Kevin Sill

Self-directed self-assembly of nanoparticle/copolymer mixtures

The organization of inorganic nanostructures within self-assembled organic or biological templates is receiving the attention of scientists interested in developing functional hybrid materials. Previous efforts have concentrated on using such scaffolds to spatially arrange nanoscopic elements as a strategy for tailoring the electrical, magnetic or photonic properties of the material. Recent theoretical arguments have suggested that synergistic interactions between self-organizing particles and a self-assembling matrix material can lead to hierarchically ordered structures. Here we show that mixtures of diblock copolymers and either cadmium selenide- or ferritin-based nanoparticles exhibit cooperative, coupled self-assembly on the nanoscale. In thin films, the copolymers assemble into cylindrical domains, which dictate the spatial distribution of the nanoparticles; segregation of the particles to the interfaces mediates interfacial interactions and orients the copolymer domains normal to the surface, even when one of the blocks is strongly attracted to the substrate. Organization of both the polymeric and particulate entities is thus achieved without the use of external fields, opening a simple and general route for fabrication of nanostructured materials with hierarchical order.

Polymeric micelles for drug delivery

Polymeric micelles have been the subject of many studies in the field of drug delivery for the past two decades. The interest has specifically been focused on the potential application of polymeric micelles in three major areas in drug delivery: drug solubilisation, controlled drug release and drug targeting. In this context, polymeric micelles consisting of poly(ethylene oxide)-b-poly(propylene oxide), poly(ethylene oxide)-b-poly(ester)s and poly(ethylene oxide)-b-poly(amino acid)s have shown a great promise and are in the front line of development for various applications. The purpose of this manuscript is to provide an update on the current status of polymeric micelles for each application and highlight important parameters that may lead to the development of successful polymeric micellar systems for individual delivery requirements.

A Versatile Polymer Micelle Drug Delivery System for Encapsulation and In Vivo Stabilization of Hydrophobic Anticancer Drugs

Chemotherapeutic drugs are widely used for the treatment of cancer; however, use of these drugs is often associated with patient toxicity and poor tumor delivery. Micellar drug carriers offer a promising approach for formulating and achieving improved delivery of hydrophobic chemotherapeutic drugs; however, conventional micelles do not have long-term stability in complex biological environments such as plasma. To address this problem, a novel triblock copolymer has been developed to encapsulate several different hydrophobic drugs into stable polymer micelles. These micelles have been engineered to be stable at low concentrations even in complex biological fluids, and to release cargo in response to low pH environments, such as in the tumor microenvironment or in tumor cell endosomes. The particle sizes of drugs encapsulated ranged between 30–80 nm, with no relationship to the hydrophobicity of the drug. Stabilization of the micelles below the critical micelle concentration was demonstrated using a pH-reversible crosslinking mechanism, with proof-of-concept demonstrated in both in vitro and in vivo models. Described herein is polymer micelle drug delivery system that enables encapsulation and stabilization of a wide variety of chemotherapeutic drugs in a single platform.

IT-141, a Polymer Micelle Encapsulating SN-38, Induces Tumor Regression in Multiple Colorectal Cancer Models.

Polymer micelles are promising drug delivery vehicles for the delivery of anticancer agents to tumors. Often, anticancer drugs display potent cytotoxic effects towards cancer cells but are too hydrophobic to be administered in the clinic as a free drug. To address this problem, a polymer micelle was designed using a triblock copolymer (ITP-101) that enables hydrophobic drugs to be encapsulated. An SN-38 encapsulated micelle, IT-141, was prepared that exhibited potent in vitro cytotoxicity against a wide array of cancer cell lines. In a mouse model, pharmacokinetic analysis revealed that IT-141 had a much longer circulation time, plasma exposure, and tumor exposure compared to irinotecan. IT-141 was also superior to irinotecan in terms of antitumor activity, exhibiting greater tumor inhibition in HT-29 and HCT116 colorectal cancer xenograft models at half the dose of irinotecan. The antitumor effect of IT-141 was dose-dependent and caused complete growth inhibition and tumor regression at well-tolerated doses. Varying the specific concentration of SN-38 within the IT-141 micelle had no detectible effect on this antitumor activity, indicating no differences in activity between different IT-141 formulations. In summary, IT-141 is a potent micelle-based chemotherapy that holds promise for the treatment of colorectal cancer.

Synthesis of heterobifunctional polyethylene glycols with azide functionality suitable for “click” chemistry

A heterobifunctional polyethylene glycol (PEG) derivative possessing both “click” and electrophilic functionalities was prepared for use in bioconjugation applications. We utilized a dibenzyl-protected amine functional initiator to prepare high purity amino-PEG-alcohol by the polymerization of ethylene oxide. Subsequent chain-end modification of the heterobifunctional PEG afforded the desired N-hydroxy succinimidyl-PEG-azide derivative in 33% overall yield. This PEG derivative allows for versatile bioconjugation chemistry where activated ester chemistry and “click” chemistry can be selectively performed, an area of orthogonal bioconjugation that has not previously been accessible.

Abstract 596: ABCG2 is upregulated in SN-38 resistant colorectal cancer cells

Irinotecan, a member of the camptothecin chemotherapeutic drug family, is a first and second line therapy for patients with colorectal cancer. However, as with most chemotherapeutic therapies, patients’ tumors eventually develop drug resistance. Therefore, identifying mechanisms of chemotherapeutic resistance will allow for the development of additional molecular therapeutic targets. In this study SN-38, the highly insoluble active metabolite of irinotecan, was used to investigate corollary resistance to irinotecan. SN-38 resistant HT-29 cells were generated and compared to parental untreated HT-29 cells. Cells were characterized following treatment with either DMSO soulubilized SN-38 or IVECT encapsulated SN-38, a triblock copolymer micelle, in aqueous solution. The ATP-binding cassette transmembrane transporter protein, ABCG2, was found to have more than 40-fold expression in the SN-38 resistant HT-29 cells compared to the parental line. The increased expression of ABCG2 protein correlated with increased resistance to SN-38 in HT-29 cells and also with increased cell efflux. Cytotoxicity studies demonstrated that SN-38 resistant HT-29 cells were affected in a similar manner by IVECT polymer encapsulated SN-38 micelles and DMSO dissolved free SN-38. Cell cycle analysis showed that SN-38 resistant cells had an abrogated S phase arrest compared to parental cells. Cell treatment with free SN-38 was limited by its solubility in addition to DMSO toxicity. Further, IVECT polymer encapsulation improved SN-38 aqueous solubility allowing for increased concentration administration. Efflux proteins such as ABCG2 have been found to be upregulated in numerous types of cancers by several classes of chemotherapeutic drugs. Since transporter proteins have an important role in cell efflux, ABCG2 overexpression could serve as an additional target for pharmacological modulation. Finally, the increased solubility of SN-38 by IVECT polymer encapsulation allows for potential in vivo clinical applications of SN-38, a molecule that is 1000 times more active than its prodrug irinotecan. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2011-596

Abstract 4453: Development of a crosslinked, daunorubicin-loaded micelle with superior pharmacokinetics compared to free daunorubicin and with tunable stability

Chemotherapeutic drugs are widely used for the treatment of cancer; however, use of these drugs is often associated with patient toxicity and poor delivery to the tumor. Poor delivery to the tumor can be attributed, in part, to rapid elimination of the drug by the reticuloendothelial system (RES), resulting in short circulation times. Nanoscopic drug carriers offer a promising approach to achieving improved delivery of chemotherapeutic drugs to tumors due to their size and ability to avoid RES uptake, but conventional nanoparticles are not stable in complex biological environments. This instability has resulted in little clinical success of conventional nanoparticles to date. Intezyne9s IVECTTM Method, which utilizes triblock copolymers to form micelles that are stabilized to dilution in complex media, was used to encapsulate daunorubicin. The resulting daunorubicin loaded micelle9s size was determined to be 60 nm by dynamic light scattering analysis. To generate stable micelles, iron (II) chloride was used to form pH-reversible iron-acetate crosslinking bonds in the outer core of the micelle. Release of daunorubicin from stabilized micelles was shown to be pH-dependent through the use of drug-release dialysis assays. Cytotoxicity studies in cancer cell lines revealed that the IC50 values of daunorubicin micelles were similar to free daunorubicin. In vivo, the AUC of daunorubicin released from stabilized daunorubicin micelles was up to 60-fold higher compared to free daunorubicin. Furthermore, the pharmacokinetic parameters were proportional to the iron (II) concentrations used for crosslinking. Repeated injections of daunorubicin micelles in rats showed little to no change in the pharmacokinetic parameters and did not exhibit accelerated blood clearance based on rat IgM ELISA measurements. These data report the development of a stabilized daunorubicin micelle that exhibits pH-dependent release and superior pharmacokinetics based on tunable crosslinking chemistry. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4453. doi:10.1158/1538-7445.AM2011-4453

Abstract 5489: The IVECT™ method, a versatile micelle for the delivery of chemotherapeutic drugs

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The development of new therapeutic agents has dramatically improved the quality of life and survival rate of patients suffering from a variety of disorders. However, drug delivery innovations are required to further improve the success rate of these treatments because approximately 40% of the newly discovered, promising small molecule therapeutics have poor aqueous solubility and cannot be delivered systemically. Rationally-designed, nanoscopic drug carriers offer a promising approach to achieving these goals due to their inherent ability to overcome multiple biological barriers. Ideally, a drug delivery system will not only be stable to systemic administration, but also be versatile enough to encapsulate a diverse range of molecules leading to decreased developmental and regulatory hurdles. These goals were the driving force behind the development of Intezynes IVECT™ Method, which is based on tri-block copolymer micelle technology; spherical nano-sized drug carriers formed by the assembly of amphiphilic block copolymers in water. This modular drug delivery platform can be tailored to accommodate a wide range of drugs, biologics or diagnostics, improve post-administration stability, vary the micelle size, control the release of the therapeutic, and target specific diseased cells based on active targeting strategies. The purpose of this study is to demonstrate encapsulation of a variety of chemotherapeutics utilizing one polymer system, and show that crosslinked micelles have improved pharmacokinetic and toxicology profiles compared to the free drugs in animal models. To this end, formulations of gemcitabine, paclitaxel, doxorubicin and SN-38 were prepared and characterized for nanoparticle size distribution by DLS and active drug weight loading by HPLC. All micelles were 30-80 nm in size with weight loadings of between 2 and 10 %. PK data from cannulated rats demonstrated significant improvement in the CMax and AUC for encapsulated crosslinked formulations, compared to the free drugs. Toxicity profiling in CD-1 mice demonstrated that the polymer alone was very safe, as an MTD was not reachable, and that the crosslinked encapsulated formulations were tolerated at significantly higher doses compared to the free drugs. In conclusion, we have demonstrated that Intezynes IVECT™ Method is extremely versatile and results in significant improvements in the overall pharmacology of chemotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5489.

Abstract 3499: Nanoparticle formulation of SN-38 by the IVECT method results in a safer, more effective treatment for colorectal cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Polymeric micelles represent an emerging class of nano-scale vehicles with unmatched potential for delivering potent, targeted chemotherapy. Intezynes advanced tri-block copolymer system, the IVECT(TM) Method, has been proven to encapsulate a wide variety of therapeutic and diagnostic agents into a proprietary micelle with unique physiochemical properties. Conventional micelles become destabilized rapidly upon dilution into the bloodstream and release a significant portion of their drug payload systemically, harming healthy tissues and limiting therapeutic effects. Intezynes IVECT Method utilizes a unique cross-linked stabilizer block that maintains micellar structure for a longer period of time post administration and is designed such that a downshift in pH at the tumor activates drug release by destroying the zinc cross-link, allowing for focused drug exposure. The IVECT Methods unmatched plasma stability allows for longer circulation times while protecting healthy tissues, and it maximizes tumor retention and efficacy through the enhanced permeability and retention (EPR) effect and pH-responsive cross-linker. Intezynes lead product IT-141 encapsulates the extremely potent active metabolite of irinotecan, SN-38. Recently, IT-141 has demonstrated 14-fold greater plasma exposure and 8-fold greater tumor accumulation vs. irinotecan. This superior pharmacokinetic profile confers 500% greater anti-tumor activity at half the dose (30mg/kg, 3X Q4D, vs. 60mg/kg irinotecan). In xenograft models, IT-141 has demonstrated tumor regression of up to 88%, with 60% of tumors regressing completely. The IVECT copolymer is safe and well tolerated at up to 6 times the therapeutic doses, with no sick mouse syndrome, and can be lyophilized for long-term storage (>1 year). Intezyne is preparing its IND for IT-141. The Company has additional programs in development that focus on delivering best-in-class chemotherapies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3499.

Abstract 599: Development of poly(Asp-DET) cationic polymers for nonviral gene delivery

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cationic polymers have received much attention as promising non-viral vectors for gene transfer. However, identification of polymers able to systemically deliver nucleic acids continues to be a significant hurdle to the success of this methodology. The purpose of this study was to develop a new biocompatible and biodegradable cationic polymer system as a non-viral gene delivery agent for plasmid DNA. Poly-aspartate-diethylene triamine (“P(Asp-DET)”) polymers were synthesized and utilized to complex plasmid DNA. The resulting polymer/DNA complexes, also known as “polyplexes,” were further modified by covalent coupling with polyethylene glycol (PEG), known as “PEGylation,” and characterized. Polyplexes were evaluated for efficiency of encapsulation, particle size, zeta potential and morphology by TEM. Stability of the complexed plasmid DNA was assessed by challenging polyplexes with nucleases. Efficacy of DNA delivery and gene expression was examined by performing transfection experiments using HCT-116 cells. In vivo DNA delivery was also investigated using tumor-bearing nude mice. Asp-DET polymers were found to bind to plasmid DNA with polymer to DNA (+/-) charge ratios of above 1.5. In addition, these polymers formed polyplex particles of approximately 50-150 nm with zeta potentials between neutral and +40 mV. TEM showed that the polyplexes were uniform and spherical in shape. It was also demonstrated that the polyplexes maintained the structural integrity of DNA following incubation in nucleases. The polyplexes were also capable of transfecting HCT-116 culture cells, with non-PEG complexes having significantly higher GFP and luciferase expression. Tail vein injections of post-PEGylated polyplexes into tumor bearing nude mice did not reveal any observable toxicities. Notably, nucleic acid accumulation was found in tumors with expression detected in lymph nodes. P(Asp-DET) cationic polymers show enormous potential as non-viral drug delivery agents. Further, the surface modification of described polyplexes with PEG allowed for systemic delivery of nucleic acids to tumors. The characterization of post-PEGylated Asp-DET/DNA polyplexes provides insights that can be used to design vectors for targeted nucleic acid delivery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 599.