Douglas A. Steeber

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.

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.

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 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.

Doxorubicin conjugated gold nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers

Water-soluble, doxorubicin (DOX) conjugated gold nanoparticles (DOX conjugated Au NPs) exhibiting a significant pH-responsive drug release profile have been prepared and characterized in this study. The Au NPs were stabilized by thiolated methoxy polyethylene glycol (MPEG-SH) and methyl thioglycolate (MTG) at an equal molar ratio. The anticancer drug DOX was conjugated to the MTG segments of the thiol-stabilized Au NPs using hydrazine as the linker. The resulting hydrazone bonds formed between the DOX molecules and the MTG segments of the thiol-stabilized Au NPs are acid cleavable, thereby providing a strong pH-responsive drug release profile. The MPEG segments attached to the Au NPs provide the Au NPs with excellent solubility and stability in an aqueous medium while potentially enhancing the circulation time. The DOX loading level was determined to be 23 wt.%. The DOX release rate from the DOX conjugated Au NPs in an acid medium (i.e., pH 5.3) was dramatically higher than that in physiological conditions (i.e., pH 7.4). The DOX conjugated Au NPs and/or the DOX released from them were found both at the perinuclear regions and the nuclei of 4T1 tumor cells after incubation in a DOX conjugated Au NPs solution for 28 h. These novel DOX conjugated Au NPs have the potential to simultaneously enhance CT imaging contrast and facilitate photothermal cancer therapy while delivering anticancer drugs to their target sites.

B Cell Depletion Delays Collagen-Induced Arthritis in Mice: Arthritis Induction Requires Synergy between Humoral and Cell-Mediated Immunity

Rheumatoid arthritis is a systemic autoimmune disease. B cells are likely to play a critical role in arthritis pathogenesis, although it is unclear whether they are necessary for disease induction, autoantibody production, or disease progression. To assess the role of B cells in inflammatory arthritis, B cells were depleted using mouse anti-mouse CD20 mAbs in a mouse model of collagen-induced arthritis. CD20 mAbs effectively depleted mature B cells from adult DBA-1 mice. When B cells were depleted using CD20 mAbs before collagen immunization, there was a delay in disease onset and autoantibody production, with significantly diminished severity of arthritis both clinically and histologically. B cell depletion further delayed disease onset if initiated before, as well as after, collagen immunization. However, in both cases, the eventual reappearance of peripheral B cells triggered autoantibody production and the subsequent development of arthritis in collagen-sensitized mice. By contrast, B cell depletion after collagen immunizations did not have a significant effect on arthritis progression or severity. Thus, disease symptoms were only induced when peripheral B cells and their autoantibody products were present in collagen-immunized mice, documenting a critical role for B cells during the elicitation phase of collagen-induced arthritis. These studies suggest that B cell depletion strategies will be most effective when initiated early in the development of inflammatory arthritis, with sustained B cell depletion required to inhibit the production of isotype-switched pathogenic Abs and the evolution of joint inflammation and destruction.

Nickel oxide hollow microsphere for non-enzyme glucose detection.

A facile strategy has been developed to fabricate nickel oxide hollow microspheres (NiO-HMSs) through a solvothermal method by using a mixed solvent of ethanol and water with the assistance of sodium dodecyl sulfate (SDS). Various techniques, including transmission electron microscopy (TEM), scanning electron microscopy (SEM), and powder X-ray diffraction (XRD), were used to characterize the morphology and the structure of as-prepared samples. It was confirmed that the products possess a hollow microsphere structure that is constructed by interconnecting porous nanoplate framework. Electrochemical studies indicate that the NiO-HMS exhibits excellent stability and high catalytic activity for electrocatalytic oxidation of glucose in alkaline solutions, which enables the NiO-HMS to be used in enzyme-free amperometric sensors for glucose determination. It was demonstrated that the NiO-HMS-based glucose biosensor offers a variety of merits, such as a wide linear response window for glucose concentrations of 1.67 μM-6.87 mM, short response time (3 s), a lower detection limit of 0.53 μM (S/N=3), high sensitivity (~2.39 mA mM(-1) cm(-2)) as well as good stability and repeatability.

Direct growth of vertically-oriented graphene for field-effect transistor biosensor.

A sensitive and selective field-effect transistor (FET) biosensor is demonstrated using vertically-oriented graphene (VG) sheets labeled with gold nanoparticle (NP)-antibody conjugates. VG sheets are directly grown on the sensor electrode using a plasma-enhanced chemical vapor deposition (PECVD) method and function as the sensing channel. The protein detection is accomplished through measuring changes in the electrical signal from the FET sensor upon the antibody-antigen binding. The novel biosensor with unique graphene morphology shows high sensitivity (down to ~2 ng/ml or 13 pM) and selectivity towards specific proteins. The PECVD growth of VG presents a one-step and reliable approach to prepare graphene-based electronic biosensors.