Matrix-Mediated Delivery of Silver Nanoparticles for Prevention of Staphylococcus aureus and Pseudomonas aeruginosa Biofilm Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a chronic health condition affecting the sinonasal cavity. CRS-associated mucosal inflammation leads to sinonasal epithelial cell death and epithelial cell barrier disruption, which may result in recurrent bacterial infections and biofilm formation. For patients who fail medical management and elect endoscopic sinus surgery for disease control, bacterial biofilm formation is particularly detrimental, as it reduces the efficacy of surgical intervention. Effective treatments that prevent biofilm formation in post-operative patients in CRS are currently limited. To address this unmet need, we report the controlled release of silver nanoparticles (AgNps) with silk-elastinlike protein-based polymers (SELPs) to prevent bacterial biofilm formation in CRS. This polymeric network is liquid at room temperature and forms a hydrogel at body temperature, and is hence, capable of conforming to the sinonasal cavity upon administration. SELP hydrogels demonstrated sustained AgNp and silver ion release for the studied period of three days, potent in vitro antibacterial activity against Pseudomonas aeruginosa (**** p < 0.0001) and Staphylococcus aureus (**** p < 0.0001), two of the most commonly virulent bacterial strains observed in patients with post-operative CRS, and high cytocompatibility with human nasal epithelial cells. Antibacterial controlled release platform shows promise for treating patients suffering from prolonged sinonasal cavity infections due to biofilms.

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.

Liquid-cell transmission electron microscopy for imaging of thermosensitive recombinant polymers.

Various polymers used in controlled release applications exhibit solution-based thermal responses. Unfortunately, very few characterization and imaging techniques permit resolution of individual polymers during their thermally-triggered phase transitions. Here, we demonstrate the use of temperature-ramp liquid-cell transmission electron microscopy (LCTEM) for real-time evaluation of the solution and interfacial behavior of elastinlike polypeptides (ELPs) and their self-assembled nanostructures over a temperature range incorporating their intrinsic lower critical solution temperatures (LCSTs). Individual polymers and supramolecular assemblies were discriminated dependent upon solubility states. The recombinant polymers were shown to adsorb to the silicon-nitride chip window from the buffered saline solution and desorb in a temperature-dependent manner. Silk-elastinlike protein block copolymers (SELPs) (composed of repeat peptide motifs of silk and elastin) differed from ELPs in thermal behavior. While both polymers were shown to cluster, only SELPs formed robust amyloid-like fibers upon heating.

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.

Translational Development of a Silk-Elastinlike Protein Polymer Embolic for Transcatheter Arterial Embolization.

Locally blocking blood flow to tumors with embolic materials is the key to transcatheter arterial embolization for treating hepatocellular carcinoma. Current microparticle agents do not deeply penetrate target tissues and are compatible with a very limited selection of therapeutic agents. Silk-elastinlike protein polymers (SELPs) combine the solubility of elastin and the strength of silk to create an easily injected liquid embolic that transition into a solid depot amenable to loading with drugs, gene therapy agents, or biologics. SELP, injected as liquid solution, penetrates the vasculature before transitioning to a solid hydrogel. The objective of this manuscript is to evaluate SELP embolization, stability, and biocompatibility at 7-, 30-, and 90-day survival intervals in a porcine model. SELP embolics selectively block blood flow in the kidneys and livers, with no off-target infarctions. As assessed with angiography, SELP renal embolization exhibits decreasing persistence for the duration of the 90-day study period. There is an increased presence of microscopic SELP emboli in the renal setting, compared to Embosphere. Histologically scored inflammatory reactions to SELP are decreased in both the renal and hepatic implantations compared to Embosphere. In conclusion, a bioresorbable SELP liquid embolic system deeply penetrates target tissue and selectively embolizes blood vessels in vivo.

A dual-functional Embolization-Visualization System for Fluorescence image-guided Tumor Resection.

Rationale: Intraoperative bleeding impairs physicians' ability to visualize the surgical field, leading to increased risk of surgical complications and reduced outcomes. Bleeding is particularly challenging during endoscopic-assisted surgical resection of hypervascular tumors in the head and neck. A tool that controls bleeding while marking tumor margins has the potential to improve gross tumor resection, reduce surgical morbidity, decrease blood loss, shorten procedure time, prevent damage to surrounding tissues, and limit postoperative pain. Herein, we develop and characterize a new system that combines pre-surgical embolization with improved visualization for endoscopic fluorescence image-guided tumor resection. Methods: Silk-elastinlike protein (SELP) polymers were employed as liquid embolic vehicles for delivery of a clinically used near-infrared dye, indocyanine green (ICG). The biophysical properties of SELP, including gelation kinetics, modulus of elasticity, and viscosity, in response to ICG incorporation using rheology, were characterized. ICG release from embolic SELP was modeled in tissue phantoms and via fluorescence imaging. The embolic capability of the SELP-ICG system was then tested in a microfluidic model of tumor vasculature. Lastly, the cytotoxicity of the SELP-ICG system in L-929 fibroblasts and human umbilical vein endothelial cells (HUVEC) was assessed. Results: ICG incorporation into SELP accelerated gelation and increased its modulus of elasticity. The SELP embolic system released 83 ± 8% of the total ICG within 24 hours, matching clinical practice for pre-surgical embolization procedures. Adding ICG to SELP did not reduce injectability, but did improve the gelation kinetics. After simulated embolization, ICG released from SELP in tissue phantoms diffused a sufficient distance to deliver dye throughout a tumor. ICG-loaded SELP was injectable through a clinical 2.3 Fr microcatheter and demonstrated deep penetration into 50-µm microfluidic-simulated blood vessels with durable occlusion. Incorporation of ICG into SELP improved biocompatibility with HUVECs, but had no effect on L-929 cell viability. Principle Conclusions: We report the development and characterization of a new, dual-functional embolization-visualization system for improving fluorescence-imaged endoscopic surgical resection of hypervascular tumors.

Location of stimuli-responsive peptide sequences within silk-elastinlike protein-based polymers affects nanostructure assembly and drug-polymer interactions.

Silk-elastinlike protein polymers (SELPs) self-assemble into nanostructures when designed with appropriate silk-to-elastin ratios. Here, we investigate the effect of insertion of a matrix metalloproteinase-responsive peptide sequence, GPQGIFGQ, into various locations within the SELP backbone on supramolecular self-assembly. Insertion of the hydrophilic, enzyme-degradable sequence into the elastin repeats allows the formation of dilution-stable nanostructures, while insertion into the hydrophobic silk motifs inhibited self-assembly. The SELP assemblies retained their lower critical solution temperature (LCST) thermal response, allowing up to eightfold volumetric changes due to temperature-induced size change. A model hydrophobic drug was incorporated into SELP nanoassemblies utilising a combination of precipitation, incubation and tangential flow filtration. While the nanoconstructs degraded in response to MMP activity, drug release kinetics was independent of MMP concentration. Drug release modelling suggests that release is driven by rates of water penetration into the SELP nanostructures and drug dissolution. In vitro testing revealed that SELP nanoassemblies reduced the immunotoxic and haemolytic side effects of doxorubicin in human blood while maintaining its cytotoxic activity.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome.

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

Matrix Mediated Viral Gene Delivery: A Review.

Polymeric matrices inherently protect viral vectors from pre-existing immune conditions, limit dissemination to off-target sites, and can sustain vector release. Advancing methodologies in development of particulate based vehicles have led to improved encapsulation of viral vectors. Polymeric delivery systems have contributed to increasing cellular transduction, responsive release mechanisms, cellular infiltration, and cellular signaling. Synthetic polymers are easily customizable, and are capable of balancing matrix retention with cellular infiltration. Natural polymers contain inherent biorecognizable motifs adding therapeutic efficacy to the incorporated viral vector. Recombinant polymers use highly conserved motifs to carefully engineer matrices, allowing for precise design including elements of vector retention and responsive release mechanisms. Composite polymer systems provide opportunities to create matrices with unique properties. Carefully designed matrices can control spatiotemporal release patterns that synergize with approaches in regenerative medicine and antitumor therapies.