Graduate Researcher, University of Utah
A thermally- and environmentally-responsive nanoparticle system to improve tumor localization
Enhancing localization of drugs in specific tissues can be achieved through engineering drug delivery systems (DDS) to respond to externally-applied stimuli and/or local environmental changes within targeted tissues in the body. Synthetic polymeric DDS can utilize specific monomers or incorporated peptide sequences to accomplish stimuli-responsiveness, but applications can be limited due to the polymers displaying high variability in size and sequence. As recombinant polymers are synthesized via natural ribosomal production processes, these polymers exhibit defined amino acid sequence and monodispersity, allowing direct control over form and function. Recent downstream processing techniques of silk-elastinlike protein polymers (SELPs) have led to the formation of nanogels with many desired properties for drug delivery applications. Incorporated silk and elastin peptide motifs allow for thermoresponsive capabilities of the nanogels, allowing for temperature-induced secondary structure changes, size contractions and expansions, and particle aggregation. If tumors are externally heated above nanogel transition temperatures, the thermoresponsiveness will likely improve bloodstream extravasation and tumor localization. Furthermore, matrix-metalloproteinase (MMP)-degradable peptide sequences that were genetically inserted into the SELP backbone provide dual-responsiveness to the nanogels. MMP concentrations are highly upregulated in cancer tissues, with enhanced disease progression leading to the greatest overexpression. When MMP-responsive sequences were inserted, the nanogels experienced a 6-fold faster degradation in the presence of tumor-relevant MMP-9 concentrations than in regular tissue concentrations. Hydrophobic drugs can be incorporated within the nanogels, and the increased degradability may enable the release of these active anticancer drugs selectively within tumors. Ultimately, improved tumor localization and selective drug release can reduce dose-limiting toxicities, decrease off-target side effects, and increase overall patient survival.
Abstract: Polymer micellar nanogels are a group of core-shell polymeric micelles with swelling properties in aqueous media. Nanogel systems have proven their potential in controlled, sustained and targetable drug delivery area with no immunological responses. This review includes a comprehensive wide range of self-assembly of polymeric nanogels as delivery systems for anticancer drugs. Nanogels are nanoparticulate drug delivery systems which are specially designed for enhanced target oriented and cellular uptake of drugs with emphasis on chemotherapeutic agents studied in this review. Self-assembling nanogels are based on natural substances or synthetic polymers including: hyaluronic acid, heparin, alginate, cyclodextrins, chondroeitin sulfate, starch, mannan, chitosan, pullulan, poly(N-isopropylacrylamide), polyvynil alcohol, Pluronic F127, polyacrylic acid, poly(hydroxylethyl methacrylate), poly[2- (dimethylamino)ethyl methacrylate and polylactide-co-glycolide-polyethylen glycol amphiphilic di or tri block copolymer used to deliver anticancer drugs are introduced and discussed.
Pub.: 13 Jul '17, Pinned: 06 Sep '17
Abstract: Silk-elastin-like protein polymers (SELPs) combine the mechanical and biological properties of silk and elastin. These properties have led to the development of various SELP-based materials for drug delivery. However, SELPs have rarely been developed into nanoparticles, partially due to the complicated fabrication procedures, nor assessed for potential as an anticancer drug delivery system. We have recently constructed a series of SELPs (SE8Y, S2E8Y, and S4E8Y) with various ratios of silk to elastin blocks and described their capacity to form micellar-like nanoparticles upon thermal triggering. In this study, we demonstrate that doxorubicin, a hydrophobic antitumor drug, can efficiently trigger the self-assembly of SE8Y (SELPs with silk to elastin ratio of 1:8) into uniform micellar-like nanoparticles. The drug can be loaded in the SE8Y nanoparticles with an efficiency around 6.5% (65 ng doxorubicin/μg SE8Y), S2E8Y with 6%, and S4E8Y with 4%, respectively. In vitro studies with HeLa cell lines demonstrate that the protein polymers are not cytotoxic (IC50 > 200 μg/mL), while the doxorubicin-loaded SE8Y nanoparticles showed a 1.8-fold higher cytotoxicity than the free drug. Confocal laser scanning microscopy (CLSM) and flow cytometry indicate significant uptake of the SE8Y nanoparticles by the cells and suggest internalization of the nanoparticles through endocytosis. This study provides an all-aqueous, facile method to prepare nanoscale, drug-loaded SELPs packages with potential for tumor cell treatments.
Pub.: 18 Feb '14, Pinned: 20 Jun '17
Abstract: Silk--elastin-like protein polymers (SELPs), consisting of the repeating units of silk and elastin blocks, combine a set of outstanding physical and biological properties of silk and elastin. Because of the unique properties, SELPs have been widely fabricated into various materials for the applications in drug delivery and tissue engineering. However, little is known about the fundamental self-assembly characteristics of these remarkable polymers. Here we propose a two-step self-assembly process of SELPs in aqueous solution for the first time and report the importance of the ratio of silk-to-elastin blocks in a SELP's repeating unit on the assembly of the SELP. Through precise tuning of the ratio of silk to elastin, various structures including nanoparticles, hydrogels, and nanofibers could be generated either reversibly or irreversibly. This assembly process might provide opportunities to generate innovative smart materials for biosensors, tissue engineering, and drug delivery. Furthermore, the newly developed SELPs in this study may be potentially useful as biomaterials for controlled drug delivery and biomedical engineering.
Pub.: 01 Oct '11, Pinned: 20 Jun '17
Abstract: Water-soluble triblock copolymers have received much attention in industrial applications and scientific fields. We here show that femtosecond mid-IR pump–probe spectroscopy is useful to study the role of water in the temperature-induced self-assembly of triblock copolymers. Our experimental results suggest two distinct subpopulations of water molecules: those that interact with other water molecules and those involved in the hydration of a triblock copolymer surface. We find that the vibrational dynamics of bulk-like water is not affected by either micellation or gelation of triblock copolymers. The increased population of water interacting with ether oxygen atoms of the copolymer during the unimer to micelle phase transition is important evidence for the entropic role of water in temperature-induced micelle formation at a low copolymer concentration. In contrast, at the critical gelation temperature and beyond, the population of surface-associated water molecules interacting with ether oxygen atoms decreases, which indicates important enthalpic control by water. The present study on the roles of water in the two different phase transitions of triblock copolymers sheds new light on the underlying mechanisms of temperature-induced self-aggregation behaviors of amphiphiles that are ubiquitous in nature.
Pub.: 14 Jun '17, Pinned: 20 Jun '17
Abstract: We evaluated the drug release capability of optically transparent recombinant silk-elastinlike protein polymer, SELP-47K, films to sustainably deliver the common ocular antibiotic, ciprofloxacin. The ciprofloxacin release kinetics from drug-loaded SELP-47K films treated with ethanol or methanol vapor to induce different densities of physical crosslinking was investigated. Additionally, the drug-loaded protein films were embedded in a protein polymer coating to further prolong the release of the drug. Drug-loaded SELP-47K films released ciprofloxacin for up to 132 h with near first-order release kinetics. Polymer coating of drug-loaded films prolonged drug release for up to 220 h. The antimicrobial activity of ciprofloxacin released from the drug delivery matrices was not impaired by the film casting process or the ethanol or methanol treatments. The mechanism of drug release was elucidated by analyzing the physical properties of the film specimens, including equilibrium swelling, soluble fraction, surface roughness and hydrophobicity. Additionally, the conformation of the SELP-47K and its physical crosslinks in the films was analyzed by FTIR and Raman spectroscopy. A three-parameter physics based model accurately described the release rates observed for the various film and coating treatments and attributed the effects to the degree of physical crosslinking of the films and to an increasing affinity of the drug with the polymer network. Together, these results indicate that optically transparent silk-elastinlike protein films may be attractive material candidates for novel ophthalmic drug delivery devices.
Pub.: 16 Aug '11, Pinned: 20 Jun '17
Abstract: Silk-elastinlike protein polymers (SELPs) are recombinant polymers consisting of tandem repeats of silk (GAGAGS) and elastin (GVGVP) units. By modification of the length and composition of these repeats, the properties of SELP hydrogels can be controlled for specific applications including nucleic acid and virus delivery and tissue engineering. Here, the structure of SELPs is further modified to include a sequence that is sensitive to matrix-metalloproteinases (MMPs). MMPs are a ubiquitous family of extracellular matrix-modifying enzymes that are commonly associated with numerous vital processes. Increased levels of MMPs are found at high levels locally in many types of solid tumors. By modifying the SELP backbone with MMP-sensitive peptide sequences, a hydrogel that is degradable by MMPs was produced. The MMP-sensitivity of the polymer was examined by incubation with MMP-2 and MMP-9, which yielded complete cleavage of all full-length polymers by 36 hours and 48 hours, respectively, with no observable effect on unmodified SELP. Hydrogel sensitivity was tested by exposure to MMP-2 or MMP-9 for 2 weeks, during which samples were taken to analyze protein loss from the hydrogel and release of 100 nm fluorescent beads. Following the incubation period, hydrogels were tested in mechanical compression to examine the loss of hydrogel stiffness due to degradation. It was found that MMP-2 and MMP-9 caused 63% and 44% increased protein loss and 65% and 95% increased release from MMP-sensitive hydrogels, while the compressive modulus decreased by 41% and 29%. These results suggest the potential of MMP-responsive SELPs for localized delivery of bioactive agents where MMPs are overexpressed.
Pub.: 02 Feb '13, Pinned: 20 Jun '17
Abstract: Silk-elastinlike protein polymers (SELPs) have been fabricated as matrices for controlled delivery of bioactive agents. In this application the need for an environmentally responsive, degradable polymer has risen to improve treatment outcomes. To satisfy this need, we have designed, synthesized, and expressed SELPs with matrix metalloproteinase (MMP) degradable sequences inserted in distinct regions of the polymer backbone. Upon characterization of the physicochemical properties of newly synthesized analogs, it was determined that conditioning of the polymers was necessary for normalization of batch properties, and to generate a more robust polymer network suitable for delivery. In this report we have examined the use of shear stress to condition synthesized material prior to application as a controlled release matrix. The application of high shear to SELPs results in significant changes in physiochemical properties as assayed by swelling ratio, soluble fraction release, rate of gel formation, stiffness of hydrogels, and nanoparticle release from matrices. These observed changes in material characteristics may be caused by the removal of semi-stable secondary and tertiary structures from single polymer strands leading to a more robust hydrogel with greater intermolecular interaction.
Pub.: 06 Aug '14, Pinned: 20 Jun '17
Abstract: Silk-elastinlike protein polymers (SELPs) have been effectively used as controlled release matrices for the delivery of viruses for cancer gene therapy in preclinical models. However, the degradability of these polymers needs to be tuned for improved localized intratumoral gene delivery. Using recombinant techniques, systematic modifications in distinct regions of the polymer backbone, namely, within the elastin blocks, silk blocks, and adjacent to silk and elastin blocks, have been made to impart sensitivity to specific matrix metalloproteinases (MMPs) known to be overexpressed in the tumor environment. In this report we investigated the structure-function relationship of MMP-responsive SELPs for viral mediated gene therapy of head and neck cancer. These polymers showed significant degradation in vitro in the presence of MMPs. Their degradation rate was a function of the location of the MMP-responsive sequence in the polymer backbone when in hydrogel form. Treatment efficacy of the adenoviral vectors released from the MMP responsive SELP analogs in a xenograft mouse model of head and neck squamous cell carcinoma (HNSCC) was shown to be polymer structure dependent. These results demonstrate the tunable nature of MMP-responsive SELPs for localized matrix-mediated gene delivery.
Pub.: 23 Jun '15, Pinned: 20 Jun '17
Abstract: The objective of this study was to utilize an on-chip degradation assay to evaluate polymer depots and the predicted drug release from the depots. We conjugated four silk-elastinlike protein (SELP) polymers including SELP-815K, SELP-815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 with a Cy5-NHS ester and fabricated SELP arrays by immobilizing the conjugated polymers onto well-type amine arrays. SELP polymer degradation rates were investigated by calculating the half-maximal effective concentration (EC50). Eight cleavage enzymes were applied, all of which exhibited distinctive EC50 values for SELP-815K and its three analogues. We successfully utilized this assay to study the in vitro release of the Cy5-conjugated C-peptide from SELP-815K hydrogel arrays. Additionally, cumulative C-peptide release from the SELP-815K depots was also demonstrated using repetitive elastase treatments. Therefore, this array-based on-chip degradation assay could potentially be used for evaluating depot degradation and controlled drug release from polymer depots at the molecular level.
Pub.: 25 Apr '16, Pinned: 20 Jun '17
Abstract: Silk and elastin are exemplary protein materials that exhibit exceptional material properties. Silk is uniquely strong, surpassing engineering materials such as Kevlar and steel, while elastin has exquisite flexibility and can reversibly fold into a more structured form at high temperatures when many other proteins would unfold and denature. This phenomenon in elastin is termed the inverse temperature transition. It is a reversible, controllable process that motivates applications in drug delivery, shape change materials, and biomimetic devices. Silk-elastinlike protein polymers (SELPs), which combine repeating B. mori silk and elastin blocks, have been introduced as biologically inspired materials that combine the distinctive properties of the component parts to achieve strong and extensible, tunable biomaterials. Here, we considered a single SELP sequence to examine temperature transition effects at the molecular scale. SELP molecular models were created using Replica Exchange Molecular Dynamics, an accelerated sampling method, and confirmed in experiment by comparing secondary structure distributions. A molecular collapse of the SELP molecule was observed with increased temperature in both molecular simulation and experiment. Temperature-specific differences were observed in the mechanical properties and the unfolding pathways of the polypeptide. Using the Bell–Evans model, we analyzed the free energy landscape associated with molecular unfolding at temperatures below and above the transition temperature range (Tt) of the polypeptide. We found that at physiological pulling rates, the energy barrier to unfold SELPs was counterintuitively higher above Tt. Our findings offer a foundational perspective on the molecular scale mechanisms of temperature-induced phase transition in SELPs, and suggest a novel approach to combine simulation and experiment to study materials for multifunctional biomimetic applications.
Pub.: 16 Mar '17, Pinned: 20 Jun '17