Adam J. Gormley

Gold nanorod mediated plasmonic photothermal therapy: A tool to enhance macromolecular delivery

Plasmonic photothermal therapy (PPTT) with gold nanostructures has been used to generate significant heat within tumors to ablate vasculature. Here we report the use of gold nanorod (GNR) mediated PPTT to induce moderate hyperthermia as a tool to enhance the delivery of macromolecules. GNRs were injected intravenously in a mouse sarcoma (S-180) tumor model. After 24h Evans blue dye (EBD) was injected and the right tumor was radiated with a laser diode for 10 min. EBD content in the right and left tumors were extracted in formamide, measured spectrophotometrically and expressed as a thermal enhancement ratio (TER). Enhanced delivery of EBD was observed (up to 1.8-fold) when tumor temperatures reached 43°C or 46°C. No statistical difference was observed between tumors at these two temperatures, though significant hemorrhage was observed in tumors and surrounding areas receiving the higher thermal dose (46°C). These results indicate that tumor directed PPTT may be used to induce moderate hyperthermia and therefore selectively increase the delivery of macromolecules with therapeutic anticancer drugs.

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.

Polymerization Amplified Detection for Nanoparticle-Based Biosensing

Efficient signal amplification processes are key to the design of sensitive assays for biomolecule detection. Here, we describe a new assay platform that takes advantage of both polymerization reactions and the aggregation of nanoparticles to amplify signal. In our design, a cascade is set up in which radicals generated by either enzymes or metal ions are polymerized to form polymers that can entangle multiple gold nanoparticles (AuNPs) into aggregates, resulting in a visible color change. Less than 0.05% monomer-to-polymer conversion is required to initiate aggregation, providing high sensitivity toward the radical generating species. Good sensitivity of this assay toward horseradish peroxidase, catalase, and parts per billion concentrations of iron and copper is shown. Incorporation of the oxygen-consuming enzyme glucose oxidase (GOx), enables this assay to be performed in open air conditions at ambient temperature. We anticipate that such a design will provide a useful platform for sensitive detection of a broad range of biomolecules through polymerization-based amplification.

Biological evaluation of RGDfK-gold nanorod conjugates for prostate cancer treatment.

Selective delivery of gold nanorods (GNRs) to sites of prostate tumor angiogenesis is potentially advantageous for localized photothermal therapy. Here, we report the cellular uptake and biodistribution of GNRs surface functionalized with the cyclic RGDfK peptide. The GNRs were synthesized to have a surface plasmon resonance (SPR) peak at 800 nm and grafted with a thiolated poly(ethylene glycol) (PEG) corona with or without RGDfK. The binding and uptake of the targeted (RGDfK) and untargeted GNRs were evaluated in DU145 prostate cancer and human umbilical vein endothelial cells (HUVEC) by high-resolution dark field microscopy, inductively coupled plasma mass spectrometry (ICP-MS), and transmission electron microscopy (TEM). The biodistribution of both GNRs was then evaluated in prostate tumor bearing mice. Targeting of the RGDfK surface-modified GNRs was confirmed in vitro due to selective binding and uptake by endothelial cells. Tumor targeting was not observed in vivo, however, due to fast clearance of the RGDfK-GNRs from the blood. Further modifications of the nanoparticle’s surface properties are needed to enhance localization of the targetable system in sites of tumor angiogenesis.

Combinatorial Low-Volume Synthesis of Well-Defined Polymers by Enzyme Degassing

The synthesis of well-defined polymers in a low-volume, combinatorial fashion has long been a goal in polymer chemistry. Here, we report the preparation of a wide range of highly controlled homo and block co-polymers by Enz-RAFT (enzyme-assisted reversible addition-fragmentation chain transfer) polymerization in microtiter plates in the open atmosphere. The addition of 1 μm glucose oxidase (GOx) to water/solvent mixtures enables polymerization reactions to proceed in extremely low volumes (40 μL) and low radical concentrations. This procedure provides excellent control and high conversions across a range of monomer families and molecular weights, thus avoiding the need to purify for screening applications. This simple technique enables combinatorial polymer synthesis in microtiter plates on the benchtop without the need of highly specialized synthesizers and at much lower volumes than is currently possible by any other technique.

Gold nanorod-mediated hyperthermia enhances the efficacy of HPMA copolymer-90Y conjugates in treatment of prostate tumors

INTRODUCTION The treatment of prostate cancer using a radiotherapeutic (90)Y labeled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer can be enhanced with localized tumor hyperthermia. An (111)In labeled HPMA copolymer system for single photon emission computerized tomography (SPECT) was developed to observe the biodistribution changes associated with hyperthermia. Efficacy studies were conducted in prostate tumor bearing mice using the (90)Y HPMA copolymer with hyperthermia. METHODS HPMA copolymers containing 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized by reversible addition-fragmentation transfer (RAFT) copolymerization and subsequently labeled with either (111)In for imaging or (90)Y for efficacy studies. Radiolabel stability was characterized in vitro with mouse serum. Imaging and efficacy studies were conducted in DU145 prostate tumor bearing mice. Imaging was performed using single photon emission computerized tomography (SPECT). Localized mild tumor hyperthermia was achieved by plasmonic photothermal therapy using gold nanorods. RESULTS HPMA copolymer-DOTA conjugates demonstrated efficient labeling and stability for both radionuclides. Imaging analysis showed a marked increase of radiolabeled copolymer within the hyperthermia treated prostate tumors, with no significant accumulation in non-targeted tissues. The greatest reduction in tumor growth was observed in the hyperthermia treated tumors with (90)Y HPMA copolymer conjugates. Histological analysis confirmed treatment efficacy and safety. CONCLUSION HPMA copolymer-DOTA conjugates radiolabeled with both the imaging and treatment radioisotopes, when combined with hyperthermia can serve as an image guided approach for efficacious treatment of prostate tumors.

RGDfK‐ functionalized gold nanorods bind only to activated platelets

Integrin-targeting peptide RGDfK-labeled gold nanorods (GNR) seek to improve hyperthermia targeted to solid tumors by exploiting the known up-regulation of integrin αvβ3 cell membrane proteins on solid tumor vasculature surfaces. Tumor binding specificity might be expected since surrounding tissues and endothelial cells have limited numbers of these receptors. However, RGD peptide binding to many proteins is promiscuous, with known affinity to several families of cell integrin receptors, and also possible binding to platelets after intravenous infusion via a different integrin receptor, αIIbβ3, on platelets. Binding of RGDfK-targeted GNR could considerably impact platelet function, ultimately leading to increased risk of bleeding or thrombosis depending on the degree of interaction. We sought to determine if RGDfK-labeled GNR could interact with platelets and alter platelet function. Targeted and untargeted nanorods exhibited little interaction with resting platelets in platelet rich plasma (PRP) preparations. However, upon platelet activation, peptide-targeted nanorods bound actively to platelets. Addition of RGDfK-GNR to unactivated platelets had little effect on markers of platelet activation, indicating that RGDfK-nanorods were incapable of inducing platelet activation. We next tested whether activated platelet function was altered in the presence of peptide-targeted nanorods. Platelet aggregation in whole blood and PRP in the presence of targeted nanorods had no significant effect on platelet aggregation. These data suggest that RGDfK-GNR alone have little impact on platelet function in plasma. However, nonspecific nanorod binding may occur in vascular beds where activated platelets are normally cleared, such as the spleen and liver, producing a possible toxicity risk for these nanomaterials.