrophilic PN segments in a wet state. This surface property significantly affects the cell attachment property of the copolymer films, and the fibroblasts cultured on the films exhibit a spheroid-like morphology. The copolymer films subcutaneously implanted in the back of rats induced milder tissue responses compared with PLLA homopolymers, because of the increased surface hydrophilicity in the former. In vivo evaluation using a uterus horn model in rats revealed that the performance of these copolymer films as an adhesion-prevention membrane is comparable to that of a conventionally utilized membrane of oxidized regenerated cellulose. These results indicate that the copolymer films are biocompatible materials with controllable mechanical properties and biodegradability as adhesion-prevention membranes.

Use of nonionic block copolymers in vaccines and therapeutics.

Nonionic block copolymers synthesized from ethylene oxide and propylene oxide were developed specifically for use as surfactants. Because the sizes and relative positions of the hydrophobic polyoxypropylene (POP) and hydrophilic polyoxyethylene (POE) blocks can be altered during synthesis, copolymers with significantly different surfactant characteristics can be produced. Copolymers of this type are currently used as excipients in a wide variety of pharmaceutical products where they act as emulsifying, wetting, thickening, stabilizing, and dispersing agents. Copolymers with unique physicochemical properties have recently been developed through the use of new manufacturing and purification techniques, and these copolymers are being used as drug-active and drug-delivery components. In this review, we summarize the current status of these new copolymers in terms of research and product development. This includes the use of new, high molecular weight copolymers as vaccine adjuvants and as vaccine-delivery vehicles. The use of purified, pharmaceutical-grade copolymers as anti-infectives and as antibiotic-delivery systems for the treatment of established bacterial and viral infections is also reviewed. These novel uses for copolymers are significantly different from the excipient uses common to this type of product and demonstrate the widespread utility of synthetic surfactant polymers.

Synthesis and characterization of segmented polyurethanes based on amphiphilic polyether diols.

Segmented polyurethanes (SPUs) based on polyethylene glycol (PEG), polypropylene glycol (PPG) and a series of Pluronics with different ethylene oxide/propylene oxide ratios (EO/PO) and molecular weights were prepared. Different diisocyanates were used for making SPUs: 4,4-diphenylmethane diisocyanate (MDI), 4,4-dicyclohexylmethane diisocyanate (MDCI), hexamethylene diisocyanate (HMDI) and isophorone diisocyanate (IPDI). 1,4-Butane diol (BD) and ethylene diamine (ED) were used as chain extenders. The polymers obtained were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). The microphase morphology (phase separation and phase mixing) is discussed in more detail.

Immunoaffinity partitioning: synthesis and use of polyethylene glycol-oxirane for coupling to bovine serum albumin and monoclonal antibodies.

Polyethylene glycol (PEG)-oxirane was synthesized by reacting aminated monomethoxy-PEG 5000 (NH2-MPEG 5000) with butanediol diglycidyl ether and used to derivatize bovine serum albumin (BSA) and monoclonal antibodies (mAb) against horseradish peroxidase (HRP) and porcine lactate dehydrogenase isoenzyme 5, respectively. Determination of oxirane end groups revealed a very high number, which arise from the chain breaks of the polymer. Covalent coupling of PEG-oxirane to BSA resulted in 30-50 times higher partition coefficients under optimized conditions. The mAb investigated could be modified with PEG-oxirane while retaining its binding properties and could be used as an affinity ligand for selective extraction of Ag in immunoaffinity partitioning. However, a high degree of modification results in a lower binding constant of mAb anti-HRP and higher [mAb]/[Ag] concentration ratios in immunoaffinity partition experiments.

[The immunomodulating properties and antitumor activity of block copolymers of ethylene oxide and propylene oxide (proxanols)]. / Immunomoduliruiushchie svoistva i protivoopukholevaia aktivnost' bloksopolimerov okisi étilena i okisi propilena (proksanolov).

Influence of block copolymers of ethylene and propylene oxides (proxanols) on the human natural killer activity was studied. Proxanols had no effect on the lymphocytes viability and increased their cytotoxic activity against target cells K 562 in vitro. Certain antitumor properties of proxanols were demonstrated in vivo (especially for ascitic tumors). The proxanols activity was chiefly determined by the membranotropic properties of its main polymer chain.