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Leslie R Walker,1 Jung Su Ryu,1 Eddie Perkins,2 Lacey R McNally,3 Drazen Raucher1 1Department of Biochemistry, 2Department of Neurosurgery, University of Mississippi Medical Center, Jackson, MS, USA; 3Division of Hematology and Oncology, University of Louisville, Louisville, KY, USAAbstract: Current therapies for the treatment of pancreatic cancer are limited. The limitations of this type of treatment are abundant. The majority of chemotherapeutic agents used in clinics are highly toxic to both tumor cells and normal tissues due to the lack of specificity. Resistance can develop due to overexposure of these agents. To address these issues, these agents must be made more exclusive toward the tumor site. We have developed a macromolecular carrier based on the sequence of the biopolymer elastin-like polypeptide (ELP) that is able to aggregate upon reaching the externally heated tumor environment. This carrier is specific to the tumor as it only aggregates at the heated tumor site. ELP is soluble below its transition temperature but will aggregate when the temperature is raised above its transition temperature. ELP was modified by p21, a cell cycle inhibitory peptide, and the addition of Bac, a cell-penetrating peptide with nuclear localization capabilities. In this study, p21-ELP-Bac and its control, ELP-p21, were used in cell proliferation studies using the pancreatic cancer cell lines Panc-1, MiaPaca-2, and S2013. ELP-p21 had little effect on proliferation, while the half maximal inhibitory concentration of p21-ELP-Bac was ~30 µM. As translocation across the plasma membrane is a limiting step for delivery of macromolecules, these polypeptides were utilized in a pancreatic xenograft model to study the plasma clearance, biodistribution, tumor accumulation, and tumor reduction capabilities of the polypeptide with and without a cell-penetrating peptide.Keywords: elastin-like polypeptide, peptide, targeted drug delivery, macromolecule

Glioblastoma multiforme (GBM), a grade IV (WHO classification) malignant brain tumor, poses significant challenges in treatment. The current standard treatment involves surgical tumor removal followed by radiation and chemotherapeutic interventions. However, despite these efforts, the median survival for GBM patients remains low. Temozolomide, an alkylating agent capable of crossing the blood-brain barrier, is currently the primary drug for GBM treatment. Its efficacy, however, is limited, leading to the exploration of combination treatments. In this study, we have investigated the synergistic effects of combining temozolomide with doxorubicin, a chemotherapeutic agent widely used against various cancers. Our experiments, conducted on both temozolomide-sensitive (U87) and -resistant cells (GBM43 and GBM6), have demonstrated a synergistic inhibition of brain cancer cells with this combination treatment. Notably, the combination enhanced doxorubicin uptake and induced higher apoptosis in temozolomide-resistant GBM43 cells. The significance of our findings lies in the potential application of this combination treatment, even in cases of temozolomide resistance. Despite doxorubicin's inability to cross the blood-brain barrier, our results open avenues for alternative delivery methods, such as conjugation with carriers like albumin or local administration at the surgical site through a hydrogel application system. Our study suggests that the synergistic interaction between temozolomide and doxorubicin holds promise for enhancing the efficacy of glioblastoma treatment. The positive outcomes observed in our experiments provide confidence in considering this strategy for the benefit of patients with glioblastoma.

Introduction: Injectable hyaluronic acid (HA) fillers are commonly used to provide tissue augmentation and combat the effects of facial aging. Ovine and human recombinant formulations of the enzyme hyaluronidase (HAse) are used interchangeably; however, it is unknown if there exists a difference in their ability to degrade HA. Objective: To compare rates at which ovine and human recombinant forms of HAse degrade various HA fillers in vitro. Methods: Increasing amounts of either ovine or human recombinant HAse were added to fixed amounts of nine unique HA filler products. Degradation rates were then analyzed using a colorimetric method by measuring absorbance levels of degraded product. Results: Human recombinant HAse degraded more HA when compared with ovine HAse overall ( p  = 0.014, confidence interval [-0.015 to -0.0018]). Conclusions: Human recombinant HAse was found to be more effective on average in degrading HA fillers when compared with ovine HAse in vitro.

Glioblastoma is a highly aggressive disease with poor patient outcomes despite current treatment options, which consist of surgery, radiation, and chemotherapy. However, these strategies present challenges such as resistance development, damage to healthy tissue, and complications due to the blood-brain barrier. There is therefore a critical need for new treatment modalities that can selectively target tumor cells, minimize resistance development, and improve patient survival. Temozolomide is the current standard chemotherapeutic agent for glioblastoma, yet its use is hindered by drug resistance and severe side effects. Combination therapy using multiple drugs acting synergistically to kill cancer cells and with multiple targets can provide increased efficacy at lower drug concentrations and reduce side effects. In our previous work, we designed a therapeutic peptide (Bac-ELP1-H1) targeting the c-myc oncogene and demonstrated its ability to reduce tumor size, delay neurological deficits, and improve survival in a rat glioblastoma model. In this study, we expanded our research to the U87 glioblastoma cell line and investigated the efficacy of Bac-ELP1-H1/hyperthermia treatment, as well as the combination treatment of temozolomide and Bac-ELP1-H1, in suppressing tumor growth and extending survival in athymic mice. Our experiments revealed that the combination treatment of Bac-ELP1-H1 and temozolomide acted synergistically to enhance survival in mice and was more effective in reducing tumor progression than the single components. Additionally, our study demonstrated the effectiveness of hyperthermia in facilitating the accumulation of the Bac-ELP1-H1 protein at the tumor site. Our findings suggest that the combination of targeted c-myc inhibitory biopolymer with systemic temozolomide therapy may represent a promising alternative treatment option for glioblastoma patients.

Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP-collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection., Competing Interests: D.R. is the CEO of Thermally Targeted Technology Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The anticancer agent doxorubicin(dox) has been widely used in the treatment of a variety of hematological malignancies and solid tumors. Despite doxorubicin's efficiency in killing tumor cells, severe damage to healthy tissues, along with cardiotoxicity, limits its clinical use. To overcome these adverse side effects, improve patient safety, and enhance therapeutic efficacy, we have designed a thermally responsive biopolymer doxorubicin carrier that can be specifically targeted to tumor tissue by locally applying mild hyperthermia (41 °C). The developed drug vehicle is composed of the following: a cell penetrating peptide (SynB1) to promote tumor and cellular uptake; thermally responsive Elastin-like polypeptide (ELP); and the (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH) containing a pH-sensitive hydrazone linker that releases doxorubicin in the acidic tumor environment. We used the in vivo imaging system, IVIS, to determine biodistribution of doxorubicin-delivered ELP in MDA-MB-231 xenografts in nude mice. Tumor bearing mice were treated with a single IV injection of 10 mg/kg doxorubicin equivalent dose with free doxorubicin, thermally responsive SynB1 ELP 1-DOXO, and a thermally nonresponsive control biopolymer, SynB1 ELP 2-DOXO. Following a 2 h treatment with hyperthermia, tumors showed a 2-fold higher uptake when treated with SynB1 ELP 1-DOXO compared to free doxorubicin. Accumulation of the thermally non-responsive control SynB1 ELP2 -DOXO was comparable to free doxorubicin, indicating that an increase in dox accumulation with ELP is due to aggregation in response to thermal targeting. Higher levels of SynB1 ELP1-DOXO and SynB1 ELP2 -DOXO with respect to free doxorubicin were observed in kidneys. Fluorescence intensity from hearts of animals treated with SynB1 ELP1-DOXO show a 5-fold decrease in accumulation of doxorubicin than the same dose of free doxorubicin. SynB1-ELP1-DOXO biopolymers demonstrated a 6-fold increase in tumor/heart ratio in comparison to free doxorubicin, indicating preferential accumulation of the drug in tumors. These results demonstrate that thermally targeted polymers are a promising therapy to enhance tumor targeting and uptake of anticancer drugs and to minimize free drug toxicity in healthy tissues, representing a great potential for clinical application.

Although doxorubicin (dox), an anthracycline antibiotic, is widely used and effective in treating cancer, its treatment efficiency is limited by low blood plasma solubility, poor pharmacokinetics, and adverse side effects, including irreversible cardiotoxicity. Moreover, cancer cells often develop drug resistance over time, which decreases the efficacy of anti-cancer drugs, including dox. In this study, we examine a macromolecular drug delivery system for its ability to specifically deliver doxorubicin to cancer cells with and without drug resistance. This drug delivery system consists of a multi-part macromolecule, which includes the following: elastin-like polypeptide (ELP), cell penetrating peptide (CPP), a cleavable linker (releasing at low pH), and a derivative of doxorubicin. ELP is thermally responsive and improves drug solubility, while the CPP mediates cellular uptake of macromolecules. We compared cytotoxicity of two doxorubicin derivatives, where one is cleavable (DOXO) and contains a pH-sensitive linker and releases dox in an acidic environment, and the other is non-cleavable (ncDox) doxorubicin. Cytotoxicity, apoptosis, cell cycle distribution and mechanism of action of these constructs were tested and compared between dox-responsive MCF-7 and dox-resistant NCI/ADR cell lines. Dox delivered by the ELP construct is comparably toxic to both sensitive and drug resistant cell lines, compared to unconjugated doxorubicin, and given the pharmacokinetic and targeting benefits conveyed by conjugation to ELP, these biopolymers have potential to overcome dox resistance in vivo.

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

The application of rationally designed therapeutic peptides (TP) may improve outcomes in cancer treatment. These peptides hold the potential to directly target proliferative pathways and stimulate cell arrest or death pathways. Elastin-like polypeptide (ELP) is an elastin derived biopolymer that undergoes a thermally mediated phase transition. This study employs p50, a nuclear localization sequence derived peptide that inhibits the activation of NFκB and is implicated in cancer cell survival and metastasis. In order to effectively delivery p50, it is conjugated to SynB1-ELP1, a thermally responsive macromolecular carrier. By applying an external heat source, mild hyperthermic conditions (41 °C) induce aggregation and therefore can be used to specifically target ELP to solid tumors in cancer therapy. The addition of a cell penetrating peptide (CPP) to the N-terminus of the macromolecular carrier enhances the cellular uptake and directs the subcellular localization of the bioactive peptide. The novel TP, p50, inhibits proliferation and induces apoptosis of breast cancer cells by blocking the intranuclear import of NFκB. By expanding the repertoire of oncogenic targets, CPPs, and ELP carrier sizes, ELP-based polypeptides may be modulated to optimize the delivery of these novel therapies and allow for the flexibility to create individualized cancer therapies., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Electrophilic fluorine-mediated dearomative spirocyclization has been developed to synthesize a range of fluoro-substituted spiro-isoxazoline ethers and lactones. The in vitro biological assays of synthesized compounds were probed for anti-viral activity against human cytomegalovirus (HCMV) and cytotoxicity against glioblastomas (GBM6) and triple negative breast cancer (MDA MB 231). Interestingly, compounds 4d and 4n showed significant activity against HCMV (IC 50 ∼ 10 μM), while 4l and 5f revealed the highest cytotoxicity with IC 50 = 36 to 80 μM. The synthetic efficacy and biological relevance offer an opportunity to further drug-discovery development of fluoro-spiro-isoxazolines as novel anti-viral and anti-cancer agents., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

To increase treatment efficiency for glioblastoma, we have developed a system to selectively deliver chemotherapeutic doxorubicin (Dox) to Glioblastoma (GBM) tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40-41 °C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the blood brain barrier and cell entry, and a 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug had partial nuclear accumulation in addition to cytoplasmic distribution. The intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the cells treated with free Dox, which positively correlates with cytotoxic activity. In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

Peptides are a promising new therapeutic approach for glioblastoma with potential for more effective targeting and fewer devastating side effects compared to conventional cancer therapies. With the specificity to target receptors which are uniquely or overexpressed on cancer cells as well as accurately targeting dysregulated signaling pathways, peptides demonstrate a high potential for the treatment of even the most aggressive cancers. By binding to these targets, peptides can be used to deliver drugs, serve as antagonists to various ligands, or, given some inherent anticancer activity, provide additional treatment options alone or in combination therapy. The highly specific targeting capacity of peptides is critical to achieve effective cancer treatment with limited side effects, and in preclinical studies peptides have shown to have both cell and blood brain barrier penetrating capacity. As tumor targeting peptides move beyond the preclinical setting, identification of additional glioblastoma-specific peptide ligands becomes imperative to expand the potential of this encouraging treatment strategy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The association between glioblastoma (GBM) and human cytomegalovirus (HCMV) infection has been the intensely debated topic over the decades for developing new therapeutic options. In this regard, the peroxides from natural and synthetic sources served as potential antiviral and anticancer agents in the past. Herein, a concise and efficient strategy has been demonstrated to access a novel class of peroxides containing a spiro-isoxazoline to primarily investigate the biological activities. The synthetic compounds were evaluated for in vitro antiviral and antiproliferative activity against HCMV and glioblastoma cell line (GBM6), respectively. While compound 13m showed moderate anti-CMV activity (IC 50 = 19 μM), surprisingly, an independent biological assay for compound 13m revealed its antiproliferative activity against the human glioblastoma cell line (GBM6) with an IC 50 of 10 μM. Hence, the unification of an isoxazoline and peroxide heterocycles could be a potential direction to initiate the HCMV-GBM drug discovery program.

This research describes a thermally responsive elastin-like polypeptide (ELP) for the delivery of dnMAML peptides that inhibit the Notch pathway. Exploiting passive targeting and a thermally active tumor-targeting technique available through the use of ELP, the dnMAML peptide was efficiently delivered to tumor tissue. Furthermore, this ELP-dnMAML was modified with the addition of a cell penetrating peptide (SynB1) for improved infiltration of ELP-dnMAML into the tumor cells. In this study, we verified that intravenously delivered SynB1-ELP-dnMAML was cleared from circulation under physiological conditions (37 °C) but accumulated at tumors grown in mice at sites to which an externally induced, local heat (40-41 °C) was applied, thereby resulting in greatly reduced tumor growth in animals. Additionally, in combination with Taxol, SynB1-ELP-dnMAML showed more potent tumor growth retardation.

Glioblastoma, the most common, aggressive brain tumor, ranks among the least curable cancers-owing to its strong tendency for intracranial dissemination, high proliferation potential, and inherent tumor resistance to radiation and chemotherapy. Current glioblastoma treatment strategies are further hampered by a critical challenge: adverse, non-specific treatment effects in normal tissue combined with the inability of drugs to penetrate the blood brain barrier and reach the tumor microenvironment. Thus, the creation of effective therapies for glioblastoma requires development of targeted drug-delivery systems that increase accumulation of the drug in the tumor tissue while minimizing systemic toxicity in healthy tissues. As demonstrated in various preclinical glioblastoma models, macromolecular drug carriers have the potential to improve delivery of small molecule drugs, therapeutic peptides, proteins, and genes to brain tumors. Currently used macromolecular drug delivery systems, such as liposomes and polymers, passively target solid tumors, including glioblastoma, by capitalizing on abnormalities of the tumor vasculature, its lack of lymphatic drainage, and the enhanced permeation and retention (EPR) effect. In addition to passive targeting, active targeting approaches include the incorporation of various ligands on the surface of macromolecules that bind to cell surface receptors expressed on specific cancer cells. Active targeting approaches also utilize stimulus responsive macromolecules which further improve tumor accumulation by triggering changes in the physical properties of the macromolecular carrier. The stimulus can be an intrinsic property of the tumor tissue, such as low pH, or extrinsic, such as local application of ultrasound or heat. This review article explores current preclinical studies and future perspectives of targeted drug delivery to glioblastoma by macromolecular carrier systems, including polymeric micelles, nanoparticles, and biopolymers. We highlight key aspects of the design of diverse macromolecular drug delivery systems through a review of their preclinical applications in various glioblastoma animal models. We also review the principles and advantages of passive and active targeting based on various macromolecular carriers. Additionally, we discuss the potential disadvantages that may prevent clinical application of these carriers in targeting glioblastoma, as well as approaches to overcoming these obstacles.

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood-brain barrier penetration. The SynB1-ELP1-dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes' expression. SynB1-ELP1-dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1-ELP1-dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.

The majority of anticancer drugs have poor aqueous solubility, produce adverse effects in healthy tissue, and thus impose major limitations on both clinical efficacy and therapeutic safety of cancer chemotherapy. To help circumvent problems associated with solubility, most cancer drugs are now formulated with co-solubilizers. However, these agents often also introduce severe side effects, thereby restricting effective treatment and patient quality of life. A promising approach to addressing problems in anticancer drug solubility and selectivity is their conjugation with polymeric carriers to form polymer-based prodrugs. These polymer-based prodrugs are macromolecular carriers, designed to increase the aqueous solubility of antitumor drugs, can enhance bioavailability. Additionally, polymer-based prodrugs approach exploits unique features of tumor physiology to passively facilitate intratumoral accumulation, and so improve chemodrug pharmacokinetics and pharmacological properties. This review introduces basic concepts of polymer-based prodrugs, provides an overview of currently emerging synthetic, natural, and genetically engineered polymers that now deliver anticancer drugs in preclinical or clinical trials, and highlights their major anticipated applications in anticancer therapies.

Cell-penetrating peptides (CPP) provide an efficient strategy for the intracellular delivery of bioactive molecules in various biomedical applications. This review focuses on recent advances in the use of CPPs to deliver anticancer therapeutics and imaging reagents to cancer cells, along with CPP contributions to novel tumor-targeting techniques. CPPs are now used extensively to deliver a variety of therapeutics, despite lacking cell specificity and having a short duration of action. Resolution of these shortcomings to enable increased cancer cell and/or tumor specificity could improve CPP-based drug delivery strategies, expand combined drug delivery possibilities, and strengthen future clinical applications of these peptides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Despite their poor specificity, small molecule drugs are considered more powerful and effective than other current chemotherapies. A promising method for targeting these anticancer drugs to tumors, elastin-like polypeptides (ELP), has recently emerged. When an anticancer drug that has been conjugated to an ELP is administered, and focal hyperthermia applied, the thermoresponsive properties and enhanced permeability and retention effects of the ELP facilitate drug aggregation within tumor tissues. By incorporating a cell penetrating peptide onto this ELP-chemotherapeutic construct, even greater drug uptake into tumor cells can be achieved., Areas Covered: The review explores the preclinical study progress of ELP-based drug delivery technology and discusses its potential in cancer therapy. Recent experimental work has shown that a delivery construct consisting of an ELP-therapeutic peptide (e.g., the c-Myc-inhibitory peptide, or the p21(WAF1/CIP1)-derived peptide), as well as ELP-small molecule drugs (e.g., doxorubicin, paclitaxel), can be thermally targeted to accumulate in tumors and diminish their growth., Expert Opinion: ELP drug delivery technology is complementary and synergistic to current drug delivery modalities and based on existing hyperthermia technology. By using this technology to achieve chemotherapeutic targeting, efficacy can be improved and side effects reduced in comparison with current regimens, providing treatment alternatives and/or augmenting current therapies for cancer treatment.

A series of structurally diverse 4-bromo spiro-isoxazolines possessing a variety of aromatic and aliphatic substituents at the 3 position, were synthesized through a 1,3-dipolar cycloaddition followed by intramolecular cyclization of a pendant hydroxyl or carboxylic acid group. The biochemical antiproliferative activity was evaluated in vitro by using two breast cancer cell lines (MCF-7 and MDA-MB-231) and two prostate cancer cell lines (PC-3 and DU-145) using the MTT viability assay, and the IC 50 values were obtained. Spiro-isoxazoline derivatives bearing a p -chloro or an o -dichloro aromatic substituent at the 3-position of the isoxazoline showed considerable antitumor activities in all four cell lines with IC 50 value ranging from 43μM to 56μM.

Elastin-like polypeptides (ELP) are thermally responsive polypeptides that are soluble in solutions at 37°C, but which aggregate above 42°C. ELP can be used as effective carrier systems of anticancer molecules, because they can be targeted to tumor sites through the application of local hyperthermia. Since molecular size largely influences how successfully therapeutic agents can cross the vasculatures of tumors, it was crucial to determine an optimal molecular size. In this study, we designed and evaluated three ELP macromolecules with varying molecular weights (43, 63, and 122 kDa), with the goal of determining which would optimize the ELP drug delivery system. The N-terminus of the ELP macromolecule was modified with the cell penetrating peptide Bac to enhance intratumoral and intracellular uptake, and it was also confirmed that each polypeptide had the target transition temperature of 37-42°C and the results of the studies, using tumor-bearing mice, showed that the tumor accumulations increased in the case of all three peptides when local hyperthermia was applied, but that the elimination patterns from these tumors varied according to peptide size. Local hyperthermia was found to produce prolonged retention of all ELP conjugates in tumors except Bac-ELP43. In addition, the pharmacokinetic analysis showed that two larger polypeptides with 63 and 122 kDa have increased AUC in comparison with the 43 kDa polypeptide. These results suggest that, when combined with local hyperthermia, the larger ELP conjugates (63 and 122 kDa) have advantages over the smaller Bac-ELP43 polypeptide in terms of enhanced permeability and higher retention effects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Spinocerebellar ataxia 1 (SCA1) results from pathologic glutamine expansion in the ataxin-1 protein (ATXN1). This misfolded ATXN1 causes severe Purkinje cell (PC) loss and cerebellar ataxia in both humans and mice with the SCA1 disease. The molecular chaperone heat-shock proteins (HSPs) are known to modulate polyglutamine protein aggregation and are neuroprotective. Since HSPs are induced under stress, we explored the effects of focused laser light induced hyperthermia (HT) on HSP-mediated protection against ATXN1 toxicity. We first tested the effects of HT in a cell culture model and found that HT induced Hsp70 and increased its localization to nuclear inclusions in HeLa cells expressing GFP-ATXN1[82Q]. HT treatment decreased ATXN1 aggregation by making GFP-ATXN1[82Q] inclusions smaller and more numerous compared to non-treated cells. Further, we tested our HT approach in vivo using a transgenic (Tg) mouse model of SCA1. We found that our laser method increased cerebellar temperature from 38 to 40 °C without causing any neuronal damage or inflammatory response. Interestingly, mild cerebellar HT stimulated the production of Hsp70 to a significant level. Furthermore, multiple exposure of focused cerebellar laser light induced HT to heterozygous SCA1 transgenic (Tg) mice significantly suppressed the SCA1 phenotype as compared to sham-treated control animals. Moreover, in treated SCA1 Tg mice, the levels of PC calcium signaling/buffering protein calbindin-D28k markedly increased followed by a reduction in PC neurodegenerative morphology. Taken together, our data suggest that laser light induced HT is a novel non-invasive approach to treat SCA1 and maybe other polyglutamine disorders.

Spinocerebellar ataxia-1 (SCA1) is a neurodegenerative disease that primarily targets Purkinje cells (PCs) of the cerebellum. The exact mechanism of PC degeneration is unknown, however, it is widely believed that mutant ataxin-1 becomes toxic because of the phosphorylation of its serine 776 (S776) residue by cAMP-dependent protein kinase A (PKA). Therefore, to directly modulate mutant ATXN1 S776 phosphorylation and aggregation, we designed a therapeutic polypeptide to inhibit PKA. This polypeptide comprised of a thermally responsive elastin-like peptide (ELP) carrier, which increases peptide half-life, a PKA inhibitory peptide (PKI), and a cell-penetrating peptide (Synb1). We observed that our therapeutic polypeptide, Synb1-ELP-PKI, inhibited PKA activity at concentrations similar to the PKI peptide. Additionally, Synb1-ELP-PKI significantly suppressed mutant ATXN1 S776 phosphorylation and intranuclear inclusion formation in cell culture. Further, Synb1-ELP-PKI treatment improved SCA1 PC morphology in cerebellar slice cultures. Furthermore, the Synb1-ELP peptide carrier crossed the blood-brain barrier and localized to the cerebellum via the i.p. or intranasal route. Here, we show the intranasal delivery of ELP-based peptides to the brain as a novel delivery strategy. We also demonstrate that our therapeutic polypeptide has a great potential to target the neurotoxic S776 phosphorylation pathway in the SCA1 disease., (© 2014 International Society for Neurochemistry.)

This work describes the effects of elastin-like polypeptide (ELP) with the p21(Waf1/Cip1)-derived cell cycle inhibitory peptide (p21) on pancreatic tumor cells with gemcitabine. The thermo-responsive property of ELP permits use of a mild, local hyperthermia to target tumors for the transport of chemotherapeutics. In this study, a p21-ELP construct with Bac cell penetrating peptide was designed, and its anticancer activities in pancreatic cancer cell lines was examined. In combination with gemcitabine, the peptide demonstrated enhanced in vitro cytotoxicity as well as tumor growth inhibition in an animal model. Our data suggest that this ELP construct, with gemcitabine, may improve pancreatic cancer therapy., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Therapeutic peptides offer important cancer treatment approaches. Designed to inhibit oncogenes and other oncoproteins, early therapeutic peptides applications were hampered by pharmacokinetic properties now addressed through tumor targeting strategies. Active targeting with environmentally responsive biopolymers or macromolecules enhances therapeutics accumulation at tumor sites; passive targeting with macromolecules, or liposomes, exploits angiogenesis and poor lymphatic drainage to preferentially accumulate therapeutics within tumors. Genetically engineered, thermally-responsive, elastin-like polypeptides use both strategies and cell-penetrating peptides to further intratumoral cell uptake. This review describes the development and application of cell-penetrating peptide-elastin-like polypeptide therapeutics for the thermally targeted delivery of therapeutic peptides.

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II β-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

Prostate cancer remains one of the most common malignancies in men. Besides surgical resection, treatments for prostate cancer include hormone therapy, chemotherapy, and radiation therapy. Advancement of prostate cancer to an androgen-independent state limits the potential of conventional therapeutic approaches. Bortezomib, an FDA-approved proteosomal inhibitor for the treatment of myeloid leukemia, has been shown to have a positive effect on the inhibition of prostate cancer growth. Unfortunately, bortezomib has a very narrow therapeutic window, which can lead to severe side effects. Elastin-like polypeptide (ELP) is a genetically engineered, thermally responsive macromolecular carrier that enables a targeted delivery of the bound molecule because of its soluble property under normal physiologic conditions. In addition, ELP aggregates in response to mild hyperthermia. Using ELP as a carrier, it is possible to improve the pharmacological properties of the therapeutic drug as well as reduce toxicity in normal tissues. In this work, we have investigated the combination treatment of androgen-independent prostate cancer cells with bortezomib and the C-terminal part of the p21(WAF1/CIP1) protein bound to the ELP carrier. We have found that combination treatment with bortezomib and ELP-bound p21(WAF1/CIP1) protein leads to increased cell cycle arrest as well as apoptosis with respect to single treatments. We believe that this approach represents a promising direction for the treatment of androgen-independent prostate cancer.

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Treatment of glioblastoma is complicated by the tumors' high resistance to chemotherapy, poor penetration of drugs across the blood brain barrier, and damaging effects of chemotherapy and radiation to normal neural tissue. To overcome these limitations, a thermally responsive polypeptide was developed for targeted delivery of therapeutic peptides to brain tumors using focused hyperthermia. The peptide carrier is based on elastin-like polypeptide (ELP), which is a thermally responsive biopolymer that forms aggregates above a characteristic transition temperature. ELP was modified with cell penetrating peptides (CPPs) to enhance delivery to brain tumors and mediate uptake across the tumor cells' plasma membranes and with a peptide inhibitor of c-Myc (H1). In rats with intracerebral gliomas, brain tumor targeting of ELP following systemic administration was enhanced up to 5-fold by the use of CPPs. When the lead CPP-ELP-fused c-Myc inhibitor was combined with focused hyperthermia of the tumors, an additional 3 fold increase in tumor polypeptide levels was observed, and 80% reduction in tumor volume, delayed onset of tumor-associated neurological deficits, and at least doubled median survival time including complete regression in 80% of animals was achieved. This work demonstrates that a c-Myc inhibitory peptide can be effectively delivered to brain tumors.

Elastin-like polypeptide (ELP) is a macromolecular carrier with thermally responsive properties that can passively accumulate in solid tumors and additionally aggregate in tumor tissue when exposed to hyperthermia. In this study, ELP was conjugated to the anticancer drug doxorubicin (DOXO) and three different cell penetrating peptides (CPP) in order to inhibit tumor growth in mice compared to free doxorubicin. Fluorescence microscopy studies in MCF-7 breast carcinoma cells demonstrated that the three different CPP-ELP-DOXO conjugates delivered doxorubicin to the cell nucleus. All CPP-ELP-DOXO conjugates showed cytotoxicity with IC(50) values in the range of 12-30 μM at 42 °C, but the ELP carrier with SynB1 as the cell penetrating peptide had the lowest intrinsic cytotoxicity. Therefore, the antitumor efficacy of SynB1-ELP-DOXO was compared to doxorubicin under hyperthermic conditions. C57BL/6 female mice bearing syngeneic E0771 murine breast tumors were treated with either free doxorubicin or the SynB1-ELP-DOXO conjugate with or without focused hyperthermia on the tumor. Under hyperthermic conditions, tumor inhibition with SynB1-ELP-DOXO was 2-fold higher than under therapy with free doxorubicin at the equivalent dose, and is thus a promising lead candidate for optimizing thermally responsive drug polymer conjugates., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Inducing apoptosis in cancer cells is an effective strategy for cancer therapy. The cationic α-helix forming KLAKLAKKLAKLAK peptide (KLAK) has been known to induce apoptosis by disrupting the mitochondria. In the present study, we have designed a thermally targeted KLAK peptide by genetically engineering the KLAK sequence to the carboxy terminus of the heat responsive biopolymer elastin-like polypeptide (ELP). The cellular internalization of ELP-KLAK was made possible by engineering a cell penetrating peptide sequence (SynB1) to the amino terminus of ELP. The SynB1-ELP1-KLAK fusion polypeptide was cytotoxic against both estrogen receptor positive and negative human breast cancer cell lines. The potency of SynB1-ELP1-KLAK was further enhanced when mild hyperthermia was added to the treatment. In response to hyperthermia, SynB1-ELP1-KLAK selectively triggered apoptosis, which was associated with disruption of the mitochondria. The thermally responsive SynB1-ELP-KLAK polypeptide can have improved tumor targeting by the application of mild hyperthermia. Furthermore, the pharmacokinetic properties of ELP can prevent degradation of KLAK in vivo, and the use of SynB1 can mediate tumor cell uptake, thereby augmenting the effect of KLAK.