Jung Su Ryu

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.

Anti-tumor efficacy of a therapeutic peptide based on thermo-responsive elastin-like polypeptide in combination with gemcitabine

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.

Elastin-like polypeptides: the influence of its molecular weight on local hyperthermia-induced tumor accumulation.

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.

Elastin-like polypeptide for improved drug delivery for anticancer therapy: preclinical studies and future applications

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 p21WAF1/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.

Penetrating the cell membrane, thermal targeting and novel anticancer drugs: the development of thermally targeted, elastin-like polypeptide cancer therapeutics

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.

Fusion of cell-penetrating peptides to thermally responsive biopolymer improves tumor accumulation of p21 peptide in a mouse model of pancreatic cancer

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.

Abstract 5129: Cell-penetrating doxorubicin released from Elastin-like polypeptide kills doxorubicin-resistant cancer cells

A drug-releasing system facilitated by external stimuli could be used to deliver cytotoxic chemotherapy agents to tumor sites selectively and safely. Among many suggested strategies, elastin-like polypeptide (ELP) exploits its characteristic of phase transitioning in response to changes in ambient temperature. This unique property permits selective targeting of the polymer to hyperthermic tumors by aggregating as it transitions. ELP therefore can be used as a thermosensitive vector for the delivery of chemotherapy agents and therapeutic peptides, resulting in a rise in drug concentration exclusively in tumors. This novel strategy introduces unprecedented options for treating cancer, with fewer concerns about indiscriminate side effects from the chemotherapy. In this study, the ELP-drug conjugate was further modified with incorporation of an enzyme-specific cleavable linker in order to trigger drug release within tumors. The suggested system is composed of ELP, a matrix metalloproteinase (MMP) substrate, a cell penetrating peptide (CPP), and 6-maleimidocaproyl amide derivative of doxorubicin (Dox). Rationale for this design is that this construct may be initially targeted to the tumor site by local application of mild heat. When the construct reaches tumor site, it is cleaved by MMP, releasing CPP conjugated Dox, which is able to more efficiently infiltrate tumor tissues and penetrate cancer cell membranes. This strategy shows up to 4-fold increase in cell penetration up to four times and results more cell death in breast cancer cells than the ELP-doxorubicin complex. Even in doxorubicin-resistant cancer cells (NCI/ADR and MES/ADR), ELP-released, cell-penetrating doxorubicin demonstrated better membrane penetration (two fold), leading to at least twice killing of the resistant cancer cells than ELP-Dox and free Dox. Citation Format: Jung Su Ryu, Drazen Raucher. Cell-penetrating doxorubicin released from Elastin-like polypeptide kills doxorubicin-resistant cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5129. doi:10.1158/1538-7445.AM2017-5129

Effects of cell penetrating Notch inhibitory peptide conjugated to elastin-like polypeptide on glioblastoma cells

Abstract 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.

Abstract 2168: Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA The anticancer agent doxorubicin is an anthracycline compound that shows high potency in treating cancer, and it is one of the most widely used chemotherapeutics. However, efficiency of doxorubicin treatment is limited by low blood plasma solubility, poor blood pharmacokinetics, and non-selective cell killing that results in serious toxicity to healthy tissues. Additionally, cancer cells often develop drug resistance, which is a significant limiting factor to the drugs effectiveness. Motivated by these problems, we have designed a drug delivery system that can specifically deliver drug to the tumor site while overcoming doxorubicin resistance in breast cancer cell lines. This drug delivery system consists of: ELP - Elastin like polypeptide, CPP - Cell penetrating peptide, a cleavable linker – to enable doxorubicin release in the targeted low pH environment, and a derivative of the anticancer agent Doxorubicin (modified by a 6-maleimidocaproyl moiety for conjugation to a terminal cysteine residue on ELP). ELP is thermally responsive and improves the complexs pharmacokinetics by prolonging its clearance rate while the CPP mediates cellular uptake of large macromolecules. The linker is an acid sensitive amino acid sequence (Gly-Phe-Leu-Gly) that serves as a substrate for lysosomal enzymes. In this study, we compared cytotoxicity of a cleavable (cDox) and non-cleavable (ncDox) doxorubicin derivative delivered by ELP biopolymer in the breast cancer cell lines MCF-7 and MCF7/ADR (doxorubicin resistant cell line). We showed that cDox had two fold higher cytotoxicity than ncDox in both cell lines. When ncDox, however, was conjugated to the ELP biopolymer containing a lysosomally degradable GFLG spacer, the drug delivery construct was equally cytotoxic to both sensitive and resistant cell lines, indicating that the construct delivers doxorubicin into cells by a mechanism that bypasses doxorubicin resistance. Confocal fluorescence microscopy showed that after two hours of exposure to the doxorubicin derivatives, cDox was predominantly localized in the nucleus in both cell lines. However, ncDox was localized in the plasma membrane and cytosol. Intracellular doxorubicin accumulation examined by flow cytometry indicated 3 fold higher uptake of ELP-cDox in sensitive MCF 7 cells compared to resistant MCF7 ADR cells. Cellular uptake of ELP-cDox was further enhanced two fold when conjugated with CPP. In conclusion, our current results indicate that ELP-doxorubicin conjugates may successfully overcome drug resistance in breast cancer cells, providing a promising approach for the use of chemotherapeutic agents such as doxorubicin in patients with drug resistant breast cancers. Citation Format: Sonja Dragojevic, Jung Su Ryu, Felix Kratz, Drazen Raucher. Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2168.

Abstract 2062: The effects of a cell cycle inhibitory peptide fused to elastin-like polypeptide on pancreatic cancer cells with hyperthermia and its combination with gemcitabine.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic cancer is the fourth most common cause of death in United States. Gemcitabine, a pyrimidine nucleoside analogue similar to cytarabine, has been used as a first line treatment for pancreatic cancer since 1997. However, its use has been limited by the side effects such as myelosuppression. So far, many efforts have been made to combine gemcitabine with other drugs, but have failed to prove superior to gemcitabine alone. This work describes the effects of genetically engineered elastin - like polypeptide (ELP) with the p21 Waf1/Cip1 - derived cell cycle inhibitory peptide (p21) on pancreatic tumor cells with or without gemcitabine. ELP is a thermally responsive polypeptide carrier that are soluble in aqueous solutions at physiological temperature (37 °C), but aggregate when the temperature is raised above its transition temperature (Tt, 42 °C). This allows it to be a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. For this study, we designed p21-ELP conjugates modified with the cell penetrating peptide, Bac, on the C-terminus of ELP. The anti-proliferative activity of p21-ELP-Bac was greater in with the presence of hyperthermia, and the combination therapy with gemcitabine showed the synergism on three pancreatic cancer cells - S2013, Panc-1, and Mia PaCa-2 in vitro. The reduced phosphorylated Rb level and the decrease of S- phase population in tumor cells treated with p21-ELP-Bac conjugates proves that the cytotoxicity which is based on cell cycle inhibition. In the S2013 pancreatic xenograft model, p21-ELP-Bac showed significant tumor growth inhibition in the presence of local hyperthermia; and this tumor growth inhibition was further enhanced upon co-treatment with gemcitabine. This combinatorial effect is expected to alleviate the toxicity of gemcitabine by reducing the dose in the clinics. Overall, our data suggest that thermal targeting of ELP-based therapeutic peptides to the pancreatic cancer cells is an effective and promising treatment strategy, especially in combination with gemcitabine. Citation Format: Jung Su Ryu, Ana-Matea Mikecin, Drazen Raucher. The effects of a cell cycle inhibitory peptide fused to elastin-like polypeptide on pancreatic cancer cells with hyperthermia and its combination with gemcitabine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2062. doi:10.1158/1538-7445.AM2013-2062