pH-Responsive Histidine-Rich Elastin-Like Polypeptides That Improve Intratumoral Spatial Distribution

Abstract

<p>The central goal of this dissertation is to develop a drug carrier capable of retaining the benefits of high molecular weight drug carriers but improving on the intratumoral spatial distribution of drug by engineering the carrier to disassemble in response to the tumor-specific endogenous signal of low pH. High molecular weight drug carriers improve clinical outcomes for cytotoxic chemotherapeutics by preventing their accumulation in healthy tissues, extending their circulation time, and allowing for gradual accumulation in solid tumors due to the leaky tumor vasculature. These carriers increase the maximum tolerated dose that can be administered, and as a result of these characteristics can exhibit improved antitumor effectiveness with reduced toxicity compared with free drug. One drawback of these formulations is that they exacerbate a problem inherent to tumor drug delivery, the poor penetration of drug into the tumor tissue due to the poor vascularization in many tumor regions coupled with the dense extracellular matrix, dense cell packing, and high interstitial fluid pressure of the tumor tissue. Therefore, a high molecular weight drug carrier that disassembles in response to a tumor-specific stimulus could retain the benefits detailed above but release the highly diffusive small molecule drug within the tumor site, improving its ability to penetrate into the tumor and treat the whole tumor volume.</p><p>This work addresses this problem by synthesizing a pH-sensitive drug carrier capable of disassembling in response to the low extracellular pH that exists in many solid tumors. The drug carrier design is a polypeptide block copolymer with a histidine-rich hydrophobic block that self-assembles at pH 7.4 but disassembles at slightly low pH as the histidine residues become charged and the hydrophobic block becomes increasingly hydrophilic. Chapter 1 briefly establishes key features of cancer and solid tumors that impact drug delivery, and then reviews the central findings of the field of tumor drug delivery. After an extensive discussion of pH-sensitive drug carriers that have been synthesized to date, an extended description of the proposed design is given.</p><p>Chapter 2 describes the synthesis and characterization of histidine-rich Elastin-Like Polypeptides (ELPs), the pH-responsive component of the material design. These polymers are synthesized using molecular biological methods and their transition temperatures are characterized using absorbance spectroscopy, demonstrating remarkable dependence on small pH changes in the physiological range of interest (pH 6-8). This chapter further demonstrates that histidine-rich ELPs are sensitive to transition metal ions known to coordinate with histidine, exhibiting a precipitous drop in transition temperature in the presence of µM concentrations of these ions. Chapter 3 characterizes histidine-rich ELP block copolymers (ELPBCs), demonstrating by dynamic light scattering that these polymers form micellar nanoparticles at body temperature and pH 7.4 that disassemble at pH 6.4. These micelles are stabilized by ZnCl2 as demonstrated by a reduced critical micelle temperature observed by light scattering and critical micelle concentration shown by pyrene fluorescence. Static light scattering, Atomic Force Microscopy, and freeze-fracture Transmission Electron Microscopy confirm the presence of these particles and demonstrate that the ELPBCs form uniform spherical micelles.</p><p>Chapter 4 attempts to develop these materials as drug delivery vehicles in several different schemes. First, drug encapsulation experiments are conducted to serve the initial design of pH-sensitive drug release, but these micelles have not yet demonstrated an ability to encapsulate chemotherapeutic drugs. An alternative scheme of disassembly-induced display of a ligand for cell entry is investigated by the inclusion of the LHRH peptide in the micelle core. Disassembly at low pH indeed increases cell uptake of the ELPBCs, but this effect does not depend on ligand presentation. Finally, based on the hypothesis that metal coordination resulted in the formation of crosslinks between ELP chains, histidine-rich ELPs are investigated as a depot formulation for intratumoral drug delivery.</p><p>Chapter 5 returns to the original ELPBC micelle design and addresses the core questions of this work: does the histidine-rich ELPBC retain the benefits of high molecular weight drug carriers, and does low pH-induced disassembly enhance the intratumoral spatial distribution? A low pH mouse tumor model is established to test the effects of pH-sensitive materials, and then fluorescently and radioactively labeled pH-sensitive and pH-insensitive ELPBCs are injected to determine their pharmacokinetics, biodistribution, and intratumoral spatial distribution. The histidine-rich ELPBCs exhibit extended blood circulation and tumor accumulation typical of high molecular weight carriers, although some healthy tissues take up significant amounts of both ELPBCs. The pH-sensitive histidine-rich ELPBCs demonstrate a more even distribution throughout the tumor volume than their pH-insensitive counterparts, indicating that stimulus-responsive nanoparticles can improve tumor penetration through a tumor-specific material response.</p><p>Finally, Chapter 6 summarizes the main findings of the work as noted here, and discusses potential future directions. The main accomplishment of this work is the demonstration that histidine-rich ELPs exhibit triple stimulus-responsiveness and achieve an improved intratumoral spatial distribution.</p>