4.1 Cell- penetrating peptides(CPP) and its Classification
One of the recent advancement in the field of molecular biology that appears to have an enormous impact on drug delivery of cancer therapy is the innovation of a tissue or cell penetration system[13, 49], Cell penetrating peptides (CPP) is a short peptides that able to pass through tissue and cell membranes via energy dependent or independent mechanisms; used to transport a wide variety of bioactive conjugates (cargoes) including proteins, peptides, DNAs, siRNAs, and small drugs, fluorescent compounds, nanoparticles and other substances into cells[50].
Besides the use of CPP as inert vectors for transportation of cargo molecules, the dual-acting CPPs which are both cell permeating and bioactive, have been emerging in now days. Studies have shown that some selected CPPs are known to produce bioactivity such able to safely modulate the intestinal paracellular barrier [51], act as neuroprotectants [52], induce apoptosis in cancer cells [53, 54] and suppresses breast tumorigenesis[55], in addition to act as cell-penetrating. More recently, a cell penetrating peptide obtained from azurin ( p28 ) has found to prevent phosphorylation of VEGFR-2, FAK and Akt, leads to inhibition of tumor growth and angiogenesis [56].
CPPs can be conjugated to cargoes either by non-covalent complex formation or by covalent bonds. Covalent conjugation of a CPP may be obtained chemically through disulfide bonds, amine bonds, or specific linkers that enable release of the cargo when internalized into the cell[54]. However, the possibility to change the bioactivities of conjugates is the main risk of the covalent conjugation of CPP [57]. Further more, the covalent methods are associated with problems such as lack of suitable reactive groups on the polymer, unstable intermediates, and inefficient coupling and purification. In these case, non-covalent approach seem more appropriate[58]. Non-covalent complex is formed by electrostatic and/or hydrophobic interactions between a positively charged CPPs and large, negatively charged cargoes [59].
CPP are extremely beneficial because they are biocompatible and the peptide sequence can be altered to fine tune hydrophobicity, affinity, charge, solubility and stability. They can also be readily synthesised in sufficient quantity [14]. However, the lack of cell specificity remains the main limitation for further clinical study of CPP[60]. Several approaches have been suggested to selectively target cancer cells using CPPs conjugated with targeting ligands. Among these, combined use of CPP with cell targeting peptides and activatable cell-penetrating peptides, are discussed in the later section.
CPPs can be classified based their origin, function, sequence, or mechanism of uptake. According to their physicalchemical properties, they can be categorized into three cationic, hydrophobic, and amphipathic peptides. Cationic CPP are the peptides with highly positive net charges at physiological pH. Primarily they are originated from the basic short strands of arginines and lysines, for example TAT48-60 (GRKKRRQRRRPPQ), Penetratin (RQIKIWFQNRRMKWKK), and DPV1047 (VKRGLKLRHVRPRVTRMDV). Hydrophobic CPP primarily contain nonpolar residues, which have amino acid groups that are vital for cellular uptake, along with a low net charge, for instance, Pep-7 (SDLWEMMMVSLACQY) and C105Y (CSIPPEVKFNKPFVYLI) are some of hydrophobic peptides A third class of CPPs is the amphipathic CPPs, which contain both polar (hydrophilic) and nonpolar (hydrophobic) regions of amino acids. Pep-1 (KETWWETWWTEWSQPKKKRKV) and pVEC (LLIILRRRIRKQAHAHSK) are the typical examples.
In another mode of classification, CPPs are categorized based on the origin of the peptide: Derived CPP, Chimeric CPP and Synthetic CPP. Derived peptides are protein-derived peptides, for example, TAT and penetratin, Chimeric peptides include two or more motifs from different peptides, such as transportan, derived from mastoparan and galanin, and its shorter analogue TP10. Synthetic peptides for example the polyarginine family[63, 64].For the detail review on the classification of CPP the reader is referred to [50, 61, 63].
4.2 Activable cell penetrating peptides
Lack of cell specificity, which is a major drawback of CPPs is mainly due to their electrostatic interaction. Generally cationic type of CPPs bind to anionic components of the plasma membrane, and this is principally accountable for their membrane transporting properties [65].
To overwhelm the problem of non-specificity, researchers have introduced the Activatable Cell penetrating peptides (ACPPs). ACPPs are novel targeting agents comprising of a polycationic CPP attached to a neutralizing polyanion unit via a cleavable linker (Fig. 2). Adsorption and uptake of CPPs into cells are prevented until the linker is cleaved [66]. In ACPP, stimulus-responsive materials are exploited to stimulate the selective display of CPPs within the pathological environment of a tumor, such as lower pH caused by building up of lactic acid or overexpression of extracellular matrix development remodeling proteases, or may be external application of heat or light to a disease site[67].
The lower pH in the tumor microenvironment compared with normal tissues can be exploited as a targeting strategies [68].The utilization of this approach has been known to improve the intracellular delivery of cargo molecules performed with CPPs. CPPs via shelter in nanocarriers with triggered exposure mechanisms, such as acid-degradable cross-links [61].
A number of studies involving different approaches of acid-activated CPP for tumor targeted delivery have been reported. For instance, Jin et al investigated acid-active peptide by amidizing the TAT lysine residues’ amines to succinyl amides (aTAT), completely inhibiting TAT’s nonspecific interactions in the blood compartment. The succinyl amides in the aTAT were quickly hydrolyzed, fully restoring TAT’s functions in the acidic tumor tissue or inside the cell lyso/endosomes. Thus, aTAT-functionalized poly(ethylene glycol)-block-poly(ε-caprolactone) micelles resulted long circulation in the blood compartment and efficiently accumulated and transported doxorubicin to tumor tissues, offering a high antitumor activity and low toxicity to cardiovascular system[69]. Moreover, Cheng et al also used pH activable cell-penetrating peptide (CR8G3PK6, ACPP) with of 2,3-dimethylmaleic anhydride (DMA) as a shielding group, and functionalized with anticancer drug doxorubicin (DOX) to produce a novel prodrug (DOX-ACPP-DMA) for tumor targeting drug delivery. The shielding group of DMA conjugated to ACPP through amide bond between DMA and the primary amines of K6, which was used to inhibit the cell-penetrating activity of the polycationic CPP (R8) by intramolecular electrostatic attraction at physiological pH 7.4. However, at tumor extracellular pH 6.8, the hydrolysis of the shielding group resulted charge reversal, activating and restoring the original function of CPP for improved cellular uptake by tumor cells. After cell internalization, the overexpressed intracellular proteases would further trigger drug release in cells [70].
Another example, Regberg et al demonstrated that Peptides (PepFect 3 ) modified with a leucine/histidine sequence have been known to be pH responsive CPP. The modified analogues PepFect 3 has shown a significantly improved cellular bioactivity than the unmodified PepFect [71].
Most recently, the group of Yao developed the acid activated CPP by introduction of some electron donating group such as Ethyl, Isopropyl, and Butyl to C-2 position of histidine of the CPP named as TH (AGYLLGHINLHHLAHL(Aib)HHIL-NH2), to form corresponding TH analogs (Ethyl-TH, Isopropyl-TH and Butyl-TH). The new TH analogs formed were linked to the antitumor drug camptothecin (CPT), and butyl-TH modified conjugate showed a remarkably stronger pH-dependent cytotoxicity to cancer cells than TH and the other conjugates [72].
Quenching of the cell-penetrating activity of the polycationic peptide by electrostatic interactions with the polyanionic domain can block the cellular uptake. However, tumor tissues, matrix metalloproteases (MMP-2/9), which are overexpressed in cancer tissues, cleaved the substrate and released the polycationic from the polyanionic domain, thereby stimulating cellular adhesion and subsequent uptake of the polycationic peptide(Fig. 2) [68, 73]. Shi et al developed a conjugate of ACPP with antitumor drug doxorubicin (DOX) which is sensitive to matrix metalloproteinase(MMP-2/9) to form ACPP-DOX conjugate consisting of the polycationic domain (CPP), the cleavable MMP-2/9-sensitive substrate, the polyanionic domain, and DOX. Activation of ACPP-DOX was found to occur by matrix metalloproteinase (MMP)-2/9 in enzyme concentration–dependent manner. The result of the flow cytometry and laser confocal microscope studies demonstrated that a higher cellular uptake of ACPP-DOX was observed by HT-1080 cells (overexpressed MMPs) than MCF-7 cells(under-expressed MMPs) after enzymatic-triggered activation [74].
The aforementioned two type of ACPP invoves the activation of CPP in vivo in tumor tissue. Another approach, that is the external illumination of tumor by near-infrared or ultraviolet light to stimulate dissociation of photosensitive groups (PG) from CPP-NP, thus controlling the release of drugs at the tumor site[75]. Shamay et al. developed photon-sensitive CPP using polymers bearing a light activated caged CPP for selective cellular uptake upon UV light illumination (365 nm), which may offers a promising approach to delivery of payload to the target cells[76]. But, the use of UV light has been limited due to its harmfull effect to tissue and low penetrability[77].
Near-IR (NIR) light able to penetrates tissues deeply and is less harmful to cells in contrast to UV light. With this line, Yang et al designed a nanostructured lipid carrier (NLC) conjugated with photon-sensitive cell penetrating peptides (psCPP) (CGRRMKWKK) and Asn-Gly-Arg (NGR) in attempt to improve targeted delivery of paclitaxel (PTX) to tumor cells. The psCPP unit facilitate specific cellular uptake after the cleavage the photon-sensitive protective group whereas, NGR moiety selectively binds to CD13-positive tumors. The results of the study demonstrated that the the tumor growth inhibition rate, and cellular up of psCPP/NGR-NLC group was significantly higher than the rest of PTX groups[77].
In this approach, the biopolymer elastin-like polypeptide (ELP),which is a heat sensitive carrier that able to undergoes a phase transition upon reaching the externally applied heat to the tumor environment can be used. This carrier is specific to the tumor as it only aggregates at the heated tumor site between 39 °C and 42 °C [61, 78].
Tumor targeting peptides (TTP)
Tumor targeting peptides (TTP) are also known as tumor homing peptides which are small peptides shorter than CPPs by 3 to 10 residues which have strong affinity and specificity to a tissue targets or tumor cell [60].There is the up-regulation of specific receptors in most of tumors and their vasculature which ared utilized by TTPs for which they show high binding ability [14]. Among a number of different receptors that are known to be overexpressed on tumor cells, integrins are the most attractive target for drug delivery because integrins have crucial roles in the process of tumor cell proliferation, migration, invasion and survival[80]
4.3.1 Peptide targeting to tumor vasculature
Tumor vasculature varies from the normal blood vessel both structurally and morphologically. For example, the tumor blood vessels are leaky and porous, unlike normal vasculature[81, 82]. In addition to the altered morphology, tumor vasculature varies from that of normal by their molecular composition [48].
Arginine–glycine–aspartic acid (RGD) and asparagine-glycine-arginine (NGR) peptides are the two most extensively studied ligands for targeting the tumor vasculature and exhbiting that their receptors are overexpressed during angiogenesis[81]. RGD peptide, can selectively target tumor vasculature expressing αγβ3 and αγβ5 integrins, and a NGR peptide (CNGRC) binds to CD13 (animopeptidase N, APN) which are specifically expressed in the tumor vasculature [83]. RGR is another peptide which was selected from phage display in pancreatic tumors that has been exhibited superior affinity to angiogenic vessels in insulinomas, and recognize various αβ integrins. RGR had been exploited as a carrier for the targeting deliver of therapeutic proteins (TNFa and IFN-g) to the targeted site in cancer therapy[84].
Another peptide thattargeting to angiogenic vasculature is the F3 peptide. The F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) is a peptide with 31-amino acid that able to targets blood vessels and tumor tissue. F3 is known to target nucleolin, which selectively expressed on the surface of endothelial cells and tumor cells [48]. After binding, F3 is internalized through receptor-mediated endocytosis, then translocated from cytoplasm to the nucleus, where it distributes itself through out organelle [85].
Peptides such as CREKA, CLT1 (CGLIIQKNEC), and CLT2 (CNAGESSKNC) are also known to target tumor blood blood vessels and represented as a novel type of homing peptides[81]. They have affinity to bind with clotted plasma proteins which exist on the walls of tumor vessels as well as in tumor stroma [86], Recently, a number of peptides homing to tumor vasculature have been identified.
Furthermore, Li et al., identified TCP-1 (CTPSPFSHC) a vasculature homing peptide by the in vivo phage library selection in an orthotopic colorectal cancer model. TCP-1 peptide was found to specifically recognize the blood vessels of orthotopic colorectal cancer in normal BABL/c mice induced by syngeneic colon cancer cells (colon 26)[87].
4.3.2 Peptides Targeting to tumor lymphatics
Peptides that home to tumor lymphatics LyP-1 (CGNKRTRGC) and LyP-2 (CNRRTKAGC)) have also been demonstrated in several reports[48]. Laakkonen et al. identified LyP-1 (CGNKRTRGC) by screening MDA-MB-435 breast carcinoma xenografts[88]. LyP-1 distinguishes lymphatics and tumor cells in MDA-MD-435 and, MMTV-PyMT breast carcinoma, and KRIB osteosarcoma xenografts, and their metastatic lesions but not C8161 melanomas. The LyP-1 receptor is a cell surface form of a mitochondrial protein p32. p32 shows unusual cell surface expression in tumor lymphatics, tumor cells, and a subset of myeloid cells, which contributes to the specificity of LyP-1 homing to tumors[89].
Simultaneously, the LyP-2 peptide (CNRRTKAGC) homes to the lymphatics of C8161 melanomas and K14-HPV16 skin and cervical carcinomas but not to the MDA-MB-435 tumors, showing heterogeneity in the molecular markers of tumor cells and lymphatic. Another peptide called RMS-II (CMGNKRSAKRPC) was identified in an in vitro screen for peptides binding to RMS cell lines and it showed some sequence similarity to LyP-1.37. However, better homing ability to the RMS xenografts in vivo was observed by RMS-II than LyP-1. Furthermore, RMS-II recognized tumor blood vessels, but not tumor lymphatic vessels which shows thdifferent specificities of these two peptides[81].
4.3.3 Peptides homing to tumor cell
Some of the homing peptides have known to possess cell-penetrating properties. For example, both the F3 and LyP-1 peptides are cell type-specific CPPs. They are able to internalize tumor cells and blood (F3) or lymphatic (LyP-1) endothelial cells in the tumors they home to[81]. CGKRK and CRGRRST (RGR) are also homing peptides that are transported to the target cell nucleus after internalization. The CGKRK and RGR peptides contain several basic amino acids, which are considered to be responsible for the internalization and may act as nuclear transport signals[90].
A number of endothelial cell-targeting peptides are found to act as tumor penetrating peptides to enable the internalization of a conjugated drug to the cancer cell. These peptides share a specific C-terminal C-end Rule (CendR) sequence, (R/K)XX(R/K), which is responsible for tissue penetration and cell internalization[91]. For example, internalizing RGD (iRGD; CRGDKGPDC, a 9-amino acid cyclic peptide where the lysine residue can also be an arginine, and the aspartic acid a glutamic acid) in addition to target the αvβ3 integrin receptor, it enable tumor penetration. iRGD is one of the most innovative tumor-targeting peptides, as it distributes much more extensively into extravascular tumor tissue than other RGD peptides, which tend to accumulate only around tumor vasculature [92].
4.4 The use of CPPs in combination with TTPs in drug delivery system
Tumor targeting peptides binds with receptors which are up-regulated on tumor cells specifically, but may not be capable to reach the target by themselves. On the other sided, CPPs penetrate plasma membrane effectively, but lack target specificity. A target specific CPP that able to both cross the plasma membrane as well as selectively deliver the drug to the desired target of action will be ideal in drug delivery system[14].
Combining a tumor homing peptide with an appropriate cell-penetrating peptide can enhance the tumor-selective internalization efficacy of the carrying cargo molecules. The conjugation of TTPs with conventional CPPs assists the translocation of the conjugate moieties to target tumor sites with improved selectivity and specificity[60]. Studies have shown that TTPs conjugated with CPPs have dramatically increased the efficiency to translocate drug molecule specifically to cancer cells as compared to TTPs alone[93]. In this study, the cyclic peptide cCPGPEGAGC (PEGA), which is a homing peptide that has been known to accumulate in breast tumor tissue in mice, and PEGA peptide that can not pass the plasma membrane were used for conjugation. Conjugation of PEGA peptide to the CPP pVEC, has been demonstrated to specifically target breast cancer cells both in vitro and in vivo. In addition, the conjugated PEGA-pVEC chimeric peptide has found to the efficacy of the anticancer drug, chlorambucil, by more than 4 times[93].
In another study, CREKA was used in combination with the CPP pVEC, as a chimeric peptide for intracellular delivery of a chlorambucil. The result of study revealed that the chlorambucil- CREKA-pVEC conjugate produced a significantly more anticancer activity in vitro than the anticancer drug alone[94] .
More recently, Fan et al., evaluated the synergistic effect of SP90 (SMDPFLFQLLQL) with a cell-penetrating peptide(C peptide) in the breast cancer homing ability. SP90 and chimeric peptide SP90-C specifically targeted cargo molecule into breast cancer cells, especially triple negative MDA-MB-231 cell, while C peptide alone had no cell-selectivity. The combined use of SP90 with C-peptide (SP90-C) has shown an increased the intracellular delivery efficiency by 12-fold or 10-fold compared to SP90 or C peptide alone, respectively. Furthermore, SP90 and SP90-C conjugation demonstrated increased the antiproliferative and apoptosis-inducing activity of HIV-1 VPR, a potential novel anticancer protein drug, to breast cancer cell without affecting normal breast cell. This finding indicated that SP90-C seems as a promising candidate vector for targeted anticancer drug delivery due to its excellent breast cancer cell-selective penetrating efficacy. Moreover, SP90-VPR-C is found as a potential novel breast cancer-targeted anticancer agent for its high anti-tumor activity and low toxicity[95].
Nowadays, researchers developed a sequence of peptides that possessed both cell penetrating property and specific cell selectivity using mRNA display technology. One of those peptides is RLW (with a sequence of RLWMRWYSPRTRAYG), which could specifically target to A549 cells. Gao t al conjugated RLW onto nanoparticles (RNPs) and then the RNPs were used for lung cancer targeting delivery, and a conventional cell penetrating peptide, R8 (RRRRRRRR), as acontrol. The in vitro cellular uptake study demonstrated that modification with RLW specifically enhanced the uptake by A549 cells rather than human umbilical vein endothelial cells, while conjugation with R8 increased the uptake by both cells, which indicates better specificity of RNP. After loaded with docetaxel, in vitro RLW could specifically target to A549 cells and enhanced the cytotoxicity of the drug [96].
4.5 Peptides for targeting multidrug resistant cancer
Most recently, anticancer drug-CPP/TTP conjugates have been one of the promising strategies to overcome tumor multidrug resistance (MDR). Sheng and coworkers designed on a dual-targeting hybrid peptide HAIYPRHGGCGMPKKKPTPIQLNP (T10-ERK), which is composed of extracellular signal-regulated kinases (ERK) peptide inhibitor MPKKKPTPIQLNP, a thiol spacer (i.e.GGCG) and transferrin receptor (TfR)-binding peptide HAIYPRH inorder to overcome the problem of drug resistance. Then, this hybrid peptide was conjugated to DOXO-EMCH (6-maleimidocaproyl) hydrazone of DOX, forming T10-ERK-DOX. The efficacy of T10-ERK-DOX to reverse drug resistance as compared with free DOX and T10-DOX was determined by using MCF-7/ADR cells and nude mice bearing MCF-7/ADR xenografts. The result of this study showed that, T10-ERK-DOX demonstrated a much lower in vitro IC50 (20.8 1.1 mM) and its in vivo extent of inhibition in mice was more evident (72.2 4.6%)[97]. Furthermore, Lelle et al. formulated a conjugate consisting of octaarginine cell-penetrating peptide (Ac-CRRRRRRRR-NH2) and a highly DNA-affine doxorubicin dimer, then tested against MCF-7, Kelly-WT as well as Kelly-ADR cells in vitro. The cytotoxic effect of the peptide-drug conjugate, studied with drug-sensitive and doxorubicin-resistant cancer cells, demonstrates that the bioconjugate can successfully overcome drug resistance in neuroblastoma cells[98]Feng et al also investigated the DOX-CPP conjugate for overcoming drug resistance in cancer. In this study, slightly acidic pH-sensitive peptide (SAPSP) with high selectivity for cancer cells was attached to DOX to obtain SAPSP-DOX prodrug. Cellular uptake studies demonstrated that SAPSP-DOX was preferably accumulated in cancer cells (Dox-sensitive MCF-7 and Dox-resistant MCF-7/ADR), followed by displaying 26-fold less toxic toward noncancerous MCF-10A cells than free DOX did[99].
