Nate Larson

Comparison of active and passive targeting of docetaxel for prostate cancer therapy by HPMA copolymer-RGDfK conjugates.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α(v)β(3) integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α(v)β(3) integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.

Anticancer and antiangiogenic activity of HPMA copolymer-aminohexylgeldanamycin-RGDfK conjugates for prostate cancer therapy.

Tumor progression is dependent on neoangiogenesis for blood supply. Neovasculature over-express α(v)β(3) integrins which recognize the Arg-Gly-Asp (RGD) sequence in the extracellular matrix. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing side chains terminated in cyclic RGD analogs such as RGDfK show increased accumulation in prostate tumors. Geldanamycin and their derivatives (e.g., aminohexylgeldanamycin (AH-GDM)) are benzoquinone ansamycins that have both antiangiogenic and antitumor activity. In this work the antiangiogenic and antitumor activities of targetable HPMA copolymer-RGDfK-AH-GDM conjugates were compared with non-targetable systems in vitro and in vivo. Copolymer-drug conjugates containing RGDfK in the side chains showed superior activity against endothelial and prostate cancer cell lines in vitro, as well as higher inhibition of angiogenesis in vivo. At equimolar doses of the drug, the RGDfK containing conjugates showed significantly higher antitumor activity in nude mice bearing DU-145 human prostate cancer xenografts. These findings suggest the utility of HPMA copolymer-RGDfK conjugates for targeted delivery of geldanamycin analogs with a dual mode of action.

Synergistic enhancement of cancer therapy using a combination of heat shock protein targeted HPMA copolymer–drug conjugates and gold nanorod induced hyperthermia

In the field of nanomedicine, selective delivery to cancer cells is a common goal, where active targeting strategies are often employed to increase tumor accumulation. In this study, tumor hyperthermia was utilized as a means to increase the active delivery of heat shock protein (HSP) targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates. Following hyperthermia, induced expression of cell surface heat shock protein (HSP) glucose regulated protein 78 kDa (GRP78) was utilized for targeted drug therapy. Conjugates bearing the anticancer agents aminohexylgeldanamycin (AHGDM), docetaxel (DOC), or cisplatin and the GRP78 targeting peptide WDLAWMFRLPVG were synthesized and characterized. Binding to cell surface expressed heat shock protein GRP78 on the surface of human prostate cancer DU145 cells was evaluated. HSP targeted AHGDM and DOC conjugates demonstrated active binding comparable to native targeting peptide. They were then assessed in vitro for the ability to synergistically induce cytotoxicity in combination with moderate hyperthermia (43 °C, 30 min). HSP targeted DOC conjugates exhibited high potency against DU145 cells with an IC₅₀ of 2.4 nM. HSP targeted AHGDM and DOC conjugates demonstrated synergistic effects in combination with hyperthermia with combination index values of 0.65 and 0.45 respectively. Based on these results, HSP targeted DOC conjugates were selected for in vivo evaluation. In DU145 tumor bearing mice, a single treatment of tumor hyperthermia, induced via gold nanorod mediated plasmonic photothermal therapy, and intravenous administration of HSP targeted HPMA copolymer-docetaxel at 10mg/kg resulted in maintained tumor regression for a period of 30 days. These results demonstrate the potential for tumor hyperthermia to increase the delivery of HSP targeted macromolecular chemotherapeutics.

Plasmonic photothermal therapy increases the tumor mass penetration of HPMA copolymers.

Effective drug delivery to tumors requires both transport through the vasculature and tumor interstitium. Previously, it was shown that gold nanorod (GNR) mediated plasmonic photothermal therapy (PPTT) is capable of increasing the overall accumulation of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers in prostate tumors. In the present study, it is demonstrated that PPTT is also capable of increasing the distribution of these conjugates in tumors. Gadolinium labeled HPMA copolymers were administered to mice bearing prostate tumors immediately before treatment of the right tumor with PPTT. The left tumor served as internal, untreated control. Magnetic resonance imaging (MRI) of both tumors showed that PPTT was capable of improving the tumor mass penetration of HPMA copolymers. Thermal enhancement of delivery, roughly 1.5-fold, to both the tumor center and periphery was observed. Confocal microscopy of fluorescently labeled copolymers corroborates these findings in that PPTT is capable of delivering more HPMA copolymers to the tumors center and periphery. These results further demonstrate that PPTT is a useful tool to improve the delivery of polymer-drug conjugates.

Biodegradable multiblock poly(N-2-hydroxypropyl)methacrylamide gemcitabine and paclitaxel conjugates for ovarian cancer cell combination treatment

The synthesis, characterization, and in vitro evaluation of a combination delivery of multiblock poly(N-2-hydroxypropyl)methacrylamide (HPMA), gemcitabine (GEM) and paclitaxel (PTX) conjugates is described in this study. Multiblock copolymer conjugates of a large molecular weight (Mw>200 kDa) were studied and compared to traditional, small molecular weight (Mw<45 kDa) conjugates. Stability of the conjugates in different pH was assessed, and their cytotoxicity in combination toward A2780 human ovarian cancer cells was evaluated by combination index analysis. Treatment duration (4 and 72 h) and sequence of addition were explored. In addition, an HPMA copolymer conjugate with both GEM and PTX in the side chains was evaluated in a similar manner and compared to a physical mixture of individual conjugates. Conjugates with narrow molecular weight distribution (Mw/Mn<1.1) were obtained via RAFT polymerization, and drug loadings of between 5.5 and 9.2 wt% were achieved. Conjugates demonstrated moderate stability with less than 65% release over 24h at pH 7.4, and near complete drug release in the presence of the lysosomal enzyme cathepsin B in 3h. In combination, the cytotoxic effects of a mixture of the conjugates were primarily additive. Synergistic effects were observed when A2780 human ovarian cancer cells were treated simultaneously for 4h with multiblock conjugates (CI<0.7). When both GEM and PTX were conjugated to the same copolymer backbone, moderate antagonism (CI 1.3-1.6) was observed. These results demonstrate that multiblock HPMA copolymer-GEM and -PTX conjugates, when delivered as a mixture of individual agents, are promising for the treatment of ovarian cancer.

Synthesis and evaluation of poly(styrene-co-maleic acid) micellar nanocarriers for the delivery of tanespimycin

Polymeric micelles carrying the heat shock protein 90 inhibitor tanespimycin (17-N-allylamino-17-demethoxygeldanamycin) were synthesized using poly(styrene-co-maleic acid) (SMA) copolymers and evaluated in vitro and in vivo. SMA-tanespimycin micelles were prepared with a loading efficiency of 93%. The micelles incorporated 25.6% tanespimycin by weight, exhibited a mean diameter of 74 ± 7 nm by dynamic light scattering and a zeta potential of -35 ± 3 mV. Tanespimycin was released from the micelles in a controlled manner in vitro, with 62% released in 24h from a pH 7.4 buffer containing bovine serum albumin. The micellar drug delivery systems for tanespimycin showed potent activity against DU145 human prostate cancer cells, with an IC(50) of 230 nM. They further exhibited potent anti-cancer activity in vivo in nu/nu mice bearing subcutaneous DU145 human prostate cancer tumor xenografts, with significantly higher anticancer efficacy as measured by tumor regression when compared to free tanespimycin at an equivalent single dose of 10mg/kg. These data suggest further investigation of SMA-tanespimycin as a promising agent in the treatment of prostate cancer.