Development of Thermoresponsive Protein Complexes for Targeting CD20 Receptors.

Therapeutics targeting cell receptors can elicit biological responses in situ. However, the ability to dictate when and where these responses occur is a current challenge. Therapeutic proteins can be combined with stimuli-responsive peptides to increase targeting and stimuli responsive behavior. To this end the authors genetically engineered an elastinlike polypeptide (ELP) fusion protein for selective ELPylation. The addition of a charged, foldable region provides these protein subunits with varied thermoresponsive properties from their parent ELPs. These subunits have responsive secondary structures, dependent on pH, indicating the capability to form coiled-coils with a complementary peptide tag. A Rituximab conjugate is generated herein, containing the complementary peptide. Upon mixing of the ELP and Rituximab subunits, the resulting protein complexes can target CD20 receptors on Raji B cells, resulting in at least twofold increase in mean fluorescent intensities. These ELP subunits fold in vitro with the complimentary generated Rituximab conjugate. This work provides the basis for the design of a therapeutic stimuli-responsive biomacromolecule for targeting receptors.

An Oligomeric Sulfated Hyaluronan and Silk-Elastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis.

Semisynthetic glycosaminoglycan ethers (SAGEs) are short, sulfated hyaluronans which combine the natural properties of hyaluronan with chemical sulfation. In a murine model, SAGEs provide protection against radiation induced proctitis (RIP), a side effect of lower abdominal radiotherapy for cancer. The anti-inflammatory effects of SAGE have been studied in inflammatory diseases at mucosal barrier sites; however, few mechanisms have been uncovered necessitating high throughput methods. SAGEs were combined with silk-elastinlike polymers (SELPs) to enhance rectal accumulation in mice. After high radiation exposure to the lower abdominal area, mice were followed for 3 days or until they met humane endpoints, before evaluation of behavioral pain responses and histological assessment of rectal inflammation. RNA sequencing was conducted on tissues from the 3-day cohort to determine molecular mechanisms of SAGE-SELP. After 3 days, mice receiving the SAGE-SELP combination yielded significantly lowered pain responses and amelioration of radiation-induced rectal inflammation. Mice receiving the drug-polymer combination survived 60% longer than other irradiated mice, with a fraction exhibiting long term survival. Sequencing reveals varied regulation of toll like receptors, antioxidant activities, T-cell signaling, and pathways associated with pain. This investigation elucidates several molecular mechanisms of SAGEs and exhibits promising measures for prevention of RIP.

Self-Assembly of Thermoresponsive Recombinant Silk-Elastinlike Nanogels.

Recombinant silk-elastinlike protein polymers (SELPs) combine the biocompatibility and thermoresponsiveness of human tropoelastin with the strength of silk. Direct control over structure of these monodisperse polymers allows for precise correlation of structure with function. This work describes the fabrication of the first SELP nanogels and evaluation of their physicochemical properties and thermoresponsiveness. Self-assembly of dilute concentrations of SELPs results in nanogels with enhanced stability over micelles due to physically crosslinked beta-sheet silk segments. The nanogels respond to thermal stimuli via size changes and aggregation. Modifying the ratio and sequence of silk to elastin in the polymer backbone results in alterations in critical gel formation concentration, stability, aggregation, size contraction temperature, and thermal reversibility. The nanogels sequester hydrophobic compounds and show promise in delivery of bioactive agents.

Bladder pain in an LL-37 interstitial cystitis and painful bladder syndrome model.

Our goal was to evaluate the pain response in an LL-37 induced murine model for interstitial cystitis/painful bladder syndrome (IC/PBS). In particular, we sought to characterize the dose dependence, time-course, and relationship of LL-37 induced bladder inflammation and pain. The IC/PBS model was induced in C57Bl/6 mice by instilling 50 µL of LL-37, an immunomodulatory human cathelicidin (anti-microbial peptide), in the bladder for 1 hr. Pain responses were measured using von Frey filaments (0.04 gm to 4.0 gm) before and after LL-37 instillation. Inflammation was evaluated using tissue myeloperoxidase (MPO) assay, gross inspection, and microscopic histologic examination. The dose response experiment demonstrated a graded pain response, with higher concentrations of LL-37 challenge yielding higher pain responses across all stimuli tested. Statistical significance was seen when comparing 1.0 gm von Frey filament results at 320 µM (68 ± 8% response) vs. 0 µM (38 ± 6% response). Interestingly, pain responses did not attenuate across time but increased significantly after 5 (p=0.0012) and 7 days (p=0.0096). Comparison with MPO data suggested that pain responses could be independent of inflammation. We demonstrated within our LL-37 induced IC/PBS model pain occurs in a dose-dependent fashion, pain responses persist beyond the initial point of insult, and our dose response and time course experiments demonstrated that pain was independent of inflammation.

Silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis.

Radiation-induced proctitis (RIP) is the most common clinical adverse effect for patients receiving radiotherapy as part of the standard course of treatment for ovarian, prostate, colon, and bladder cancers. RIP limits radiation dosage, interrupts treatment, and lowers patients' quality of life. A prophylactic treatment that protects the gastrointestinal tract from deleterious effects of radiotherapy will significantly improve patient quality of life and may allow for higher and more regular doses of radiation therapy. Semi-synthetic glycosaminoglycan (GAG), generated from the sulfation of hyaluronic acid, are anti-inflammatory but have difficulty achieving therapeutic levels in many tissues. To enhance the delivery of GAG, we created an in situ gelling rectal delivery system using silk-elastinlike protein polymers (SELPs). Using solutions of SELP 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit) with GAG GM-0111, we created an injectable delivery platform that transitioned in <5min from a liquid at room temperature to a hydrogel at body temperature. The hydrogels released 50% of their payload within 30min and enhanced the accumulation of GAG in the rectum compared to traditional enema-based delivery. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21days by 53±4%, 47±10%, and 12±6%, respectively. Matrix-mediated delivery of GAG by SELP represents an innovative method for more effective treatment of RIP and promises to improve quality of life of cancer patients by allowing higher radiotherapy doses with improved safety.

Silk-Elastinlike Protein Polymer Liquid Chemoembolic for Localized Release of Doxorubicin and Sorafenib.

Locoregional therapies for cancer are minimally invasive procedures in which the treatment is administered directly into cancerous tissue. Transarterial chemoembolization (TACE) is used to treat intermediate stage hepatocellular carcinoma (HCC). TACE uses an embolic material to block blood flow while coadministering a chemotherapeutic to the neoplastic tissue. Liquid embolics capable of drug loading are at the forefront of development as they allow for deeper permeation of tumor vasculature, increase neoplasm exposure to therapeutics, and resist revascularization by occupying both large and small diameter vessels. In this work, two chemotherapeutics used in the treatment of HCC, doxorubicin and sorafenib, were incorporated into the in situ gelling liquid embolic composed of a silk-elastinlike protein polymer (SELP-815 K). The base forms of the drugs had no significant effect on the viscosity, the gelation kinetics, and the gel stiffness of the SELP: all properties essential for the successful performance of an injectable liquid embolic. In vitro release studies indicated that the SELP liquid embolic delivered doxorubicin and sorafenib, either alone or in combination, at therapeutically relevant concentrations for a minimum of 14 and 30 days, respectively.

Polymeric materials for embolic and chemoembolic applications.

Percutaneous transcatheter embolization procedures involve the selective occlusion of blood vessels. Occlusive agents, referred to as embolics, vary in material characteristics including chemical composition, mechanical properties, and the ability to concurrently deliver drugs. Commercially available polymeric embolics range from gelatin foam to synthetic polymers such as poly(vinyl alcohol). Current systems under investigation include tunable, bioresorbable microspheres composed of chitosan or poly(ethylene glycol) derivatives, in situ gelling liquid embolics with improved safety profiles, and radiopaque embolics that are trackable in vivo. This article reviews commercially available materials used for embolization as well as polymeric materials that are under investigation.