Silk-elastinlike protein polymers (SELP's) are block copolymers of silk-like and elastin-like tandem repeats. With appropriate sequence and composition SELPs can be liquid at room temperature and form hydrogels at body temperature. The influence of polymer structure and concentration on biodegradation in vitro and controlled delivery of adenoviruses carrying reporter genes to head and neck tumors in vivo was evaluated using hydrogels made from three SELP analogs. SELP-815K, with eight silk and fifteen elastin units and a lysine (K) modified elastin, was compared to SELP-415K and SELP-47K. Hydrogels with higher silk content and concentration degraded at a slower rate compared to other analogs. Intratumoral injection of adenoviruses with SELPs enhanced gene expression in tumor tissue up to 10 fold compared to viral injection without polymer. Viruses delivered with SELP-815K at 4 wt% polymer concentration showed the highest level of gene expression. Tumor to liver ratio of expression was up to 55 fold higher for SELP-mediated systems. This study demonstrates the influence of exquisite control over polymer structure using recombinant techniques on spatial and temporal control over adenoviral delivery and establishes the utility of SELP matrices as biodegradable systems for gene therapy of head and neck cancer.
Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase 1 trial employing repeated locoregional B7-H3CARs to children with recurrent/refractory CNS tumors and DIPG. Here, we report results of the first 3 evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3CARs. Targeted mass spectrometry of CSF biospecimens revealed modulation in B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3CAR dosing, and that intracranial delivery may induce local immune activation.
Phagocytes are important players in host exposure to nanomaterials. Macrophages in particular are believed to be among the “first responders” and primary cell types that uptake and process nanoparticles, mediating host biological responses by subsequent interactions with inflammatory signaling pathways and immune cells. However, variations in local microenvironmental cues can significantly change the functional and phenotype of these cells, impacting nanoparticle uptake and overall physiological response. Herein we focus on describing the response of specific RAW 264.7 macrophage phenotypes (M1, INF-gamma/LPS induced and M2, IL-4 induced) to Stöber silica nanoparticle exposure in vitro and how this response might correlate with macrophage response to nanoparticles in vivo. It was observed that variations in macrophage phenotype produce significant differences in macrophage morphology, silica nanoparticle uptake and toxicity. High uptake was observed in M1, versus low uptake in M2 cells. M2 cells also displayed more susceptibility to concentration dependent proliferative effects, suggesting potential M1 involvement in in vivo uptake. Nanoparticles accumulated within liver and spleen tissues, with high association with macrophages within these tissues and an overall Th1 response in vivo. Both in vitro and in vivo studies are consistent in demonstrating that silica nanoparticles exhibit high macrophage sequestration, particularly those with Th1/M1 phenotype and in clearance organs. This sequestration and phenotypic response should be a primary consideration when designing new Stöber silica nanoparticle systems, as it might affect the overall efficacy.
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.
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