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.

Anti-adhesive antimicrobial peptide coating prevents catheter associated infection in a mouse urinary infection model.

Catheter-associated urinary tract infections (CAUTIs) represent one of the most common hospital acquired infections with significant economic consequences and increased patient morbidity. CAUTIs often start with pathogen adhesion and colonization on the catheter surface followed by biofilm formation. Current strategies to prevent CAUTIs are insufficiently effective and antimicrobial coatings based on antimicrobial peptides (AMPs) hold promise in curbing CAUTIs. Here we report an effective surface tethering strategy to prepare AMP coatings on polyurethane (PU), a common biomedical plastic used for catheter manufacture, by using an anti-adhesive hydrophilic polymer coating. An optimized surface active AMP, labeled with cysteine at the C-terminus (RRWRIVVIRVRRC), was used. The coated PU surface was characterized using ATR-FTIR, XPS and atomic force microscopy analyses. The tethered peptides on the PU catheter surface displayed broad spectrum antimicrobial activity and showed long term activity in vitro. The surface coating prevented bacterial adhesion by up to 99.9% for both Gram-positive and -negative bacteria, and inhibited planktonic bacterial growth by up to 70%. In vivo, the coating was tested in a mouse urinary catheter infection model; the AMP-coated PU catheter was able to prevent infection with high efficiency by reducing the bacteria adhesion on catheter surface by more than 4 logs (from 1.2 × 106 CFU/mL to 5 × 101 CFU/mL) compared to the uncoated catheter surface, and inhibit planktonic bacterial growth in the urine by nearly 3 logs (1.1 × 107 CFU/mL to 1.47 × 104 CFU/mL). The AMP-brush coating also showed good biocompatibility with bladder epithelial cells and fibroblast cells in cell culture. The new coating might find clinical applications in preventing CAUTIs.

[Biological properties study and animal experiment of polylactide/polytrimethylene carbonate blends membrane].

Polylactide is a biodegradable and biocompatible biomaterial. Based on the PLA (Polylactide) membrane, we have produced a new PLA/PTMC (Polytrimethylene carbonate) blends membrane. The biological properties of this membrane were studied by cell toxicity experiment, acute toxicity experiment, skin irritant experiment, sensitization test, hemolytic test, micronucleus test and subcutaneous implantation test. The results demonstrated that the blends membrane has no toxicity and it does not cause skin irritation, hypersensitive reaction and hemolysis. The micronucleus ratio of the membrane is 1.3% +/- 1.0%, being less than 3%. The result of medullary micronucleus test was reported negative. The wounds were free from suppuration and necrosis after subcutaneous implantation in all periods. In the experimental application of this member to preventing adhesion after rabbit intestine operation, the membrane demonstrated good effect. In conclusion, PLA/PTMC blends membrane is a material with good biocompatibility.

Choline phosphate functionalized cellulose membrane: A potential hemostatic dressing based on a unique bioadhesion mechanism.

UNLABELLED: Wound dressings are a key component in provision of optimal conditions for bleeding control and wound healing. For absorbent dressings, electrostatic interactions are frequently utilized as one of the mechanisms driving dressing adhesion. Herein, a choline phosphate functionalized biocompatible cellulose membrane that can efficiently arrest human red blood cells was developed to have potential application in wound dressing. The bioadhesion is based on the unique multivalent electrostatic interaction between the head groups of phosphatidyl choline based lipids on the cell membrane and its inverse orientation but virtually identical structure, choline phosphate, coupled to the cellulose membrane. For functionalization, the cellulose membrane was decorated with polymer brushes bearing multiple choline phosphate groups via surface-initiator atom transfer radical polymerization followed by click chemistry. The modified cellulose membranes were characterized by ATR-FTIR and the molecular weight and the grafting density of polymer brushes grafted from the cellulose membrane surface were thoroughly evaluated by calibrated force-distance measurements with atomic force microscopy (AFM). This new method provides an approach to estimating polymer brush parameters on rough surfaces of unknown surface area based on the dependence of brush thickness on brush density and polymer molecular weight for a calibration set of brushes. The dependence of binding of human red blood cells (RBCs) to the cellulose membrane surface on the number density of choline phosphate groups (e.g. molecular weight) and the grafting density were investigated using this AFM-based approach. Bound RBCs showed "pseudopodia"-like membrane projections under scanning electron microscopy where cells contacted the microfibers of the cellulose, distorting the RBC shape, reflecting the multivalent interactions between the RBCs and the choline phosphate-doped cellulose membrane. We believe this efficient strategy provides a promising approach to blood conservation and trauma management. STATEMENT OF SIGNIFICANCE: Uncontrolled bleeding can dramatically affect morbidity and mortality. Absorptive wound dressings provide either adherent or non-adherent layers to control bleeding. Our new adherent material is based on a universal adhesion reaction between cell membrane phosphatidyl choline (PC) headgroups and cellulose membranes (CM) decorated with polymer brushes carrying a CP group per monomer. The CP-PC multivalent interactions provide adherence to cut tissue margins and blood cells, blocking bleeding. We here demonstrate the strong specific binding of red cells to CM-CP but not CM-PC membranes and determine the requisite brush molecular weight and surface concentration via a new approach using atomic force microscopy, applicable to rough surfaces. We believe this strategy provides a promising approach to blood conservation and trauma management.

Abnormal blood clot formation induced by temperature responsive polymers by altered fibrin polymerization and platelet binding.

Thermoresponsive polymers (TRPs) have been extensively investigated as smart devices, drug delivery systems and protein conjugates due to their unique phase transition properties. Here, we report the unusual influence of TRPs in blood clotting and the mechanism by which TRPs change the three dimensional organization of blood clot structure. Ten different TRPs with lower critical solution temperatures ranged from 26 to 80 °C are studied. TRPs altered the fibrin polymerization by increasing the rate of protofibril aggregation, decreased the fibrin fiber diameter and changed the platelet integration within the clot. The mechanical properties of the clot decreased considerably in presence of TRPs due to the poor platelet binding. The poor integration of platelets within the clot is not due to the inhibition of platelet activation by TRPs but may due to the unusual organization of fibrin structure. The plasma phase of the blood coagulation is not affected in presence of TRPs. We anticipate that our results will have significant implications on the use of TRPs in applications where blood contact is essential. These observations may also open up new avenues, for example, in the design of new generation antithrombotics.

A thermoreversible poly(choline phosphate) based universal biomembrane adhesive.

A new monomer, 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl methacrylate (AEO4 MA), and its direct atom transfer radical polymerization (ATRP) into poly(AEO4 MA), then "clicked" with prop-2-ynyle choline phosphate (CP) to produce a poly(choline phosphate) are described. This polymer exhibits a lower critical solution temperature (LCST) at ≈ 32 °C, and provides a universal thermally reversible biomembrane adhesive, which can rapidly both bind to any mammalian cell membrane and internalize into the cytoplasm of nucleated cells below the LCST. Moving above the LCST reverses cell surface binding. The use of ATRP implies that such polymers can be applied to modify the surfaces of a wide range of biomaterials. The capacity to bind and immobilize cells at room temperature and release them above the LCST should be particularly useful for in vitro cell manipulation and tissue engineering applications.

ATRP synthesis of poly(2-(methacryloyloxy)ethyl choline phosphate): a multivalent universal biomembrane adhesive.

A new monomer, 2-(methacryloyloxy)ethyl choline phosphate, and its direct polymerization into a polyvalent choline phosphate are described, providing a universal biomembrane adhesive exhibiting rapid, strong attachment to any mammalian cell membrane and fast internalization, properties of great value in applications such as tissue engineering and drug delivery.

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.

Folate-functionalized polymeric micelle as hepatic carcinoma-targeted, MRI-ultrasensitive delivery system of antitumor drugs.

Targeted delivery is a highly desirable strategy to improve the diagnostic imaging and therapeutic outcome because of enhanced efficacy and reduced toxicity. In the current research, anticancer drug doxorubicin (DOX) and contrast agent for magnetic resonance imaging (MRI), herein superparamagnetic ion oxide Fe(3)O(4) (SPIO), were accommodated in the core of micelles self-assembled from amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(epsilon-caprolactone) (PCL) with a targeting ligand (folate) attached to the distal ends of PEG (Folate-PEG-PCL). The in vitro tumor cell targeting efficacy of these folate functionalized and DOX/SPIO-loaded micelles (Folate-SPIO-DOX-Micelles) was evaluated upon observing cellular uptake of micelles by human hepatic carcinoma cells (Bel 7402 cells) which overexpresses surface receptors for folic acid. In the Prussian blue staining experiments, cells incubated with Folate-SPIO-DOX-Micelles showed much higher intracellular iron density than the cells incubated with the folate-free SPIO-DOX-Micelles. According to the flow cytometry data, cellular DOX uptake observed for the folate targeting micelle was about 2.5 fold higher than that for the non-targeting group. Furthermore, MTT assay showed that Folate-SPIO-DOX-Micelles effectively inhibited cell proliferation, while the folate-free SPIO-DOX-Micelles did not show the same feat at comparable DOX concentrations. The potential of Folate-SPIO-DOX-Micelle as a novel MRI-visible nanomedicine platform was assessed with a 1.5 T clinical MRI scanner. The acquired MRI T (2) signal intensity of cells treated with the folate targeting micelles decreased significantly. By contrast, T (2) signal did not show obvious decrease for cells treated with the folate-free micelles. Our results indicate that the multifunctional polymeric micelles, Folate-SPIO-DOX-Micelles, have better targeting tropism to the hepatic carcinoma cells in vitro than their non-targeting counterparts, and the cell targeting events of micelles can be monitored using a clinical MRI scanner.

Interactions between an anticancer drug and polymeric micelles based on biodegradable polyesters.

Interactions between the anticancer drug quercetin and biodegradable polyesters within micelles were investigated by DSC, WAXD, and UV analyses. For micelles based on poly(ethylene glycol) methyl ether-block-poly(epsilon-caprolactone) (MPEG-PCL), DSC analysis indicated that the interactions were between the hydrophobic core and the drug within the micelle. For micelles based on poly(ethylene glycol) methyl ether-block-poly(L-lactide) (MPEG-PLLA), the interactions were between the hydrophobic core and the drug and between hydrophilic segments and the drug. WAXD results indicated that no crystalline phase of the drug was found in either of the micelle types. Based on the DSC and WAXD results, two probable micelle structures were proposed. The UV spectra revealed the presence of hydrogen bonding as the main interaction between the drug and the polyesters. In vitro studies demonstrated that quercetin release from micelles was sustained and was affected by the polymer-drug interaction.

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.

FG020326-loaded nanoparticle with PEG and PDLLA improved pharmacodynamics of reversing multidrug resistance in vitro and in vivo.

AIM: FG020326, a novel imidazole derivative, is a potent multidrug-resistance (MDR) modulator in vitro and in vivo. However, FG020326 is insoluble. PEDLLA-FG020326 is a FG020326-loaded nanoparticle formed with diblock copolymers of poly (ethylene glycol)-block-poly (D,L-lactic acid) (PEG:PDLLA, PEDLLA) that can solubilize FG020326. This work was intended to evaluate the pharmacodynamics of PEDLLA-FG020326 on reversing MDR in vitro and in vivo. METHODS: Cytotoxicity was determined by tetrazolium assay. The intracellular accumulation and efflux of doxorubicin (Dox) were detected by fluorescence spectrophotometry. The function of P-glycoprotein was examined by Rhodamine 123 (Rh123) accumulation detected by flow cytometry. The KBv200 cell xenograft model was established to investigate the effect of PEDLLA-FG020326 on reversing MDR in vivo. RESULTS: PEDLLA-FG020326 and FG020326 exhibited 56.4- and 35.9-fold activity in reversing KBv200 cells to vincristine (VCR) resistance, respectively and 14.98- and 7.64-fold to Dox resistance, respectively. PEDLLA-FG020326 was much stronger than FG020326, resulting in the increase of Dox and Rh123 accumulation and the decrease of intracellular Dox extrusion in KBv200 cells. Importantly, PEDLLA-FG020326 exhibited more powerful activity than FG020326 in enhancing the effect of VCR against KBv200 cell xenografts in nude mice, but did not appear more toxic. CONCLUSION: The pharmacodynamics of FG020326 was improved by incorporating it into a micellar nanoparticle formed with PEG-block-PDLLA copolymers.