- Email: [email protected]
Tumor-penetrating peptides
Tumor-penetrating peptides Ruirui Qiao*,1, Kun Wang2 and Jian Zhong*,3 1 Monash University, Parkville, VIC, Australia, 2Chinese Academy of Sciences, Beijing, China, 3Shanghai Ocean University, Shanghai, China
14.1
14
Introduction
Tumor targeting is attractive in anticancer therapy since it largely improves the therapeutic efficacy from increased local accumulation and lower systemic exposure. Tumor cells express molecules that can be markers for differentiating them from normal cells. Historically, such molecules were recognized and detected by antibodies but certain peptides have shown great potential through targeting and penetrating the tumor cell [1 4]. Cargo molecules can be attached to such peptides and then delivered into tumor tissue and enable enhanced drug delivery. The major obstacle for targeting of solid tumor is that drugs can only penetrate a few cell diameters into the extravascular tumor tissue from blood vessels [5]. Limited drug distribution within tumors is mainly due to the poorly organized vascular architecture, irregular perfused blood vessels, and high interstitial pressure which causes tissue fluid to flow out of the tumor [6 8]. The poor penetration from the leakiness of tumor vessels gives rise to the so-called enhanced permeability and retention (EPR) effect for passive targeting, but the EPR effect still suffers from variability from tumor to tumor and efficiency is still very low [9,10]. The discovery of cell-penetrating peptides (CPPs) provided enhanced permeability for passive targeting of payloads due to the capability of transporting the varying cargo across cell membranes. However, the nonspecific targeting to normal tissues can result in severe toxicity. Targeting the specific biology of tumor tissue by using different ligands could improve their efficacy due to more extensive penetration of the tumor tissue [2]. The targeted receptors can be on the surface of tumor cells, vascular cells, and stromal cells within tumors. Peptides that can specifically target tumor and tumor-associated microenvironments are often called “tumor homing peptides” (THPs) which can largely improve the selectivity of drug delivery. In 2002, Ruoslahti and collaborators discovered a type of peptide that can specifically recognize the endothelium of tumor vessels, extravagate, and penetrate deep into the extravascular tumor tissue and this type of peptide has been called a tumorpenetrating peptide (TPP). This chapter will focus on the discovery of TPPs, the mechanism for tumor targeting and the biomedical applications of TPPs for imaging and/or drug-delivery applications.
Corresponding authors: [email protected]; [email protected]
Peptide Applications in Biomedicine, Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-08-100736-5.00014-4 Copyright © 2018 Elsevier Ltd. All rights reserved.
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14.2
Peptide Applications in Biomedicine, Biotechnology and Bioengineering
Discovery of TPPs
14.2.1 Phage display In 1985, the discovery of a technology that enabled filamentous bacteriophages to be genetically engineered to express peptide sequences at the surface of phage particles lead to the development of libraries of phages which contains up to 109 different peptides [4,11]. Phage screening libraries can be used in vitro to isolate clones displaying peptides with the desired activity and selectivity to the target. In vivo phage screening allows the sequencing and identification of peptides in live animals, therefore, more specific binding at the target tissue can be achieved [12]. Since being introduced by Smith [11], phage display peptide libraries and the screening technique have uncovered many specific peptides for tumor targeting. Phage display is particularly suitable for the discovery of markers on the surface of tumor cells and tumor endothelial cells since the method relies on recognition of the accessible targets rather than overall expression levels. Tumor blood vessels express various cell surface and extracellular matrix proteins which are different from those expressed on the surface of normal vessels. The expressed proteins in cancer cells mostly result from neoangiogenesis, and the proteins are functionally important in this distinctive biological process [13,14]. In vivo phage screening mostly probes the molecular heterogeneity in the vasculature of normal and tumor tissues due to the reason that the phage particles (40 600 nm) do not readily penetrate the vascular wall [15]. Using this technique, Ruoslahti and collaborators have discovered a tumor-homing peptide, LyP-1, which was specifically accumulated in tumor lymphatics [16]. The receptor of LyP-1 is a p32 protein (gC1q receptor, hyaluronic acid-binding protein), expressed at the cell surface of lymphatic, myeloid, and cancer cells in tumors [17]. In vivo studies revealed that the peptide primarily targets myeloid/macrophages in tumors after intravenous injection into tumorbearing mice [17].
14.2.2 Markers in tumor vasculature Specific activations are present in vascular cells during specific responses and processes such as tumor growth and inflammation. For example, expression of a set of proteins including P-selectin, E-selectin, ICAM-1, and VCAM-1 can be turned on during the process of inflammation in venular endothelial cells allowing for leukocyte rolling and adhesion/migration in response to inflammation [18,19]. Another set of molecules including integrins, growth factor receptors, extracellular proteases, and extracellular matrix proteins, are expressed during angiogenesis, which makes tumor vasculature distinguishable from vasculature in normal tissues [18]. Among these, integrins have been intensively studied and are considered to be prime targets for tumor targeting since they are readily expressed on the surface of the endothelium and are available for the binding of bloodborne compounds. Integrins are the primary cell matrix adhesion molecules and each integrin is composed of noncovalently associated α and β subunits that combine to activate
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signaling pathways by coclustering with one or several extracellular matrix (ECM) components [20]. In particular, αvβ3 and αvβ5 integrins, which are highly expressed in the vessels of most malignant tumors, recognize the Arg-Gly-Asp (RGD) sequence in their respective substrates including fibronectin, vitronectin, von Willebrand factor, tenascin, osteopontin, fibrillin, fibrinogen, and thrombospondin [21,22]. Integrin-binding RGD motif-containing peptides, which have been used for enhanced drug delivery to tumors, have strongly stimulated the field of tumor targeting of diagnostics and therapeutic strategies [23].
14.3
Molecular structure of TPPs
14.3.1 CendR motif By using phage display technology, Ruoslahti and collaborators discovered that a peptide that combines the RGD sequence with a peptide motif for neuropilin-1 (NRP-1) can lead to both selective tumor vascular targeting and facilitate penetration through the vascular wall into extravascular tumor tissue [24,25]. The discovery of the CendR motif (arginine/lysine-X-X-arginine/lysine [R/KXXR/K]) by the same group revealed the mechanism for spreading within tumors. Two different peptides, LyP-1 and iRGD, both contain three independent modules including a vascular homing motif, a CendR tissue penetration motif, and a protease recognition site, were identified through phage display. The amino acid sequence and structure in the peptides allows for multistep penetration into tumors: First, the RGD motif recognizes and binds to αvβ3 and αvβ5 integrins; second, the CendR motif is cut off by a protease and the internal R/KXXR/K motif at the C-terminus of the truncated peptide is exposed; third, the CendR motif binds to neuropilin-1 (NRP-1) or neuropilin-2 (NRP-2) followed by the endocytotic/ exocytotic transport, leading to the enhanced transport of payloads into the tumor parenchyma (Fig. 14.1). This mechanism is called the CendR pathway. NRP1 is a cell-surface protein identified as a receptor for both the collapsin/semaphorin family of receptors and vascular endothelial growth factor 165 (VEGF165) (a key regulator of angiogenesis) [26]. The presence of free C-terminal arginine or lysine residues is required for the binding and internalization of the R/KXXR/K motif since the addition of other amino acid residues to the motif or amidation of the carboxyl terminus resulted in loss of activity [24]. Furthermore, the internalization of the NRP1 receptor after binding with CendR peptide can stimulate cell penetration [24]. Molecular modeling studies show that peptides with a C-terminal CendR motif, such as RPARPAR, fit well to the binding pocket at the b1b2 domain of NRP-1, but the role of the penultimate arginine residue outside the binding pocket is unclear (Fig. 14.2) [27,28]. Whereas the role of NRP-1 is to act as a coreceptor for various receptors to be recruited and presented to their receptors, overexpression of NRP-1 in tumor cells and stroma is believed to be implicated in the development and maintenance of tumor vessels and in tumor growth and progression [29]. Meanwhile, NRP-1 has been reported to be a target of anticancer therapy using
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Figure 14.1 Schematic representation of the CendR transport pathway [4]. The process of tumor penetration with TPPs: (1) binding with a primary receptor on tumor endothelial cells; (2) peptide cleavage by protease to expose the CendR motif; and (3) binding with neuropilin-1 (NRP-1) and mediating vascular and tissue permeability [4].
Figure 14.2 Ribbon representation of the NRP-1 RPAR complex [27].
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antibodies or peptides as targeting ligands [30 36]. However, targeting of NRP-1 suffers from limited tumor specificity due to expression in normal vessels and the plasma protease carboxypeptidases in the bloodstream can remove C-terminal arginine residues. Therefore, the efficacy of systemically active CendR peptides in tumor drug delivery is largely limited. Benefiting from the localized tumor-specific proteolytic activation, the exposed CendR motif in TPPs provides tumor-specific activation of cell and tissue penetration (Table 14.1).
14.3.2 Tumor-penetrating CendR peptides Another design of TPPs is based on a concept with two motifs for proteolytic activation and tumor-homing. The CendR motif containing the RGD peptide, iRGD (CRGDKGPDC), has been demonstrated to be able to carry 10 times more drug cargo into a tumor than the conventional RGD peptide [25]. Apart from the iRGD, other tumor-homing CendR peptides, such as F3 [64] and LyP-1 [16], have been shown to increase the extravasation of cargo loading and drug uptake to tumors [65,66]. The NGR tumor-homing motif, which binds to endothelial CD13 in angiogenic tumor vessels, was converted to a CendR motif (RNGR) and embedded in the iRGD framework to form a new peptide (CRNGRGPDC, iNGR). In comparison to NRG, the iNGR showed greater tumor penetration of coupled nanoparticles and coadministered compounds [59]. It has been demonstrated that the tumor penetration of CendR peptides is through an endosomal route and the process of tumor penetrating has been identified to be energy dependent, indicating an active transport pathway [18]. The CendR pathway has been characterized as starting with an endocytosis step and it is different from other endocytosis pathways. Initiated by an NRP receptor, the CendR endocytic vesicles resemble macropinocytotic vesicles with an average diameter of about 200 nm. A considerable volume of extracellular fluid and payloads within this size can be swept through into the pathway, leading to the remarkable feature of a coadministered effect. Under this mechanism, the therapeutic agent can be transported into the tumor parenchyma without conjugating to the CendR peptide [43]. A slight advantage of the coadministered modality was observed, whereas the conjugated modality gained better selective tumor penetration for drugs ranging in size from low-molecular-weight chemicals to nanoparticles and antibodies [43].
14.3.3 Cell-penetrating peptides Different from TPPs, CPPs generally refers to peptides that are internalized into cells. CPPs are small polypeptides that contain several positively charged amino acids, such as lysine or arginine, or having sequences that contain a pattern of alternating polar and nonpolar, hydrophobic amino acids [1]. There are two main types of structures: polycationic and amphipathic. The mechanism of cell uptake of CPPs still remains controversial since the methods employed by different labs are often not comparable. However, it has been shown that positively charged CPPs can target and penetrate into tumor cells in a receptor-independent manner [3,67].
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Table 14.1
Peptide Applications in Biomedicine, Biotechnology and Bioengineering
Tumor-penetrating peptides
Type of peptide
Type of application
References
iRGD
Cisplatin in nanoparticles (coadministration drug delivery)
[37]
iRGD
Gemcitabine (coadministration drug delivery)
[38,39]
iRGD
Doxorubicin, sorafenib (coadministration drug delivery)
[40,41]
iRGD
Nanoparticles containing doxorubicin (coadministration drug delivery)
[42]
iRGD
Nab-paclitaxel (conjugates drug delivery)
[25,43]
iRGD
Paclitaxel in nanoparticles (conjugates drug delivery)
[44]
iRGD
Thymopentin fused to iRGD (conjugates drug delivery)
[45]
iRGD
Doxorubicin in chitosan nanoparticles (conjugates drug delivery)
[46]
iRGD
Doxorubicin liposomes (conjugates drug delivery)
[47]
iRGD
Doxorubicin multilamellar liposomes (conjugates drug delivery)
[48]
iRGD
Paclitaxel nanoparticles (conjugates drug delivery)
[48]
iRGD
Oncolytic viruis (conjugates drug delivery)
[49]
iRGD
Paclitaxel liposomes (conjugates drug delivery)
[50]
iRGD
Paclitaxel and surviving siRNA in nanoparticles (conjugates drug delivery)
[51]
iRGD
Doxorubicin-containing exosomes (conjugates drug delivery)
[52]
iRGD
ATAP mitochondrial targeting peptide (conjugates drug delivery)
[53]
iRGD
Anti-EGFR (conjugates drug delivery)
[41]
iRGD
Nanomicelles containing salinomycin (conjugates drug delivery)
[54]
iRGD
Microbubbles for ultrasound imaging and photothermal/ photodynamic therapy
[55]
iRGD
Liposomal doxorubicin (conjugates drug delivery)
[56]
iRGD
Paclitaxel nanocrystallite (conjugates drug delivery)
[57]
iRGD
Sorafenib in porous silicon nanoparticles (conjugates drug delivery)
[58]
iNGR
Doxorubicin (coadministration drug delivery)
[59]
tLyP-1
Paclitaxel nanoparticles (coadministration drug delivery)
[60]
tLyp-1
Tumor targeted nanoparticles (coadministration drug delivery)
[61]
RPARPAR
Doxorubicin liposomes (conjugates drug delivery)
[62]
RGERPPR
Doxorubicin liposomes (conjugates drug delivery)
[63]
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Although CPPs have been widely applied as delivery vehicles in tumor diagnosis and therapy studies, in vivo application is still limited for two reasons. Firstly, the positively charged peptides can be easily taken up by the reticuloendothelial system (RES), leading to fast clearance from the blood circulation. Secondly, CPPs suffer from low specificity for targeting tumor cells versus normal cells, resulting in toxicity and insufficient therapy. To solve this problem, multistage nanocomplexes which carry targeting ligands that respond to the tumor microenvironment were designed including the activatable cell-penetrating peptide (ACPP) systems, deshielding systems, “pop-up” systems, and trojan-horse targeting systems. The cationic CPPs may also contain the CendR sequence motifs and it is conceivable that they may use the CendR pathway to enter cells. One such example is a herpes virus protein, VP22, which has been shown to have similar tumorpenetrating function [68,69]. However, the tissue-penetrating function in other CPPs is unclear. The mechanism of cell uptake can occur through different endocytosis routes including caveolae [70], micropinocytosis [71], or clathrin-dependent pathway [72], cholesterol-dependent clathrin-mediated pathway [73], or the transGolgi network [74]. The Tat peptide, which is a prototypic CPP, is commonly applied in delivering payloads into cells through the endocytic pathway [71,75]. Generally, the cell uptake is considered to be heparan sulfate-dependent and it can be inhibited with an excess of free heparan sulfate [76]. Different from CPPs, typical CendR peptides do not bind to heparan sulfate but only use the CendR pathway, even though the endocytic vesicles containing the two different pathway cargoes are morphologically similar. The distinctive feature of the two different pathways is that the CendR receptor, i.e., NRP1, is overexpressed in tumors while the heparan sulfate is widely expressed in other cell surfaces. Therefore, higher tumor specificity can be achieved with TPPs.
14.4
Tumor-penetrating CendR peptides in drug delivery
14.4.1 The drug penetration problem Drugs have to cross the vascular barrier and penetrate into the tumor stroma to reach tumor and tumor-associated parenchymal cells to effectively kill cancer cells. However, it has been demonstrated in many cases that drugs penetrate the equivalent of only a few cell diameters from blood vessels into the extravascular tumor tissue [77]. Due to the highly heterogeneous features of the cancerous tissue including (1) regional differences in tumor structure such as leaky vasculature and (2) defective lymphatics, and physiology such as inflammation, fibrosis, acidity, and hypoxia, the uptake and distribution of drugs can be entirely different in different regions. Small-molecule drugs, such as doxorubicin, may distribute up to a distance of about 40 μm [77], whereas larger drug molecules such as trastuzumab (herceptin antibody) are not detected inside many tumors [78]. As a result, low concentrations of drugs often cause drug resistance and the efficacy of chemotherapy to be low.
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The reasons for the poor penetration of drugs into tumor are: First, the tumor microenvironment constitutes a dense extracellular matrix which creates a physical barrier to the penetration of anticancer drugs into tumor tissue; and second, the abnormal structure and function of tumor vessels and the poorly functional lymphatic drainage leading to high interstitial pressure [77,78]. Dissolving extracellular matrix with enzymes such as collagenase or hyaluronidase and inhibiting the activity of tumor-associated fibroblasts can partially destroy the physical barrier and increase drug delivery [79,80]. To lower the interstitial pressure, vasodilatory compounds such as bradykinin, endothelin, and calcium channel antagonists can be used to allow better tumor perfusion and therefore increase the drug concentration at the tumor site [81]. However, tumor penetration still remains a challenge in cancer drug delivery, especially with nanoparticle drugs [4].
14.4.2 TPP-enhanced drug delivery Drug delivery with tumor-homing CendR peptides may overcome the tumor penetration problem. Drugs attached to CendR peptides can be delivered to the tumor tissue more effectively than the drug alone. Tumor penetration with fluorescein (FAM)-labeled peptides showed a fast distribution in tumor parenchyma within several minutes after intravenous administration in comparison to FAM-labeled control peptides. Meanwhile, the THPs that lack a CendR motif only showed binding to blood vessels [16,25,59,82]. Studies on iRGD and conventional RGD peptides, such as CRGDC and cRGDfK, demonstrated that iRGD peptides can deliver the payloads to tumor parenchyma, whereas the conventional RGD peptides only target tumor vessels [25,43]. The coadministration of CendR peptides with drugs can also overcome drug resistance in tumors. Ruoslahti et al. reported a study in a breast cancer xenograft model (BT474), which is essentially resistant to paclitaxel, showing effectiveness after the coadministration of nab-paclitaxel (Abraxane) nanoparticles together with iRGD or with iRGD coupled to the nanoparticle surface [43]. This finding may be explained by the fact that the drugs with the iRGD tumor endothelium-targeting domain enter cells through the CendR pathway rather than directly through the cell membrane [83,84]. Apart from iRGD, other CendR peptides also showed enhanced tumor penetration for drug delivery. For example, the LyP-1 peptide coupled with Abraxane nanoparticles, which is a clinically approved paclitaxel-albumin nanoparticle, have been demonstrated to produce more significant inhibition of tumor growth in mice bearing MDA-MB-435 human cancer model compared to the Abraxane nanoparticles without the tumor-targeting peptide [66]. Another CendR peptide, iNGR, possesses the same targeting ability as iRGD for delivery of doxorubicin in a mouse model [59]. Interestingly, drug delivery using CendR peptides can be achieved under the condition that the therapeutic agent is not linked to the peptides and can be enhanced by the peptides’ stimulatory effect (bystander effect). This strategy offers a number of advantages. First, a faster route for clinical translation can be provided since no new chemical entities are synthesized; second, there is no limitation to the
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number of available receptors, which is usually quite low on the surface of cells. Therefore, significant work has been performed to allow for the clinical translation of iRGD peptides. However, the bystander effect of intravenously injected iRGD can only last for a very short time since the blood half-life of the peptide is only about 8 min [75]. Obviously, extending the iRGD half-life will offer better effect for drug delivery. A more recent study by Jindˇrich Kopeˇcek et al. demonstrated that a stimuliresponsive drug-delivery system with iRGD conjugated to a HPMA copolymer DOX conjugate via a MMP-2 cleavable spacer can enhance cell accumulation and penetration of DOX in both monolayer and multicellular spheroid models of prostate cancer [85]. The main goal of this design was to develop a smart drug-delivery system to conquer the problem that the conjugated form of iRGD may lose its cell-penetrating/targeting ability from lack of exposure of the CendR motif. iRGD in the resulting HPMA copolymer was linked with a peptide sequence that can be released into the tumor microenvironment. Therefore, drug delivery and penetration were achieved through the coadministration effect from the free iRGD [86]. Moreover, this nanosystem can also prolong the half-life of iRGD as well as preserve its penetrating/targeting ability.
14.5
Tumor-penetrating CendR peptides in tumor imaging
CendR peptides can also be used to enhance tumor diagnosis using techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET). Superparamagnetic iron oxide nanoworms with sizes of about 80 nm long and 30 nm thick were conjugated with iRGD peptide and injected into a tumor-bearing mouse model. The results showed enhanced T2-weighted contrast in MR images 3 h postinjection (Fig. 14.3) [25]. CRGDC nanoworms caused decreased signal intensity in the tumor vasculature, whereas the untargeted nanoworms showed no significant contrast enhancement at the tumor site (Fig. 14.3). To some extent, the successful delivery of molecular imaging probes offers a promising tool for validating the drug-delivery efficacy of CendR peptides. A similar diagnostic platform was also developed using LyP-1 peptide as a targeting regent and it was tested in an atherosclerotic plaque model. After intravenous injection of the LyP-1-labeled superparamagnetic iron-oxide nanoworms, the plaques showed decreased signals in the interior of plaques which was also validated by the colocalization of p32 and CD68 markers in the plaques [87]. More sensitive detection was also achieved with PET imaging, which showed a specific accumulation of the LyP-1-labeled probe within the aortic plaque. Xiaoyuan Chen et al. investigated the imaging ability of iRGD peptide using a novel design of an optical coupling structure [88]. Significant tumor-targeting imaging was observed 4 h postinjection in an orthotopic MDA-MB-435 xenograft model, suggesting the importance of the iRGD targeting peptide for tumor imaging [88].
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Figure 14.3 Tumor imaging with iRGD-coated iron oxide nanoworms [25].
Recently, Zheng et al. constructed a multifunctional near-infrared (NIR) probe by using iRGD-modified indocyanine green (ICG) liposomes (iRGD ICG-LPs) for molecular imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) against breast tumors. The results from optical imaging suggested that iRGD ICG-LPs can target breast tumor with sensitive detection of breast tumor through NIR fluorescence molecular imaging. In vivo studies in a 4T1 tumor-bearing model showed significant tumor inhibition and almost complete tumor regression upon treatment of the animals with the iRGD ICG-LP formulations in comparison to control animals. Therefore, theranostic application for both tumor diagnosis and therapy can be achieved by integrating molecular imaging into PTT/PDT therapy [55].
14.6
Conclusion
TPPs have shown significant efficiency in delivering both covalently coupled and coadministered drug payloads deep into tumor tissues. Evidence from in vivo animal studies demonstrated that drug distribution in tumor tissue is significantly increased upon the use of such TPPs. Activated by the CendR motif with the NRP-
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1 receptor on the vasculature surface, TPPs can activate the process for the form of resembling micropinocytosis that can uptake the drug link to the peptide or be coadministered with it. Due to overexpression of the NRP-1 receptor in tumor tissues, CendR peptides are generally more specific than traditional CPP or vascular targeting peptides. However, the short blood half-life of the peptide largely reduces its overall efficacy. Therefore, future improvements will involve the rational design of TPP with increased blood circulation. Nevertheless, TPPs, such as iRGD, have been poised for clinical studies. Their applications in molecular imaging or tumors and in theranostics clearly suggest that the system is very promising not only for the functionalization of nanoparticle-based diagnostic tools but also for the treatment of several types of cancer.
Acknowledgments This work has been supported by research grants from the Special National Key Research and Development Plan (No. 2016YFD0400202-8) and the National Natural Science Foundation of China (No. 81571746).
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[33] K. Karjalainen, D.E. Jaalouk, C.E. Bueso-Ramos, et al., Targeting neuropilin-1 in human leukemia and lymphoma, Blood 117 (3) (2011) 920 927. [34] D. Bechet, L. Tirand, F. Plenat, et al., Targeting of neuropilin-1 to improve the antivascular effect of photodynamic therapy in xenograft human malignant glioma, EJC Suppl. 6 (9) (2008) 25 26. [35] L. Tirand, C. Frochot, R. Vanderesse, et al., A peptide competing with VEGF(165) binding on neuropilin-1 mediates targeting of a chlorin-type photosensitizer and potentiates its photodynamic activity in human endothelial cells, J. Control. Release 111 (1 2) (2006) 153 164. [36] C. Nasarre, M. Roth, L. Jacob, et al., Peptide-based interference of the transmembrane domain of neuropilin-1 inhibits glioma growth in vivo, Oncogene 29 (16) (2010) 2381 2392. [37] W.T. Song, M.Q. Li, Z.H. Tang, et al., Methoxypoly(ethylene glycol)-block-poly(Lglutamic acid)-loaded cisplatin and a combination with iRGD for the treatment of nonsmall-cell lung cancers, Macromol. Biosci. 12 (11) (2012) 1514 1523. [38] Y. Akashi, T. Oda, Y. Ohara, et al., Anticancer effects of gemcitabine are enhanced by co-administered iRGD peptide in murine pancreatic cancer models that overexpressed neuropilin-1, Br. J. Cancer 110 (6) (2014) 1481 1487. [39] Q. Zhang, Y. Zhang, K. Li, et al., A novel strategy to improve the therapeutic efficacy of gemcitabine for non-small cell lung cancer by the tumor-penetrating peptide iRGD, PLoS One 10 (6) (2015) e0129865. [40] C. Schmithals, V. Koberle, H. Korkusuz, et al., Improving drug penetrability with iRGD leverages the therapeutic response to sorafenib and doxorubicin in hepatocellular carcinoma, Cancer Res. 75 (15) (2015) 3147 3154. [41] H.Z. Sha, Z.Y. Zou, K. Xin, et al., Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy, J. Control. Release 200 (2015) 188 200. [42] X.L. Cun, J.T. Chen, S.B. Ruan, et al., A novel strategy through combining iRGD peptide with tumor-microenvironment-responsive and multistage nanoparticles for deep tumor penetration, ACS Appl. Mater. Interfaces 7 (49) (2015) 27458 27466. [43] K.N. Sugahara, T. Teesalu, P.P. Karmali, et al., Coadministration of a tumorpenetrating peptide enhances the efficacy of cancer drugs, Science 328 (5981) (2010) 1031 1035. [44] Z.S. Zhu, C. Xie, Q. Liu, et al., The effect of hydrophilic chain length and iRGD on drug delivery from poly(epsilon-caprolactone)-poly(N-vinylpyrrolidone) nanoparticles, Biomaterials 32 (35) (2011) 9525 9535. [45] X.Z. Lao, M. Liu, J. Chen, et al., A tumor-penetrating peptide modification enhances the antitumor activity of thymosin alpha 1, PLoS One 8 (8) (2013) e72242. [46] X. Wang, X. Zhen, J. Wang, et al., Doxorubicin delivery to 3D multicellular spheroids and tumors based on boronic acid-rich chitosan nanoparticles, Biomaterials 34 (19) (2013) 4667 4679. [47] K.F. Yu, W.Q. Zhang, L.M. Luo, et al., The antitumor activity of a doxorubicin loaded, iRGD-modified sterically-stabilized liposome on B16-F10 melanoma cells: in vitro and in vivo evaluation, Int. J. Nanomedicine 8 (2013) 2473 2485. [48] G.Z. Gu, X.L. Gao, Q.Y. Hu, et al., The influence of the penetrating peptide iRGD on the effect of paclitaxel-loaded MT1-AF7p-conjugated nanoparticles on glioma cells, Biomaterials 34 (21) (2013) 5138 5148. [49] C. Puig-Saus, L.A. Rojas, E. Laborda, et al., iRGD tumor-penetrating peptide-modified oncolytic adenovirus shows enhanced tumor transduction, intratumoral dissemination and antitumor efficacy, Gene Ther. 21 (8) (2014) 767 774.
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[50] R. Du, T. Zhong, W.Q. Zhang, et al., Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid-paclitaxel (CLA-PTX) on B16-F10 melanoma, Int. J. Nanomedicine 9 (2014) 3091 3105. [51] J.N. Shen, Q.S. Meng, H.P. Sui, et al., iRGD conjugated TPGS mediates codelivery of paclitaxel and survivin shRNA for the reversal of lung cancer resistance, Mol. Pharm. 11 (8) (2014) 2579 2591. [52] Y.H. Tian, S.P. Li, J. Song, et al., A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy, Biomaterials 35 (7) (2014) 2383 2390. [53] G.J. De, J.K. Ko, T. Tan, et al., Amphipathic tail-anchoring peptide is a promising therapeutic agent for prostate cancer treatment, Oncotarget 5 (17) (2014) 7734 7747. [54] X.L. Mao, J.J. Liu, Z.R. Gong, et al., iRGD-conjugated DSPE-PEG2000 nanomicelles for targeted delivery of salinomycin for treatment of both liver cancer cells and cancer stem cells, Nanomedicine (Lond.) 10 (17) (2015) 2677 2695. [55] F. Yan, H. Wu, H.M. Liu, et al., Molecular imaging-guided photothermal/ photodynamic therapy against tumor by iRGD-modified indocyanine green nanoparticles, J. Control. Release 224 (2016) 217 228. [56] W.B. Dai, Y.C. Fan, H. Zhang, et al., A comprehensive study of iRGD-modified liposomes with improved chemotherapeutic efficacy on B16 melanoma, Drug Deliv. 22 (1) (2015) 10 20. [57] D.Z. Ni, H. Ding, S. Liu, et al., Superior intratumoral penetration of paclitaxel nanodots strengthens tumor restriction and metastasis prevention, Small 11 (21) (2015) 2518 2526. [58] M.Q. Li, Z.H. Tang, D.W. Zhang, et al., Doxorubicin-loaded polysaccharide nanoparticles suppress the growth of murine colorectal carcinoma and inhibit the metastasis of murine mammary carcinoma in rodent models, Biomaterials 51 (2015) 161 172. [59] L. Alberici, L. Roth, K.N. Sugahara, et al., De novo design of a tumor-penetrating peptide, Cancer Res. 73 (2) (2013) 804 812. [60] D.Y. Miao, M.Y. Jiang, Z.Y. Liu, et al., Co-administration of dual-targeting nanoparticles with penetration enhancement peptide for antiglioblastoma therapy, Mol. Pharm. 11 (1) (2014) 90 101. [61] Q.Y. Hu, G.Z. Gu, Z.Y. Liu, et al., F3 peptide-functionalized PEG-PLA nanoparticles co-administrated with tLyp-1 peptide for anti-glioma drug delivery, Biomaterials 34 (4) (2013) 1135 1145. [62] Z.Q. Yan, Y.Y. Yang, X.L. Wei, et al., Tumor-penetrating peptide mediation: an effective strategy for improving the transport of liposomes in tumor tissue, Mol. Pharm. 11 (1) (2014) 218 225. [63] Y.Y. Yang, Z.Q. Yan, D.X. Wei, et al., Tumor-penetrating peptide functionalization enhances the anti-glioblastoma effect of doxorubicin liposomes, Nanotechnology 24 (40) (2013) 405101. [64] V. Fogal, K.N. Sugahara, E. Ruoslahti, et al., Cell surface nucleolin antagonist causes endothelial cell apoptosis and normalization of tumor vasculature, Angiogenesis 12 (1) (2009) 91 100. [65] K. Porkka, P. Laakkonen, J.A. Hoffman, et al., A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo, Proc. Natl. Acad. Sci. U. S. A. 99 (11) (2002) 7444 7449. [66] P.P. Karmali, V.R. Kotamraju, M. Kastantin, et al., Targeting of albumin-embedded paclitaxel nanoparticles to tumors, Nanomedicine 5 (1) (2009) 73 82.
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[67] J.Q. Fu, C.M. Yu, L. Li, et al., Intracellular delivery of functional proteins and native drugs by cell-penetrating poly(disulfide)s, J. Am. Chem. Soc. 137 (37) (2015) 12153 12160. [68] N.D. Brewis, A. Phelan, N. Normand, et al., Particle assembly incorporating a VP22BH3 fusion protein, facilitating intracellular delivery, regulated release, and apoptosis, Mol. Ther. 7 (2) (2003) 262 270. [69] X.D. Xue, J.H. Huang, H.S. Wang, The study of the intercellular trafficking of the fusion proteins of herpes simplex virus protein VP22, PLoS One 9 (6) (2014) e100840. [70] A. Fittipaldi, A. Ferrari, M. Zoppe, et al., Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins, J. Biol. Chem. 278 (36) (2003) 34141 34149. [71] I.M. Kaplan, J.S. Wadia, S.F. Dowdy, Cationic TAT peptide transduction domain enters cells by macropinocytosis, J. Control. Release 102 (1) (2005) 247 253. [72] J.P. Richard, K. Melikov, H. Brooks, et al., Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors, J. Biol. Chem. 280 (15) (2005) 15300 15306. [73] R.E. Vandenbroucke, S.C. De Smedt, J. Demeester, et al., Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes, Biochim. Biophys. Acta 1768 (3) (2007) 571 579. [74] R. Fischer, K. Kohler, M. Fotin-Mleczek, et al., A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides, J. Biol. Chem. 279 (13) (2004) 12625 12635. [75] H.B. Pang, G.B. Braun, T. Friman, et al., An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability, Nat. Commun. 5 (2014) 4904. [76] J.M. Gump, S.F. Dowdy, TAT transduction: the molecular mechanism and therapeutic prospects, Trends Mol. Med. 13 (10) (2007) 443 448. [77] A.J. Primeau, A. Rendon, D. Hedley, et al., The distribution of the anticancer drug doxorubicin in relation to blood vessels in solid tumors, Clin. Cancer Res. 11 (24) (2005) 8782 8788. [78] C.H. Heldin, K. Rubin, K. Pietras, et al., High interstitial fluid pressure: an obstacle in cancer therapy, Nat. Rev. Cancer 4 (10) (2004) 806 813. [79] I. Tufto, R. Hansen, D. Byberg, et al., The effect of collagenase and hyaluronidase on transient perfusion in human osteosarcoma xenografts grown orthotopically and in dorsal skinfold chambers, Anticancer Res. 27 (3b) (2007) 1475 1481. [80] F. Xing, J. Saidou, K. Watabe, Cancer associated fibroblasts (CAFs) in tumor microenvironment, Front. Biosci. (Landmark Ed.) 15 (2010) 166 179. [81] O. Feron, Targeting the tumor vascular compartment to improve conventional cancer therapy, Trends Pharm. Sci. 25 (10) (2004) 536 542. [82] P. Laakkonen, M.E. Akerman, H. Biliran, et al., Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells, Proc. Natl. Acad. Sci. U. S. A. 101 (25) (2004) 9381 9386. [83] C.M.J. Hu, L.F. Zhang, Therapeutic nanoparticles to combat cancer drug resistance, Curr. Drug Metab. 10 (8) (2009) 836 841. [84] J. Huwyler, A. Cerletti, G. Fricker, et al., By-passing of P-glycoprotein using immunoliposomes, J. Drug Target. 10 (1) (2002) 73 79. [85] Z.H. Peng, J. Kopecek, Enhancing accumulation and penetration of HPMA copolymerdoxorubicin conjugates in 2D and 3D prostate cancer cells via iRGD conjugation with an MMP-2 cleavable spacer, J. Am. Chem. Soc. 137 (21) (2015) 6726 6729.
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[86] Z.H. Peng, J. Kopecek, Synthesis and activity of tumor-homing peptide iRGD and histone deacetylase inhibitor valproic acid conjugate, Bioorg. Med. Chem. Lett. 24 (8) (2014) 1928 1933. [87] J. Hamzah, V.R. Kotamraju, J.W. Seo, et al., Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice, Proc. Natl. Acad. Sci. U. S. A. 108 (17) (2011) 7154 7159. [88] Y.P. Ye, L. Zhu, Y. Ma, et al., Synthesis and evaluation of new iRGD peptide analogs for tumor optical imaging, Bioorg. Med. Chem. Lett. 21 (4) (2011) 1146 1150.
14.1
14
Introduction
Tumor targeting is attractive in anticancer therapy since it largely improves the therapeutic efficacy from increased local accumulation and lower systemic exposure. Tumor cells express molecules that can be markers for differentiating them from normal cells. Historically, such molecules were recognized and detected by antibodies but certain peptides have shown great potential through targeting and penetrating the tumor cell [1 4]. Cargo molecules can be attached to such peptides and then delivered into tumor tissue and enable enhanced drug delivery. The major obstacle for targeting of solid tumor is that drugs can only penetrate a few cell diameters into the extravascular tumor tissue from blood vessels [5]. Limited drug distribution within tumors is mainly due to the poorly organized vascular architecture, irregular perfused blood vessels, and high interstitial pressure which causes tissue fluid to flow out of the tumor [6 8]. The poor penetration from the leakiness of tumor vessels gives rise to the so-called enhanced permeability and retention (EPR) effect for passive targeting, but the EPR effect still suffers from variability from tumor to tumor and efficiency is still very low [9,10]. The discovery of cell-penetrating peptides (CPPs) provided enhanced permeability for passive targeting of payloads due to the capability of transporting the varying cargo across cell membranes. However, the nonspecific targeting to normal tissues can result in severe toxicity. Targeting the specific biology of tumor tissue by using different ligands could improve their efficacy due to more extensive penetration of the tumor tissue [2]. The targeted receptors can be on the surface of tumor cells, vascular cells, and stromal cells within tumors. Peptides that can specifically target tumor and tumor-associated microenvironments are often called “tumor homing peptides” (THPs) which can largely improve the selectivity of drug delivery. In 2002, Ruoslahti and collaborators discovered a type of peptide that can specifically recognize the endothelium of tumor vessels, extravagate, and penetrate deep into the extravascular tumor tissue and this type of peptide has been called a tumorpenetrating peptide (TPP). This chapter will focus on the discovery of TPPs, the mechanism for tumor targeting and the biomedical applications of TPPs for imaging and/or drug-delivery applications.
Corresponding authors: [email protected]; [email protected]
Peptide Applications in Biomedicine, Biotechnology and Bioengineering. DOI: http://dx.doi.org/10.1016/B978-0-08-100736-5.00014-4 Copyright © 2018 Elsevier Ltd. All rights reserved.
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14.2
Peptide Applications in Biomedicine, Biotechnology and Bioengineering
Discovery of TPPs
14.2.1 Phage display In 1985, the discovery of a technology that enabled filamentous bacteriophages to be genetically engineered to express peptide sequences at the surface of phage particles lead to the development of libraries of phages which contains up to 109 different peptides [4,11]. Phage screening libraries can be used in vitro to isolate clones displaying peptides with the desired activity and selectivity to the target. In vivo phage screening allows the sequencing and identification of peptides in live animals, therefore, more specific binding at the target tissue can be achieved [12]. Since being introduced by Smith [11], phage display peptide libraries and the screening technique have uncovered many specific peptides for tumor targeting. Phage display is particularly suitable for the discovery of markers on the surface of tumor cells and tumor endothelial cells since the method relies on recognition of the accessible targets rather than overall expression levels. Tumor blood vessels express various cell surface and extracellular matrix proteins which are different from those expressed on the surface of normal vessels. The expressed proteins in cancer cells mostly result from neoangiogenesis, and the proteins are functionally important in this distinctive biological process [13,14]. In vivo phage screening mostly probes the molecular heterogeneity in the vasculature of normal and tumor tissues due to the reason that the phage particles (40 600 nm) do not readily penetrate the vascular wall [15]. Using this technique, Ruoslahti and collaborators have discovered a tumor-homing peptide, LyP-1, which was specifically accumulated in tumor lymphatics [16]. The receptor of LyP-1 is a p32 protein (gC1q receptor, hyaluronic acid-binding protein), expressed at the cell surface of lymphatic, myeloid, and cancer cells in tumors [17]. In vivo studies revealed that the peptide primarily targets myeloid/macrophages in tumors after intravenous injection into tumorbearing mice [17].
14.2.2 Markers in tumor vasculature Specific activations are present in vascular cells during specific responses and processes such as tumor growth and inflammation. For example, expression of a set of proteins including P-selectin, E-selectin, ICAM-1, and VCAM-1 can be turned on during the process of inflammation in venular endothelial cells allowing for leukocyte rolling and adhesion/migration in response to inflammation [18,19]. Another set of molecules including integrins, growth factor receptors, extracellular proteases, and extracellular matrix proteins, are expressed during angiogenesis, which makes tumor vasculature distinguishable from vasculature in normal tissues [18]. Among these, integrins have been intensively studied and are considered to be prime targets for tumor targeting since they are readily expressed on the surface of the endothelium and are available for the binding of bloodborne compounds. Integrins are the primary cell matrix adhesion molecules and each integrin is composed of noncovalently associated α and β subunits that combine to activate
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signaling pathways by coclustering with one or several extracellular matrix (ECM) components [20]. In particular, αvβ3 and αvβ5 integrins, which are highly expressed in the vessels of most malignant tumors, recognize the Arg-Gly-Asp (RGD) sequence in their respective substrates including fibronectin, vitronectin, von Willebrand factor, tenascin, osteopontin, fibrillin, fibrinogen, and thrombospondin [21,22]. Integrin-binding RGD motif-containing peptides, which have been used for enhanced drug delivery to tumors, have strongly stimulated the field of tumor targeting of diagnostics and therapeutic strategies [23].
14.3
Molecular structure of TPPs
14.3.1 CendR motif By using phage display technology, Ruoslahti and collaborators discovered that a peptide that combines the RGD sequence with a peptide motif for neuropilin-1 (NRP-1) can lead to both selective tumor vascular targeting and facilitate penetration through the vascular wall into extravascular tumor tissue [24,25]. The discovery of the CendR motif (arginine/lysine-X-X-arginine/lysine [R/KXXR/K]) by the same group revealed the mechanism for spreading within tumors. Two different peptides, LyP-1 and iRGD, both contain three independent modules including a vascular homing motif, a CendR tissue penetration motif, and a protease recognition site, were identified through phage display. The amino acid sequence and structure in the peptides allows for multistep penetration into tumors: First, the RGD motif recognizes and binds to αvβ3 and αvβ5 integrins; second, the CendR motif is cut off by a protease and the internal R/KXXR/K motif at the C-terminus of the truncated peptide is exposed; third, the CendR motif binds to neuropilin-1 (NRP-1) or neuropilin-2 (NRP-2) followed by the endocytotic/ exocytotic transport, leading to the enhanced transport of payloads into the tumor parenchyma (Fig. 14.1). This mechanism is called the CendR pathway. NRP1 is a cell-surface protein identified as a receptor for both the collapsin/semaphorin family of receptors and vascular endothelial growth factor 165 (VEGF165) (a key regulator of angiogenesis) [26]. The presence of free C-terminal arginine or lysine residues is required for the binding and internalization of the R/KXXR/K motif since the addition of other amino acid residues to the motif or amidation of the carboxyl terminus resulted in loss of activity [24]. Furthermore, the internalization of the NRP1 receptor after binding with CendR peptide can stimulate cell penetration [24]. Molecular modeling studies show that peptides with a C-terminal CendR motif, such as RPARPAR, fit well to the binding pocket at the b1b2 domain of NRP-1, but the role of the penultimate arginine residue outside the binding pocket is unclear (Fig. 14.2) [27,28]. Whereas the role of NRP-1 is to act as a coreceptor for various receptors to be recruited and presented to their receptors, overexpression of NRP-1 in tumor cells and stroma is believed to be implicated in the development and maintenance of tumor vessels and in tumor growth and progression [29]. Meanwhile, NRP-1 has been reported to be a target of anticancer therapy using
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Figure 14.1 Schematic representation of the CendR transport pathway [4]. The process of tumor penetration with TPPs: (1) binding with a primary receptor on tumor endothelial cells; (2) peptide cleavage by protease to expose the CendR motif; and (3) binding with neuropilin-1 (NRP-1) and mediating vascular and tissue permeability [4].
Figure 14.2 Ribbon representation of the NRP-1 RPAR complex [27].
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antibodies or peptides as targeting ligands [30 36]. However, targeting of NRP-1 suffers from limited tumor specificity due to expression in normal vessels and the plasma protease carboxypeptidases in the bloodstream can remove C-terminal arginine residues. Therefore, the efficacy of systemically active CendR peptides in tumor drug delivery is largely limited. Benefiting from the localized tumor-specific proteolytic activation, the exposed CendR motif in TPPs provides tumor-specific activation of cell and tissue penetration (Table 14.1).
14.3.2 Tumor-penetrating CendR peptides Another design of TPPs is based on a concept with two motifs for proteolytic activation and tumor-homing. The CendR motif containing the RGD peptide, iRGD (CRGDKGPDC), has been demonstrated to be able to carry 10 times more drug cargo into a tumor than the conventional RGD peptide [25]. Apart from the iRGD, other tumor-homing CendR peptides, such as F3 [64] and LyP-1 [16], have been shown to increase the extravasation of cargo loading and drug uptake to tumors [65,66]. The NGR tumor-homing motif, which binds to endothelial CD13 in angiogenic tumor vessels, was converted to a CendR motif (RNGR) and embedded in the iRGD framework to form a new peptide (CRNGRGPDC, iNGR). In comparison to NRG, the iNGR showed greater tumor penetration of coupled nanoparticles and coadministered compounds [59]. It has been demonstrated that the tumor penetration of CendR peptides is through an endosomal route and the process of tumor penetrating has been identified to be energy dependent, indicating an active transport pathway [18]. The CendR pathway has been characterized as starting with an endocytosis step and it is different from other endocytosis pathways. Initiated by an NRP receptor, the CendR endocytic vesicles resemble macropinocytotic vesicles with an average diameter of about 200 nm. A considerable volume of extracellular fluid and payloads within this size can be swept through into the pathway, leading to the remarkable feature of a coadministered effect. Under this mechanism, the therapeutic agent can be transported into the tumor parenchyma without conjugating to the CendR peptide [43]. A slight advantage of the coadministered modality was observed, whereas the conjugated modality gained better selective tumor penetration for drugs ranging in size from low-molecular-weight chemicals to nanoparticles and antibodies [43].
14.3.3 Cell-penetrating peptides Different from TPPs, CPPs generally refers to peptides that are internalized into cells. CPPs are small polypeptides that contain several positively charged amino acids, such as lysine or arginine, or having sequences that contain a pattern of alternating polar and nonpolar, hydrophobic amino acids [1]. There are two main types of structures: polycationic and amphipathic. The mechanism of cell uptake of CPPs still remains controversial since the methods employed by different labs are often not comparable. However, it has been shown that positively charged CPPs can target and penetrate into tumor cells in a receptor-independent manner [3,67].
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Table 14.1
Peptide Applications in Biomedicine, Biotechnology and Bioengineering
Tumor-penetrating peptides
Type of peptide
Type of application
References
iRGD
Cisplatin in nanoparticles (coadministration drug delivery)
[37]
iRGD
Gemcitabine (coadministration drug delivery)
[38,39]
iRGD
Doxorubicin, sorafenib (coadministration drug delivery)
[40,41]
iRGD
Nanoparticles containing doxorubicin (coadministration drug delivery)
[42]
iRGD
Nab-paclitaxel (conjugates drug delivery)
[25,43]
iRGD
Paclitaxel in nanoparticles (conjugates drug delivery)
[44]
iRGD
Thymopentin fused to iRGD (conjugates drug delivery)
[45]
iRGD
Doxorubicin in chitosan nanoparticles (conjugates drug delivery)
[46]
iRGD
Doxorubicin liposomes (conjugates drug delivery)
[47]
iRGD
Doxorubicin multilamellar liposomes (conjugates drug delivery)
[48]
iRGD
Paclitaxel nanoparticles (conjugates drug delivery)
[48]
iRGD
Oncolytic viruis (conjugates drug delivery)
[49]
iRGD
Paclitaxel liposomes (conjugates drug delivery)
[50]
iRGD
Paclitaxel and surviving siRNA in nanoparticles (conjugates drug delivery)
[51]
iRGD
Doxorubicin-containing exosomes (conjugates drug delivery)
[52]
iRGD
ATAP mitochondrial targeting peptide (conjugates drug delivery)
[53]
iRGD
Anti-EGFR (conjugates drug delivery)
[41]
iRGD
Nanomicelles containing salinomycin (conjugates drug delivery)
[54]
iRGD
Microbubbles for ultrasound imaging and photothermal/ photodynamic therapy
[55]
iRGD
Liposomal doxorubicin (conjugates drug delivery)
[56]
iRGD
Paclitaxel nanocrystallite (conjugates drug delivery)
[57]
iRGD
Sorafenib in porous silicon nanoparticles (conjugates drug delivery)
[58]
iNGR
Doxorubicin (coadministration drug delivery)
[59]
tLyP-1
Paclitaxel nanoparticles (coadministration drug delivery)
[60]
tLyp-1
Tumor targeted nanoparticles (coadministration drug delivery)
[61]
RPARPAR
Doxorubicin liposomes (conjugates drug delivery)
[62]
RGERPPR
Doxorubicin liposomes (conjugates drug delivery)
[63]
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Although CPPs have been widely applied as delivery vehicles in tumor diagnosis and therapy studies, in vivo application is still limited for two reasons. Firstly, the positively charged peptides can be easily taken up by the reticuloendothelial system (RES), leading to fast clearance from the blood circulation. Secondly, CPPs suffer from low specificity for targeting tumor cells versus normal cells, resulting in toxicity and insufficient therapy. To solve this problem, multistage nanocomplexes which carry targeting ligands that respond to the tumor microenvironment were designed including the activatable cell-penetrating peptide (ACPP) systems, deshielding systems, “pop-up” systems, and trojan-horse targeting systems. The cationic CPPs may also contain the CendR sequence motifs and it is conceivable that they may use the CendR pathway to enter cells. One such example is a herpes virus protein, VP22, which has been shown to have similar tumorpenetrating function [68,69]. However, the tissue-penetrating function in other CPPs is unclear. The mechanism of cell uptake can occur through different endocytosis routes including caveolae [70], micropinocytosis [71], or clathrin-dependent pathway [72], cholesterol-dependent clathrin-mediated pathway [73], or the transGolgi network [74]. The Tat peptide, which is a prototypic CPP, is commonly applied in delivering payloads into cells through the endocytic pathway [71,75]. Generally, the cell uptake is considered to be heparan sulfate-dependent and it can be inhibited with an excess of free heparan sulfate [76]. Different from CPPs, typical CendR peptides do not bind to heparan sulfate but only use the CendR pathway, even though the endocytic vesicles containing the two different pathway cargoes are morphologically similar. The distinctive feature of the two different pathways is that the CendR receptor, i.e., NRP1, is overexpressed in tumors while the heparan sulfate is widely expressed in other cell surfaces. Therefore, higher tumor specificity can be achieved with TPPs.
14.4
Tumor-penetrating CendR peptides in drug delivery
14.4.1 The drug penetration problem Drugs have to cross the vascular barrier and penetrate into the tumor stroma to reach tumor and tumor-associated parenchymal cells to effectively kill cancer cells. However, it has been demonstrated in many cases that drugs penetrate the equivalent of only a few cell diameters from blood vessels into the extravascular tumor tissue [77]. Due to the highly heterogeneous features of the cancerous tissue including (1) regional differences in tumor structure such as leaky vasculature and (2) defective lymphatics, and physiology such as inflammation, fibrosis, acidity, and hypoxia, the uptake and distribution of drugs can be entirely different in different regions. Small-molecule drugs, such as doxorubicin, may distribute up to a distance of about 40 μm [77], whereas larger drug molecules such as trastuzumab (herceptin antibody) are not detected inside many tumors [78]. As a result, low concentrations of drugs often cause drug resistance and the efficacy of chemotherapy to be low.
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The reasons for the poor penetration of drugs into tumor are: First, the tumor microenvironment constitutes a dense extracellular matrix which creates a physical barrier to the penetration of anticancer drugs into tumor tissue; and second, the abnormal structure and function of tumor vessels and the poorly functional lymphatic drainage leading to high interstitial pressure [77,78]. Dissolving extracellular matrix with enzymes such as collagenase or hyaluronidase and inhibiting the activity of tumor-associated fibroblasts can partially destroy the physical barrier and increase drug delivery [79,80]. To lower the interstitial pressure, vasodilatory compounds such as bradykinin, endothelin, and calcium channel antagonists can be used to allow better tumor perfusion and therefore increase the drug concentration at the tumor site [81]. However, tumor penetration still remains a challenge in cancer drug delivery, especially with nanoparticle drugs [4].
14.4.2 TPP-enhanced drug delivery Drug delivery with tumor-homing CendR peptides may overcome the tumor penetration problem. Drugs attached to CendR peptides can be delivered to the tumor tissue more effectively than the drug alone. Tumor penetration with fluorescein (FAM)-labeled peptides showed a fast distribution in tumor parenchyma within several minutes after intravenous administration in comparison to FAM-labeled control peptides. Meanwhile, the THPs that lack a CendR motif only showed binding to blood vessels [16,25,59,82]. Studies on iRGD and conventional RGD peptides, such as CRGDC and cRGDfK, demonstrated that iRGD peptides can deliver the payloads to tumor parenchyma, whereas the conventional RGD peptides only target tumor vessels [25,43]. The coadministration of CendR peptides with drugs can also overcome drug resistance in tumors. Ruoslahti et al. reported a study in a breast cancer xenograft model (BT474), which is essentially resistant to paclitaxel, showing effectiveness after the coadministration of nab-paclitaxel (Abraxane) nanoparticles together with iRGD or with iRGD coupled to the nanoparticle surface [43]. This finding may be explained by the fact that the drugs with the iRGD tumor endothelium-targeting domain enter cells through the CendR pathway rather than directly through the cell membrane [83,84]. Apart from iRGD, other CendR peptides also showed enhanced tumor penetration for drug delivery. For example, the LyP-1 peptide coupled with Abraxane nanoparticles, which is a clinically approved paclitaxel-albumin nanoparticle, have been demonstrated to produce more significant inhibition of tumor growth in mice bearing MDA-MB-435 human cancer model compared to the Abraxane nanoparticles without the tumor-targeting peptide [66]. Another CendR peptide, iNGR, possesses the same targeting ability as iRGD for delivery of doxorubicin in a mouse model [59]. Interestingly, drug delivery using CendR peptides can be achieved under the condition that the therapeutic agent is not linked to the peptides and can be enhanced by the peptides’ stimulatory effect (bystander effect). This strategy offers a number of advantages. First, a faster route for clinical translation can be provided since no new chemical entities are synthesized; second, there is no limitation to the
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number of available receptors, which is usually quite low on the surface of cells. Therefore, significant work has been performed to allow for the clinical translation of iRGD peptides. However, the bystander effect of intravenously injected iRGD can only last for a very short time since the blood half-life of the peptide is only about 8 min [75]. Obviously, extending the iRGD half-life will offer better effect for drug delivery. A more recent study by Jindˇrich Kopeˇcek et al. demonstrated that a stimuliresponsive drug-delivery system with iRGD conjugated to a HPMA copolymer DOX conjugate via a MMP-2 cleavable spacer can enhance cell accumulation and penetration of DOX in both monolayer and multicellular spheroid models of prostate cancer [85]. The main goal of this design was to develop a smart drug-delivery system to conquer the problem that the conjugated form of iRGD may lose its cell-penetrating/targeting ability from lack of exposure of the CendR motif. iRGD in the resulting HPMA copolymer was linked with a peptide sequence that can be released into the tumor microenvironment. Therefore, drug delivery and penetration were achieved through the coadministration effect from the free iRGD [86]. Moreover, this nanosystem can also prolong the half-life of iRGD as well as preserve its penetrating/targeting ability.
14.5
Tumor-penetrating CendR peptides in tumor imaging
CendR peptides can also be used to enhance tumor diagnosis using techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET). Superparamagnetic iron oxide nanoworms with sizes of about 80 nm long and 30 nm thick were conjugated with iRGD peptide and injected into a tumor-bearing mouse model. The results showed enhanced T2-weighted contrast in MR images 3 h postinjection (Fig. 14.3) [25]. CRGDC nanoworms caused decreased signal intensity in the tumor vasculature, whereas the untargeted nanoworms showed no significant contrast enhancement at the tumor site (Fig. 14.3). To some extent, the successful delivery of molecular imaging probes offers a promising tool for validating the drug-delivery efficacy of CendR peptides. A similar diagnostic platform was also developed using LyP-1 peptide as a targeting regent and it was tested in an atherosclerotic plaque model. After intravenous injection of the LyP-1-labeled superparamagnetic iron-oxide nanoworms, the plaques showed decreased signals in the interior of plaques which was also validated by the colocalization of p32 and CD68 markers in the plaques [87]. More sensitive detection was also achieved with PET imaging, which showed a specific accumulation of the LyP-1-labeled probe within the aortic plaque. Xiaoyuan Chen et al. investigated the imaging ability of iRGD peptide using a novel design of an optical coupling structure [88]. Significant tumor-targeting imaging was observed 4 h postinjection in an orthotopic MDA-MB-435 xenograft model, suggesting the importance of the iRGD targeting peptide for tumor imaging [88].
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Figure 14.3 Tumor imaging with iRGD-coated iron oxide nanoworms [25].
Recently, Zheng et al. constructed a multifunctional near-infrared (NIR) probe by using iRGD-modified indocyanine green (ICG) liposomes (iRGD ICG-LPs) for molecular imaging-guided photothermal therapy (PTT) and photodynamic therapy (PDT) against breast tumors. The results from optical imaging suggested that iRGD ICG-LPs can target breast tumor with sensitive detection of breast tumor through NIR fluorescence molecular imaging. In vivo studies in a 4T1 tumor-bearing model showed significant tumor inhibition and almost complete tumor regression upon treatment of the animals with the iRGD ICG-LP formulations in comparison to control animals. Therefore, theranostic application for both tumor diagnosis and therapy can be achieved by integrating molecular imaging into PTT/PDT therapy [55].
14.6
Conclusion
TPPs have shown significant efficiency in delivering both covalently coupled and coadministered drug payloads deep into tumor tissues. Evidence from in vivo animal studies demonstrated that drug distribution in tumor tissue is significantly increased upon the use of such TPPs. Activated by the CendR motif with the NRP-
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1 receptor on the vasculature surface, TPPs can activate the process for the form of resembling micropinocytosis that can uptake the drug link to the peptide or be coadministered with it. Due to overexpression of the NRP-1 receptor in tumor tissues, CendR peptides are generally more specific than traditional CPP or vascular targeting peptides. However, the short blood half-life of the peptide largely reduces its overall efficacy. Therefore, future improvements will involve the rational design of TPP with increased blood circulation. Nevertheless, TPPs, such as iRGD, have been poised for clinical studies. Their applications in molecular imaging or tumors and in theranostics clearly suggest that the system is very promising not only for the functionalization of nanoparticle-based diagnostic tools but also for the treatment of several types of cancer.
Acknowledgments This work has been supported by research grants from the Special National Key Research and Development Plan (No. 2016YFD0400202-8) and the National Natural Science Foundation of China (No. 81571746).
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