Tumor-targeting, pH-responsive, and stable unimolecular micelles as drug nanocarriers for targeted cancer therapy.

A new type of multifunctional unimolecular micelle drug nanocarrier based on amphiphilic hyperbranched block copolymer for targeted cancer therapy was developed. The core of the unimolecular micelle was a hyperbranched aliphatic polyester, Boltorn H40. The inner hydrophobic layer was composed of random copolymer of poly(ε-caprolactone) and poly(malic acid) (PMA-co-PCL) segments, while the outer hydrophilic shell was composed of poly(ethylene glycol) (PEG) segments. Active tumor-targeting ligands, i.e., folate (FA), were selectively conjugated to the distal ends of the PEG segments. An anticancer drug, i.e., doxorubicin (DOX) molecules, was conjugated onto the PMA segments with pH-sensitive drug binding linkers for pH-triggered drug release. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis showed that the unimolecular micelles were uniform with a mean hydrodynamic diameter around 25 nm. The drug loading content was determined to be 14.2%. The drug release profile, cell uptake and distribution, and cytotoxicity of the unimolecular micelles were evaluated in vitro. The folate-conjugated micelles can be internalized by the cancer cells via folate-receptor-mediated endocytosis; thus, they exhibited enhanced cell uptake and cytotoxicity. At pH 7.4, the physiological condition of bloodstream, DOX conjugated onto the unimolecular micelles exhibited excellent stability; however, once the micelles were internalized by the cancer cells, the pH-sensitive hydrazone linkages were cleavable by the intracellular acidic environment, which initially caused a rapid release of DOX. These findings indicate that these unique unimolecular micelles may offer a very promising approach for targeted cancer therapy.

Multifunctional polymeric vesicles for targeted drug delivery and imaging.

Multifunctional polymeric vesicles were developed for targeted drug delivery and imaging. To fabricate this system, a biodegradable amphiphilic diblock copolymer, folate-poly(ethylene glycol)-poly(D,L-lactide) was designed and synthesized through sequential anionic polymerization in a well-controlled manner. Hydrophobic superparamagnetic iron oxide nanoparticles were loaded into the hydrophobic membrane for ultra-sensitive magnetic resonance imaging. Meanwhile, the anticancer drug, doxorubicin was encapsulated in the aqueous core of the vesicles. Cell culture experiments demonstrated the potential of polymeric vesicles as an effective targeting nanoplatform for the delivery of anticancer drugs due to the folate attached to the surface of the vesicles.

Multifunctional SPIO/DOX-loaded wormlike polymer vesicles for cancer therapy and MR imaging.

Stable and tumor-targeting multifunctional wormlike polymer vesicles simultaneously loaded with superparamagnetic iron oxide (SPIO) nanoparticles (NPs) as magnetic resonance imaging (MRI) contrast agent and anticancer drug doxorubicin (DOX) were developed for targeted cancer therapy and ultrasensitive MR imaging. These multifunctional wormlike polymer vesicles were formed by heterobifunctional amphiphilic triblock copolymers R (R = methoxy or folate (FA))-PEG(114)-PLA(x)-PEG(46)-acrylate using a double emulsion method. The long PEG segments bearing methoxy/folate groups (CH(3)O/FA-PEG(114)) were mostly segregated to the outer hydrophilic PEG layers of the wormlike vesicles thereby providing active tumor-targeting ability, while the short PEG segments bearing acrylate groups (PEG(46)-acrylate) were mostly segregated onto the inner hydrophilic PEG layers of the wormlike vesicles thereby allowing the inner PEG layers to be crosslinked via free radical polymerization for enhanced in vivo stability. The hydrophobic anticancer drug, DOX, was loaded into the hydrophobic membrane of the wormlike vesicles. Meanwhile, a cluster of hydrophilic SPIO NPs was encapsulated into the aqueous cores of the stable wormlike vesicles with crosslinked inner PEG layers for ultrasensitive MRI detection. Cellular uptake of the FA-conjugated wormlike vesicles facilitated by the folate receptor-mediated endocytosis process was higher than that of the FA-free vesicles thereby leading to high cytotoxicity against the HeLa human cervical tumor cell line. Moreover, the SPIO/DOX-loaded wormlike vesicles with crosslinked inner PEG layers demonstrated a much higher r(2) relaxivity value than Feridex, a commercially available T(2) agent, which can be attributed to the high SPIO NPs loading level as well as the SPIO clustering effect. These unique stable and tumor-targeting multifunctional SPIO/DOX-loaded wormlike polymer vesicles would make targeted cancer theranostics possible thereby paving the road for personalized medicine.

Multifunctional stable and pH-responsive polymer vesicles formed by heterofunctional triblock copolymer for targeted anticancer drug delivery and ultrasensitive MR imaging.

A multifunctional stable and pH-responsive polymer vesicle nanocarrier system was developed for combined tumor-targeted delivery of an anticancer drug and superparamagnetic iron oxide (SPIO) nanoparticles (NPs). These multifunctional polymer vesicles were formed by heterofunctional amphiphilic triblock copolymers, that is, R (folate (FA) or methoxy)-poly(ethylene glycol)(M(w):5000)-poly(glutamate hydrozone doxorubicin)-poly(ethylene glycol) (M(w):2000)-acrylate (i.e., R (FA or methoxy)-PEG(114)-P(Glu-Hyd-DOX)-PEG(46)-acrylate). The amphiphilic triblock copolymers can self-assemble into stable vesicles in aqueous solution. It was found that the long PEG segments were mostly segregated into the outer hydrophilic PEG layers of the vesicles, thereby providing active tumor targeting via FA, while the short PEG segments were mostly segregated into the inner hydrophilic PEG layer of the vesicles, thereby making it possible to cross-link the inner PEG layer via the acrylate groups for enhanced in vivo stability. The therapeutic drug, DOX, was conjugated onto the polyglutamate segment, which formed the hydrophobic membrane of the vesicles using a pH-sensitive hydrazone bond to achieve pH-responsive drug release, while the hydrophilic SPIO NPs were encapsulated into the aqueous core of the stable vesicles, allowing for ultrasensitive magnetic resonance imaging (MRI) detection. The SPIO/DOX-loaded vesicles demonstrated a much higher r(2) relaxivity value than Feridex, a commercially available SPIO-based T(2) contrast agent, which was attributed to the high SPIO NPs loading level and the SPIO clustering effect in the aqueous core of the vesicles. Results from flow cytometry and confocal laser scanning microscopy (CLSM) analysis showed that FA-conjugated vesicles exhibited higher cellular uptake than FA-free vesicles which also led to higher cytotoxicity. Thus, these tumor-targeting multifunctional SPIO/DOX-loaded vesicles will provide excellent in vivo stability, pH-controlled drug release, as well as enhanced MRI contrast, thereby making targeted cancer therapy and diagnosis possible.

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.

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 approximately 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.

Folate-conjugated amphiphilic hyperbranched block copolymers based on Boltorn H40, poly(L-lactide) and poly(ethylene glycol) for tumor-targeted drug delivery.

Folate-conjugated amphiphilic hyperbranched block copolymer (H40-PLA-b-MPEG/PEG-FA) with a dendritic Boltorn H40 core, a hydrophobic poly(l-lactide) (PLA) inner shell and a hydrophilic methoxy poly(ethylene glycol) (MPEG) and folate-conjugated poly(ethylene glycol) (PEG-FA) outer shell was synthesized as a carrier for tumor-targeted drug delivery. The block copolymer was characterized using (1)H NMR and gel permeation chromatography (GPC) analysis. Due to its core-shell structure, this block polymer forms unimolecular micelles in aqueous solutions. The micellar properties of H40-PLA-b-MPEG/PEG-FA block copolymer were extensively studied by dynamic light scattering (DLS), fluorescence spectroscopy, and transmission electron microscopy (TEM). An anticancer drug, doxorubicin in the free base form (DOX) was encapsulated into H40-PLA-b-MPEG/PEG-FA micelles. The DOX-loaded micelles provided an initial burst release (up to 4h) followed by a sustained release of the entrapped DOX over a period of about 40 h. Cellular uptake of the DOX-loaded H40-PLA-b-MPEG/PEG-FA micelles was found to be higher than that of the DOX-loaded H40-PLA-b-MPEG micelles because of the folate-receptor-mediated endocytosis, thereby providing higher cytotoxicity against the 4T1 mouse mammary carcinoma cell line. In vitro degradation studies revealed that the H40-PLA-b-MPEG/PEG-FA block copolymer hydrolytically degraded into polymer fragments within six weeks. These results indicated that the micelles prepared from the H40-PLA-b-MPEG/PEG-FA block copolymer have great potential as tumor-targeted drug delivery nanocarriers.

Amphiphilic multi-arm block copolymer based on hyperbranched polyester, poly(L-lactide) and poly(ethylene glycol) as a drug delivery carrier.

A novel type of biodegradable/biocompatible amphiphilic hyperbranched copolymer (H40-PLA-b-MPEG) was synthesized. Its micellar properties were studied by DLS, fluorescence spectroscopy and TEM. The drug release profile showed that the H40-PLA-b-MPEG micelles provide an initial burst release, followed by a sustained release of the entrapped hydrophobic model drug over a period of 4 to 58 h. The copolymer degraded hydrolytically within 6 weeks under physiological conditions. The MTT assay showed no obvious cytotoxicity against a human endothelial cell line at a concentration range of 0-400 microg x mL(-1). These results indicate that the H40-PLA-b-MPEG micelles have great potential as hydrophobic drug delivery carriers.

Biodegradable hydrogels based on novel photopolymerizable guar gum-methacrylate macromonomers for in situ fabrication of tissue engineering scaffolds.

Guar gum (GG) is a non-ionic polysaccharide that is found abundantly in nature and has many properties desirable for biomedical applications. In the present work GG with molecular weights ranging from 74 to 210 kDa was modified with glycidyl methacrylate (GMA) to produce a series of water-soluble photopolymerizable guar gum-methacrylate (GG-MA) macromonomers of different molecular weights. We investigated the effects of molecular weight of GG-MA macromonomers from 102 to 216 kDa and with percent degree of methacrylation (%DM) ranging from 14% to 56% on the properties of GG-MA hydrogels. GG-MA hydrogels exhibited a three-dimensional open cell microstructure with an average pore size ranging from approximately 10 to 55 microm and an average pore density of from approximately 2.4 x 10(6) to 8.6 x 10(7) pores cm(-3). The hydrogels exhibited equilibrium swelling ratios ranging from approximately 22% to 63%. The degree of in vitro enzymatic biodegradation of the hydrogels decreased linearly with increasing gel content and the degree of methacrylation of the respective macromonomers. The human endothelial cell line EA.hy926 was photo-encapsulated in the GG-MA hydrogels. Cells remained viable at low macromonomer concentrations, but cell viability decreased sequentially as the macromonomer concentration increased. GG-MA hydrogels with a 0.05 wt.% GG-MA macromonomer concentration revealed excellent endothelial cell proliferation, similar to that of the Matrigel control.

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 degrees C. At 37 degrees 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 degrees 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.

Stimuli-responsive chitosan-graft-poly(N-vinylcaprolactam) as a promising material for controlled hydrophobic drug delivery.

A novel type of pH- and thermo-responsive copolymer, chitosan-graft-poly(N-vinylcaprolactam) (chitosan-g-PNVCL), was prepared by grafting carboxyl-terminated poly(N-vinylcaprolactam) (PNVCL-COOH) chains onto a chitosan backbone as a drug-delivery carrier. The formation of chitosan-g-PNVCL was confirmed by FT-IR and 1H NMR techniques. Chitosan-g-PNVCL showed a definite phase transition at 32 degrees C as occurs in pure PNVCL. The swelling degree of the chitosan-g-PNVCL beads was found to be higher at pH 2.2 than at pH 7.4. Moreover, the swelling degree of the beads decreased with increased environmental temperature. Compared to the chitosan beads, the release profile of chitosan-g-PNVCL beads showed a slower and more controlled release of the entrapped ketoprofen. The release behavior of the chitosan-g-PNVCL beads was influenced by both the pH and temperature of the medium. The MTT assay showed no obvious cytotoxicity of chitosan-g-PNVCL against a human endothelial cell line over a concentration range of 0-400 microg x mL(-1). These results suggest that chitosan-g-PNVCL could be a potential stimuli-responsive material for controlled drug delivery, and it may improve the bioavailability, efficacy, and compliance of the encapsulated drugs. [Reaction: see text].

Polymer semiconductors as a biosensing platform: peroxidase activity of enzyme bound to organic semiconducting films.

Organic polymer semiconductors have unique electronic properties which make them attractive for use in microelectronic and optoelectronic devices fabricated using inexpensive manufacturing processes. In addition, novel chemical and biological sensors have been proposed which make use of the photophysical and electrical properties of conjugated polymer semiconducting films. The work described herein illustrates one such biosensing application by demonstrating successful immobilization of horseradish peroxidase enzyme onto a thin film of the semiconducting polymer MDMO-PPV. Validation of bound peroxidase activity is accomplished through the use of a substrate solution of 3,3'-diaminobenzidine and hydrogen peroxide, which yields a dark brown precipitate in the presence of peroxidase. Photometric measurements are used to derive a quantitative assay of bound peroxidase concentration. This work supports the feasibility of organic semiconducting polymer films as a biosensing platform in microelectronic sensor devices.