Bifunctional Elastin-like Polypeptide Nanoparticles Bind Rapamycin and Integrins and Suppress Tumor Growth in Vivo.

Recombinant protein-polymer scaffolds such as elastin-like polypeptides (ELPs) offer drug-delivery opportunities including biocompatibility, monodispersity, and multifunctionality. We recently reported that the fusion of FK-506 binding protein 12 (FKBP) to an ELP nanoparticle (FSI) increases rapamycin (Rapa) solubility, suppresses tumor growth in breast cancer xenografts, and reduces side effects observed with free-drug controls. This new report significantly advances this carrier strategy by demonstrating the coassembly of two different ELP diblock copolymers containing drug-loading and tumor-targeting domains. A new ELP nanoparticle (ISR) was synthesized that includes the canonical integrin-targeting ligand (Arg-Gly-Asp, RGD). FSI and ISR mixed in a 1:1 molar ratio coassemble into bifunctional nanoparticles containing both the FKBP domain for Rapa loading and the RGD ligand for integrin binding. Coassembled nanoparticles were evaluated for bifunctionality by performing in vitro cell-binding and drug-retention assays and in vivo MDA-MB-468 breast tumor regression and tumor-accumulation studies. The bifunctional nanoparticle demonstrated superior cell target binding and similar drug retention to FSI; however, it enhanced the formulation potency, such that tumor growth was suppressed at a 3-fold lower dose compared to an untargeted FSI-Rapa control. This data suggests that ELP-mediated scaffolds are useful tools for generating multifunctional nanomedicines with potential activity in cancer.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.

From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 µm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 µm) with a reduced diffusion coefficient of 1.1 µm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains.

Biodegradation of elastin-like polypeptide nanoparticles.

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin-like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (T(t)). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.

Design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain.

Hepatocytes and acinar cells exhibit high-efficiency, fiber-dependent internalization of adenovirus; however, viral capsids have unpredictable immunological effects and are challenging to develop into targeted drug carriers. To exploit this internalization pathway and minimize the use of viral proteins, we developed a simple gene product that self assembles nanoparticles decorated with the knob domain of adenovirus serotype 5 fiber protein. The most significant advantages of this platform include: (i) compatibility with genetic engineering; (ii) no bioconjugate chemistry is required to link fusion proteins to the nanoparticle surface; and (iii) it can direct the reversible assembly of large nanoparticles, which are monodisperse, multivalent, and biodegradable. These particles are predominantly composed from diblock copolymers of elastin-like polypeptide (ELP). ELPs have unique phase transition behavior, whereby they self-assemble above a transition temperature that is simple to control. The diblock ELP described contains two motifs with distinct transition temperatures, which assemble nanoparticles at physiological temperatures. Analysis by non-denaturing-PAGE demonstrated that the purified knob-ELP formed trimers or dimers, which is a property of the native knob/fiber protein. Dynamic light scattering indicated that the diblock copolymer, with or without knob, is able to self assemble into nanoparticles ~40 nm in diameter. To examine the functionality of knob-ELP, their uptake was assessed in a hepatocyte cell-line that expresses the receptor for adenovirus serotype 5 fiber and knob, the coxsackievirus and adenovirus receptor (CAR). Both plain ELP and knob-ELP were bound to the outside of hepatocytes; however, the knob-ELP fusion protein exhibits more internalization and localization to lysosomes of hepatocytes. These findings suggest that functional fusion proteins may only minimally influence the assembly temperature and diameter of ELP nanoparticles. These results are a proof-of-principal that large fusion proteins (>10 kDa) can be assembled by diblock ELPs without the need for bioconjugate chemistry, which greatly simplifies the design and evaluation of targeted drug carriers.

Imaging and drug delivery using theranostic nanoparticles.

Nanoparticle technologies are significantly impacting the development of both therapeutic and diagnostic agents. At the intersection between treatment and diagnosis, interest has grown in combining both paradigms into clinically effective formulations. This concept, recently coined as theranostics, is highly relevant to agents that target molecular biomarkers of disease and is expected to contribute to personalized medicine. Here we review state-of-the-art nanoparticles from a therapeutic and a diagnostic perspective and discuss challenges in bringing these fields together. Major classes of nanoparticles include, drug conjugates and complexes, dendrimers, vesicles, micelles, core-shell particles, microbubbles, and carbon nanotubes. Most of these formulations have been described as carriers of either drugs or contrast agents. To observe these formulations and their interactions with disease, a variety of contrast agents have been used, including optically active small molecules, metals and metal oxides, ultrasonic contrast agents, and radionuclides. The opportunity to rapidly assess and adjust treatment to the needs of the individual offers potential advantages that will spur the development of theranostic agents.

Environmentally responsive peptides as anticancer drug carriers.

The tumor microenvironment provides multiple cues that may be exploited to improve the efficacy of established chemotherapeutics; furthermore, polypeptides are uniquely situated to capitalize on these signals. Peptides provide: 1) a rich repertoire of biologically specific interactions to draw upon; 2) environmentally responsive phase behaviors, which may be tuned to respond to signatures of disease; 3) opportunities to direct self-assembly; 4) control over routes of biodegradation; 5) the option to seamlessly combine functionalities into a single polymer via a one-step biosynthesis. As development of cancer-targeted nanocarriers expands, peptides provide a unique source of functional units that may target disease. This review explores potential microenvironmental physiology indicative of tumors and peptides that have demonstrated an ability to target and deliver to these signals.