Dan E. Meyer

Drug targeting using thermally responsive polymers and local hyperthermia.

We report a new thermal targeting method in which a thermally responsive drug carrier selectively accumulates in a solid tumor that is maintained above physiological temperature by externally applied, focused hyperthermia. We synthesized two thermally responsive polymers that were designed to exhibit a lower critical solution temperature (LCST) transition slightly above physiological temperature: (1) a genetically engineered elastin-like polypeptide (ELP) and (2) a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm). The delivery of systemically injected polymer-rhodamine conjugates to solid tumors was investigated by in vivo fluorescence video microscopy of ovarian tumors implanted in dorsal skin fold window chambers in nude mice, with and without local hyperthermia. When tumors were heated to 42 degrees C, the accumulation of a thermally responsive ELP with a LCST of 40 degrees C was approximately twofold greater than the concentration of the same polymer in tumors that were not heated. Similar results were also obtained for a thermally responsive poly(NIPAAM-co-AAm), though the enhanced accumulation of this carrier in heated tumors was lower than that observed for the thermally responsive ELP. These results suggest that enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers combined with local heating of tumors.

Targeted drug delivery by thermally responsive polymers.

This review article summarizes recent results on the development of macromolecular carriers for thermal targeting of therapeutics to solid tumors. This approach employs thermally responsive polymers in conjunction with targeted heating of the tumor. The two thermally responsive polymers that are discussed in this article, poly(N-isopropylacrylamide-co-acrylamide) (poly(NIPAAm)) and an artificial elastin-like polypeptide (ELP), were designed to exhibit a soluble-insoluble lower critical solution transition in response to increased temperature slightly above 37 degrees C. In vivo fluorescent videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately two-fold increase in tumor localization compared to the same polypeptide without hyperthermia. Similar results were also obtained for poly(NIPAAm) though the extent of accumulation was somewhat lesser than observed for the ELP. The endocytotic uptake of a thermally responsive ELP was also observed to be significantly enhanced by the thermally triggered phase transition of the polypeptide in cell culture for three different tumor cell lines. Preliminary cytotoxicity studies of an ELP-doxorubicin conjugate indicate that the ELP-doxorubicin conjugate has near equivalent cytotoxicity as free doxorubicin in a cell culture assay.

Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery.

In this article, we review recombinant DNA methods for the design and synthesis of amino acid-based biopolymers, and briefly summarize an approach, recursive directional ligation (RDL), that we have employed to synthesize oligomeric genes for such biopolymers. We then describe our ongoing research in the use of RDL to synthesize recombinant polypeptide carriers for the targeted delivery of radionuclides, chemotherapeutics and biomolecular therapeutics to tumors. The targeted delivery system uses a thermally responsive, elastin-like polypeptide (ELP) as the drug carrier to enhance the localization of ELP-drug conjugates within a solid tumor that is heated by regional hyperthermia. In the context of this drug delivery application, we discuss the design of ELPs and their recombinant synthesis, which enables the molecular weight and the thermal properties of the polypeptide to be precisely controlled. Finally, our results pertaining to the in vivo targeting of tumors with ELPs are briefly summarized.

Protein Purification by Fusion with an Environmentally Responsive Elastin‐Like Polypeptide: Effect of Polypeptide Length on the Purification of Thioredoxin

Elastin‐like polypeptides (ELPs) undergo a reversible, soluble‐to‐insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (Tt). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin‐ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4‐fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of ∼2 μm form as the temperature is raised to above the Tt. These aggregates persist at all temperatures above the Tt for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of ∼12 nm begin to form at the expense of the larger, micron‐size aggregates as the temperature is further raised above the Tt. Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron‐size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.

A genetically-engineered polypeptide carrier for thermal targeting of therapeutics

Reports the synthesis of a environmentally-responsive polypeptide carrier for thermally-targeted delivery of radionuclide therapeutics. The authors hypothesized that systemically-injected radionuclide-polypeptide conjugates are likely to be cleared from circulation under physiological conditions, while externally-induced local hyperthermia at tumor sites will result in radionuclide accumulation because of carrier aggregation. The authors report preliminary in vivo results which support this hypothesis.