Drazen Raucher

Targeted drug delivery by thermally responsive polymers.

This review article summarizes recent results on the development of macromolecular carriers for thermal targeting of therapeutics to solid tumors. This approach employs thermally responsive polymers in conjunction with targeted heating of the tumor. The two thermally responsive polymers that are discussed in this article, poly(N-isopropylacrylamide-co-acrylamide) (poly(NIPAAm)) and an artificial elastin-like polypeptide (ELP), were designed to exhibit a soluble-insoluble lower critical solution transition in response to increased temperature slightly above 37 degrees C. In vivo fluorescent videomicroscopy and radiolabel distribution studies of ELP delivery to human tumors implanted in nude mice demonstrated that hyperthermic targeting of the thermally responsive ELP for 1 h provides a approximately two-fold increase in tumor localization compared to the same polypeptide without hyperthermia. Similar results were also obtained for poly(NIPAAm) though the extent of accumulation was somewhat lesser than observed for the ELP. The endocytotic uptake of a thermally responsive ELP was also observed to be significantly enhanced by the thermally triggered phase transition of the polypeptide in cell culture for three different tumor cell lines. Preliminary cytotoxicity studies of an ELP-doxorubicin conjugate indicate that the ELP-doxorubicin conjugate has near equivalent cytotoxicity as free doxorubicin in a cell culture assay.

Evaluation of an elastin-like polypeptide-doxorubicin conjugate for cancer therapy.

Thermally responsive elastin-like polypeptides (ELPs) were synthesized by recombinant DNA techniques and conjugated to doxorubicin through an acid-labile hydrazone bond to enable release of the drug in the acidic environment of lysosomes. The thermal properties, intracellular localization and cytotoxicity of the conjugate were investigated in this study. The conjugation procedure resulted in a mixed population of free ELP and ELP-doxorubicin (ELP-dox) conjugates that exhibit a broader transition than the parent ELP. A simple centrifugation procedure was developed to purify the ELP-dox conjugate from other reactants and resulted in a sharper thermal transition, similar to the parent ELP. The ELP was endocytosed by squamous cell carcinoma cells (FaDu) and trafficked into lysosomes, as observed by the colocalization of the ELP with a lysosome-specific dye through confocal fluorescence microscopy. Interestingly, both the ELP-dox conjugate and free drug exhibited near equivalent in vitro cytotoxicity, although their subcellular localization was significantly different. The free drug was largely concentrated in the nucleus, while the conjugate was dispersed throughout the cytoplasm with limited nuclear accumulation. These differences are significant because they suggest a different mechanism of cytotoxicity for the conjugate as compared with the free drug.

Thermal Targeting of an Acid-Sensitive Doxorubicin Conjugate of Elastin-like Polypeptide Enhances the Therapeutic Efficacy Compared with the Parent Compound In Vivo

Elastin-like polypeptides (ELP) aggregate in response to mild hyperthermia, but remain soluble under normal physiologic conditions. ELP macromolecules can accumulate in solid tumors because of the enhanced permeability and retention effect. Tumor retention of ELPs can be further enhanced through hyperthermia-induced aggregation of ELPs by local heating of the tumor. We evaluated the therapeutic potential of ELPs in delivering doxorubicin in the E0771 syngeneic mouse breast cancer model. The ELP-Dox conjugate consisted of a cell-penetrating peptide at the N-terminus and the 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensured release of doxorubicin in the lysosomes/endosomes after cellular uptake of the drug conjugate. ELP-Dox dosed at 5 mg doxorubicin equivalent/kg, extended the plasma half-life of doxorubicin to 5.5 hours. In addition, tumor uptake of ELP-Dox increased 2-fold when hyperthermia was applied, and was also enhanced compared to free doxorubicin. Although high levels of doxorubicin were found in the heart of animals treated with free doxorubicin, no detectable levels of doxorubicin were found in ELP-Dox–treated animals, indicating a correlation between tumor targeting and reduction of potential cardiac toxicity by ELP-Dox. At an optimal dose of 12 mg doxorubicin equivalent/kg, ELP-Dox in combination with hyperthermia induced a complete tumor growth inhibition, which was distinctly superior to free drug that only moderately inhibited tumor growth. In summary, our findings show that thermal targeting of ELP increases the potency of doxorubicin underlying the potential of exploiting ELPs to enhance the therapeutic efficacy of conventional anticancer drugs. Mol Cancer Ther; 11(7); 1547–56. ©2012 AACR.

Cell Penetrating Peptides Fused to a Thermally Targeted Biopolymer Drug Carrier Improve the Delivery and Antitumor Efficacy of an Acid-sensitive Doxorubicin Derivative

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.

Application of thermally responsive polypeptides directed against c-Myc transcriptional function for cancer therapy

Elastin-like polypeptides are biopolymers composed of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly. Elastin-like polypeptides are soluble in aqueous solution below their transition temperature, but they hydrophobically collapse and aggregate when the temperature is raised above the transition temperature. Previous studies have suggested that the aggregation of these polypeptides in response to externally applied hyperthermia may be exploited in the use of elastin-like polypeptide for thermally targeted drug delivery. This work shows the application of elastin-like polypeptide as a delivery vehicle for a short peptide that can inhibit the transcriptional function of a specific oncogene. The coding sequence for elastin-like polypeptide was modified by the addition of the membrane translocating sequence penetratin and a peptide derived from helix 1 of the helix-loop-helix region of c-Myc (H1-S6A,F8A), known to inhibit c-Myc transcriptional function. The designed polypeptide (Pen-ELP-H1) was then expressed and purified from Escherichia coli. Cellular uptake of Pen-ELP-H1 is enhanced by both the penetratin sequence and by the hyperthermia-induced phase transition as shown by flow cytometry studies. Using immunofluorescence and reverse transcription-PCR, we show that Pen-ELP-H1 is able to disrupt the nuclear localization of c-Myc and inhibit transcriptional activation by c-Myc. Cell proliferation studies showed that Pen-ELP-H1 inhibits growth of MCF-7 cells. Furthermore, the use of hyperthermia increased the antiproliferative effect of a thermally responsive Pen-ELP-H1 ∼2-fold compared with a nonthermally responsive control polypeptide. These studies show that genetically engineered elastin-like polypeptide carriers may provide a new way to thermally target specific oncogene inhibitors to solid tumors.

A thermally targeted elastin-like polypeptide-doxorubicin conjugate overcomes drug resistance

SummaryThe ability of cancer cells to become simultaneously resistant to different drugs, a trait known as multidrug resistance, remains a major obstacle for successful anticancer therapy. One major mechanism of resistance involves cellular drug efflux by expression of P-glycoprotein (P-gp), a membrane transporter with a wide variety of substrates. Anthracyclines are especially prone to induction of resistance by the P-gp mechanism. P-gp mediated resistance is often confronted by use of P-gp inhibitors, synthesis of novel analogs, or conjugating drugs to macromolecular carriers in order to circumvent the efflux mechanism. In this report, the effect of free and Elastin-like polypeptide (ELP) bound doxorubicin (Dox) on the viability of sensitive (MES-SA and MCF-7) and multidrug resistant (MES-SA/Dx5 and NCI/ADR-RES) human carcinoma cells was studied in vitro. The resistant MES-SA/Dx5 cells demonstrated about 70 times higher resistance to free Dox than the sensitive MES-SA cells, and the NCI/ADR-RES cells were about 30 fold more resistant than the MCF-7 cells. However, the ELP-bound Dox was equally cytotoxic in both sensitive and resistant cell lines. The ELP-bound Dox was shown to accumulate in MES-SA/Dx5 cells, as opposed to free Dox, which was rapidly pumped out by the P-gp transporter. Since ELP is a thermally responsive carrier, the effect of hyperthermia on the cytotoxicity of the ELP-Dox conjugate was investigated. Both cytotoxicity and apoptosis were enhanced by hyperthermia in the Dox resistant cells. The results suggest that ELP-Dox conjugates may provide a means to thermally target solid tumors and to overcome drug resistance in cancer cells.

Cell penetrating elastin-like polypeptides for therapeutic peptide delivery

Current treatment of solid tumors is limited by side effects that result from the non-specific delivery of drugs to the tumor site. Alternative targeted therapeutic approaches for localized tumors would significantly reduce systemic toxicity. Peptide therapeutics are a promising new strategy for targeted cancer therapy because of the ease of peptide design and the specificity of peptides for their intracellular molecular targets. However, the utility of peptides is limited by their poor pharmacokinetic parameters and poor tissue and cellular membrane permeability in vivo. This review article summarizes the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutic peptides (TP), and the use of cell penetrating peptides (CPP) to enhance the intracellular delivery of the ELP-fused TPs. CPP-fused ELPs have been used to deliver a peptide inhibitor of c-Myc function and a peptide mimetic of p21 in several cancer models in vitro, and both polypeptides are currently yielding promising results in in vivo models of breast and brain cancer.

Thermally targeted delivery of a c-Myc inhibitory polypeptide inhibits tumor progression and extends survival in a rat glioma model.

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.

Application of Thermally Responsive Elastin-like Polypeptide Fused to a Lactoferrin-derived Peptide for Treatment of Pancreatic Cancer

A well characterized, peptide derivative of bovine lactoferrin, L12, has been shown to possess anticancer properties in multiple cell lines. However, adverse side effects in normal tissues and poor plasma kinetics that hinder the clinical effectiveness of current chemotherapeutics also deter the potential for effective delivery of this L12 peptide. To overcome these limitations, we have developed an Elastin-like polypeptide (ELP) carrier that has the potential to thermally target therapeutic peptides and chemotherapeutics to a tumor site. The coding sequence of ELP was modified with the L12 peptide at the C-terminus and a membrane transduction domain derived from the HIV-1 Tat protein at the N-terminus (Tat-ELP-L12). The thermally responsive Tat-ELP1-L12 is soluble in aqueous solutions at 37°C but aggregates near 41°C, which makes Tat-ELP1-L12 ideal for targeting to solid tumors on application of focused hyperthermia. We observed that under hyperthermia conditions at 42°C, Tat-ELP1-L12 mediated cytotoxicity in MIA PaCa-2 pancreatic adenocarcinoma cells was enhanced by nearly thirty-fold. We investigated the mechanisms of cell death and found evidence of mitochondrial membrane depolarization and caspase activation, which are characteristic of apoptosis, as well as, increased membrane permeability, as shown by LDH release. These results suggest that Tat-ELP1-L12 possesses cytotoxic properties to cancer cells in vitro and may have the potential to provide an effective vehicle to thermally target solid tumors.

Cell-penetrating peptides: strategies for anticancer treatment

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.