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Tumor Targeting Strategies Advances and Limitations

Info: 7532 words (30 pages) Dissertation
Published: 10th Dec 2019

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Tags: MedicineCancer


Cancer has been becoming among the leading causes of mortality globally. The major challenges of conventional cancer therapy were the failure of most chemotherapeutic agents to accumulate selectively in the tumor cells and severe systemic side effects. In the past three decades, a number of drug delivery approaches have been discovered to overwhelm the obstacles. Among these, nanocarriers have gained a lot of attention for their excellent and efficient drug delivery systems to improve specific tissue/organ/cell targeting. To further enhance the targeting effect, and reduce the some limitation associated with NPs, the surfaces of NPs were modified with different ligands. Nowadays, several kinds of ligand-modified nanomedicines have been reported. Recently, Cell-penetrating peptides (CPP) are emerging drug delivery system and attracting the attention of researchers due to their ability to transport bioactive molecules intracellularly. Currently, nanocarriers functionalized with a scell penetrating and tumor targeting peptides have shown dramatically enhanced cellular uptake and specific cytotoxicity to cancer cells.

Therefore, in this review we focus on recent advances of tumor targeting strategies, cell penetrating peptides and its limitation as delivery systems, different classes of CPP; tumor targeting peptides. Furthermore, we discuss the application of CPP and/ or TTP in delivery of plant derived chemotherapeutic agents.


Cancer, Nanocarriers, cell penetrating peptide, targeting drug delivery, herb-based drug.

  1. Introduction

Despite huge efforts in development of anticancer drugs, cancer nevertheless remain one of the main reasons of morbidity and mortality worldwide. In 2015, cancer was accountable for 8.8 million deaths. Globally, from 6 deaths one is due to cancer. Within the coming two decades, it is estimated to be increased by about 70%. In addition, the significant and increasing economic impact of cancer has been observed. In 2010, the overall annual economic cost of cancer was estimated approximately 1.16 trillion US$  [1].

Chemotherapy, radiation, and surgery with chemotherapy are the major treatment protocol of cancer.  Utilization of chemotherapy has demonstrated to enhance the survival rate of patients with malignant to some degree. In spite of the fact that surgery and radiotherapy are the most effective treatments for local tumor and non-metastatic cancers, they are beneficial for cancer that has not been dissiminated throughout the body. Therefore, chemotherapy is the treatment decision for the metastatic malignancies, since they are well distributed to every organ in the body [2]. However, most of conventional chemotherapeutic agents currently in clinical use are limited by their several undesirable properties, including poor solubility and bioavailability, rapid elimination from systemic circulation, narrow therapeutic index, and non-selective site of action after intravenous / oral administration and cytotoxicity to normal tissues, which might be the main reason for treatment  failure in cancer therapy[3].

Furthermore, the conventional chemotherapeutic agents often unable to penetrate and reach the internal part of solid tumors, leading to inefficient  cytotoxicity to the cancerous cell. The other problem of traditional chemotherapy is associated with Pglycoprotein, a multidrug resistance protein(MRP) that is overexpressed in  cancer cells, which is acting as the efflux pump and inhibit drug accumulation inside the tumor, and often mediates the development of resistance against anticancer drugs. Thus the administered drugs remain unsuccessful to produce the desired response [3, 4]. Various drug delivery approaches constituting antibodies, growth factors, hormones, etc. have been designed and administered timely, however a significant internalization the cancerous cells could not be achieved due to the presence reticuloendothelial system (RES) and intracellular enzymes[5]. Consequently, in the late decades tremendous endeavors have been given to overcome the major drawbacks of the conventional cancer chemotherapy.

Various novel drug delivery strategies have been designed to date, including polymeric nanoparticles[6], polymeric micelles [7], dendrimers [8], liposomes [9], viral nanparticles [10], carbon-based systems (carbon nanotubes and grapheme oxide), magnetic nanoparticles, silica and gold nanoparticles [11] and lipid nanoparticles (solid lipid nanoparticles, and nanstructured lipid carriers). Nanoparticles possess several advantages including, the larger surface area in contrast to bigger particles, that can be easily modified to accommodate large quantity of drug, increase the circulation time of the drug in the blood and improve the accumulation of drugs in solid tumors through the enhanced permeability and retention (EPR) effect and passive targeting of tumor cells. Nanocarriers have been also known to improve the solubility, bioavailability and pharmacokinetics profile of chemotherapeutic agents[9].

To date, decorating a surface of nanocarrier with different ligands targeting specific receptors that are over-expressed on the cancer cell have been developed for active targeting of chemotherapeutic agent to the cell [12]. Most importantly, nanocarriers functionalized with a cell penetrating or/and tumor targeting peptides have been a highly promising strategies and attracting the attention of researchers. These peptides are very advantageous as they have efficiently delivered a broad variety of cargo intracellularly [13]. In addition, CPPs are biocompatible and the amino acids sequence can be easily modifiable to fine tune hydrophobicity, affinity, charge, solubility and stability. They can also be readily synthesized in large quantity [14]. CPPs-mediated drug delivery are achieved either by the formation of stable, non-covalent complexes or the covalent bond with the cargo [15]. In this review we focuses on recent advances in tumor targeting approach of herbal based anticancer bioactive substances using cell penetrating and/or tumor targeting peptides modified nanocarriers.

  1. Overview of tumor targeting drug delivery approaches

Targeted cancer therapy is viewed as an irreplaceable component of current anticancer drug development [16]. The best strategy to improve the efficacy and reduce the toxicity of a cancer drug is directing the drug to its target and maintaining its concentration for a sufficient time at the site to produce the desired therapeutic effect. Cancer cells can be targeted by the two approaches: such as passive targeting and active targeting.

Passive targeting involves the extravagation of drug preparation through leaky vasculature/capilary of tumor that results from abnormal angiogenesis at the site of tumor, resulting in accumulation and retention. This phenomenon was recognized as the Enhanced Permeation and Retention (EPR effect) (Fig. 1) [3]. In addition, administration of pH-sensitive drug release in the acidic microenvironment inside the cancer cell [5, 17] and particulate carrier phagocytosis by mononuclear phagocytosis systems (MPS) and privileged localization in the organs of the reticuloendothelial system (RES) are considered as passive targeting [18].

The size of drug carriers and the abnormal and permeable vasculature of the tumor are the base for passive targeting [19]. Most of tumors manifest an abnormally dense and leaky vasculature formed via stimulation by vascular endothelial growth factor (VEGF). In normal vasculature, particles larger than 2 nm are prevented from crossing between endothelial cells due to tight junction [20]. However, in tumor vasculature, the tight junctions and basement membrane are disordered, allowing passage of particles ranged from 10 to 200 nm through the leaky neovasculature of the tumor and then retain in tumor site. Furthermore, the poor venous and lymphatic clearance system in tumor created an opportunity for the NPs to accumulate in the tumor with high concentration for a long time [3]. However, the passive targeting occurs to almost all nanocarriers, deprived of a selective delivery, and insufficient tumor cell uptake.

The other, more advanced approach of targeting for oncology applications is the modification of surface of nanoparticles with a specific tumor-homing ligands [21]. The ligands are known to bind to receptors that can be overexpressed on the surface of cancer cell. The ligands, with a selctive affinity toward a specific receptor or molecule differentially expressed at the target site, are presented on the surface of nanocarriers, resulting in the selective accumulation and cellular uptake at the site of action [22]. This strategy  significantly increases accumulation and retention of NPs in the tumor vasculature and specific and successful internalization by target tumor cells, which is known as “active tumor targeting”[23]. The ligands that have been used to modify NPs includes monoclonal antibodies, folic acid, hyaluronic acid, albumin, vitamins (folate, vitamin B12, thiamine, and biotin), transferrin,  lectins,  aptamers,  and peptides [5, 18, 19, 24].

Depending on the degree of penetration, active targeting may occur at the tissue, cell, or subcellular level. In solid tumors it is vital to remember that, the active targeting process starts with the accumulation of the drug delivery system in the tumor tissue by passive targeting, therefore, any actively targeted carrier need to fulfill the basic requirements mentioned for passively targeted systems [17].

Effective  targeting drug to tumors is achieved by using a combination of different independent concepts such as the events of EPR effect, the design and properties of nanoparticles, increased the circulation time by PEGylation, and ligand– receptor type interactions. [25]. The other important aspect in cancer treatment is targeting circulating tumor cells (CTCs). The existence of circulating tumor cells (CTCs) is closely related to tumor metastasis, which is accountable for more than 90% of deaths due to cancer [26].  CTCs often express sialylated carbohydrate ligands that bind to selectin protein on their surfaces. Therefore, selective targeting as well as killing of CTCs could be achieved by using E-selectin functionalized nanocarriers[27].

  1. Nanocarriers in tumor targeting delivery system

Recently, carrier-based strategies have been employed to circumvent the majority of the obstacles in the delivery of anticancer drug. Among the numerous approaches, nanocarriers particularly in the size range of 10 to 100 nm offer some unique properties [3], and  capable of transporting anticancer agents (drugs with small molecular weight or macromolecules as genes or proteins) to tumor tissue and achieving a cytotoxic concentration several-fold higher in the tumors with a reduced toxicity to the rest part of the body compared with free drugs [2, 28].

The emerging of nanotechnology in cancer therapy is to overcome the limitations that are inherent with conventional drug delivery methods, which are nonspecific biodistribution, inefficient cellular uptake, and low therapeutic[28]. In addition, nanocarriers offer protection of the drug from degradation and, decrease the renal elimination and increase its half- life in the circulation, augment the payload of cytotoxic drugs, allow the controlled release kinetics of the drugs, improve the solubility, and also possess the potential ability to bypass multidrug-resistance mechanisms (MDR) [29], by various approaches [2].

The unique properties of nanoparticles (NPs), such as large surface-to-volume ratio,

small size, the potential to encapsulate wide varieties of drugs, and modifiable surface chemistry,  offer them several advantages, including multivalent surface modification with targeting ligands, efficient navigation of the complex in vivo environment, improve internalization by the cancer cell, and allow the sustained release of drug payload. These benefits make NPs a potentially superior approach of treatment to traditional cancer therapies [19]. The size and surface charge of NPs are known to affect the half-life and biodistribution of NPs significantly. The larger NPs (>100 nm) are usually cleared from the circulation by phagocytosis. Similarly, the very small nanoparticles of particle size less than 10 nm has a high rate of clearance. The positive charge on the surface of particles assists internalization into the cancer cells. Moreover, surface modification of NPs with some polymers such as polyethylene glycol (PEGylation) can also increase the circulation time of particles via inhibition of clearance by reticuloendothelial system and increasing the accumulation of NPs in tumor site but, it can affect the cellular uptake by cancer cells [30].

To date, several types of nanocarriers have been emerged for the drug delivery of chemotherapeutic agents  including magnetic and metallic nanoparticles, such as iron oxide or gold nanoparticles, silver NPs [31], nanodiamond[32], carbon based structures(graphene sheets and carbon nanotubes), polymeric nanoparticles, dendrimers, quantum dots, hydrogel-based delivery systems, and silica-based nanoparticles[33], lipid based NPs (liposomes(LP), solid lipid nanoparticles(SLN) and nanostructured lipid carrier(NLC)) and Viral NPs and Hybrid NPs (combination of two or more of above) [34, 35].

Though nanoparticles are commonly considered as a promising concept for  delivery of drugs to specific tissues or cancer tumors,   the delivery efficiency in vivo remains low, and only a few of them are in clinical use including Abraxane®, Doxil®, and Myocet® that are approved by FDA [36, 37]. The main reason for the clinical failure of NPs could be due to the presence of different barriers which hinder internalization into the cancer cells after they penetrate into the tumor vasculature. Furthermore, in rapidly growing tumors, cancer cells are located adjacent to the endothelial barrier, and nanocarriers with targeting moieties will bind to the first receptors they find, not penetrating into the rest of the tumor There are also the issues associated with the possible toxicity and long-term effects of NPs made from non-biodegradable materials [2].

Chan et al, recently reviewed data from published studies conducted during 2005 – 2015 regarding tumor targeting with nanoparticles (passively or actively targeted), and concluded that, on average, nanoparticles demonstrate low delivery efficiency to solid tumors [38]. It is clear that new novel strategies should be inaugurated [39]. In attempt  to overcome the above mentioned limitation of NPs,  a wide variety of ligands have been used to modify the surface of NPs, permitting a selective recognition of different receptors or antigens overexpressed on the surfaces of tumor cell, improving the cytotoxicity of the anticancer agents in tumors and minimizing their toxic effects [40]. There are several targeting moieties that have been attached to the surface of NPs, and some  examples are mentioned as follows.

Transferrin is a membrane glycoprotein  which transports iron to rapidly growing cells [18]. Transferrin binds to the transferrin receptor (Tfr) and gets internalized by the cells via endocytosis and finally dissociates the iron inside the acidic pH of the endosomes. Tfr  are known to overexpressed in cancer cells up to 100-fold, which makes it as a promising candidate in targeted cancer therapy [41]. Albumin-receptors on the plasma membrane are responsible for albumin uptake by cell  [42]. Albumin-bound nanoparticles have found to accumulate in the  tumors by the EPR effect, and also by binding to glycoprotein 60  receptor that support the endothelial transcytosis[2].

The vitamins used for targeting potential includes folate, vitamin B12, thiamine, and biotin [18]. Their receptor have been known to be upregulated in numerous human cancers[43]. Particularly, the folate has been extensively used as a targeting moiety for nano­carriers.

Lectins are a group of sugar-binding  cell surface protein receptors that distinguishes and binds to specific sugar moieties. Some of these receptors were well-known to be overexpressed in specifc cancer cells, e.g asialoglycoprotein receptor in hepatic cancers [44]. It is suggested that binding of surface carbohydrates with their ligands (lectins) leads to accumulation of glycans into the cells via the endocytotic process[30].

The  conjugation of  monoclonal antibodies (mAb) to the surface of nanoparticles (immuno-nanoparticles) can specifically target antigens or receptors overexpressed in  tumors.  EGFR is one of the mAb targets with a higher clinical relevance, which is known to be overexpressed in several type cancers [2]. Aptamer is a single-stranded  RNA or DNA molecules, with three-dimensional structures, having a specific high affinity to nucleolin protein on the surface of cancer cells [45].  Additionally, E3 is known to targets and internalizes into a variety of cancer, including lung, breast, ovarian, melanoma, colon, glioblastoma, leukemia, and liver cancers[46].

Most recently, peptide-based targeting ligands has been attracting the attention of

the researchers and is the main concern of this review. It provides several advantages over other ligands in terms of cellular targeting,  precise chemical structure and chemical stability. Manufacturing of  peptides is inexpensive, and easy to scale up. Furthermore, peptide ligands show good biocompatibility and  their proteolytic degradation can be inhibited by chemical modifications, such as the cyclization or addition of D-amino acids or [47]. Moreover, peptide ligands are highly selective for target tissues or cells and multiple ligands can be conjugated to a single drug carrier to offer multivalent conjugation, thus increasing binding affinity to the target [48]. The detail of cell penetrating peptide is presented in the following section.

  1. Peptides as targeting and enhancing anticancer delivery to cell

 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 the release of the cargo when internalized into the cell [54]. However, the possibility to change the bioactivities of the conjugates is the main risk of the covalent conjugation of CPP [57]. Furthermore, 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 synthesized 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 activable 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 physicochemical properties, they can be categorized into three cationic, hydrophobic, and amphipathic peptides. Cationic CPPs 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 containing 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 detailed 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 the 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].

4.2.1 Acid-activated CPP

The lower pH in the tumor microenvironment compared with normal tissues can be exploited as a targeting strategy [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 the 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].

4.2.2 Enzyme activated CPP

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].

4.2.3 Photon-sensitive CPP

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 harmful 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 tumor growth inhibition rate, and cellular up of psCPP/NGR-NLC group was significantly higher than the rest of PTX groups[77].

4.2.4 Thermally-activated CPP

In this approach, the biopolymer elastin-like polypeptide (ELP), which is a heat sensitive carrier that able to undergo 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].

3 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 a strong affinity and specificity to a tissue  target or tumor cell [60]. There is the up-regulation of specific receptors in most of tumors and their vasculature which are 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  exhibiting 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 that is targeting to angiogenic vasculature is the F3 peptide. The F3 (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK) is a peptide with 31-amino acid that able to target 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 the cytoplasm to the nucleus, where it distributes itself throughout organelle [85].

Peptides such as CREKA, CLT1 (CGLIIQKNEC), and  CLT2 (CNAGESSKNC) are also known to target tumor blood vessels and represented as a novel type of homing peptides[81]. They have an 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. The TCP-1 peptide was found to selectively recognize the vasculature 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

LyP-1 (CGNKRTRGC) and LyP-2 (CNRRTKAGC)) are the peptides that home to tumor lymphatics and have also been demonstrated in several reports[48].  Laakkonen et al., identified LyP-1 (CGNKRTRGC) by screening breast carcinoma xenografts (MDA-MB-435)  [88]. p32 is a mitochondrial protein which is  known to be the receptorfor LyP-1. LyP-1, selectively binds  to p32, which shows unusual expression on the surface tumor lymphatics, tumor cells, and a subset of myeloid cells, which contributes to the specificity of LyP-1 homing to tumors[89]. LyP-1 has found to distinguish lymphatics and tumor cells in MDA-MD-435 and, MMTV-PyMT breast carcinoma, and KRIB osteosarcoma xenografts, and their metastatic lesions, however unable  recognize C8161 melanomas[90]. 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, RMS-II (CMGNKRSAKRPC), which has some sequence similarity with LyP-1 was also identified in an in vitro screen for peptides binding to RMS cell lines. Better targeting ability to the RMS xenografts was observed by RMS-II than LyP-1in vivo. Furthermore, RMS-II recognized tumor blood vessels, but not tumor lymphatic vessels which show the different 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, F3 and LyP-1 peptides are cell type-specific CPPs. These peptides have the ability to enter into tumor cells and blood (F3) or lymphatic endothelial cells(LyP-1)  in the tumors they home [81]. CRGRRST (RGR) and CGKRK are another homing peptides that are conveyed into a nucleus of the targeted cell after the cellular uptake. These peptides consist a number of basic amino acids, which are considered to be accountable for interacellular and intranuclear transportation[91].

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[92]. For example, internalizing RGD (iRGD; CRGDKGPDC, one of the most innovative tumor-targeting peptides which is a 9-amino acid cyclic peptide where the lysine residue can also be an arginine, and the aspartic acid a glutamic acid. bIn addition to targeting the αvβ3 integrin receptor, it is able to penetrate tumor. As compared to other RGD peptides, iRGD can distribute much more extensively into extravascular tumor tissue [93].

4.4 The use of CPPs in combination with TTPs in tumor targeting delivery

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 side, CPPs penetrate plasma membrane effectively, but lack target specificity. A target specific CPP that able to both penetrate the plasma membrane and selectively transport the drug to the desired target of the action is will be considered an ideal type of CPP [14].

Combining a TTP with a suitable CPP has known to facilitates the translocation of the conjugate moieties to target tumor sites with better 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 [94, 95]. The study conducted by Mae et al., using the cyclic peptide cCPGPEGAGC (PEGA), which is a homing peptide that has been known to target the breast tumor tissue in mice, and  PEGA peptide is a CPP that can not pass the plasma membrane. The combined use of this two peptide by conjugation of PEGA to pVEC has exhibited to specifically target breast cancer cells both in vitro and in vivo. In addition, the conjugated PEGA-pVEC chimeric peptide has found to increase the efficacy of chlorambucil by more than 4 times[95].

The further study by the same group, used the combination of CREKA with the pVEC, as a chimeric peptide for delivery of a chlorambucil intracellularly. The result of the study revealed that the chlorambucil- CREKA-pVEC conjugate produced a significantly better anticancer activity in vitro than the anticancer drug alone. The study also showed that CREKA-pVEC is better in translocating cargo molecules
inside cancer cells as compared to the PEGA–pVEC peptid [96, 97].

More recently, Fan et al., evaluated the synergistic effect of SP90 (SMDPFLFQLLQL)  with C peptide (GPGLWERQAREHSERKKRRRESECKAA) in the breast cancer homing ability. SP90 is able to bind specifically into breast cancer cells (MDA-MB-231cell),  while C peptide alone had no cell-selectivity. The combined use of SP90 with C-peptide (SP90-C) has shown a 12-fold or 10-fold increase in efficiency of the intracellular delivery as compared to SP90 or C peptide alone, respectively. Furthermore, SP90 and SP90-C conjugation have been used for delivery of HIV-1 VPR, which is a potential novel anticancer protein drug to breast cancer cell.  SP90-VPR-C has demonstrated an improved apoptosis-inducing and antiproliferative activity of HIV-1 VPR, without affecting normal breast cell. [98].

Nowadays, a sequence of peptides that possessed both cell penetrating property and specific cell selectivity have been developed using mRNA display technology. For instance, RLW (RLWMRWYSPRTRAYG) is known to exhibit both properties and could target A549 cells specifically.  Gao et al.,  conjugated RLW with nanoparticles to form RNPs for targeting delivery into lung cancer. Cells(A549), and a conventional CPP(R8, RRRRRRRR), was used as a control. The result in vitro cellular uptake study has shown that NPs functionalized with RLW specifically improved the uptake by A549 cells as compared to human umbilical vein endothelial cells. However, the conjugation with R8 increased the uptake by both cells, which indicates a better targeting specificity of RNP.  The RNP loaded with docetaxel, has demonstrated RLW could specifically target to A549 cells and enhanced the cytotoxicity of the drug in vitro [99].

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 et al., designed a dual-targeting hybrid peptide HAIYPRHGGCGMPKKKPTPIQLNP (T10-ERK)  which is composed of extracellular signal-regulated kinases (ERK) peptide inhibitor (MPKKKPTPIQLNP), a thiol linker (i.e.GGCG) and transferrin receptor (TfR)-binding peptide (HAIYPRH) inorder to overwhelmed the problem of drug resistance. Then, T10-ERK was conjugated to DOXO-EMCH (a prodrug of DOX), resulting T10-ERK-DOX. The efficacy of T10-ERK-DOX in reversing 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 the study indicated that  T10-ERK-DOX could efficiently inhibit drug resistance and improve the cytotoxicity of DOX by blocking P-gp
mediated drug efflux and inducing apoptosis [100].

Furthermore, Lelle et al. formulated a conjugate consisting of octaarginine cell-penetrating peptide (Ac-CRRRRRRRR-NH2) and doxorubicin dimer with a high DNA affinity. The cytotoxicity of the peptide-drug conjugate was evaluated against drug-sensitive and doxorubicin-resistant cancer cells, and showed that that the conjugate can overcome drug resistance in neuroblastoma cells efficiently [101]. Feng et al also studied the DOX- SAPSP (slightly acidic pH-sensitive peptide) conjugate in order to interfere drug resistance in cancer therapy. In this study, DOX was attached to SAPSP, CHGAHEHAGHEHAAGEHHAHE-NH2 to achieve SAPSP-DOX prodrug. The cellular uptake studies have demonstrated that SAPSP-DOX  selectively accumulated in both Dox sensitive and DOX resistance cells cancer cells along with 26-fold less toxic toward noncancerous MCF-10A cells than free DOX did[102].

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