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

Background: Elastin-like polypeptide (ELP) undergoes its characteristic of phase transitioning in response to ambient temperature. ELP therefore has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be targeted to hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer, with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of ELP, a matrix metalloproteinase (MMP) substrate, a cell penetrating peptide (CPP), and 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This construct may be initially targeted to the tumor by application of mild heat after administration. Within the hyperthermic tumor, then this construct is cleaved by MMP, releasing CPP-Dox, which can infiltrate tumor tissues and penetrate cell membranes. Methods: We produced the construct in E.coli and examined its cleavage by MMP enzymes in vitro. Flow cytometry and confocal analysis were used to verify the facilitated uptake of the digested cell-penetrating Dox by breast cancer cells and Dox-resistant cells. Cytotoxicity tests further demonstrated improvements in bioavailability of cell-penetrating Dox following the enhanced cellular uptake of the cancer cells. Comparisons with the non-cleavable ELP counterpart were paralleled. Results: This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells than the ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES/ADR), ELP-released, cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells than ELP-Dox and free Dox. Conclusion: MMP-digested CPP-Dox shows better membrane penetration and induces more cancer cell death in vitro . This CPP-complexed Dox released from ELP kills even dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox. for polypeptides, and the are an of at least 3 with representing in GM001 MMP digestion, and undigested ELP-mmpL-CPP-rho likely washed off the cells during the rinsing These results demonstrate that ELP-mmpL-CPP-rho is digested by intrinsic MMP released from HT-1080, and the resultant cleaved CPP-rho penetrates the HT-1080 cells.


Background
It is acknowledged that current conventional chemotherapy is comprised of mostly cytotoxic drugs. These drugs have a strong anticancer efficacy, but cause collateral damage to non-tumor tissues. These unwanted side effects usually render a dose-limiting factor for chemotherapy and are a main reason for unsatisfactory prognosis of the therapy. Many efforts have been tried to resolve these problems, usually by attempting to raise the therapeutic index of the chemotherapy.
Mostly these efforts have aimed at selectively exterminating cancer cells. Molecular targeted therapy, sophisticated radiation, and immunotherapy such as monoclonal antibody therapy are good examples of such efforts. Another strategy, a drug delivery technology, is to specifically deliver drugs to tumor tissue so that the concentration of drugs in tumor tissue will increase compared to the concentration in other normal tissues. This allows more opportunity for a drug to express its activity on tumor cells, resulting in selective death of cancer cells with tolerable side effects.
Elastin-like polypeptide (ELP), a thermo-responsive biopolymer, is a bio-polymeric carrier for targeted drug delivery. ELP, as a derivative from tropoelastin, consists of repeats of pentapeptides (valine, proline and glycine) (1,2). The repeats of these hydrophobic amino acids permit ELP to have a unique rearrangement of molecules responding to a surrounding temperature, which is a thermo-responsive phase transition. At low temperature, ELP remains as a monomer and soluble in solution; however, it coacervates and precipitates in solution when ambient temperature rises above its phase transition temperature (3)(4)(5). This co-acervation can also be reversed decreasing the temperature of the ELP solution. Thus, this reversible phase transition of ELP is mainly under the control of surrounding temperatures, and ELP are highlighted as a controllable carrier for anticancer drugs in active tumor targeting strategies.
Additionally, ELP also exploits an 'enhanced permeability and retention (EPR) effect' and can progressively accumulate in tumor tissue due to abnormal histological structures of tumor. These unique properties render ELP itself a drug carrier that can exploit both the EPR effect and tumor targeting using the hyperthermic technique (6). Furthermore, ELP has been modified by the addition of cell penetrating peptides (CPP) to allow enhanced cellular uptake, improved penetration of physiological barriers like the blood-brain barrier, and preferential intracellular distribution such as in the cytoplasm or the nucleus (7,8).
Many previous researchers have verified the potential of this polymer (9)(10)(11)(12), and animal studies that include the use of local hyperthermia have demonstrated that ELP is able to deliver a sufficient amount of drugs to the tumor area showing significant tumor reduction efficacy. In this study, we further modified the ELP drug delivery system to release drugs in response to an enzyme which is abundant in tumor tissue. The suggested system is composed of ELP; a matrix metalloproteinase (MMP) substrate, mmpL; a cell penetrating peptide, CPP; and a 6-maleimidocaproyl amide derivative of doxorubicin, Dox. In this study, we generated an MMP-responsive ELP drug delivery system that releases CPP-Dox, and we report the usefulness of this strategy to overcome Dox-resistance by investigating cellular uptake and anti-proliferation properties of the proposed system.

Design of construct and protein preparation
ELP has 150 repeats of VPGXG with guest residues (amino acid at position X) of Val, Gly, and Ala in a 5:3:2 ratio. ELP coding sequences were modified by the addition of the "Tat" cell penetrating peptide sequence (YGRKKRRQRRR), an MMP cleavable sequence (PLGALG) and three Gly-Gly-Cys residues to the C-terminus of ELP for the conjugation with Dox (Table 1). For an MMP uncleavable control, six Gly residues were used instead of the MMP cleavable sequence. All constructs were expressed in the Escherichia coli strain BLR(DE3) using pET 25b as an expression vector and were purified by repeated inverse transition cycling.

Conjugation of ELP constructs with doxorubicin or fluorescent probes
100 uM protein in PBS was reduced with 1 mM of tris-(2-carboxyethyl) phosphine (TCEP, Molecular probes) for 30 min at room temperature. Conjugation with 200 uM of the 6-maleimidocaproyl amide derivative of doxorubicin or tetramethylrhodamine-5-iodoacetamide dihydroiodide (Molecular probes, Eugene, OR) was followed by incubation at 4 °C overnight. Conjugated peptides were purified by inverse transition purification as described previously (3), and the concentration and the labeling efficiency were assessed by UV-visible spectrometry (UV-1600, Shimadzu). Concentrations of labeled ELP polypeptides were determined using the following equations:

Cleavage assays
Recombinant-human pro-MMP-2 (Enzo life science) was activated with 2.5 mM 4-aminophenylmercuric acetate at 37°C for 2 h. Then 1ug of each ELP construct was incubated with the pre-activated MMP-2 (10 pmol) for 4 h in reaction buffer (50 mM Tris, 200 mM NaCl, 10 mM, CaCl2, and 10 mM ZnCl2, pH 7.5). After reactions were loaded and separated on an SDS-PAGE gel, each peptide's cleavage pattern was confirmed by silver staining and by scanning the fluorescence of the gel with the IVIS Live Animal Imager (Caliper Lifesciences).

Flow cytometry analysis of cellular uptake
Cells were incubated with each treatment for 2 h at 37°C. Cells were rinsed with PBS and collected by trypsinization. Intracellular fluorescence was measured using a Gallios Flow Cytometer (Beckman Coulter) after trypan blue quenching as described previously (13). Forward versus side scatter gating was used to remove cell debris from the analysis, and the mean cellular fluorescence intensity was recorded. The mean cellular fluorescence was corrected for differences in labeling efficiencies among polypeptides, and the results shown are an average of at least 3 experiments with bars representing SEM.

Cytotoxicity test
Cells were plated in a 96-well plate and treated with a range of concentrations of each treatment for 24 h at 37°C. After further incubation with fresh media for 48h, cell viability was assessed using the MTT assay (Sigma). Briefly, a 0.5 mg/ml solution of thiazolyl blue tetrazolium bromide dissolved in PBS was added to each well and the plates incubated for 4 h at 37°C. Formazan formed by mitochondrial reduction was dissolved in 100 uL of DMSO and its absorbance was read at 570 nm. Survival rate of each group was calculated in comparison to a vehicle-treated control group.

Confocal microscopy
Cells (10 3 cells/chamber) were plated in 2-well Lab-Tek CC2 chamber slides (Nunc). After 24 h incubation at 37°C, the cells were treated with each treatment for two hours at 37°C. The cells were washed three times with PBS, fixed with cold 4% paraformaldehyde and stained with DAPI (Molecular probe) for 10 min at RT to visualize the nucleus. Distribution of each molecule was investigated by laser scanning confocal microscopy with a 60x oil immersion objective (Leica). Figure 2A depicts how ELP-mmpL-CPP would be cleaved by MMP, producing ELP (60KDa) and cleaved CPP (Tat peptide, 3KDa), while the other construct, ELP-CPP-Dox (63KDa), will not be digested by MMP. This hypothesis was verified by the following experiments. MMP-2 was used for the digestion in this experiment since MMP-2 is involved in the degradation of extracellular matrices in tumors and is overexpressed in most tumors compared with normal tissues (14). After incubation of each rhodamine (rho)-labeled construct (ELP-mmpL-CPP-rho and ELP-CPP-rho) with MMP-2, the reactant was run on SDS-PAGE and analyzed by both silver staining and fluorescence scanning ( Figure 2B). Silver-stained gels

MMP-2 digestion increases the cellular uptake of Tat-rhodamine in breast cancer cells
MMP-digestion will produce CPP-rhodamine (rho), which is smaller than the whole construct, ELP-mmpL-CPP-rho. This small size would be a primary contributor for facilitated uptake by cells. Cells treated with MMP-digested ELP-mmpL-CPP-rho and ELP-CPP-rho, respectively, were analyzed for uptake ability via flow cytometry. In Figure 3A, cells treated with cleaved CPP-rho (from ELP-mmpL-CPP-rho) showed up to five times higher uptake rates than the ELP-CPP-rho treated group in three cancer cell lines. This improved cellular uptake was also evident in observation with a fluorescence microscope ( Figure 3B).

Cleaved Tat-Dox kills breast cancer cells more efficiently than non-cleaved ELP-Tat
Rhodamine was replaced by doxorubicin to investigate whether improved uptake of cleaved Tat will contribute to cytotoxocity. Figure 3C compares the cytotoxicities of MMP-2 digested ELP-mmpL-CPP-Dox and ELP-CPP-Dox against three cancer cell lines. Improved cytotoxicity was observed in MMP-2 digested ELP-mmpL-CPP-Dox treated cells over ELP-CPP-Dox. These results suggest that MMP digestion of ELP-mmpL-CPP-Dox results in increased uptake of cargo molecules and facilitated death of cancer cells by cleaved CPP-Dox.

Cleaved CPP-Dox deposits in and kills Dox-resistant cancer cells.
To investigate whether cleaved CPP-Dox is able to penetrate and kill even Dox-resistant cancer cells, comparison of cytoxotoxicities and uptake rates of MMP-cleaved CPP-Dox were made between Doxresistant cells (NCI-ADR, MESSA-5DX) and Dox-sensitive cells (MCF7, MES-SA). Figure 4A shows the validated Dox resistance in MCF/ADR and MESSA-5DX, and cleaved CPP-Dox from ELP-mmpL-CPP-Dox showed more cell killing than ELP-CPP-Dox at 4uM Dox equivalence. Confocal microscopic images of MCF/ADR cells show that cleaved CPP-Dox from ELP-mmpL-CPP-Dox were taken up by MCF/ADR more than the other groups; free Dox and ELP-CPP-Dox ( Figure 4B). This was also confirmed by flow cytometry ( Figure 5C). The uptake rate of MMP-digested CPP-Dox in MCF/ADR was almost doubled compared with those of free Dox and ELP-CPP-Dox. These results suggest that MMPcleaved CPP-Dox can penetrate and kill even Dox-resistant cancer cells probably with help of a CPP (Tat peptide).

MMP-releasing HT-1080 can cleave ELP-mmpL-CPP-rho and take up cleaved CPP-rho.
This experiment shows that the ELP-mmpL-CPP construct can also be digested by endogenous MMP enzyme and release CPP-cargo molecules. MMP-releasing HT1080 cells were incubated with either ELP-mmpL-CPP-rho or ELP-CPP-rho for 4hrs, and each group of treated cells was processed either for flow cytometry or fluorescence microscopy. In flow cytometry, cells incubated with the ELP-mmpL-CPP-rho group had twice the rhodamine signal than did the ELP-CPP-rho group. However, this increased uptake was reversed by pretreatment with GM001, an MMP catalytic inhibitor ( Figure 5A). This finding was further confirmed by fluorescence microscopy, with the rhodamine particles being found in the nucleus of HT1080 cells treated with ELP-mmpL-CPP-rho ( Figure 5B). Uptake of these particles, as in the flow cytometry experiment, were also abolished by GM001 pretreatment. GM001 prevents MMP digestion, and undigested ELP-mmpL-CPP-rho was likely washed off the cells during the rinsing step. These results demonstrate that ELP-mmpL-CPP-rho is digested by intrinsic MMP released from HT-1080, and the resultant cleaved CPP-rho penetrates the HT-1080 cells.

Discussion
Our tumor-targeted drug delivery system using ELP delivers anticancer cargo molecules specifically to the tumor site by exploiting the enhanced permeation and retention (EPR) effect along with the active thermal targeting approach. This thereby increases the relative concentration of the cargo drugs in tumors and improves the therapeutic index of the drugs, alleviating unacceptable toxicity to the patients (6,15). A striking example of this targeting can be found in previous research (16) in which fluorescentlylabeled CPP-ELPs were administered into S2013 tumor-bearing mice. One group of animals received hyperthermic treatment with infrared (IR) lasers on tumors immediately after injection of CPP-ELP so that the temperature in the tumor core reached 42 °C, while the other group were exempt from hyperthermic treatment. This study demonstrated that the IR heating of tumors created 2-3 times greater tumor accumulation of CPP-ELP. Given the thermo-responsive behavior of ELP, the aggregation of CPP-ELP in hyperthermic tumors resulted in an increase of the construct's concentration in the tumors. These results strongly suggest that ELP preferentially accumulates in tumors in response to local hyperthermia.
We have further renovated the ELP system in this study to release payloads in response to additional external stimuli, matrix metalloproteinases. This novel system is comprised of four components; ELP, MMP-2-cleavable linker, CPP (Tat peptide) and doxorubicin as a payload. The linker is a substrate of MMP-2, designed to be cleaved by MMP-2 so that the ELP system can eventually release a complex of the payload (doxorubicin) and a CPP (CPP-Dox) in tumor tissues. Involvement of MMPs, zinccontaining endopeptidases, in cancer biology has been extensively discussed in a variety of publications (17)(18)(19)(20). Especially, increased expression and activity of MMP-2 and MMP-9 subtypes in tumors are known to be related to the degradation of basement membranes, an essential step in tumor invasion, and enhancing angiogenesis. For example, Tutton et al. reported that MMP-2 expression was significantly increased in colorectal cancer tissues compared to matched normal colon as measured by ELISA (21). High levels of MMPs in tumors will facilitate the release of CPP-Dox out of the renovated ELP complex and provide the additional secondary tumor targeting opportunity compared to the previous ELP delivery system. This system, thus, becomes a triple-targeting strategy when used along with the EPR effect and local hyperthermia. Specifically, this cleavable ELP construct still contains the ELP molecule until just before being digested by MMPs at tumor tissues. This late cleavage process will allow CPP-Dox to benefit from ELP by EPR and thermo-targeting. ELP is expected to allow the proposed construct to be initially targeted to the tumor site by local application of mild heat. Then, ELP-mmpL-CPP-Dox will be fully digested by MMP to release CPP-Dox, followed by improved cellular uptake by cancer cells and increased cancer cell death.
This MMP-cleavable system displays a couple of other advantages in delivering chemotherapeutic molecules. First, when the MMP-cleavable ELP-CPP-drug is digested by MMP in tumor tissue, small fragments (CPP-Dox) will be produced. Since the molecular weight of the released CPP-Dox (< 3kDa) is twenty times less than the parental ELP construct (60kDa), it will quickly infiltrate into adjacent tumor cells, as can be seen in other studies (22,23). This hypothesis was examined by cell uptake assays in this study. Cells treated with CPP-rho, which is a digested product from ELP-mmpL-CPP-rho, showed more rhodamine uptake than the cells treated with undigested ELP-CPP-rho. It was also demonstrated in Figure 5 that the ELP-mmpL-CPP-rho can be digested by endogenous MMPs and taken up by HT1080 cells. Reversal of this uptake by GM001, an MMP inhibitor, indicates that the cell uptake of rhodamine by HT1080 relied on the catalytic activity of MMP. This increased uptake was reflected in the enhanced cytotoxicity of MMP-cleaved CPP-Dox in breast cancer cells. After MMP-2 digestion, ELP-mmpL-CPP-Dox killed more cancer cells than ELP-CPP-Dox ( Figure 3C).
A second advantage of this system is that the released CPP-Dox still takes advantage of the abilities of the CPP to facilitate uptake by the cells and to penetrate physiological barriers like the blood-brain barrier. More importantly, there are an increasing number of studies reporting the role of CPPs on overcoming the multidrug resistance (MDR) of cancer cells, which has been one of main hurdles that doxorubicin has faced. The use of doxorubicin, one of the most effective chemotherapy agents since the 1960s , has been compromised by the development of MDR in patients (24,25). MDR involves increased efflux, decreased uptake, and enzymatic deactivation of chemotherapeutic drugs such as doxorubicin. An elevated expression of active drug transporters in cancer cells is known to be a major resistance mechanism (26). The coupling of chemotherapeutic drugs to peptides such as CPPs has been suggested as the solution for these problems, since this strategy may alter the cellular uptake pathway and circumvent ABC-transporter-mediated drug efflux, allowing drugs to accumulate at high concentrations in drug-resistant cells, leading to an improved therapeutic index and fewer adverse effects (23,(27)(28)(29). Specifically, Tat-Dox developed by Liang et al. (29) showed a 59% uptake rate in Dox-resistant MCF7 cells, while 90% of free dox was lost during cell penetration, leading to considerable improvement in the IC50 of doxorubicin. In line with these reports, our current study also demonstrates that cleaved CPP-Dox showed facilitated cellular uptake by Dox-resistant NCI-ADR in comparison with free Dox and ELP-CPP -Dox ( Figure 4C). Like Dox-sensitive breast cancer cells, this increased uptake also led to enhanced cytotoxicity of cleaved CPP-Dox against NCI-ADR and MESSA-5DX ( Figure 4B).

Conclusion
In summary, the modified ELP-CPP-Dox is cleaved by incubation with intrinsic or extrinsic MMP enzyme. MMP-digestion produces CPP-Dox (rhodamine), which shows better membrane penetration and induces more cancer cell death in vitro. This CPP-complexed Dox released from ELP kills even dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Conflicts of Interest
Drazen Raucher is a founder/owner of a 'Thermally Targeted Therapeutics, Inc.'. Felix Kratz is Vice president of CytRx Pharmaceuticals.