Cancer is a complex disease and, as it progresses, it can become aggressive, manifested by the invasion of cells from the primary tumor to the liver, lungs, brain and other organs. Tumor cell metastasis is a major cause of death amongst cancer patients, and in order to prevent this process, it is necessary to effectively treat primary solid tumors. However, current treatment for solid tumors is limited by the fact that only a small percentage of the administered dose of drug reaches the tumor site, while the rest of the drug is distributed throughout the body, which leads to increased toxicity in normal tissues.
Tumor tissues differ from normal tissues in anatomical and structural characteristics. They have a heterogeneous distribution of blood vessels and usually lack effective lymphatic drainage. These factors lead to an uneven and slowed blood flow and abnormal fluid dynamics in the tumor tissue. As a result, macromolecules and soluble polymeric carriers penetrate and accumulate preferentially in tumors relative to normal tissues. This phenomenon is called the enhanced permeability and retention (EPR) effect [1
], and it is the key to the clinical success of anti-cancer macromolecular carrier systems. With the goals of increasing specificity and lowering systemic toxicity, many different systems such as macromolecular prodrugs, liposomes, and micro- and nano-particles have been developed (reviewed in [4
]) to treat solid tumors. Several natural and synthetic water-soluble polymers, such as N
-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, dextrans, poly(ethylene glycol) (PEG), and poly(l-glutamic acid) have been utilized successfully in clinical research or are in human clinical trials (reviewed in [3
]). While clinically useful anti-tumor activity has been achieved by exploiting the EPR effect and using passive targeting by macromolecular drug delivery systems, further selectivity is possible by active targeting.
Bioconjugation of targeting moieties, such as peptide sequences or antibodies which have specific affinity to cancer cells, to the polymer backbone can further exploit differences between cancer and normal cells through selective receptor-mediated endocytosis. Another way of active targeting can be achieved using thermally responsive biopolymers, such as elastin-like polypeptide (ELP), that undergo an inverse phase transition [9
]. When intravenously delivered, these thermally responsive polypeptides are likely to be cleared under physiological conditions (37 °C). However, they will aggregate and selectively accumulate in tumors where externally induced focused heat (40-42 °C) is applied. Work in the lab of Chilkoti using human tumors implanted in nude mice has clearly demonstrated that hyperthermia of the tumor results in increased accumulation of ELP polypeptides [12
]. This method combines the established advantages of macromolecular carriers with the additional advantage of active thermal targeting, and it also introduces other synergistic effects of hyperthermia treatment. Hyperthermia preferentially increases the permeability of endothelial tumor vasculature to macromolecular drug carriers, which can further enhance the delivery of drugs to tumors [15
]. Furthermore, hyperthermia enhances the cytotoxicity of some chemotherapeutic agents [18
]. The clinical application of hyperthermia to increase tissue temperatures to 40 - 43 °C has been integrated in multimodal anti-cancer strategies [19
], and due to substantial technical improvements, hyperthermia is becoming more accepted clinically. Selected increase of temperatures in superficial and deep-seated tumors is accomplished using microwave, radio-frequency, and high-intensity focused ultrasound. Therefore, the effect of hyperthermia combined with the therapeutic effect of a polymeric drug carrier might offer further synergistic advantages in treatment of localized tumors.
In order to deliver therapeutic cargo to intracellular molecular targets and demonstrate therapeutic efficacy, the ELP carrier must successfully overcome transport barriers to drug delivery that are posed by unique structural and physiological characteristics of tumors. Despite the beneficial EPR effect, which favors accumulation of macromolecular carriers in tumors, there are many factors opposing the delivery of drugs to tumors. A solid tumor does not simply exist as a mass of malignant cells, but contains tumor cells, normal cells, extracellular matrix, and tumor blood vessels. Leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and other cell types. Tumor and stromal cells produce and assemble the extracellular matrix, which consists of a meshwork of collagens, proteoglycans, and other molecules that together represent significant barriers to penetration by therapeutics agents [22
]. Although the lack of lymphatic drainage reduces efflux of the drug away from the tumor, it also reduces redistribution and transport of the drug within the tumor by generating higher pressure in the interstitium than in the vasculature, which makes macromolecular uptake less efficient [24
]. Furthermore, once macromolecular carriers reach the cancer cell, there is the additional obstacle of plasma membrane impermeability. The plasma membrane of eukaryotic cells is generally impermeable to therapeutic macromolecules such as oligonucleotides and proteins due to the large size and inherently poor penetration capabilities of these molecules. Cell penetrating peptides (CPP) can be used to overcome these transport barriers to drug delivery and permit noninvasive delivery of polypeptides to their appropriate intracellular molecular target.
CPPs are short peptides that can be cationic, amphipathic, or hydrophobic. These peptides have the ability to efficiently cross cellular plasma membranes and enter the cytoplasm. Furthermore, when CPPs are linked to oligonucleotides, proteins, or nanoparticles, they facilitate the transport of these entities across the cell membrane [25
]. Thus, a number of investigators have assessed the use of CPPs for intracellular delivery of macromolecules (reviewed in [28
]). CPPs have shown efficacy in vivo
for delivery of macromolecules to tumors and even across the blood brain barrier [30
]. As discussed in this review, addition of CPPs to the ELP carrier may not only enhance its uptake into the tumor cells in vitro
], but the CPPs also mediate escape of the polypeptide from the tumor vasculature and entry into the tumor cells.
In addition to physically targeting therapeutic agents to cancer cells, another method of specifically inhibiting cancer cell proliferation is to exploit their genetic abnormalities. In some cancer types, cancer cells have overexpressed and/or permanently active oncogenes, causing hyperactive growth and division and/or protection against apoptosis. Some cancer cells have inactivated or missing tumor suppressor genes, resulting in deregulation of the apoptotic pathway and loss of control over the cell cycle and DNA replication. Recent characterization of the genetic alterations that occur during carcinogenesis has identified many potential molecular targets for which to develop new therapeutics. One of the major advantages of therapeutic peptides is that they are much easier to design using a rational approach than small molecule drugs for stimulation or inhibition of a given protein/protein interaction. These peptides are derived from high-throughput screening or by using NMR or crystal structures of their molecular target and further optimized by a rational drug design approach. Such therapeutic peptides can be designed to bind almost any protein of interest with high affinity and specificity and can interfere with molecular pathways that are deregulated in cancer cells [39
]. The use of peptides to specifically inhibit aberrant oncogenic or tumor suppressor proteins should be more effective and have fewer side effects than current nonspecific cytotoxic drug treatments. However, the clinical efficacy of therapeutic peptides is limited by pharmacodynamic properties. When applied in vivo
, therapeutic peptides are rapidly degraded in circulation, and their relatively large size and often charged nature makes them impermeable to cancer cell membranes [41
]. Therefore, in order to advance therapeutic peptides into the clinical setting, a suitable carrier system that can overcome these limitations and target the peptide to the tumor site and into the tumor cells is needed.
Attention is being focused on peptide delivery using macromolecular carriers. Micro- and nanospheres are being investigated for their ability to deliver bioactive peptides via the oral route, stabilizing and delivering them through absorption barriers in the gastrointestinal tract [43
]. Liposome - peptide conjugates have been investigated, but the focus of this field is the conjugation of cell penetrating peptides to the surface of liposomes to enhance fusion with the cell membrane [44
]. To overcome limitations of other macromolecular carriers and improve delivery of peptide therapeutics to solid tumors, our lab is working to develop an ELP-based thermally targeted peptide vector. Such a carrier would have all of the characteristics and advantages of existing soluble macromolecular carriers, but it would be also be capable of active targeting by application of local hyperthermia.
ELP has been used for thermally targeted delivery of small molecule drugs [37
], plasmid DNA [48
], therapeutic peptides [35
], and proteins [51
] in various cancer models. This review focuses on the use of thermally responsive polypeptide carriers for hyperthermia targeted delivery of therapeutic peptides. In this article, we present a summary of results from our lab comparing cellular uptake mechanisms and efficiency of several different CPP-fused ELPs and discuss their potential therapeutic utility. We also describe use of ELP to deliver two therapeutic peptides, one that inhibits the function of the oncogenic transcription factor c-Myc [35
] and one that mimics the Cdk inhibitor p21 [38