Raucher, Dražen

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.

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.

AbstractThe 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.