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

lastin-

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Therapeutic Delivery

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.

The significance of peptide therapy in cancer treatments Since 1982, when the first US FDA-approved peptide therapeutic, insulin, appeared on the market, a gamut of peptide drugs has followed suit. Developed in response to urgent clinical needs, drugs such as exenatide, goserelin and octreotide now provide effective treatments for a variety of conditions, ranging from diabetes to cardiovascular disease and cancer. At the time of this writing, more than 100 peptide drugs have obtained approval for clinical use [1] and generate annual sales of US$13 billion in 2011 [2] . Therapeutic peptide (TP) development came to prominence in biomedical research along with an increased understanding of the decisive role played by peptides in regulating critical physiological and pathological cell function processes. The ease with which finely tuned TPs could be synthesized and modified, in contrast to chemically synthesized small molecule drugs, rendered them yet more attractive [3,4] , as did their amenability to tailored designs for specific targets [3,5–6] . This amenability provided a definitive therapeutic sensitivity and specificity: finely tuned TPs bind selectively to their spe-

10.4155/TDE14.14 © 2014 Future Science Ltd

Jung Su Ryu, Marija Kuna & Drazen Raucher* Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA *Author for correspondence: Tel.: +1 601 984 1510 Fax: +1 601 984 1501 [email protected]

cific target, yielding both high potency and reduced off-target toxicity. Thus, TPs can circumvent two important problems now besetting antibody cancer treatment: obstacles posed by therapeutic agent size; and nonspecific uptake into the reticuloendothelial system, resulting in dose-limiting liver and bone marrow toxicity [6] . Other strategies have been proffered to address these problems. Small molecule drugs, for example, have better pharmacokinetic features than therapeutic peptides – but their specificity is limited as they cannot differentiate between the sequence similarities of multiple proteins within a target’s active site [7] ; thus, only a small fraction of administered doses actually reach their actual target [8] . Many newly developed, highly specific biotechnological drugs, such as protein ligands and antibodies, pose other drawbacks associated with relatively larger size and stronger immune responsiveness [9] . In contrast, the smaller size of TPs prevents serious immune responses, as they are metabolically cleaved and rapidly cleared from the body, lessening accumulation in critical organs and minimizing toxic side effects [10] .

Therapeutic Delivery (2014) 5(4), 429–445

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Key Terms Therapeutic peptide: Class of peptides that are capable of eliciting a therapeutic response by modulation of targets within cells. Cell-penetrating peptide: Typically composed of 5–30 amino acids are noteworthy for their ability to cross cellular membranes. Enhanced permeability and retention effect: Phenomenon that macromolecules tend to accumulate in the tumor tissues over time due to impaired lymphatic drainage, and sloppy vasculature in tumor. Elastin-like polypeptide: Thermoresponsive biopolymers that undergo reversible-phase transition depending on ambient temperature. Mild hyperthermia: Mild hyperthermia involves raising temperature of focal area in body to approximately 42°C, which can be exploited in tumor-targeted drug delivery.

TP’s inhibition of protein–protein/DNA interactions helps regulate both oncogene and tumor suppressor gene function [11–13] . TP agents also block tumor angiogenesis and signal transductions to prevent tumor-cell proliferation [14,15] . Thus while only a few TPs have reached clinical anticancer trials, many researchers consider them important alternatives for cancer therapy. Nevertheless, treatment approaches that use peptides are currently restricted by their metabolic instability, low bioavailability, poor membrane permeability, and cost [1] . The pharmacokinetic problems in particular, including a short half-life and biodegradability [1] , have posed such hurdles that peptide drug candidate development has been largely blocked for wider markets. A number of strategies have attempted to address these pharmacokinetic challenges. PEG has been conjugated to peptide drugs to prolong the half-life of peptides [16] . Liposomes have also been used to improve peptide pharmacokinetic properties [17] . To protect enzymatic degradation and increase the bioavailability of GLP-1, a liposomal formulation of GLP-1 was prepared resulting in improved pharmacokinetics and insulinotropic action in rats [18] . In experimental models, Woodle et al. prolonged the biological activity of a TP by conjugating liposomes with vasopressin, showing improved anti-uretic action [19] . Finally, Lindqvist et al. demonstrated that liposomes can increase the half-life of an opioid peptide in the blood of rats and deliver it to the brain [20] . In 1988, a new class of peptides with the capacity to improve bioavailability, facilitate membrane permeability, and enhance targeting was introduced through the efforts of Frankel et al. [21] . This group discovered that the HIV-1 viral protein transactivator of transcription could cross cell membranes and efficiently trans-

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activate the viral promoter. The discovery of this and other cell-penetrating peptides (CPPs) greatly facilitated the development of targeted cancer therapeutics. CPPs for the delivery of therapeutic peptides Typically composed of 5–30 amino acids that are most often arranged in positively charged sequences, CPPs are noteworthy for their ability to cross cellular membranes [22] and thus, to improve access to molecular targets. Various CPPs have been utilized to address the hurdles of macromolecule internalization and of delivery to the appropriate intracellular compartment. Although the mechanism of CPP-mediated transport is not yet fully understood, it is generally classified as direct translocation through electrostatic interaction and endocytosis. In direct translocation, heparin sulfate proteoglycan plays a crucial role in ionic interactions between the amino acids of CPPs and the cellular membrane [23] . Endocytosis, the major CPP mechanism for cell entry, involves the formation of endosomes or lysosomes, able to store internalized CPP vesicles. Four manifestations of endocytosis have been identified: macropinocytosis, clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, and clathrin/caveolae-independent endocytosis [24,25] . Energy-dependent macropinocytosis, a primary endocytotic pathway, provides the means by which a CPP can pass through a cell membrane [25,26] . Even when conjugated with large cargoes (molecular weight > 30 KDa), CPPs can bypass low intracellular bioavailability and improve cargo molecule activity. Through this mechanism, CPPs have been reported to deliver such therapeutic cargos as peptides, proteins [27] , nucleic acids [28] , imaging agents [29,30] , and small molecules [31] , leading cargo translocation into such intracellular compartments as mitochondria [32] , the nucleus, and cytoplasm [33] . CPPs can also be designed for specificity to certain types of cells, so as to minimize side effects. For example, Lim et al. developed a buforin IIb, BR2, derivative that targets HeLa, HCT-116, and B16/F10 cancer cells, while sparing normal cells [34] . These researchers found that the CPP cargo-conjugate BR2-scFV could itself induce apoptosis in HCT-116 [34] . Another laboratory showed that a novel CPP sequence, TD2.2, could selectively transduce human glial cells, but not non-glial cell types in brain [35] . This CPP, fused to an EGFP, successfully trafficked to mature oligodendrocytes, demonstrating a potential pathway toward the treatment of glial-related disease (Table 1). In addition, some neuroprotectants were delivered by using this CPP strategy. The Tat-NEMO binding domain was developed to inhibit NF-κB and to reduce brain damage in a cerebral hypoxia-ischemia

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The development of thermally targeted, elastin-like polypeptide cancer therapeutics

model [45] . In other research GDNF was conjugated with Tat to help the blood–brain barrier penetration of GDNF and to diminish brain injury in experimental animal models [46] .

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CPPs have also been used to improve oral bioavailability, along with intracellular bioavailability. For example, poor absorption occurs with oral administration of nanostructured lipid carriers for the delivery

Table 1. Antitumor peptides conjugated with elastin-like polypeptide. Peptide name

Targets

Peptide sequence

CPPs conjugated to peptide sequence

Description of the studies

c-Myc inhibitory c-Myc peptide (H1S6A, F8A, H1)

NELKRAFAALRDQI

Bac, Pen, Tat

[8,36,37,38,39] H1 peptide can [8,36,37] inhibit the c-Myc [37] signaling pathway by interacting with c-Myc. CPP-ELP-H1-S6A induced apoptosis and inhibited proliferation of MCF7 in vitro. BacELP1–H1, targeted to tumors via focused hyperthermia, significantly reduced tumor growth in an orthotopic mouse model of breast cancer (E0771) and in a rat glioma model

p21WAF1/CIP1 p21WAF1/CIP1derived peptide (p21)

GRKRRQTSMTDFYHSKRRLIFSKRKP Bac, Antp

Bac-ELP1-p21 displayed a cytoplasmic and nuclear distribution in the SKOV-3 cells, decreased Rb phosphorylation levels, and induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/Mphase. Its combination with bortezomib led to increased cell-cycle arrest and apoptosis in prostate cancer cells (PC-3, Du-145)

[40]

LactoferrinMitochondrial derived peptide membrane (L12)

GPAWRKAFRWAKRMLKKAA

Under conditions of hyperthermia (42°C), Tat-ELP1-L12 mediated cytotoxicity in MIA PaCa-2. Mitochondrial membrane depolarization, caspase activation, and increased membrane permeability are the mechanisms of apoptosis

[42]

Tat

Ref.

[41]

CPP: Cell-penetrating peptides; ELP: Elastic-like polypeptide.


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Table 1. Antitumor peptides conjugated with elastin-like polypeptide (cont.). Peptide name

Targets

Peptide sequence

CPPs conjugated to peptide sequence

Description of the studies

Ref.

Mitochondria disruption peptide (KLAK)

Mitochondrial membrane

(KLAKLAK) 2

SynB1

SynB1-ELP1-KLAK fusion polypeptide was cytotoxic against both estrogen receptor positive and negative human breast cancer cell lines, and triggered apoptosis associated with disruption of the mitochondria

[43]

Peptide inhibitor of arginine methylation (GRG)

The survival of GRGRGRGRGRGR motor neurons protein (SMN)

SynB1

SynB1–ELP1–GRG was found to localize to the cytoplasm. It inhibited proliferation, and induced apoptosis in HeLa cells in combination with hyperthermia

[44]

CPP: Cell-penetrating peptides; ELP: Elastic-like polypeptide.

of tripterine. However, tripterine’s oral bioavailability dramatically improves when a new CPP, Ste-R6L2, is added to tripterine-loaded nanostructured lipid carriers [47] . In cancer research, many other applications of CPPs for peptide delivery can be found. Harada et al. developed a Tat-oxygen-dependent degradation-caspase-3 fusion protein to sensitize tumors to radio- and chemotherapy [48] . Tat was also used to develop an Akt inhibitor for use in treating T-cell leukemia. For this work, Hiromura et al. showed that Tat-Akt inhibitor could inhibit a human T-cell line, achieving in vitro as well as in vivo tumor growth inhibition [49] . In an experimental model of pancreatic cancer, the Antennapedia carrier sequence coupled with p16 suppressed tumor growth [50] . CPPs are a beneficial peptide in delivering molecules into the cells, but several issues remain to overcome [51] . First, the functions of the CPPs depend on cell type or membrane components [52,53] , suggesting that CPPs may not provide a sufficient master key for the intracellular delivery of macromolecules. Secondly, in vivo applicability of CPPs is still under investigation, with low metabolic stability [54] , low permeability [55] , and CPP interference with the activity of cargo molecules [56] . Passive & active transport of therapeutic peptides The exploitation of the tumor enhanced permeability and retention effect (EPR) to achieve macromolecule

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delivery, pioneered by Matsumura and Maeda in 1986, is a renowned example of passive targeting [57] . Solid tumors possess rather permeable structures, impaired lymphatic drainage, and a sloppy tumor vasculature with poorly aligned endothelial cells. These anomalies result in a leaky and narrow structure, abnormal fluid dynamics, and consequently, a tendency of macromolecules to accumulate in the tumor tissues over time [58] . Matsumura and Maeda showed that passive targeting, based on these features, could achieve an increased tumor concentration of albumin-bound styrenemaleic acid polymer-functionalized neocarzinostatin, a prolonged duration of action, and increased therapeutic efficacy. Based on their findings, such macromolecular carriers as styrene-co-maleic acid anhydride [59] , hydroxypropylmethacrylate [60] , liposomes [61] , micelles, and nanoparticles have been developed [58] . These strategies exploiting the EPR effect are attractive for systemic-drug delivery owing to their tendency to permit a longer circulation time and to accumulate preferentially within tumors, providing a significantly lower systemic toxicity as compared with free drug. Active tumor transport to a targeted site also generally exploits an intrinsic cell characteristic to obtain drug delivery. For example, a drug conjugate may be constructed with tumor monoclonal antibodies or ligands that are appropriately matched to specific tumor-cell antigens or receptors, respectively [62,63] . Environmentally responsive macromolecular drug carriers can be also targeted to tumors by using such



external stimuli as heat, light, ultrasound, and magnetic field. These carriers, including thermosensitive liposomes, release their cargo drugs upon application of an external stimulus. As a result, effective local concentrations of the drug are increased and side effects to surrounding healthy tissues are reduced. Elastin-like polypeptides (ELP; see below) are able by virtue of their size and thermoresponsive properties to exploit the EPR effect. ELPs can thus be targeted to tumors by both active and passive transport mechanisms. These dual capacities convey significant advantages for the delivery of TPs and have rendered the ELP macromolecule an important, emerging tool for cancer drug delivery. ELPs ELPs, a class of biopolymers systematically characterized in the 1980s by Urry and colleagues [64] , have received considerable attention over the past decade owing to increased recognition and exploitation of their capacity to transport therapeutic agents. These polypeptides, produced by using an E. coli hyperexpression protocol [65,66] , are now applied in studies of targeted delivery models and in experimental models of cancer treatment [8] . An ELP polymer consists of five amino acid repeats (pentamers) of the form Val–Pro–Gly–Xaa–Gly, wherein Xaa can be any amino acid except Proline. In tandem with other factors, this amino acid sequence contributes to the characteristic transition of ELPs at an appropriate thermal trigger between soluble and aggregated phases. In solution, the tertiary structure of an ELP is not ordered: for example, neither α-coils nor β-sheets form. However, when an ELP transitions and begins to form aggregates, its structure is likely to change into a dynamic ordered structure, a β-spiral with one β-turn per pentamer [67,68] . By slightly modifying the DNA that encodes a specific amino acid sequence, ELP transition properties can be manipulated. Recursive directional ligation provides a relatively easy tool for manipulating ELP to achieve desired modifications, including increased construct size [69] . Thus, oligonucleotide cassettes, which encode 10–16 pentapeptide repeats, can be sequentially introduced into a vector so that they will be fused both directionally and in frame. By repeating this process several times, a desired ELP length can be obtained. After Urry’s early work [70] , Chilkoti and colleagues explored a number of ELP constructs and methods for their synthesis [65,71] . The focus of their research was thermally targeted delivery of radioisotopes and small molecule drugs, such as doxorubicin [72,73] . A variety of other applications exist for ELPs. They have been used as tools for recombinant-protein puri-


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fication [65] , in tissue engineering [74,75] , and as carriers for pharmaceutical therapeutics [76] . Sarangthem et al. have reported that conjugation of ELPs with the AP1 peptide efficiently targeted IL-4 receptor-expressing MDA-MB-231 cells in a nonthermal-sensitive way [77] . Kojima et al. developed a temperature-sensitive elastinmimetic dendrimer [78] that successfully encapsulated a rose Bengal, which can be released by temperature stimulus. Amruthwar et al. used ELPs as a depot for the release of proteins and antibiotics by taking advantage of their temperature-responsive transition, which allowed high drug loading and gradual release [79,80] . Despite these uses, few publications report the use of ELPs as a thermally targeted drug-delivery carrier for anticancer drugs. Hyperthermia & anticancer therapy The use of ELPs within cancer treatment approaches requires a discussion of ELP thermal properties and therapeutic hyperthermia. Mild hyperthermia involves raising a local, regional, or whole body temperature from its normal physiological range near 37°C to approximately 42°C [81,82] . A typical procedure lasts for a few hours and is well tolerated by patients. Localized hyperthermia is currently achieved through external energy sources, such as ultrasound waves, radiofrequencies, or microwaves [83] . For deeply seated tumors, ultrasonography can be used to help place the antennas or electrodes delivering the heat waves directly within a tumor mass. However, the recent development of high-intensity focused ultrasound, which employs a frequency greater than 20 kHz, now permits a minimal or noninvasive means of creating a high degree of heat at a focal point in a matter of seconds [84] . Currently, clinical high-intensity focused ultrasound procedures are most often performed in conjunction with guidance by MRI, both to localize the heat waves and to monitor the temperature of heated tissues [85] . Hyperthermia preferentially increases tumor-blood flow and vascular permeability compared with those observed in normal tissue. As tumors lack a perfused microvasculature, a hyperthermia-driven increase in tumoral perfusion rates can be used to deliver chemotherapeutic agents to tumor regions [86] . Hyperthermia can further induce perforation of tumor blood vessels and cancer cell membranes to work synergistically with anticancer drugs or radiation treatment [81] . For these reasons, hyperthermia has been implemented in treatments for glioblastoma, head and neck cancer, breast cancer, cancer of the gastrointestinal or urogenital tract, and sarcoma [87] . The use of thermal targeting to deliver a range of novel anticancer drug constructs is under active


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Review Ryu, Kuna & Raucher investigation. Liposome delivery of TPs, for example, though promising, has not yet been adequately researched. Thermosensitive liposomes are a technological innovation that employ lipid components with thermal sensitivity in the physiological temperature range [88] . Upon application of hyperthermia, the lipid membrane undergoes a phase transition and becomes more permeable, thus, releasing a drug that has been loaded inside. In animal models, this approach has been used successfully to deliver such chemotherapeutic drugs as methotrexate [89] and cisplatin [90] to solid tumors. The use of thermosensitive liposomes, however, is limited by the requirement of drug diffusion from the liposome under hyperthermic conditions; they can thus, deliver only small, relatively hydrophobic molecules. Even under hyperthermic conditions, TPs are too large and hydrophilic to escape liposomes. Given these limitations, ELPs currently remain the most promising vehicles for an optimized peptide delivery. Thermally targeted-peptide delivery using ELPs Chilkoti et al. [72,91–92] investigated the feasibility of targeting ELPs to solid tumors by local hyperthermia. Using human tumors implanted in nude mice, they demonstrated that hyperthermia in the tumor resulted in a twofold increase in tumor localization of a thermally responsive ELP, compared with localization without hyperthermia [92] . Over half of the increased accumulation could be attributed to the thermally triggered phase transition and concomitant ELP aggregation. These results suggest that an enhanced delivery of drugs to solid tumors can be achieved by conjugation to thermally responsive polymers, with local heating of tumors. Although tumor localization results for ELPs are promising, their therapeutic efficacy can be demonstrated only in so far as they successfully overcome the unique structural barriers to transport and drug delivery posed by tumor properties. The major impediments posed by solid tumors include the high permeability of tumor vessels and the absence of a functional lymphatic system. This combination results in an elevated interstitial pressure that retards convective transport of high-molecular weight drugs. Additional impediments include the tumor cell plasma membranes, which are generally impermeable to high-molecular weight drug carriers. To address these issues, the ELP coding sequence was modified by adding both a CPP, to promote the polypeptide’s tumoral and cellular uptake, and a TP, to inhibit cancer cell proliferation (Figure 1) . These redesigned polypeptides not only have targeting capacity, but also overcome delivery transport barriers, reach

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molecular sites of action within cancer cells and inhibit cancer cell growth. Thermal targeting of c-Myc inhibitory polypeptides

In 2005, Bidwell et al. reported the first application of an ELP to the delivery of a TP that had been derived from helix 1 of the helix-loop-helix region of c-Myc (H1), known to inhibit c-Myc transcriptional function and breast cancer cell proliferation [8] . In this study, a penetratin (Pen) CPP introduced to the ELP conjugate achieved an improved cellular uptake rate that resulted in increased cytotoxicity. In subsequent work, Pen-ELP-H1 was tested for its use in a combinational therapy with doxorubicin to treat MCF7 breast cancer in vitro [36] . Pretreatment of cancer cells with Pen-ELP-H1 lowered the doxorubicin and etoposide IC50 values in cotreated cells to nearly half their respective single treatment values. Using immunofluorescence, Pen-ELP-H1 was shown to be able to sequester endogenous c-Myc to the cytoplasm. The effect of inhibiting the c-Myc transcriptional function by Pen-ELP-H1 was also confirmed by assaying mRNA levels of the c-Myc controlled genes lactate dehydrogenase-A and ornithine decarboxylase and by using RT-PCR. Proliferation studies showed that Pen-ELP-H1 inhibited growth of MCF-7 cells, and that the use of mild heat increased the antiproliferative effect of the thermally responsive Pen-ELP-H1 by approximately twofold, compared with a thermally nonresponsive control polypeptide. In a later study, Pen CPP was replaced with Bac [93] . This resulted in a primary localization of ELP-H1 to the nucleus and a greater cell cytotoxicity than seen with Pen-ELP-H1 and Tat-ELPH1 in MCF7. The increase was likely due to ELP-H1’s direct ability to prevent c-Myc and Max dimerization in the nucleus [37] . CPP-ELP-p21 cell cycle inhibitory peptide

The effort to deliver an anticancer TP to cancer cells continued with the use of a p21WAF1/CIP1 derived-peptide (p21) [41] , which was demonstrated to mimic the C-terminus of p21WAF1/CIP1, interfere with PCNA function and inhibit cyclin–CDK activity [12,94] . The ELP-p21 peptide, conjugated with a CPP from Antp, further showed greater cytotoxicity against HeLa and SK-OV-3 at 20 μM than the Antp-ELP, ELP-p21 peptide, or those groups treated only with the p21 peptide. This study revealed that the Antp CPP can deliver the ELP-p21 conjugate into cells and effectively inhibit cancer cells proliferation. The p21 peptide has been further conjugated to such CPPs as Bac [40] . When either CPP was attached to



Review

A

CPP

Therapeutic peptide

ELP

Cell penetrating peptide Mediates uptake of large aggregates

Thermal targeting Hydrophobically collapses and aggregates when T>Tt

Inhibition of cell proliferation

B

T

T

TTt

Figure 1. Elasin-like peptide-based drug delivery vector. (A) This delivery system consists of a CPP SynB1 or Bac, which promotes cellular uptake of the polypeptide, a thermally responsive ELP and a drug that inhibits cancer cell proliferation. (B) The ELP remains a soluble monomer when the solution temperature is at or below body temperature. However, when the solution temperature is raised above body temperature (T>Tt), it hydrophobically collapses and forms aggregates. CPP: Cell-penetrating peptide; ELP: Elasin-like peptide.

ELP-p21, the resulting construct showed and increased antiproliferative effects as compared with a thermally unresponsive CPP-ELP2-p21. Importantly, nuclear localized Bac-ELP-p21 produced enhanced inhibition of Rb phosphorylation, along with cell cycle arrests in S and G2/M phase. These results show that this conjugate works in the same manner as does p21WAF1/CIP1 in tumor cells. Other CPP-ELP-antitumor peptides

In 2009, Massodi et al. conjugated a peptide derivative of bovine lactoferrin-based lytic peptide, L12, with ELP [42] . Lactoferrin is known to induce apoptosis in cancer cells [95] ; a shortened version, L12, has been shown to inhibit the growth of MethA, HT-29 and of MT1 cancer cell lines [96] . In the Massodi investigation, the antiproliferative activities against several cancer cell lines of L12 conjugated with ELP and Tat was studied in combination with hyperthermia [42] . Through a fluorescein isothiocyanate-dextran uptake experiment, Tat-ELP-L12 was found to induce enhanced permeability of the cellular mem-


brane, with more cancer cell death observed at 42°C than at 37°C. Moktan et al. conjugated ELP to the cationic α-helix that forms the KLAKLAKKLAKLAK peptide (KLAK) [43] . They then further modified ELPKLAK with the CPP SynB1, derived from antimicrobial peptides called protegrins. The previously reported anticancer activity of KLAK [97] was then investigated to determine whether or not the introduction of ELP and hyperthermia would potentiate KLAK activity. Indeed, a heat-induced aggregation of SynB1-ELP-KLAK resulted in increased cellular uptake compared with a control, characterized by a temperature-insensitive ELP control and no CPP. These results showed that a higher cellular uptake rate may increase KLAK cellular concentrations and induce greater apoptosis in both estrogen-receptor positive and negative human breast cancer cell lines. ELP technology was also applied to the delivery of TPs to cancer cell target splicing machinery components. The rationale for this study was that cancer cells must carry out both transcription and RNA


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Review Ryu, Kuna & Raucher splicing at levels higher than those of normal cells in order to produce the quantity of proteins needed to maintain their high rate of proliferation. Inhibiting these processes should, thus, have a more prominent effect on cancer cells than on normal senescent or slowly dividing cells. The inhibitory peptide symmetric dimethylarginine, which can interfere with the splicing machinery, was attached to ELP to increase its stability and facilitate thermal targeting. The resulting fusion polypeptide inhibited interactions of the survival of the motor neuron complex with the SmB protein, as well as inhibiting the splicing machinery, reducing cell proliferation, and inducing apoptosis in HeLa cells [44] . In vivo studies Results from the studies described above confirm that TPs are effective in cell cultures. However, clinical application requires the in vivo demonstration of these polypeptides’ capacity to be targeted, to overcome delivery-transport barriers, to reach molecular sites of action within cancer cells and to inhibit their growth. Inhibition of breast cancer proliferation in vivo by H1 peptide

The first in vivo application of an ELP modified with the Bac CPP and H1 was performed in a breast cancer animal model [8,98] . Mice bearing orthotopic syngeneic breast tumors were administered an intravenous (iv.) or intraperitoneal (ip.) dose of Bac-ELP-H1, with hyperthermia provided in tandem at the tumor site. The hyperthermia protocol resulted in tumor region polypeptide levels as much as 2.8-fold higher than those seen in controls without hyperthermia. Moreover, treatment with Bac-ELP-H1 and hyperthermia induced a 70% reduction in tumor volume as compared with controls lacking either the H1 inhibitory peptide or concommitant hyperthermia [99,100] . No significant adverse side effects were seen in the animals treated with both Bac-ELP-H1 and hyperthermia. Bidwell et al. replaced the use of water bath immersion, an earlier means of producing hyperthermia [101,102] , with an infrared LED device that shields areas surrounding the tumor from illumination, but prompts a high degree of infrared (950 nm) light absorption by tumor tissue. This noninvasive method, applied in a number of preclinical animal cancer models, obtained increases in tumor core tissue temperatures up to 42°C. To achieve better ELP accumulation, Bidwell et al. also used a thermal cycling protocol [102] consisting of four 20-min cycles of continuous heating, each followed by a 10-min cooling period (Figure 2) [102]. This method permitted a focused increase to 42°C

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of tumor temperature to enhance ELP aggregation, while sparing healthy tissue from heat damage [102] . In these experiments, two constructs with different thermal properties, Bac-ELP1-H1 and Bac-ELP2-H1, were purified as previously reported [8] . The polypeptides, labeled with Alexa Fluor ®750 C5-maleimide (Invitrogen™; CA, USA) as described [8] , were administered by iv. and ip. and their pharmacokinetics, tumor uptake, and biodistribution subsequently examined. The reported half-life of ELP polypeptides not modified by adding a CPP, at 8.37 h [101] , differed notably from those that are modified, with the halflife for Bac-ELP1-H1 at 100 min [38] . Comparing the iv. and ip. routes of administration used in this model revealed the same level of measured fluorescence. In addition, microscopic analysis and in vivo fluorescence imaging revealed ELP polypeptides in tumor tissue; the highest levels were seen at 6 h after iv. administration, with a threefold increase of Bac-ELP1-H1 levels in tumor tissue after injection and hyperthermia relative to nonhyperthermia controls. The polypeptide was cleared from the tumors within 24 h of administration. These results support the capacity of ELPs to help deliver TPs, which inhibit tumor progression, as well as the ability of hyperthermia to further facilitate intratumor delivery. The blood–brain barrier: a further obstacle to the delivery of therapeutics to solid tumors

With peptide delivery shown to be a successful therapeutic method for solid tumors, attention has shifted to a further obstacle: the blood–brain barrier (BBB). This barrier, a direct result of a layer of endothelial cells connected by tight junctions [103] , restricts exchange between the peripheral circulation and the CNS. Several methods have attempted to overcome the BBB either by changing the barrier integrity [104] or by modifying drug characteristics [105] . Recently, Hearst et al. explored the possibility of delivering treatment across the BBB by using ELP as a delivery system [106] . Their research focused on treatment options for spinocerebellar ataxia type 1 (SCA1), neurodegenerative disorder caused by a CAG repeat mutation in the ATXN1 gene on chromosome 6p23, inherited as an autosomal-dominant trait. The illness is manifest as a progressive ataxia resulting from the loss of cerebellar Purkinje cells (PCs) and neurons in the brainstem [107–110] . Bergmann glial protein S100B has been identified in cytoplasmic PC vacuoles that develop in association with abnormal PC morphology in SCA1 patients [111–113] and its increased levels are thought to be a causative factor for neurodegeneration [114–117] . The S100B protein, part of the EF-hand



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45 43

Temperature (°C)

41 39 37

Tumor temperature

35

Body temperature

33

Skin temperature

31 29 27 25

0

20

40

60

80

100

120

Time (min) Figure 2. Heating tumors with infrared light. Tumor temperature (as monitored by a needle thermocouple in the tumor core), body temperature and skin temperature over the heated site were recorded while illuminating the tumor with a 950 nm light from an LED light source. Data represent the mean of three mice bearing 250 mm3 E0771 mammary tumors, the bars show the SD. Reproduced with permission from [38] .

family of calcium binding proteins, functions as a signaling molecule that gives neutrophic or neurotoxic signals to neighboring neurons [115,118–128] . The SynB1 peptide, derived from naturally occurring protegrin peptides, can assist transport across the BBB, the most critical limitation to the delivery of therapeutics to the brain [129,130] . Recently, Hearst et al. developed a construct using SynB1 as the CPP, an ELP and, on the C-terminus, the inhibitory peptide TRTK12, with its high binding affinity for S100B that can block S100B interactions with its target proteins [130–136] . The group’s most notable discovery was in demonstrating that ELP delivery through the BBB is possible. A control polypeptide labeled with Alexa Fluor ®750 C5-maleimide was administered to the in vivo model, heat applied to the cerebellum, and polypeptide concentration measured by fluorescence level [106] . As Figure 3 clearly shows, animals whose treatment included hyperthermia show higher fluorescence levels compared with the control and unheated animals. This promising study demonstrates that ELPs can be used to overcome the BBB and may provide a delivery system for the treatment of brain tumors. Delivery of a H1 inhibitory peptide to brain tumor in an in vivo model

Glioblastoma multiforme is the most common and aggressive form of malignant brain tumor [136] . Glioblastoma multiforme is highly resistant to che-


motherapeutics, but the greatest obstacle to its successful treatment remains the BBB [137,138] . The task of delivering effective therapeutics across the BBB has proved extremely difficult [139] . To address these problems, the breast cancer inhibitor of CPP-ELP-H1 combined with heat were expanded to a brain cancer animal model [8,37–38] . A rat glioma model, generated by intracranial implantation of C6 glioma cells, which share histological features of human gliomas and their aggressive nature [140] , was used to assess the potential of ELP therapeutics delivery to brain tumors. Moreover, as C6 [141] cells express c-Myc in culture and in orthotopic xenografts [141,142] , they provide a suitable model for examining peptide inhibitory effects against c-Myc/ Max dimerization. To increase the efficiency of BBB and tumor penetration, an amphipathic CPP derived from protegrins, SynB1 [129] , and a basic CPP derived from bovine neutrophils, Bac [143] , were fused to ELP constructs. Both CPPs were analyzed, with SynB1 targeting the ELP polypeptide to the cell cytoplasm and Bac targeting it to the cell nucleus [37] , to determine any increase in efficiency for reaching respective molecular targets and inhibiting cancer cell proliferation. Pharmacokinetics and biodistribution studies after systemic administration of the two CPP-ELPs showed that ELP brain-tumor targeting could be enhanced up to fivefold by the CPPs. Figure 4 compares the results


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Control

Unheated

Heated Epi-fluorescence

Photo

5.0

4.0

Fluorescence

3.0

2.0

Overlay

1.0

Radiant efficiency x 107

4.0 mm

Figure 3. Scanned in vivo imaging systems image displaying a photograph of the brains and the fluorescent uptake of fluorescent labeled Synb1-ELP-GGC taken from a control animal, an unheated animal and an animal where the cerebellum was heated by thermal cycling [106] .

obtained for hyperthermia in combination with BacELP1-H1, Bac-ELP1-H1 without hyperthermia, and Bac-ELP2-H1 (a thermally unresponsive control) with hyperthermia. Bac-ELP1-H1 with hyperthermia showed both an enhanced uptake and the most efficient tumor reduction, at 80%. This treatment also delayed neurological deficits and doubled the survival rate. [39] . This study serves as the first proof of principle for the use of thermally targeted therapeutic in the treatment of solid tumors in the brain. Future perspective The studies reviewed here support the great potential of ELPs for assisting the targeted delivery of TPs. As ELP biopolymers are based on a simple genetic code, they can be easily modified to include both CPPs, to mediate tumor cell uptake of a macromolecular ELP carrier, and

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TPs. Inclusion of the latter in the ELP macromolecular carrier can overcome many limitations, such as peptide susceptibility to degradation and renal clearance. Different targeting approaches permit the site-specific delivery of peptides to tumor tissues so as to yield an increased efficiency of tumor reduction, as well as reduced side effects in healthy tissue. The first targeting feature is passive targeting (the EPR effect), which can result in the preferential accumulation of ELP macromolecular TP carriers within tumor tissue. The second targeting feature is an active one: ELP accumulation can be further enhanced in tumor tissue due to the construct’s thermoresponsive properties. With a careful choice of a molecular target or signaling pathway, one uniquely or preferentially expressed in a particular cancer, TPs delivered by ELP can be designed to act preferentially on cancer cells. Future studies should



focus on expanding molecular targets to pathways apart from those involving oncogenes, cell cycles, and apoptosis; molecular pathways for cancer stem cells may provide a more effective treatment target. Similarly, an ELP can be redesigned to include imaging agents to help in monitoring TP biodistribution and the effectiveness of therapeutic action. MRI contrast imaging agents are of special interest, as the currently employed high-intensity focused ultrasound is usually MRI guided. It is generally accepted that cancer cannot be cured with a single drug therapy. However, combined therapies now provide important treatment opportunities for many cancers. Thermally responsive ELPs can be designed to deliver a number of drugs that have nonoverlapping toxicities, possess a documented single-

Tumor volume (mm3)

A

Review

agent activity, and confer synergistic mechanisms of action. These targeted therapies have the potential to be combined with significantly lower doses than conventional systemic treatment, and may support both more effective treatment and a sparing of the severe toxicity of current therapies. Finally, thermal targeting with ELPs relies on hyperthermia technology currently in clinical use. Thus, currently available methods of clinical hyperthermia can be implemented to augment the novel targeting approaches here described by assisting the selective, intratumoral accumulation of a broad range of anticancer drugs and TPs, sparing healthy tissue, and thereby conserving patient health and function throughout cancer treatment.

3000 2500

Saline

2000

Saline + Hyperthermia

1500

Bac-ELP1 Bac-ELP1-H1

1000

Bac-ELP1-H1+ Hyperthermia

500 0 0

5

10

15

Days after first injection

Tumor volume (mm3)

B

4000 Saline

3000

Bac-ELP1-H1

2000

Bac-ELP2-H1 Bac-ELP2-H1+ Hyperthermia

1000

0 0

5

10

15

Days after first injection Figure 4. Tumor volume reduction by Bac-ELP-H1. E0771 Tumor volume (A & B) after seven daily treatments (arrows) with saline, saline + hyperthermia, (A) Bac-ELP1, Bac-ELP1-H1, Bac-ELP1-H1 + hyperthermia, (B) BacELP2-H1, and Bac-ELP2-H1 + hyperthermia. Data represent the mean ± SD of six mice/group. *Tumor volumes are statistically significant from the control (one-way ANOVA, p = 0.0017, post hoc Bonferroni, 95% CI). Reproduced with permission from [38] .



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Review Ryu, Kuna & Raucher Acknowledgement The authors thank JA Fordham and S Porter for critical reading and editorial assistance of the manuscript.

Financial & competing interests disclosure The authors would like to acknowledge the National Science Foundation (IIP-1321375), National Science Foundation (CBET-

0931041), and iCorp program of National Science Foundation (IIP-1264214). 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. No writing assistance was utilized in the production of this manuscript.

Executive summary Therapeutic peptides in cancer treatment • Therapeutic peptides (TPs) can be designed to specifically inhibit the interaction of oncogenes and other proteins implicated in tumorigenesis and cancer progression. • The challenge to a clinical application of TPs lies in their short half-lives within systemic circulation, due to degradation by proteases and clearance by the reticuloendothelial system. • Pharmacokinetics properties of TPs can be improved by utilizing liposomes and macromolecules as a delivery vehicles.

Elastin-like polypeptides & tumor-targeted drug delivery • Elastin-like polypeptides (ELPs) are a thermally responsive pentamer of the form Val–Pro–Gly–Xaa–Gly, wherein Xaa can be any amino acid except Proline. • These biopolymers are genetically engineered, allowing incorporation of a TP sequence in the ELP carrier by simple molecular biology techniques. • To ensure tumor and cellular uptake, ELPs may be modified with cell-penetrating peptides (CPPs), which mediate tumor and cellular uptake. • Modification of an ELP with CPPs allows targeting of TPs to different tissues and intracellular compartments. • CPP-ELP-drug constructs can overcome the blood–brain barrier.

Applications of ELP system in peptide delivery to tumors • TPs that target oncogenic, cell cycle, and apoptotic pathways have been conjugated with the ELP delivery system and have been shown to result in enhanced cellular uptake rates, increased cancer cell death, and apoptosis. • In animal tumor models, hyperthermia induces the aggregation of ELPs (polypeptides) at their phase transition, permitting the thermal targeting of ELP TP conjugates to solid tumors. • Following systemic administration, an ELP-delivered c-Myc inhibitory peptide targeted to tumors through localized hyperthermia reduced tumor growth in an orthotopic mouse model of breast cancer. • The thermally targeted delivery of a c-Myc inhibitory polypeptide inhibited tumor progression, delayed the onset of tumor-associated neurological deficits and extended survival in a rat glioma model. • The thermally targeted delivery of TPs can be implemented with a clinically available hyperthermia technology. • This promising method for selectively inducing the tumoral accumulation of a broad range of anticancer drugs and TPs has the potential to augment the effectiveness and reduce the toxicity of traditional treatment regimens for cancers.

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Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics.

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Development of synthetic lethality anticancer therapeutics.

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