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Biomaterial-based approaches for a combination of radiotherapy and immunotherapy can improve outcomes in metastatic cancer through local delivery of both therapeutic modalities to the primary tumor to control local tumor growth and distant metastases. This study describes an injectable depot for sustained intratumoral (i.t.) delivery of an iodine-131 ( 131 I) radionuclide and a CpG oligodeoxynucleotide immunostimulant, driven by the thermally sensitive phase transition behavior of elastin-like polypeptides (ELPs). We synthesized and characterized an ELP with an oligolysine tail (ELP-K 12 ) that forms an electrostatic complex with CpG for delivery from an ELP depot and evaluated the ability of the complex to enhance local and systemic tumor control as a monotherapy and in combination with 131 I-ELP brachytherapy. I.t delivery of CpG from an ELP-K 12 depot dramatically prolongs i.t. retention to more than 21 days as compared to soluble CpG that is only retained within the tumor for <24 h. ELP-K 12 also enhances CpG delivery by increasing cellular uptake of CpG to generate greater toll-like receptor 9 (TLR9) activation than CpG alone. I.t. treatment with an ELP-K 12 /CpG depot slows primary tumor growth and reduces lung metastases in a poorly immunogenic 4 T1 syngeneic breast cancer model whereas i.t treatment of CpG alone has no significant effect on primary tumor growth or metastases. Notably, a combination of 131 I-ELP brachytherapy and ELP-K 12 /CpG delivered i.t. inhibited 4 T1 tumor growth and strongly decreased the development of lung metastases, leading to a synergistic improvement in mouse survival. These preclinical results demonstrate that injectable ELP depots may provide a useful approach for the delivery of combination radio- and immuno-therapy to treat metastatic disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. We developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates at around 30-31 °C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells, including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastase in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds., (Copyright © 2021. Published by Elsevier B.V.)

As a potent macrolide immunosuppressant, cyclosporine A (CsA) is used to treat multiple autoimmune diseases, including non-autoimmune and autoimmune-mediated dry eye disease, rheumatoid arthritis and psoriasis. Despite its potency, CsA has poor solubility, poor bioavailability, and can cause serious adverse reactions such as nephrotoxicity and neurotoxicity. To overcome these limitations, we invented a new strategy to carry CsA by fusing its cognate human receptor, cyclophilin A (CypA), to a 73 kDa elastin-like polypeptide (ELP) termed A192 using recombinant protein expression. Derived from human tropoelastin, ELPs are characterized by the ability to phase separate above a temperature that is a function of variables including concentration, molecular weight, and hydrophobicity. The resultant fusion protein, termed CA192, which assembles into a dimeric species in solution, effectively binds and solubilizes CsA with a K d of 189 nM, comparable to that of endogenous CypA with a K d of 35.5 nM. The release profile of CsA from CA192 follows a one phase decay model with a half-life of 957.3 h without a burst release stage. Moreover, CA192-CsA inhibited IL-2 expression induced in Jurkat cells through the calcineurin-NFAT signaling pathway with an IC 50 of 1.2 nM, comparable to that of free CsA with an IC 50 of 0.5 nM. The intravenous pharmacokinetics of CA192 followed a two-compartment model with a mean residence time of 7.3 h. Subcutaneous administration revealed a bioavailability of 30% and a mean residence time of 15.9 h. When given subcutaneously for 2 weeks starting at 14 weeks in male non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis used to study Sjögren's syndrome (SS), CA192-CsA (2.5 mg/kg, every other day) significantly (p = 0.014) increased tear production relative to CA192 alone. Moreover, CA192 delivery reduced indications of CsA nephrotoxicity relative to free CsA. CA192 represents a viable new approach to deliver this effective but nephrotoxic agent in a modality that preserves therapeutic efficacy but suppresses drug toxicity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21. First, the ELP acts as a solubility enhancer during E. coli expression, yielding active fusion protein from the soluble cell lysate fraction and eliminating the protein refolding steps that are required for purification of FGF21 from inclusion bodies. Second, the ELP's phase transition behavior is exploited for facile chromatography-free purification of the ELP-FGF21 fusion. Third, the composition and molecular weight of the ELP are designed such that the ELP-FGF21 fusion undergoes a phase transition triggered solely by body heat, resulting in an immiscible viscous phase upon subcutaneous (s.c.) injection and thereby creating an injectable depot. Indeed, a single s.c. injection of ELP-FGF21 affords up to five days of sustained glycemic control in ob/ob mice. The ELP fusion partner massively streamlines production and purification of FGF21, while providing a controlled release method for delivery that reduces the frequency of injection, thereby enhancing the pharmacological properties of FGF21 as a protein drug to treat metabolic disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intratumoral radiation therapy - 'brachytherapy' - is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide (131)I to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 min; second, the high energy β-emissions of (131)I further stabilize the depot by introducing crosslinks within the ELP depot over 24h. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3 M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72h. The (131)I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p=0.001, ANOVA) and enhanced median survival to 27 days over controls., (Published by Elsevier B.V.)

Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry-eye disease that affect the vision of millions worldwide. To overcome this challenge, this manuscript explores a novel strategy to reach the ocular surface via receptor-mediated transcytosis across the lacrimal gland (LG), which produces the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR); furthermore, we recently reported a peptide-based nanoparticle (KSI) that targets CAR on liver cells. This manuscript reports the unexpected finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in lumen formed by LG acinar cells. LG transduction with a myosin Vb tail, which is dominant negative towards transcytosis, inhibits lumenal accumulation. Transcytosis of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following administration of KSI nanoparticles. These findings suggest that it is possible to target nanomaterials to the tears by targeting certain receptors on the LG. This design strategy represents a new opportunity to overcome barriers to ocular delivery., (Copyright © 2014 Elsevier B.V. All rights reserved.)

L4F, an alpha helical peptide inspired by the lipid-binding domain of the ApoA1 protein, has potential applications in the reduction of inflammation involved with cardiovascular disease as well as an antioxidant effect that inhibits liver fibrosis. In addition to its biological activity, amphipathic peptides such as L4F are likely candidates to direct the molecular assembly of peptide nanostructures. Here we describe the stabilization of the amphipathic L4F peptide through fusion to a high molecular weight protein polymer. Comprised of multiple pentameric repeats, elastin-like polypeptides (ELPs) are biodegradable protein polymers inspired from the human gene for tropoelastin. Dynamic light scattering confirmed that the fusion peptide forms nanoparticles with a hydrodynamic radius of approximately 50nm, which is unexpectedly above that observed for the free ELP (~5.1nm). To further investigate their morphology, conventional and cryogenic transmission electron microscopy were used to reveal that they are unilamellar vesicles. On average, these vesicles are 49nm in radius with lamellae 8nm in thickness. To evaluate their therapeutic potential, the L4F nanoparticles were incubated with hepatic stellate cells. Stellate cell activation leads to hepatic fibrosis; furthermore, their activation is suppressed by anti-oxidant activity of ApoA1 mimetic peptides. Consistent with this observation, L4F nanoparticles were found to suppress hepatic stellate cell activation in vitro. To evaluate the in vivo potential for these nanostructures, their plasma pharmacokinetics were evaluated in rats. Despite the assembly of nanostructures, both free L4F and L4F nanoparticles exhibited similar half-lives of approximately 1h in plasma. This is the first study reporting the stabilization of peptide-based vesicles using ApoA1 mimetic peptides fused to a protein polymer; furthermore, this platform for peptide-vesicle assembly may have utility in the design of biodegradable nanostructures., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system., (© 2013 Elsevier B.V. All rights reserved.)

Numerous nanocarriers of small molecules depend on either non-specific physical encapsulation or direct covalent linkage. In contrast, this manuscript explores an alternative encapsulation strategy based on high-specificity avidity between a small molecule drug and its cognate protein target fused to the corona of protein polymer nanoparticles. With the new strategy, the drug associates tightly to the carrier and releases slowly, which may decrease toxicity and promote tumor accumulation via the enhanced permeability and retention effect. To test this hypothesis, the drug Rapamycin (Rapa) was selected for its potent anti-proliferative properties, which give it immunosuppressant and anti-tumor activity. Despite its potency, Rapa has low solubility, low oral bioavailability, and rapid systemic clearance, which make it an excellent candidate for nanoparticulate drug delivery. To explore this approach, genetically engineered diblock copolymers were constructed from elastin-like polypeptides (ELPs) that assemble small (<100nm) nanoparticles. ELPs are protein polymers of the sequence (Val-Pro-Gly-Xaa-Gly)n, where the identity of Xaa and n determine their assembly properties. Initially, a screening assay for model drug encapsulation in ELP nanoparticles was developed, which showed that Rose Bengal and Rapa have high non-specific encapsulation in the core of ELP nanoparticles with a sequence where Xaa=Ile or Phe. While excellent at entrapping these drugs, their release was relatively fast (2.2h half-life) compared to their intended mean residence time in the human body. Having determined that Rapa can be non-specifically entrapped in the core of ELP nanoparticles, FK506 binding protein 12 (FKBP), which is the cognate protein target of Rapa, was genetically fused to the surface of these nanoparticles (FSI) to enhance their avidity towards Rapa. The fusion of FKBP to these nanoparticles slowed the terminal half-life of drug release to 57.8h. To determine if this class of drug carriers has potential applications in vivo, FSI/Rapa was administered to mice carrying a human breast cancer model (MDA-MB-468). Compared to free drug, FSI encapsulation significantly decreased gross toxicity and enhanced the anti-cancer activity. In conclusion, protein polymer nanoparticles decorated with the cognate receptor of a high potency, low solubility drug (Rapa) efficiently improved drug loading capacity and its release. This approach has applications to the delivery of Rapa and its analogs; furthermore, this strategy has broader applications in the encapsulation, targeting, and release of other potent small molecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Radiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold., (© 2013.)

We developed a novel temperature-sensitive liposome, STL composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000), cholesterol, and a fatty acid conjugated elastin-like polypeptide (ELP). The STL had a unilamellar spherical shape with a mean diameter of 160 nm. Doxorubicin (DOX) was encapsulated by the STL using an ammonium sulfate gradient method with a lipid to drug ratio of 1:0.2 (w/w), resulting in 95% loading efficiency. The STL exhibited better stability than conventional low temperature sensitive liposome (LTSL-lysolipid-based temperature sensitive liposomes; DPPC:MSPC:DSPE-PEG-2000=90:10:4) at 37 °C in the presence of serum; there was rapid release of doxorubicin in the range of 39-42 °C (≥95% release at 42 °C within 10s). A confocal microscope revealed that DOX encapsulated in STL (STL-DOX) was taken up much better by cell nuclei at 42 °C than at 37 °C. The difference in cell viability between 37 and 42 °C was 63% relative to STL-DOX and 18% for LTSL-DOX. The pharmacokinetics (PK) and antitumor effect of STL-DOX combined with high-intensity focused ultrasound (HIFU) were studied, and compared with LTSL. An in vivo study demonstrated that STL-DOX is highly stable, with a long circulating property (half life=2.03±0.77 h) in HIFU-untreated mice, and resulted in significant tumor regression for 2 days after intravenous injection of STL-DOX at 5 mg DOX/kg in combination with HIFU. These results are better than conventional LTSL, for which the blood circulation time is short (0.92±0.17 h) and inhibition of tumor growth is weak. These results indicate that the properties of stability at 37 °C and burst release at 42 °C of STL-DOX act synergistically against tumors., (Copyright © 2013 Elsevier B.V. All rights reserved.)