Taming Cell Penetrating Peptides: Never Too Old To Teach Old Dogs

Aug 3, 2015 - POSS-cored and peptide functionalized ternary gene delivery systems with enhanced endosomal escape ability for efficient intracellular ...
0 downloads 0 Views 3MB Size
Subscriber access provided by EMORY UNIV

Review

Taming cell penetrating peptides: Never too old to teach old dogs new tricks Qianyu Zhang, Huile Gao, and Qin He Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00428 • Publication Date (Web): 03 Aug 2015 Downloaded from http://pubs.acs.org on August 12, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Taming cell penetrating peptides: Never too old to teach old dogs new tricks Qianyu Zhang, Huile Gao, Qin He*.

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, and State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P. R. China *

Corresponding author Qin He Tel/Fax: +86-28-85502532. E-mail: [email protected]

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

Abstract Cell penetrating peptides (CPPs) received substantial attention due to their intrinsic property to cross plasma membranes or even as helpers to facilitate the cellular entry of drug molecules, macromolecules and nanoparticles. Although CPPs and CPP-like peptides provided versatile platforms for drug delivery, their non-selectivity or lack of delivery efficiency is stirring up debates as to the tactics for the optimizing the CPPs themselves. The good news is that, as spurred by the recent progress in the understanding of tumor micro-environment as well as biochemistry and material sciences, we have made attempts in working on perfecting or even “taming” CPPs and CPP-functionalized drug vectors for tumor delivery and some of them afforded gratifying results. Due to the fact that these peptides are mainly short peptides made up of amino acids (5-30 amino acids), the addition, modification or replacement on amino

acids

might

lead

to

surprisingly

improved

performance.

Several

novel

environment-responsive CPPs or CPP-like peptides have also been discovered. In this review we will discuss on the measures taken to harness the power of CPPs and the discovery of environment-responsive peptides with CPP properties. Keywords: Cell penetrating peptides (CPPs), responsive, selectivity, drug delivery systems, improved efficiency

ACS Paragon Plus Environment

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1. Introduction: to be selective or not to be? ? It has been over twenty years since Frank and Pabo observed for the first time that the transcription-transactivating protein of HIV-1 (TAT) could traverse cross cellular membranes and even into the cellular nuclei [1]. It was then that the concept of protein transduction domain (PTD) and cell penetrating peptides (CPPs) made the “debut” and started to be greeted by public acceptance. Over the years, CPPs with great structure and mechanism diversity have been discovered, including cationic CPPs (such as TAT, polyarginines, penetratin, etc. [2-5]), amphipathic CPPs (such as Pep-1, transportan, MAP, etc. [6-8]) and hydrophobic CPPs (such as PFVYLI [9]). Whether derived from natural resources or synthesized from peptide libraries, it has been well-recognized that CPPs were peptides with 5-30 amino acids that can cross cellular plasma membranes [10]. They themselves can serve as vectors for small molecules, proteins, siRNA and nucleotides; moreover, they can also orient nano-carriers into cells after proper surface modification [10-14], propelling the progress of novel drug delivery systems. Indeed CPPs have taken the center stage and have been researchers’ favorites for years; however, the limitation and impediment as to the application of CPPs-related drug delivery systems has also gained concerns. One of the most notorious setbacks for them should be the non-specificity (as CPPs were also depicted as “Trojan horse”) [15-17]. Although highly efficient and most of the times non-toxic in vitro, their indiscriminative distribution and strong penetration into normal tissues severely hampered the in vivo applications [16-18]. The systemic cytotoxicity was therefore left to be more or less unpredictable and inevitable due to the off-target effect of most CPPs and CPP-modified nano-carriers. Of course, everything could be toxic as long as it reaches a certain threshold of concentration or exposure time, therefore the therapeutic windows should be determined with great care. Furthermore, although positive charge was necessary for the translocation of common cationic CPPs into cells, once applied in vivo, the high positive charge of CPPs could possibly deteriorate the drug delivery efficacy due to the existence of the negatively charged serum protein in the blood circulation [19]. Therefore, promising in drug delivery as they could be, CPPs were also challenged and impeded by their non-selectivity towards targeted and non-relevant cells. Nevertheless, some researchers argued otherwise and showed that some peptides, including CPPs could preferentially enter tumor cells instead of normal cells [20-25]. Part of this was because cellular membranes of tumor and healthy cells differ both in glycosaminoglycans (GAGs) and lipid composition. The tumor cells exposed more GAGs and anionic lipids such as phosphatidylserine (PS) on their surface, leaving their cellular membranes more negatively charged, which could attract more positively-charged CPPs towards them than healthy cells [25]. This is especially true for arginine- or lysine-rich CPPs with high positive charge, and the deletion of GAGs from cellular membrane surface decreased cellular internalization of CPPs [26, 27]. Jobin et al. reported that a penetratin-derived peptide, RW16, was more inclined to interact and perturb liposomes composed of anionic lipids than zwitterionic lipids [28]. Moreover, the membrane fluidity of tumorigenic cells was also altered, and it might interfere with the insertion and crossing of membranes of amphipathic peptides [26, 29]. Although this propensity of CPPs towards tumor cells did exist according to previous reports, the strong cell penetrating effect over normal cells and the extensive in vivo distribution was still ineluctable and jeopardized their role as drug delivery vectors [30]. One

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

recently published work pointed out that the fatty acid played decisive role in the cellular internalization of guanidinium-rich molecules, such as arginine-rich TAT [31]. This process was universal in almost all kinds of cells including cells from animals, plants or insects [31]. On the other hand, this also signified the restrictions when it comes to the application of some of the positively-charged CPPs. Other than the natural component difference in the cellular membranes, tumor cells expressed higher level of certain receptors and markers, which could be employed in the so-called “active targeting”. Dual ligand and multi-functional carriers have been established when CPPs were combined with other specific ligand or antibody, increasing the selectivity and exerting synergistic effect on tumor targeting [32-36]. This could evolve further into a tandem peptide which was to link CPP with specific ligand, and more details will be discussed later in 3.5. Besides the difference in phenotypes between tumor and normal cells, the discrepancy in the microenvironment between tumors and healthy tissues also shouldn’t be neglected. Angiogenesis, hypoxia and inflammation were all crucial hallmarks in cancer development [37, 38]. Intelligent CPP-based strategies could thus be deployed in response to these unique features such as low pH, highly and pathologically-expressed cytokines or enzymes in the tumor milieu, etc. In this review we will summarize and elaborate on the approaches to engineer multi-functional or responsive CPPs or CPP-like peptides and the nano-carriers oriented by them for drug delivery (Tab.1).

ACS Paragon Plus Environment

Page 4 of 38

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Molecular Pharmaceutics

Table.1: Summary for representative “tamed” CPPs described in this review CPP sequence

Action mechanism

Ref

Stimuli-responsive

R8 (RRRRRRRR);

CPP were protected by cleavable PEG layer, which could detach and expose

[39-

steric protections

TAT (AYGRKKRRQRRR), etc.

CPP in a responsive manner

42]

a

CPP could be masked by modification group on amino acid in peptide

[50-

were amidized), etc.

sequence

52]

PenArg (RQIRIWFQNRRMRWRR);

Arginine replacement over lysine promoted CPP penetrating capacity

[56]

r8 (made of eight D-formed arginines)

D-formed amino acid were more stable than L-form counterparts

[65]

TH (AGYLLGHINLHHLAHL(Aib)HHIL)

Histidines could deprotonate under acid environment, activating CPP

[72-

Functional group modification Amino acid substitution

Addition of histidines

TAT (AYGRKKRRQRRR, underlined amino acids

R6H4 (RRRRRRHHHH), etc. Charge coverage

End-tethering with Tailoring of CPPs

functional amino acid sequences tumor lineage-homing

ACPP (Suc-e8-(Aop)-PLGC(Me)AG-r9-c-NH2)

Arginines were masked by glutamates by MMP-responsive linker, which

[82-

could be cleaved by enzymes

92]

R8-RGD [RRRRRRRR-c(RGDfK)];

Synergistic or combined effect of both CPP and functioning amino acid

[101-

CM18-TAT11(KWKLFKKIGAVLKVLTTGYGRKKRRQ

sequence

105]

Screened by mRNA display technology, which could specifically home to

[106-

certain tumor cells such as A549.

107]

RRR), etc. RLW (RLWMRWYSPRTRAYG), etc.

CPPs Other responsive

GALA

77]

WEAALAEALAEALAEHLAEALAEALEALAA

A stable α-helix could be formed at low pH which could cross lipid bilayer.

with CPP

[117, 118]

peptides pHLIPs

characteristics iRGD (CendR peptides)

ACEQNPIYWARYADWLFTTPLLLLDLALLVDADE

Forming helical structure under acidic environment, which was favorable for

[127-

GT

membrane insertion and traversing.

135]

CRGDKGPDC

Upon peptide cleavage by cell surface-associated protease(s), RGD

[137-

mediated integrin binding and R/KXXR/K CendR motif mediated binding with

142]

neuropilin-1, penetrating cells and tissues.

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2. Stimuli-responsive steric protections of CPPs Steric protection for CPP was accomplished by the addition of polymers, usually the “PEG clouds”, onto the surface of nano-carriers. CPP remained stealth in the blood circulation, and once upon reaching the targeted site, it could be exposed in a stimuli-responsive manner. In the past years, researchers have come a long way to establish “smart” drug delivery systems where cleavable PEG with stimuli-responsive linkers and CPPs were both incorporated into the nano-carriers [39-42]. Cleavable PEG could be detached from liposome surface under the stimuli of pH, enzymes or reducing agent and exposed the shielded CPPs. Our group has optimized liposomes simultaneously modified with TAT and cleavable PEG for systemic delivery of cargoes into tumors (Fig.1) [41], and they could increase tumor accumulation compared with liposomes modified only with TAT. Intravenous injection of cysteine could cleave PEG from the surface of liposomes, and the exposure of TAT facilitated intracellular delivery of liposomes within tumors [41]. Similar technique was also wielded in composing a multistage liposome made up of cleavable PEG, RGD and TAT. Although it was such a classic and typical PEG-deshielding technique that showed distinct improvement over nano-carriers modified only with CPPs, it should also be noted that the cleavage was usually not efficient and complete enough at desired site, therefore the “on-demand effect” would somehow be delayed or crippled [43, 44].

Fig.1: Schematic illustrations of TAT-SL, TAT and cleavable PEG comodified liposomal system (C-TAT-SL). Adapted from Ref.41 with permission. Besides detachable PEG, “super pH-sensitive multifunctional polymeric micelles” has also emerged lately [45-47]. Bae and his co-workers anchored TAT onto the surface of micelles via pH-sensitive polyhistidine (polyHis). polyHis was hydrophobic at pH 7.4, and since the core of the micelles was made up of hydrophobic materials, TAT was pulled closer to the core by hydrophobic interaction between polyHis and the core, and was therefore buried by the PEG corona. While reaching tumor, as the pH decreased, polyHis would be ionized and stretched out, and TAT was “popped out” out of the PEG corona, promoting cellular entry of

ACS Paragon Plus Environment

Page 6 of 38

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

micelles only at tumors. Hansen et al. proposed a liposomal drug delivery system with constrained cell penetrating peptides (TAT) for intracellular delivery [48]. TAT was incorporated into a loop on the surface of PEGylated liposomes via two alkyl-chains, one of which contained a UV-cleavable linker. TAT was temporarily “locked” on the inner layer of the corona of liposomal surface; once irradiated, UV-responsive linker was cleaved, opening the loop and exposing TAT. However, the in vivo application of this liposome might be limited due to the potential hazard posed by UV irradiation to individuals and the poor penetration of UV light deeper into the tissues at the desired site. 3. Strategies for CPPs tailoring Instead of fabricating drug delivery platforms mediated by CPPs protected by “PEG clouds”, some researchers have diverted their focus onto optimizing CPPs themselves recently. Ideal CPPs should be able to penetrate cells or tissues of target only, thus minimizing the off-target effect. Sometimes their cell penetrating capacity could also be optimized after improvement. In this review, we will discuss on the strategies working on “perfecting” CPPs themselves, including the addition, modification or replacement of the amino acids in traditional CPPs. Some innovative CPPs were also designed artificially with tumor cell-specific penetration. 3.1 Functional group modification on amino acids In order to endue responsiveness to CPP, chemical modification was often conducted on the amino acid functional groups by environment-responsive linkages or masking groups. These linkages should be labile or cleavable under certain circumstances, regenerating the original structure of the initial CPPs. As a typical cationic CPP, TAT is rich in arginines (pKa=12.5) and lysines (pKa=10.2) [49], both of which are responsible for the high positive net charge of TAT. In order to shield its positive charge, modifications have been made on the amino acids in the peptide sequence [50-52]. Jin et al. [50] applied succinyl amide to amidize two lysine residue amines as well as a

the glutamine amide in the sequence, leading to the modified version of TAT, denoted as TAT (Fig.2). The guanidinyl groups in the arginines were not affected in the amidization. Compared to the fast clearance of TAT-PEG-PCL, aTAT-PEG-PCL micelles had no specific interaction with the blood components and could circulate in the bloodstream just as the nonfunctionalized PEG-PCL micelles. Upon reaching the tumor interstitium or cellular endo/lysosomes, the acid-labile amides would be hydrolyzed and the function of TAT could thus be restored (Fig.2), delivering micelles into tumor cells or even into the cell nuclei. Zhang et al. devised stepwise-acid-active multifunctional mesoporous silica nanoparticle for doxorubicin delivery [51], in which TAT and K11 (constituted by 11 lysines) were masked with succinic anhydride (SA) and dimethylmaleic anhydride (DMA) respectively at first, and the re-exposure of TAT and K11 with cell penetrating function could be triggered gradually by the acidified environment within tumors and endo/lysosomes. In another work done by Liu et al. [52], the substrate of endoprotease legumain, alanine-alanine-asparagine (ANN) moiety was added to the fourth lysine in the TAT, and the modified TAT was anchored to the liposomal surface. They have confirmed that legumain expression was up-regulated in a variety of solid tumors and was positively correlated with the level of malignancy; and the ANN moiety could be recognized and cleaved by the legumain highly expressed in tumors and exposed the

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

original TAT peptide. TAT subsequently could enhance the internalization of liposomes and the cargo at tumor site. This was actually quite ingenious due to the fact that ANN could act both as the targeting moiety for tumors and the shrouding moiety for TAT, and it could be removed once liposomes accumulated in tumors, leaving intact TAT to play its role.

Fig.2: Amidization of TAT’s primary amines to succinyl amides and their acid-triggered hydrolysis. Adapted from Ref.50 with permission. Instead of being considered as a targeting moiety, CPPs themselves were also favorable vectors for drug delivery. M918K was a CPP studied by Lee et al. which showed the optimal gene transfection potency compared with other CPPs [53]. Peptide nucleic acid (PNA) with a terminal lysine as the nucleic acid analogue payload was conjugated to the cysteine in M918K, and PEG chains were then coupled to the primary amines in the peptide sequence (each lysine owns a primary amine, and there is one N-end with a primary amine in M918K) via azobenzene bond to shield the positive charge of M918K. Azobenzene bond could be reduced and cleaved in the colon environment, and after the attachment of PEG chains, TAT-linked PNA could enter diseased cells in colons for colon-targeted delivery (Fig. 3).

Fig.3: Schematic illustration of colon-specific delivery with activatable CPP-PNA (After the reductive cleavage of azobenzene bond, a self-immolative moiety releases the intact lysine residue on the CPP backbone). Adapted from Ref. 53 with permission.

ACS Paragon Plus Environment

Page 8 of 38

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

3.2 Amino acid substitution Replacement of amino acid was another strategy for revamping CPPs, which was mainly for three goals: one was to increase the intracellular internalization, another was to improve the stability of CPP and the last was to accord pH-responsiveness to CPP (mainly by substitution by histidines, which will be discussed separately later in 3.3). Arginines have strong positive charge and strong affinity to cellular surface, and studies showed that the numbers of arginines could also affect the internalization profile and efficiency [54, 55]. There was also study to substitute lysine in the peptide sequence with arginine to augment the membrane activity of CPP [56]. Amand et al showed that the arginine-rich (PenArg) and histidine-rich (PenLys) analogs of penetratin showed different uptake efficiency on cells, as in the order of PenArg>penetratin>PenLys [56]. Kamide et al. discovered that novel hydrophobic CPPs could be unearthed using in vitro virus library, and found that both arginines and tryptophans were important for the cell penetrating activity of the CPP [57]. Not only charge but also the amphipathicity of CPP were dominant in the membrane translocation of CPPs, especially for amphipathic CPPs. Song et al. showed that when composing transportan 10 (TP10) variants, simple replacement of lysines with arginines significantly decreased the cell penetrating activity of TP10, which might be due to fact that the introduction of arginines influenced the amphipathicity of the amphipathic TP10 [58]. To settle this, the positive charge and the amphipathicity should be both considered and balanced when conceiving new CPP analogs. The stability of peptides is fundamental for CPP-mediated delivery of cargoes. However, this was usually compromised by the CPP cleavage by plasma proteases before reaching targeted area [18, 59, 60]. Other than protection by PEGylation, D-amino acid configuration was also adopted in CPP modification since D-form amino acids were less susceptible to protease activity than their natural L-amino acid counterparts [61-63]. A novel peptide, Xentry was synthesized as a D-isomer to increase its resistance to protease [64]. Results suggested that D-isomeric Xentry was cell-permeable, and it was stable in serum for 4h, much longer than L-isomer which turned inactive rapidly within 1h [64]. Nakase et al. found that D-form amino acid-constituted R8 (r8) significantly increased its accumulation in tumors compared with R8, which might be owing to the reason that arginine-rich CPPs composed of D-amino acids were more resistant to proteolysis than L-isomers, favoring the prolonged retention of D-isomer peptide structures within tumors [65]. 3.3 The power of histidines The introduction of histidines into peptide sequences be they CPPs, anti-microbial peptides (AMPs) or lytic peptides, has emerged lately and proved itself as a facile means to tackle and tame peptides. Histidine is a major amino acid responsible for the buffering capacity of biological systems and has been investigated by various researchers to constitute pH-responsive drug delivery systems [66-68]. With a pKa around 6.5 [69], histidines could remain slightly negatively charged under physiological condition and protonate into positive charge in acidic environment. Meanwhile, histidines were also known for their endo/lysosome escaping capacity for they are excellent receivers and donors of protons (proton sponge effect) [70, 71]. As mentioned before, lysine (pKa=10.5) was responsible for the positive charge of some cationic peptides; therefore, researchers replaced lysines with histidines to fabricate pH-responsive peptides, including CPPs [72-77]. Zhang et al. [72] designed peptide TH from

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TK

(analog

of

TP10

[58]),

(AGYLLGKINLKKLAKL(Aib)LLIL-NH2)

replacing with

all

the

Page 10 of 38

lysines

histidines

into

in

peptide peptide

TK TH

(AGYLLGHINLHHLAHL(Aib)HHIL-NH2), and TH-conjugated camptothecin (TH-CPT) could preferentially enter cells in acidic environment. Our group also utilized this pH-responsive CPP TH to establish TH-mediated liposomes (TH-Lip) for tumor delivery (Fig. 4) [73]. Results showed that the zeta potential of TH-Lip could switch from negative (-4.8±1.2mV, pH 7.4) in normal tissues to positive in acidified tumor-mimicking microenvironment (10.8±1.3mV, pH 6.3) with low pH. In vivo study showed that the modification of TH onto the surface of PEGylated liposomes hardly sacrificed its long circulating property, yet it could attain higher cellular internalization at tumor site while avoiding non-specific penetration in other organs such as livers.

Fig.4: Surface charge of TH-Lip was shifted from negative to positive under tumor microenvironment where the pH value declined, and it was internalized into tumor cells efficiently via instant electrostatic attraction between protonated TH peptide and cellular membrane and then the subsequent endocytosis. Adapted from Ref.73 with permission. In spite of the effectiveness of the histidine-replacing strategy, it has to be mentioned that the activity of the original CPPs could not be fully recovered after protonation of histidine in tumor environment or even in the endo/lysosome environment (ref. 71, and also according to our unpublished results). Besides remolding already-existed CPPs, some researchers turned to fabricating novel CPPs rich in histidines for drug delivery [78-81]. LAH4 is an amphipathic peptide rich in alanines and leucines containing four histidines in the central region of the sequence [78]. It demonstrated strong gene delivery efficiency comparable to commercially available reagents, and researchers found that the transfection efficiency depended upon the number and position of histidines [78]. Some other peptides were mainly consisted of arginines and histidines, as the arginines could act as the cell penetrating motif and histidines

ACS Paragon Plus Environment

Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

could act as the pH-responsive motif that can influence the surface property of the drug carriers (micelles or liposomes). Zhang et al. [80] have screened a series of histidine- and arginine-rich peptides to balance the pH-sensitivity and cell penetrating efficiency, and peptide R6H4 was determined as the optimal one. The strong positive charge produced by arginine could not be shielded by other histidines; so a layer around liposomes was formed consisted of hyaluronic acid to protect the pH-responsive CPP-modified liposomes in blood circulation. Due to the contribution of histidines, it has been evidenced by them that the proton sponge effect accompanied by membrane penetrating ability of R6H4 produces endosomal/lysosomal escape for efficient intracellular delivery (Fig. 5). In another contribution by Zhang et al. [81], peptide H7K(R2)2 was fabricated and the seven histidines could temporarily “lock” the branched cell-penetrating motif -(R2)2 onto the core of the polymeric micelles by hydrophobic attraction. Once in the tumor microenvironment (in their set, pH 6.8), histidines could then be ionized, and as the hydrophobic interaction between histidines and the core of micelles weakened, the released and expanded histidines could then push the -(R2)2 out of the PEG shell and enhance tumor cell internalization. It was noteworthy that the protonation and deprotonation of histidines under different pH conditions were prompt, meaning that CPPs could respond swiftly to outside environment [73], demonstrating the superiority of this strategy.

Fig. 5: Surface HA could be degradated by HAase in HAase-rich tumor milieu and exposed peptide R6H4, where it could be applied for enhanced cell penetration to mildly acidic tumor microenvironment for improved cellular uptake. Upon internalization, R6H4 could facilitate liposomal endosomal/lysosomal escape. Adapted from Ref.80 with permission. 3.4 Positive charge coverage by anionic counterparts Masking the cationic nature of CPPs abundant in basic residues in non-targeted organs

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

or tissues has always being the priority for researchers trying to harness the power of CPPs. Jiang et al. found that the cellular association with CPPs could be effectively blocked when an inhibitory domain made up of negatively charged residues (six to nine consecutive glutamates) were fused with the cationic CPP sequences (polyarginines), naming it activatable CPPs (ACPPs) [82]. The linker was a peptide sequence cleavable with MMP (matrix metalloproteinases)-responsiveness. After the cleavage of the MMP-responsive linker in tumors, the inhibitory polyanionic domains diffused and CPPs could be thus unleashed and take its cargo into cells. Since then, this strategy has quickly aroused researchers’ attention and ACPP-based drug delivery systems have been thoroughly investigated and applied to tumor imaging or tumor management [83-92]. This measure could even be adopted in fluorescence-guided surgery in tumor models [90, 91]. Savariar et al. discovered a family of novel ratiometric activatable cell penetrating peptides, which contained Cy5 as far red fluorescent donor and and Cy7 as near infrared fluorescent acceptor [92]. Cy5 was quenched by the fluorescence resonance energy transfer (FRET) with Cy7. After cleavage of the MMP-responsive linker in tumor area, the FRET was disrupted and the contrast of Cy5 could be improved for cancer detection in metastatic lymph nodes. 3.5 End-tethering with functional amino acid sequences The conjugation of additional amino acid sequences onto the C-end or N-end of CPP was mainly for two causes: one was to endow the specificity towards diseased tissues to CPPs by the introduction of tumor homing peptides [93-100], and the other was to improve the endo/lysosome destabilization capacity of traditional CPPs such as TAT [101-105]. As mentioned in the Introduction, one of the shortcomings of CPPs was the lack of specificity towards diseased tissues or organs. But this could be alleviated by the conjugation of tumor homing peptides for targeted delivery. Under this circumstance, CPP could serve either as the drug delivery vehicle for delivery (especially genes) [93-96] or as cell penetrating module [97-100]. Ren et al. synthesized a library of tandem peptides bearing a tumor-specific domain and cell-penetrating domain, and formed nanocomplexes with siRNA and tested their delivery efficiency [93]. They identified that myristoylated tandem peptide myr-TP-Lyp-1 could effectively deliver siRNA in a cell-type specific manner, and it could seek out cancer cells and deliver therapeutics to deep-seeded tumors [93]. Myrberg et al. [98] found that combining a tumor-specific vascular homing peptide PEGA sequence with a well-studied CPP pVEC (PEGA-pVEC) could result in colocalization of the chimeric peptide with tumor vascular marker and increased the tumor cell accumulation in vivo afterwards. PEGA-pVEC was able to translocate breast tumor cell membrane and elevate the cytotoxicity of chlorambucil towards tumor cells after its conjugation with chlorambucil [98]. Our group has also synthesized tandem peptides based on this principle [99]; cyclic RGD peptide was conjugated to octaarginine

through

amide

bond

to

obtain

a

tandem

peptide

R8-RGD

[RRRRRRRR-c(RGDfK)]. R8-RGD can be used as a promising ligand for BBB transporting, glioma targeting and tumor penetrating (Fig.6) owing to the overexpression of integrin αvβ3 receptor on both brain capillary endothelial cells and glioma cells and the strong cell penetrating ability of R8. R8-RGD-mediated liposomes could more effectively cross the BBB in vitro and also accumulate more in the glioma area in vivo compared with liposomes modified with only R8 or RGD, displaying its potential in glioma-targeted delivery as a multifunctional

ACS Paragon Plus Environment

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

CPP with specificity.

Fig.6: Liposomes modified with R8-RGD could specifically bind to integrin avβ3 receptors expressed on the brain capillary endothelial cells and transport across the BBB through a synergetic effect of both R8 and RGD. Adapted from Ref.99 with permission. Whether arginine-rich CPPs such as TAT and penetratin could escape from endocytic vesicles remained highly controversial [101-104]. Several studies suggested that these CPP-linked nano-carriers were sequestered in endocytic vesicles, impairing their gene drug delivery efficiency [104]. However, this drawback could be ameliorated by fusing CPP sequence (such as TAT) to membrane-disruptive motifs. Salomone et al. devised a novel chimeric peptide, in which TAT11 was fused to CM18 hybrid (KWKLFKKIGAVLKVLTTG, residues 1–7 of Cecropin-A and 2–12 of Melittin, both of which were membrane-perturbing AMPs) [103]. They showed that this chimeric peptide could combine the highly efficient uptake properties of CPPs with the membrane-disruption ability of linear cationic AMPs, and allow the intracellular localization of diverse membrane-impermeable molecules (including Tat11-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with hardly any cytotoxicity. It has already been pointed out previously that histidines were especially helpful when it comes to engineering pH-responsive cell penetrating peptides due to their function in proton exchange. Lo and Wang [105] constructed endosomolytic TAT peptide after incorporation of ten histidine and two cycsteine residues into the TAT sequence (C-5H-TAT-5H-C), with histidines for the endosomolytic effect and cysteines for the stabilization effect brought by the disulfide bonds form by the thiol groups. Results showed that the gene transfection efficiency was comparable to PEI 25kDa with no obvious sign of cytotoxicity, demonstrating its superiority in gene delivery. 3.6 Screening for tumor lineage-homing CPPs Matsushita et al. developed a tumor homing CPP-screening protocol with mRNA display technology [106, 107]. Novel tumor lineage-homing CPPs were obtained aiming at different

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

tumor cell lines (including Hela cells, A549 cells and K562 cells, etc.) [106]. As one of the representative CPPs, CPP44 showed high level of transduction in myelogenous leukaemia and hepatic tumor cells. It could specifically home to metastatic leukaemic tumors in vivo, without significant retention in the liver or other normal organs including brains, lungs, hears, intestines or kidneys. These tumor lineage-homing CPPs could noninvasively target their corresponding tumors while minimizing transduction into normal tissues. It was verified by us that one of the peptide identified in ref.108, RLW (RLWMRWYSPRTRAYG), after functionalized onto nanoparticles, could specifically target to A549 cells compared with HUVEC cells while R8 as a control showed no difference between these two cell lines [108]. In another study, they found that peptide1-NS△ could selectively incorporate and deliver payload into glioblastoma multiforme (GBM) cell lines compared with other cell lines, and the GBM-selective recognition mechanism of the tumor-homing CPP peptide1-NS△ remained yet to be clarified [107]. However, for these tumor-homing CPPs, it should be noted that although they were claimed to be termed as CPPs, some of their property actually resemble that of a specific ligand. Take CPP44 for example, mechanism studies showed that cellular entry of CP44 was mediated by dynamin-dependent and clathrin-independent endocytic pathway, and M160 (CD163L1; scavenger receptor cysteine-rich type I) mRNA knockdown in AML cells (primary myelogenous leukemic cells) significantly impaired intracellular uptake of CPP44, implying that endogenous M160 expression was critically involved in CPP44 penetration [106], and this M160-related pathway governed the internalization of CPP44. These might seem inconsistent to what we perceive as traditional CPPs which could cross cellular membrane of almost any kind with no specific receptors on the cellular membranes. However, it should also be borne in mind that specific ligand could provide targeting ability but not necessarily strong cellular entry of nano-carriers [32], and surprisingly the cellular uptake efficiency of CPP44 was quite high on leukemic cell lines, even much higher than TAT and R9, imparting a rather fascinating nature of the combination of both tumor cell-targeting and tumor cell-penetrating capacity to CPP44. With regard to the mechanisms of these tumor lineage-homing CPPs, more work will be needed in the future. 4. Discovering responsive peptides with CPP characteristics The term CPP was not born until about two decades ago, as we have pointed out before; however, the studies on peptides with cell penetrating ability could be dated way back. These peptides included membrane-active peptides and anti-microbial peptides (AMP) [109-111]. These peptides shared important structural resemblance and membrane-destabilization features with CPPs [112, 113], and some of them even demonstrated inter-changeable activities with CPPs [114]. They can also aid in delivering cargoes into cells both in vitro and in vivo. For example, we have proved that histidine-rich anti-microbial peptide [D]-H6L9 could be applied in liposomal paclitaxel and antagomir delivery [115], and CPP-like effect could also be attained [116]. In this sense, it was highly controversial to classify peptides into a definite category. Therefore, in this review, we will also discuss on some of the peptides with potential responsive CPP properties, for the discoveries and application of these peptides may help further our understanding in designing novel stimuli-responsive CPPs in the future. 4.1 GALA peptide and its family GALA was a 30 amino acid-peptide (WEAALAEALAEALAEHLAEALAEALEALAA) synthesized

by

Szoka

Jr

et

al.

with

a

repeated

ACS Paragon Plus Environment

sequence

of

glutamic

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

acid-alanine-lucine-alanine (EALA) [117, 118]. A stable α-helix could be formed at low pH which was long enough to cross a lipid bilayer [119]. Actually, the design of GALA and GALA-related peptides (such as KALA) was inspired by influenza virus hemagglutinin (HA) and some other natural α-helical toxins of its like, in which a loop-helix conformational change could be induced under low pH, ultimately leading to the complete fusion of both viral and host membranes [119]. Whether GALA should be labeled as fusogenic peptide or cell penetrating peptide remained in disputation, however, its marvelous drug delivery capacity should never be ignored; GALA and GALA-related peptide (such as cationic KALA and RALA, etc.) could escape endosome entrapment and improve gene transfection [120-125].Harashima et al. demonstrated that after incorporating cholesteryl-GALA into multifunctional envelop-type nano device (MEND), endosomal escape was enhanced following internalization of MENDs via endocytosis [120]. It was worth noticing that the penetrating and fusion property was concealed until these peptides reached acidic environment such as endosomes, minimizing its cytotoxicity to normal tissues. Among them, GALA peptide should be counted as the most classic one since it showed high endosomal activity and low cytotoxicity [125]. Recently, Harashima et al. discovered that GALA peptide could recognize the sialic acid-terminated sugar chains on pulmonary endothelium, which could aid in the cellular uptake of GALA-functionalized MEND by lung endothelial cells, and the nucleic acid delivery could be further improved by the endosomal membrane fusion by GALA peptide [126]. This phenomenon revealed the tissue-targeting potency of GALA peptide. 4.2 pH low insertion peptides (pHLIPs) As the name suggests, pHLIPs could insert itself across membranes under low pH. Unlike the amphipathic nature of GALA, pHLIPs was moderately hydrophobic, containing the sequence of the bacteriorhodopsin C helix [127]. In low-pH environment, the protonation of negatively charged residues (Asp or Glu) could enhance hydrophobicity, increasing the affinity between peptide with lipid bilayers [128, 129]. Similar to GALA, conformation changes were also evoked by acidic environment and helical structure could also be formed within pHLIPs [127-129], which would be favorable for membrane insertion and traversing. Plenty of pHLIP variants were devised to improve its delivery property, which would be beneficial to selectively targeting tissues with various degrees of acidity [130, 131]. GALA peptide was chosen mostly for its endosomal disruptive capacity; while pHLIPs were usually applied in tumor extracellular pH-targeting [129, 132-135] for they were active at pH 6.0~6.5 (depend on pHLIP sequence [136]), which was within the range of tumor environmental pH. Also, it has been ascertained that the insertion of pHLIP only relied on physical properties of the membrane instead of the active process from cells, displaying a simple path for pHLIP to cross the cellular membrane (Fig.7) [136]. Conjugated with fluorescent probes, pHLIP could accumulate within various types of tumors for imaging which was directly dependent on the extra cellular acidity of tumor environment [134]. pHLIP can guide nanogold into tumors and distribute throughout the tumor mass, enhancing local concentration and retention in tumor mass, achieving improved radiation therapeutic effect [129]. When decorated on PEG-liposomes, results showed that the cellular uptake of pHLIP-mediated liposomes (fusogenic or non-fusogenic liposomes) was promoted at pH 6.0, and more cell death could be induced when liposomes were encapsulated with C6-ceramide compared with PEG-liposomes [135]. Other than promoting tumor cellular membrane fusion under tumor environment, more pHLIP might insert into endosomes where

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the pH was even lower (pH 5.0-5.5), leading to lipid mixing and fusion and liposomal payload was thus released into the cytoplasm as liposomal lipids could be found in different cellular compartment [135], suggesting its important role in endosomal escape as well. (Fig.8)

Fig.7: pHLIP peptides exhibit three distinct states. In State I, pHLIPs are soluble and unstructured in aqueous solution. In State II, pHLIPs bind reversibly to the outer leaflet of membrane bilayers, remaining largely unstructured at physiological pH. In acidic extracellular environments a pHLIP inserts its C-terminus across the membrane to form State III, a stable transmembranea-helix. Adapted from Ref. 136 with permission.

Fig.8: Schematic presentation of pHLIP/PEG coated liposomes (A) and their interaction with cellular membranes (B). Adapted from Ref. 135 with permission. 4.3 iRGD and peptides with CendR motif (CendR peptides) Strictly speaking, iRGD and peptides of the same category were often referred to as

ACS Paragon Plus Environment

Page 16 of 38

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

tumor-penetrating peptides instead of cell penetrating peptides [137-142]. However, their cellular internalization was caused by the cell-penetrating property of CendR sequences which led to vascular leakage and tissue penetrating in vivo [137], therefore CendR peptides was also included in this review for their potential in cell penetrating. CendR peptides such as iRGD usually contained three independent modules: a vascular homing motif, an R/KXXR/K tissue penetration motif, and a protease recognition site [140]. After in vivo administration, iRGD peptide would preferentially bind with αvβ3/5 integrins overexpressed on tumor cells. Then proteolytic cleavage could take place and expose the CendR motif, which would interact with neurophilin-1 (NRP-1) and trigger the internalization process (Fig. 9) [140, 141]. This process allowed for the activation of CendR motif only in targeted tissues, sparing the normal tissue from unspecific penetration. When co-administered with small molecule, nanoparticle or monoclonal antibody, these CendR peptides could improve their therapeutic index significantly by its selective accumulation and penetration into tumors, triggering tumor-specific penetration of the co-administered compounds [139]. On the other hand, these peptides could also help deliver nano-sized carriers into tumor tissues [143-146], rendering themselves as outstanding tumor-homing ligands for various drug delivery platforms. iRGD also showed anti-metastatic activity, which provided additional benefit in metastatic tumor therapy [147]. Based on the basic structure of CendR peptides proposed by Ruoslahti et al. [137, 139-142], researchers developed other peptides with similar property to iRGD targeting neuropilin-1 on tumor cells [148-152]. The structure of CendR peptide was apparently quite simple, accelerating the designing of novel peptides of its kind. It was worth noting that although the applicability of CendR peptides has been validated in various types of tumors, the mechanisms underlying proteolytic cleavage were yet to be determined. Whether there could be more than one pathway for CendR sequences remained unknown. Pang et al. suggested that endocytosis of CendR peptides is distinct from known endocytic pathways, and CendR endocytosis and subsequent intercellular transport of CendR cargo were both stimulated by nutrient depletion [153]. More endeavors should be made in unveiling the mysteries of CendR peptides.

Fig.9: Multistep Binding and Penetration Mechanism of iRGD. Adapted from Ref. 139 with permission. 5. Conclusions

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

It has been almost thirty years since the naissance of the concept of cell penetrating peptides, namely CPPs, but the research on CPP-like peptides was an even longer story, and it is continuing now and will carry on in the future. The non-selectivity of CPPs summoned both fortunes and challenges for drug vector designers. On the plus side, their strong and almost indiscriminate penetration into cells makes them attractive choice that seems kind of omnipresent in drug delivery, and they still hold great potential for us to exploit. On the down side, it also created obstacles when establishing targeted drug delivery systems whose spot of action was rather focalized. Their charming yet unruly nature makes it challenging to rein them, but the comforting message is that we are right now getting closer to mastering and taming this wild “old dog” and teaching it new tricks. The secret lies in the difference between targeted area (such as tumors, about which we were most concerned in this article) and non-targeted tissues (such as normal tissues). As researches on tumor microenvironment as well as tumor cells advance forward day by day, a vivid picture about the tumorigenesis and its progression is being portrayed, with all the dots being connected and missing pieces being collected. Utilizing the heterogeneity of tumors, novel CPPs emerged as powerful means in drug delivery. Whether it’s pH-responsive, enzyme-responsive, reduction-responsive, or just exhibiting higher affinity towards tumor cells than normal cells, the designing of novel CPPs will ultimately come down to two things: satisfactory selectivity and considerable efficacy. Intriguingly, they seemed somewhat contradictory to each other in most cases but hopefully not completely incompatible. Delicate balancing of these two elements could help us attain the desired CPPs. Besides treating tumors, CPPs and CPPs-based drug delivery systems could be employed for other purposes as well, such as anti-microbial or anti-inflammation, etc. [154]. For example, pHLIPs have been found to target acidic conditions in vivo including ischemic heart muscles and localized rheumatoid arthritis [136]. Of course, the simple secret again lies in the difference between targeted tissues with non-targeted areas. For the existing CPPs, their potential tissue-targeting ability (such as GALA peptide for pulmonary endothelium delivery) remained underestimated in most cases and perhaps this untapped field called for more attention. We have shown numerous approaches that could successfully tame CPPs and put them to fair use, and these strategies paved the way for future development and application of CPPs. 6. Acknowledgements The work was funded by the National Basic Research Program of China (973 Program, 2013CB932504) and the National Natural Science Foundation of China (81373337).

References 1. Frankel A. D.; Pabo C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988, 55, 1189–1193. 2. Richard J. P.; Melikov K.; Vives E.; Ramos C.; Verbeure B.; Gait M. J.; Chernomordik L. V.; Lebleu B. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem. 2003, 278(1), 585-590. 3. Drin G.; Cottin S.; Blanc E.; Rees A. R.; Temsamani J.Studies on the internalization mechanism of cationic cell-penetrating peptides. J Biol Chem. 2003, 278(33), 31192-31201. 4. Terrone D.; Sang S. L.; Roudaia L.; Silvius J. R. Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer

ACS Paragon Plus Environment

Page 18 of 38

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

potential. Biochemistry. 2003, 42(47), 13787-13799. 5. Patel L. N.; Zaro J. L.; Shen W. C.Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm Res. 2007, 24(11), 1977-1992. 6. Morris M. C.; Depollier J.; Mery J.; Heitz F.; Divita G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 2001, 19, 1173–1176 7. Pooga M.; Hällbrink M.; Zorko M.; Langel U. Cell penetration by transportan. FASEB J. 1998, 12, 67–77 8. Deshayes S.; Morris M. C.; Divita G.; Heitz F. Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cell Mol Life Sci. 2005, 62(16), 1839-1849. 9. Watkins C. L.; Brennan P.; Fegan C.; Takayama K.; Nakase I.; Futaki S.; Jones A. T. Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD. J Control Release. 2009, 140(3), 237-244. 10. Milletti F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today. 2012, 17(15-16), 850-860. 11. Gupta B.; Levchenko T. S.; Torchilin V. P. Intracellular delivery of large molecules and small particles by cell-penetrating proteins andpeptides. Adv Drug Deliv Rev. 2005, 57(4), 637-651. 12. Stewart K. M.; Horton K. L.; Kelley S. O. Cell-penetrating peptides as delivery vehicles for biology and medicine. Org Biomol Chem. 2008, 6(13), 2242-2255. 13. Temsamani J.; Vidal P. The use of cell-penetrating peptides for drug delivery. Drug Discov Today. 2004, 9(23), 1012-1019. 14. Said Hassane F.; Saleh A. F.; Abes R.; Gait M. J.; Lebleu B. Cell penetrating peptides: overview and applications to the delivery of oligonucleotides. Cell Mol Life Sci. 2010, 67(5), 715-26. 15. Vives E. Present and future of cell-penetrating peptide mediated delivery systems: “Is the Trojan horse too wild to go only to Troy?” J Control Release. 2005, 109(1-3), 77-85. 16. Huang Y.; Jiang Y.; Wang H.; Wang J.; Shin M. C.; Byun Y.; He H.; Liang Y.; Yang V. C. Curb challenges of the “Trojan Horse” approach: Smart strategies in achieving effective yet safe cell-penetrating peptide-based drug delivery. Adv Drug Deliv Rev. 2013, 65(10), 1299-1315. 17. Shi N. Q.; Qi X. R.; Xiang B.; Zhang Y. A survey on “Trojan Horse” peptides: Opportunities, issues and controlled entry to “Troy”. J Control Release. 2014, 194, 53-70 18. Koren E.; Torchilin V. P. Cell-penetrating peptides: breaking through to the other side. Trends Mol Med. 2012, 18(7), 385-393. 19. Buyens K.; De Smedt S. C.; Braeckmans K.; Demeester J.; Peeters L.; van Grunsven L. A.; de Mollerat du Jeu X.; Sawant R.; Torchilin V.; Farkasova K.; Ogris M.; Sanders N. N. Liposome based systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design. J Control Release. 2012, 158(3), 362-70. 20. Farkhani SM.; Valizadeh A.; Karami H.; Mohammadi S.; Sohrabi N.; Badrzadeh F. Cell penetrating peptides: Efficient vectors for delivery of nanoparticles, nanocarriers therapeutic and diagnostic molecules. Peptides. 2014, 57, 78-94. 21. Nakase I.; Takeuchi T.; Tanaka G.; Futaki S. Methodological and cellular aspects that govern the internalization mechanisms of arginine-rich cell-penetrating peptides. Adv Drug Deliv Rev. 2008, 60(4-5), 598-607.

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

22. Bechara C.; Pallerla M.; Zaltsman Y.; Burlina F.; Alves I. D.; Lequin O.; Sagan S. Tryptophan

within

basic

peptide

sequences

triggers

glycosaminoglycan-dependent

endocytosis. FASEB J. 2013, 27(2), 738-749. 23. Schröder-Borm H.; Bakalova R.;

Andrä J. The NK-lysin derived peptide NK-2

preferentially kills cancer cells with increased surface levels of negatively charged phosphatidylserine. FEBS Lett. 2005, 579(27), 6128-6134. 24. Papo N.; Seger D.; Makovitzki A.; Kalchenko V.; Eshhar Z.; Degani H.; Shai Y. Inhibition of Tumor Growth and Elimination of Multiple Metastasesi n Human Prostate and Breast Xenografts by Systemic Inoculation of a Host Defense–Like Lytic Peptide. Cancer Res. 2006, 66(10), 5371-5378. 25. Jobin M. L.; Alves I. D. On the importance of electrostatic interactions between cell penetrating

peptides

and

membranes:

A pathway

toward

tumor

cell

selectivity?

Biochimie. 2014, 107 Pt A, 154-159. 26. Amand H. L.; Rydberg H. A.; Fornander L. H.; Lincoln P.; Nordén B.; Esbjörner E. K. Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochim Biophys Acta. 2012, 1818(11), 2669-2678. 27. Ziegler A.; Seelig J. Contributions of glycosaminoglycan binding and clustering to the biological uptake of the nonamphipathic cell-penetrating peptide WR9. Biochemistry. 2011, 50(21), 4650-4664. 28. Jobin ML.; Bonnafous P.; Temsamani H.; Dole F.; Grélard A.; Dufourc E. J.; Alves I. D. The enhanced membrane interaction and perturbation of a cell penetrating peptide in the presence of anionic lipids: Toward an understanding of its selectivity for cancer cells. Biochim Biophys Acta. 2013, 1828(6), 1457-1470. 29. Riedl S.; Zweytick D.; Lohner K. Membrane-active host defense peptides – Challenges and perspectives for the development of novel anticancer drugs. Chem Phys Lipids. 2011, 164(8), 766-781. 30. Kersemans V.; Kersemans K.; Cornelissen B. Cell penetrating peptides for in vivo molecular imaging applications. Curr Pharm Des. 2008, 14(24), 2415-2447. 31. Herce HD.; Garcia A. E.; Cardoso M. C. Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules. J Am Chem Soc. 2014, 136(50), 17459-17467. 32. Takara K.; Hatakeyama H.; Ohga N.; Hida K.; Harashima H. Design of a dual-ligand system using a specific ligand and cell penetrating peptide, resulting in a synergistic effect on selectivity and cellular uptake. Int J Pharm. 2010, 396(1-2), 143-148. 33. Kibria G.; Hatakeyama H.; Ohga N.; Hida K.; Harashima H. Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery. J Control Release. 2011, 153(2), 141-148. 34. Tang J.; Zhang L.; Liu Y.; Zhang Q.; Qin Y.; Yin Y.; Yuan W.; Yang Y.; Xie Y.; Zhang Z.; He Q. Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified with cholesterol anchored transferrin and TAT. Int J Pharm. 2013, 454(1), 31-40. 35. Zong T.; Mei L.; Gao H.; Cai W.; Zhu P.; Shi K.; Chen J.; Wang Y.; Gao F.; He Q. Synergistic dual-ligand doxorubicin liposomes improve targeting and therapeutic efficacy of brain glioma in animals. Mol Pharm. 2014, 11(7), 2346-2357. 36. Yang Y.; Yang Y.; Xie X.; Wang Z.; Gong W.; Zhang H.; Li Y.; Yu F.; Li Z.; Mei X.

ACS Paragon Plus Environment

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Dual-modified liposomes with a two-photon-sensitive cell penetrating peptide and NGR ligand for siRNA targeting delivery. Biomaterials. 2015, 48, 84-96. 37. Danhier F.; Feron O.; Préat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release. 2010, 148(2), 135-146. 38. Quail D. F.; Joyce J. A. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013, 19(11), 1423-1437. 39. Sawant R. R.; Torchilin V. P. Liposomes as 'smart'pharmaceutical nanocarriers. Soft Matter 2010, 6, 4026-4044. 40. Koren E.; Apte A.; Jani A.; Torchilin V. P. Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity. J Control Release. 2012, 160(2), 264-273. 41. Kuai R.; Yuan W.; Li W.; Qin Y.; Tang J.; Yuan M.; Fu L.; Ran R.; Zhang Z.; He Q. Targeted Delivery of Cargoes into a Murine Solid Tumor by a Cell-Penetrating Peptide and Cleavable Poly(ethylene glycol) Comodified Liposomal Delivery System via Systemic Administration. Mol Pharm. 2011, 8(6), 2151-2161. 42. Hatakeyama H.; Akita H.; Harashima H. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv Drug Deliv Rev. 2011, 63(3), 152-160. 43. Zhu L.; Kate P.; Torchilin V. P. Matrix metalloprotease 2-responsive multifunctional liposomal nanocarrier for enhanced tumor targeting. ACS Nano. 2012, 6(4), 3491-3498. 44. He L.; Lai H.; Chen T. Efficient delivery of payload into tumor cells in a controlled manner by TAT and thiolytic cleavable PEG co-modified liposomes. Biomaterials. 2015, 51, 30-42. 45. Lee E. S.; Na K.; Bae Y. H. Super pH-Sensitive Multifunctional Polymeric Micelle. Nano Lett. 2005, 5(2), 325-329. 46. Lee E. S.; Gao Z.; Kim D.; Park K.; Kwon I. C.; Bae Y. H. Super pH-sensitive multifunctional polymeric micelle for tumor pHespecific TAT exposure and multidrug resistance. J Control Release. 2008, 129(3), 228-326. 47. Gao ZG.; Tian L.; Hu J.; Park I. S.; Bae Y. H. Prevention of metastasis in a 4T1 murine breast cancer model by doxorubicin carried by folate conjugated pH sensitive polymeric micelles. J Control Release. 2011, 152(1), 84-89. 48. Hansen M. B.; van Gaal E.; Minten I.; Storm G.; van Hest J. C.; Löwik D. W. Constrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomes. J Control Release. 2012, 164(1), 87-94. 49. Gilar M.; Olivova P.; Daly A. E.; Gebler J. C. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J Sep Sci. 2005, 28(14), 1694-1703. 50. Jin E.; Zhang B.; Sun X.; Zhou Z.; Ma X.; Sun Q.; Tang J.; Shen Y.; Van Kirk E.; Murdoch W. J.; Radosz M. Acid-Active Cell-Penetrating Peptides for in Vivo Tumor-Targeted Drug Delivery. J Am Chem Soc. 2013, 135(2) 933-940. 51. Li ZY.; Liu Y.; Hu J. J.; Xu Q.; Liu L. H.; Jia H. Z.; Chen W. H.; Lei Q.; Rong L.; Zhang X. Z. Stepwise-Acid-Active Multifunctional Mesoporous Silica Nanoparticles for Tumor-Specific Nucleus-Targeted Drug Delivery. ACS Appl Mater Interfaces. 2014, 6(16), 14568-14575.

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

52. Liu Z.; Xiong M.; Gong J.; Zhang Y.; Bai N.; Luo Y.; Li L.; Wei Y.; Liu Y.; Tan X.; Xiang R. Legumain

protease-activated

TAT-liposome

cargo

for

targeting

tumours

and

their

microenvironment. Nat Commun. 2014, 5, 4280. 53. Lee S. H.; Moroz E.; Castagner B.; Leroux J. C. Activatable Cell Penetrating Peptide−Peptide Nucleic Acid Conjugate via Reduction of Azobenzene PEG Chains. J Am Chem Soc. 2014, 136(37), 12868-12871. 54. Wender P. A.; Mitchell D. J.; Pattabiraman K.; Pelkey E.T.; Steinman L.; Rothbard J. B. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters. Proc Natl Acad Sci U S A. 2000, 97(24), 13003–13008. 55. Nakase I.; Niwa M.; Takeuchi T.; Sonomura K.; Kawabata N.; Koike Y.; Takehashi M.; Tanaka S.; Ueda K.; Simpson J. C.; Jones A. T.; Sugiura Y.; Futaki S. Cellular Uptake of Arginine-Rich Peptides: Roles for Macropinocytosis and Actin Rearrangement. Mol Ther. 2004, 10(6), 1011-1022. 56. Amand H. L.; Fant K.; Nordén B.; Esbjörner E. K. Stimulated endocytosis in penetratin uptake: effect of arginine and lysine. Biochem Biophys Res Commun. 2008, 371(4), 621-625. 57. Kamide K.; Nakakubo H.; Uno S.; Fukamizu A. Isolation of novel cell-penetrating peptides from a random peptide library using in vitro virus and their modifications. Int J Mol Med. 2010, 25(1), 41-51. 58. Song J.; Kai M.; Zhang W.; Zhang J.; Liu L.; Zhang B.; Liu X.; Wang R. Cellular uptake of transportan 10 and its analogs in live cells: Selectivity and structure–activity relationship studies. Peptides. 2011, 32(9), 1934-1941. 59. Rizzuti M.; Nizzardo M.; Zanetta C.; Ramirez A.; Corti S. Therapeutic applications of the cell-penetrating HIV-1 Tat peptide. Drug Discov Today. 2015, 20(1), 76-85. 60. Grunwald J, Rejtar T, Sawant R, Wang Z, Torchilin VP. TAT peptide and its conjugates: proteolytic stability. Bioconjug Chem. 2009, 20(8), 1531-1537. 61. Elmquist A.; Langel U. In vitro uptake and stability study of pVEC and its all-D analog. Biol Chem. 2003, 384(3), 387-393. 62. Pujals S.; Sabidó E.; Tarragó T.; Giralt E. All-D proline-rich cellpenetrating peptides: a preliminary in vivo internalization study. Biochem Soc Trans. 2007, 35(Pt 4), 794-796. 63. Verdurmen W. P.; Bovee-Geurts P. H.; Wadhwani P.; Ulrich A. S.; Hällbrink M.; van Kuppevelt T. H.; Brock R. Preferential Uptake of L- versus D-Amino Acid Cell-Penetrating Peptides in a Cell Type-Dependent Manner. Chem Biol. 2011, 18(8), 1000-1010. 64. Montrose K.; Yang Y.; Sun X.; Wiles S.; Krissansen G. W. Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs. Sci. Rep. 2013, 3, 1661. 65. Nakase I.; Konishi Y.; Ueda M.; Saji H.; Futaki S. Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug deliveryin vivo. J Control Release. 2012, 159(2), 181-188. 66. Yao X.; Chen L.; Chen X.; He C.; Zheng H.; Chen X. Intercellular pH-responsive histidine modified dextran-g-cholesterol micelle for anticancer drug delivery. Colloids Surf B Biointerfaces. 2014, 121, 36-43. 67. Toriyabe N.; Hayashi Y.; Harashima H. The transfection activity of R8-modified nanoparticles and siRNA condensation using pH sensitive stearylated-octa histidine. Biomaterials 2013, 34, 1337-1343.

ACS Paragon Plus Environment

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

68. Wu H.; Zhu L.; Torchilin V. P. pH-sensitive poly(histidine)-PEG/DSPE-PEG co polymer micelles for cytosolic drug delivery. Biomaterials 2013, 34, 1213-1222. 69. Tian L.; Bae Y. H. Cancer nanomedicines targeting tumor extracellular pH. Colloids Surf B Biointerfaces. 2012, 99, 116–126 70. Wen Y.; Guo Z.; Du Z.; Fang R.; Wu H.; Zeng X.; Wang C.; Feng M.; Pan S. Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives. Biomaterials. 2012, 33(32), 8111-8121. 71. Cai X.; Zhu H.; Dong H.; Li Y.; Su J.; Shi D. Suppression of VEGF by reversible-PEGylated histidylated polylysine in cancer therapy. Adv Healthc Mater. 2014, 3(11), 1818-1827. 72. Zhang W.; Song J.; Zhang B.; Liu L.; Wang K.; Wang R. Design of acid-activated cell penetrating peptide for delivery of active molecules into cancer cells. Bioconjug. Chem. 2011, 22, 1410–1415. 73. Zhang Q.; Tang J.; Fu L.; Ran R.; Liu Y.; Yuan M.; He Q. A pH-responsiveα-helical cell penetrating peptide-mediated liposomal delivery system. Biomaterials 2013, 34, 7980–7993. 74. Tu Z.; Volk M.; Shah K.; Clerkin K.; Liang J. F. Constructing bioactive peptides with pH dependent activities. Peptides 2009, 30, 1523–1528. 75. Li L.; He J.; Eckert R.; Yarbrough D.; Lux R.; Anderson M.; Shi W. Design and characterization of an acid-activated antimicrobial peptide. Chem. Biol. Drug Des. 2010, 75, 127–132. 76. Makovitzki A.; Fink A.; Shai Y. Suppression of human solid tumor growth in mice by intratumor and systemic inoculation of histidine-rich and pH-dependent host defense–like lytic peptides, Cancer Res. 2009, 69, 3458–3463 77. Midoux P.; Kichler A.; Boutin V.; Maurizot J. C.; Monsigny M. Membrane permeabilization and efficient gene transfer by a peptide containing several histidines. Bioconjug Chem. 1998, 9(2), 260-267. 78. Kichler A.; Mason A. J.; Bechinger B. Cationic amphipathic histidine-rich peptides for gene delivery. Biochim Biophys Acta. 2006, 1758(3), 301-317. 79. Tanaka K.; Kanazawa T.; Horiuchi S.; Ando T.; Sugawara K.; Takashima Y.; Seta Y.; Okada H. Cytoplasm-responsive nanocarriers conjugated with a functional cell-penetrating peptide for systemic siRNA delivery. Int J Pharm. 2013, 455(1-2), 40-47. 80. Jiang T.; Zhang Z.; Zhang Y.; Lv H.; Zhou J.; Li C.; Hou L.; Zhang Q. Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery. Biomaterials, 2012, 33, 9246–9258. 81. Zhao B. X.; Zhao Y.; Huang Y.; Luo L. M.; Song P.; Wang X.; Chen S.; Yu K. F.; Zhang X. Zhang Q. The efficiency of tumor-specific pH-responsive peptide-modified polymeric micelles containing paclitaxel, Biomaterials 2012, 33, 2508–2520 82. Jiang T.; Olson E. S.; Nguyen Q. T.; Roy M.; Jennings P. A.; Tsien R. Y. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc Natl Acad Sci U S A. 2004, 101(51), 17867-17872. 83. Aguilera T. A.; Olson E. S.; Timmers M. M.; Jiang T.; Tsien R. Y. Systemic in vivo distribution of activatable cell penetrating peptides is superior to cell penetrating peptides. Integr Biol (Camb). 2009, 1(5-6), 371-381. 84. Olson ES.; Aguilera T. A.; Jiang T.; Ellies L. G.; Nguyen Q. T.; Wong E. H.; Gross L. A.; Tsien R. Y. In vivo characterization of activatable cell penetrating peptides for targeting

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

protease activity in cancer. Integr Biol (Camb). 2009, 1(5-6), 382-393. 85. Olson E. S.; Jiang T.; Aguilera T. A.; Nguyen Q. T.; Ellies L. G.; Scadeng M.; Tsien R. Y. Activatable cell penetrating peptides linked to nanoparticles as dual probes for in vivo fluorescence and MR imaging of proteases. Proc Natl Acad Sci U S A. 2010, 107(9), 4311-4316. 86. Huang S.; Shao K.; Liu Y.; Kuang Y.; Li J.; An S.; Guo Y.; Ma H.; Jiang C. Tumor-Targeting and Microenvironment-Responsive Smart Nanoparticles for Combination Therapy of Antiangiogenesis and Apoptosis. ACS Nano. 2013, 7(3), 2860-2871. 87. Gao H.; Zhang S.; Cao S.; Yang Z.; Pang Z.; Jiang X. Angiopep-2 and Activatable Cell-Penetrating Peptide DualFunctionalized Nanoparticles for Systemic Glioma-Targeting Delivery. Mol Pharm. 2014, 11(8), 2755-2763. 88. van Duijnhoven S. M.; Robillard M. S.; Hermann S.; Kuhlmann M. T.; Schäfers M.; Nicolay K.; Grüll H. Imaging of MMP Activity in Postischemic Cardiac Remodeling Using Radiolabeled MMP-2/9 Activatable Peptide Probes. Mol Pharm. 2014, 11(5), 1415-1423. 89. van Duijnhoven S. M.; Robillard M. S.; Nicolay K.; Grüll H.Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation. J Nucl Med. 2011, 52(2), 279-286. 90. Nguyen Q. T.; Olson E. S.; Aguilera T. A.; Jiang T.; Scadeng M.; Ellies L. G.; Tsien R. Y. Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival. Proc Natl Acad Sci U S A. 2010, 107(9), 4317-4322. 91. Metildi C. A.; Felsen C. N.; Savariar E. N.; Nguyen Q. T.; Kaushal S.; Hoffman R. M.; Tsien R. Y.; Bouvet M. Fluorescence-guided surgery of pancreatic cancer using activatable cell penetrating peptides (ACPPs) in orthotopic mouse models. Ann Surg Oncol. 2015, 22(6), 2082-2087. 92. Savariar E. N.; Felsen C. N.; Nashi N.; Jiang T.; Ellies L. G.; Steinbach P.; Tsien R. Y.; Nguyen Q. T. Real-time In Vivo Molecular Detection of Primary Tumors and Metastases with Ratiometric Activatable Cell-Penetrating Peptides. Cancer Res. 2013, 73(2), 855-864. 93. Ren Y.; Hauert S.; Lo J. H.; Bhatia S. N. Identification and Characterization of Receptor-Specific Peptides for siRNA Delivery. ACS Nano. 2012, 6(10), 8620-8631. 94. Karagiannis E. D.; Alabi C. A.; Anderson D. G. Rationally Designed Tumor-Penetrating Nanocomplexes. ACS Nano. 2012, 6(10), 8484-8487. 95. Gong C.; Li X.; Xu L.; Zhang Y. H. Target delivery of a gene into the brain using the RVG29-oligoarginine peptide. Biomaterials. 2012, 33(12), 3456-3463. 96. Youn P.; Chen Y.; Furgeson D. Y. A Myristoylated Cell-Penetrating Peptide Bearing a Transferrin

Receptor-Targeting

Sequence

for

Neuro-Targeted

siRNA

Delivery.

Mol

Pharm. 2014, 11(2), 486-495. 97. Svensen N.; Walton J. G.; Bradley M. Peptides for cell-selective drug delivery. Trends Pharmacol Sci. 2012, 33(4), 186-192. 98. Myrberg H.; Zhang L.; Mäe M.; Langel U. Design of a Tumor-Homing Cell-Penetrating Peptide. Bioconjug Chem. 2008 , 19(1), 70-75. 99. Liu Y.; Ran R.; Chen J.; Kuang Q.; Tang J.; Mei L.; Zhang Q.; Gao H.; Zhang Z.; He Q. Paclitaxel loaded liposomes decorated with a multifunctional tandem peptide for glioma targeting. Biomaterials. 2014, 35(17), 4835-4847.

ACS Paragon Plus Environment

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

100. Kang M. H.; Park M. J.; Yoo H. J.; Hyuk K. Y.; Lee S. G.; Kim S. R.; Yeom D. W.; Kang M. J.; Choi Y. W. RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delivery to hepsin-expressing cancer cells. Eur J Pharm Biopharm. 2014, 87(3), 489-499. 101. Wadia J. S.; Stan R. V.; Dowdy S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 2004, 10, 310–315. 102. Ye SF; Tian M. M.; Wang T. X.; Ren L.; Wang D.; Shen L. H.; Shang T. Synergistic effects of cell-penetrating peptide Tat and fusogenic peptide HA2-enhanced cellular internalization and gene transduction of organosilica nanoparticles. Nanomedicine. 2012, 8(6), 833-841. 103. Salomone F.; Cardarelli F.; Di Luca M.; Boccardi C.; Nifosì R.; Bardi G.; Di Bari L.; Serresi M.; Beltram F. A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape. J Control Release. 201, 163(3), 293-303. 104. El-Sayed A.; Futaki S.; Harashima H. Delivery of Macromolecules Using Arginine-Rich Cell-Penetrating Peptides: Ways to Overcome Endosomal Entrapment. AAPS J. 2009, 11(1), 13-22. 105. Lo S. L.; Wang S. An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection. Biomaterials. 2008, 29(15), 2408-2414. 106. Kondo E.; Saito K.; Tashiro Y.; Kamide K.; Uno S.; Furuya T.; Mashita M.; Nakajima K.; Tsumuraya

T.; Kobayashi

N.; Nishibori

M.; Tanimoto

M.; Matsushita

M.

Tumour

lineage-homing cell-penetrating peptides as anticancer molecular delivery systems. Nat Commun. 2012, 3, 951. 107. Higa M.; Katagiri C.; Shimizu-Okabe C.; Tsumuraya T.; Sunagawa M,.; Nakamura M.; Ishiuchi S.; Takayama C.; Kondo E.; Matsushita M. Identification of a novel cell-penetrating peptide targeting human glioblastoma cell lines as a cancer-homing transporter. Biochem Biophys Res Commun. 2015, 457(2), 206-212. 108. Gao H.; Zhang Q.; Yang Y.; Jiang X.; He Q. Tumor homing cell penetrating peptide decorated nanoparticles used for enhancing tumor targeting delivery and therapy. Int J Pharm. 2014, 478(1), 240-250. 109. Jones A. T.; Sayers E. J. Cell entry of cell penetrating peptides: tales of tails wagging dogs. J Control Release. 2012, 161(2), 582-591 110. Raghuraman H.; Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep. 2007, 27(4-5), 189-223. 111. Kaiser E. T.; Kézdy F. J. Peptides with affinity for membranes. Annu Rev Biophys Biophys Chem. 1987, 16, 561-581. 112. Ferrer-Miralles N.; Vázquez E.; Villaverde A. Membrane-active peptides for non-viral gene therapy: making the safest easier. Trends Biotechnol. 2008, 26(5), 267-275. 113. Henriques S. T.; Melo M. N.; Castanho M. A. Cell-penetrating peptides and antimicrobial peptides: how different are they? Biochem J. 2006 , 399(1), 1-7. 114. Wadhwani P.; Reichert J.; Bürck J.; Ulrich A. S. Antimicrobial and cell-penetrating peptides induce lipid vesicle fusion by folding and aggregation. Eur Biophys J. 2012, 41(2), 177-187. 115. Zhang Q.; Ran R.; Zhang L.; Liu Y.; Mei L.; Zhang Z.; Gao H.; He Q. Simultaneous delivery of therapeutic antagomirs with paclitaxel for the management of metastatic tumors by

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

a pH-responsive anti-microbial peptide-mediated liposomal delivery system. J Control Release. 2015, 197, 208-218. 116. Zhang Q.; Tang J.; Ran R.; Liu Y.; Zhang Z.; Gao H.; He Q. Development of an anti-microbial peptide-mediated liposomal delivery system: a novel approach towards pH-responsive anti-microbial peptides. Drug Deliv. 2015, 19, 1-8. 117. Subbarao N. K.; Parente R. A.; Szoka F. C Jr.; Nadasdi L.; Pongracz K. pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry. 1987, 26(11), 2964-2972. 118. Parente R. A.; Nir S.; Szoka F. C. Jr. pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide. J Biol Chem. 1988, 263(10), 4724-4730. 119. Li W.; Nicol F.; Szoka F. C. Jr. GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery. Adv Drug Deliv Rev. 2004, 56(7), 967-985. 120. Kakudo T.; Chaki S.; Futaki S.; Nakase I.; Akaji K.; Kawakami T.; Maruyama K.; Kamiya H.; Harashima H. Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system. Biochemistry. 2004, 43(19), 5618-5628. 121. Sasaki K.; Kogure K.; Chaki S.; Nakamura Y.; Moriguchi R.; Hamada H.; Danev R.; Nagayama K.; Futaki S.; Harashima H.An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives. Anal Bioanal Chem. 2008, 391(8), 2717-2727. 122. Hatakeyama H.; Ito E.; Akita H.; Oishi M.; Nagasaki Y.; Futaki S.; Harashima H. A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. J Control Release. 2009, 139(2), 127-132. 123. McCarthy H. O.; McCaffrey J.; McCrudden C. M.; Zholobenko A.; Ali A. A.; McBride J. W.; Massey A. S.; Pentlavalli S.; Chen K. H.; Cole G.; Loughran S. P.; Dunne N. J.; Donnelly R. F.; Kett V. L.; Robson T. Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery. J Control Release. 2014, 189, 141-149. 124. Shaheen S. M.; Akita H.; Nakamura T.; Takayama S.; Futaki S.; Yamashita A.; Katoono R.; Yui N.; Harashima H. KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine. Biomaterials. 2011, 32(26), 6342-6350. 125. Nouri F. S.; Wang X.; Dorrani M.; Karjoo Z.; Hatefi A. A Recombinant Biopolymeric Platform for Reliable Evaluation of the Activity of pH-Responsive Amphiphile Fusogenic Peptides. Biomacromolecules. 2013, 14(6), 2033-2040. 126. Kusumoto K.; Akita H.; Ishitsuka T.; Matsumoto Y.; Nomoto T.; Furukawa R.; El-Sayed A.; Hatakeyama H.; Kajimoto K.; Yamada Y.; Kataoka K.; Harashima H. Lipid Envelope-Type Nanoparticle Incorporating a Multifunctional Peptide for Systemic siRNA Delivery to the Pulmonary Endothelium. ACS Nano. 2013, 7(9), 7534-7541. 127. Hunt J. F.; Rath P.; Rothschild K. J.; Engelman D. M. Spontaneous, pH-Dependent Membrane Insertion of a Transbilayer α-Helix. Biochemistry. 1997, 36(49), 15177-15192. 128.

Andreev

O.

A.; Engelman

D.

M.; Reshetnyak

Y.

K.

pH-sensitive

membrane peptides (pHLIPs) as a novel class of delivery agents. Mol Membr Biol. 2010, 27(7), 341-352. 129.

Yao

L.; Daniels

J.; Moshnikova

A.; Kuznetsov

S.; Ahmed

ACS Paragon Plus Environment

A.; Engelman

D.

Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

M.; Reshetnyak Y. K.; Andreev O. A. pHLIP peptide targets nanogold particles to tumors. Proc Natl Acad Sci U S A. 2013, 110(2), 465-470. 130. Weerakkody D.; Moshnikova A.; Thakur M. S.; Moshnikova V.; Daniels J.; Engelman D. M.; Andreev O. A.; Reshetnyak Y. K. Family of pH (low) insertion peptides for tumor targeting. Proc Natl Acad Sci U S A. 2013, 110(15), 5834-5839. 131. Musial-Siwek M.; Karabadzhak A.; Andreev O. A.; Reshetnyak Y. K.; Engelman D. M. Tuning the insertion properties of pHLIP. Biochim Biophys Acta. 2010, 1798(6), 1041-1046. 132. Andreev O. A.; Dupuy A. D.; Segala M.; Sandugu S.; Serra D. A.; Chichester C. O.; Engelman D. M.; Reshetnyak Y. K. Mechanism and uses of a peptide that targets tumors and other acidic tissue in vivo. Proc Natl Acad Sci U S A. 2007, 104(19), 7893-7898. 133. Vāvere A. L.; Biddlecombe G. B.; Spees W. M.; Garbow J. R.; Wijesinghe D.; Andreev O. A.; Engelman D. M.; Reshetnyak Y. K.; Lewis J. S. A novel technology for the imaging of acidic prostate tumors by positron emission tomography. Cancer Res. 2009, 69(10), 4510-4516. 134. Andreev O. A.; Engelman D. M.; Reshetnyak Y. K. Targeting acidic diseased tissue: New technology based on use of the pH (Low) Insertion Peptide (pHLIP). Chim Oggi. 2009, 27(2), 34-37. 135. Yao L.; Daniels J.; Wijesinghe D.; Andreev O. A.; Reshetnyak Y. K. pHLIP®-mediated delivery of PEGylated liposomes to cancer cells. J Control Release. 2013, 167(3), 228-237. 136. Deacon J. C.; Engelman D. M.; Barrera F. N. Targeting acidity in diseased tissues: Mechanism and applications of the membrane-inserting peptide, pHLIP. Arch Biochem Biophys. 2015, 565, 40-48. 137. Teesalu T.; Sugahara K. N.; Kotamraju V. R.; Ruoslahti E. C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration. Proc Natl Acad Sci U S A. 2009, 106(38), 16157-16162. 138. Feron O. Tumor-penetrating peptides: a shift from magic bullets to magic guns. Sci Transl Med. 2010, 2(34), 34ps26. 139. Sugahara K. N.; Teesalu T.; Karmali P. P.; Kotamraju V. R.; Agemy L.; Greenwald D. R.; Ruoslahti E. Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs. Science. 2010, 328(5981), 1031-1035. 140. Teesalu T.; Sugahara K. N.; Ruoslahti E. Tumor-penetrating peptides. Front Oncol. 2013, 3, 216. 141. Sugahara K. N.; Teesalu T.; Karmali P. P.; Kotamraju V. R.; Agemy L.; Girard O. M.; Hanahan D.; Mattrey R. F.; Ruoslahti E. Tissue-Penetrating Delivery of Compounds and Nanoparticles into Tumors. Cancer Cell. 2009, 16(6), 510-520. 142. Alberici L.; Roth L.; Sugahara K. N.; Agemy L.; Kotamraju V. R.; Teesalu T.; Bordignon C.; Traversari C.; Rizzardi G. P.; Ruoslahti E. De Novo Design of a Tumor-Penetrating Peptide. Cancer Res. 2013, 73(2), 804-812. 143. Wang K.; Zhang X.; Liu Y.; Liu C.; Jiang B.; Jiang Y. Tumor penetrability and anti-angiogenesis

using

iRGD-mediated

delivery of

doxorubicin-polymer

conjugates.

Biomaterials. 2014, 35(30), 8735-8747. 144. Kang T.; Gao X.; Hu Q.; Jiang D.; Feng X.; Zhang X.; Song Q.; Yao L.; Huang M.; Jiang X.; Pang Z.; Chen H.; Chen J. iNGR-modified PEG-PLGA nanoparticles that recognize tumor vasculature and penetrate gliomas. Biomaterials. 2014, 35(14), 4319-4332. 145. Zhao C.; Liu X.; Liu J.; Yang Z.; Rong X.; Li M.; Liang X.; Wu Y. Targeting peptide

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

iRGD-conjugated amphiphilic chitosan-co-PLA/DPPE drug delivery system for ennced tumor therapy. Colloids Surf B Biointerfaces. 2014, 123, 787-796. 146. Li M.; Tang Z.; Zhang D.; Sun H; Liu H.; Zhang Y.; Zhang Y.; Chen X. Doxorubicin-loaded polysaccharide nanoparticles suppress the growth of murine colorectal carcinoma and inhibit the metastasis of murine mammary carcinoma in rodent models. Biomaterials. 2015, 51, 161-172. 147. Sugahara K. N.; Braun G. B.; de Mendoza T. H.; Kotamraju V. R.; French R. P.; Lowy A. M.; Teesalu T.; Ruoslahti E. Tumor-Penetrating iRGD Peptide Inhibits Metastasis. Mol Cancer Ther. 2015, 14(1), 120-128. 148. Paoli E. E.; Ingham E. S.; Zhang H.; Mahakian L. M.; Fite B. Z.; Gagnon M. K.; Tam S.; Kheirolomoom A.; Cardiff R. D.; Ferrara K. W. Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes. J Control Release. 2014, 178, 108-117. 149. Wang J.; Lei Y.; Xie C.; Lu W.; Wagner E.; Xie Z.; Gao J.; Zhang X.; Yan Z.; Liu M. Retro-Inverso

CendR

Peptide-Mediated

Polyethyleneimine

for

Intracranial

Glioblastoma-Targeting Gene Therapy. Bioconjug Chem. 2014, 25(2), 414-423. 150. Roth L.; Agemy L.; Kotamraju V. R.; Braun G.; Teesalu T.; Sugahara K. N.; Hamzah J.; Ruoslahti E. Transtumoral targeting enabled by a novel neuropilin-binding peptide. Oncogene. 2012, 31(33), 3754-3763. 151. Hu Q.; Gu G.; Liu Z.; Jiang M.; Kang T.; Miao D.; Tu Y.; Pang Z.; Song Q.; Yao L.; Xia H.; Chen H.; Jiang X.; Gao X.; Chen J. F3 peptide-functionalized PEG-PLA nanoparticles co-administrated with tLyp-1 peptide for anti-glioma drug delivery. Biomaterials. 2013, 34(4), 1135-1145. 152. Wang J.; Lei Y.; Xie C.; Lu W.; Yan Z.; Gao J.; Xie Z.; Zhang X.; Liu M. Targeted gene delivery

to

glioblastoma

using

a

C-end

rule

RGERPPR

peptide-functionalised

polyethylenimine complex. Int J Pharm. 2013, 458(1), 48-56. 153. Pang H. B.; Braun G. B.; Friman T.; Aza-Blanc P.; Ruidiaz M. E.; Sugahara K. N.; Teesalu T.; Ruoslahti E. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability. Nat Commun. 2014, 5, 4904. 154. Vasconcelos L.; Pärn K.; Langel U. Therapeutic potential of cell-penetrating peptides. Ther Deliv. 2013, 4(5), 573-591.

ACS Paragon Plus Environment

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

For Table of Contents Only

Taming cell penetrating peptides: Never too old to teach old dogs new tricks. Qianyu Zhang, Huile Gao, Qin He*.

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.1: Schematic illustrations of TAT-SL, TAT and cleavable PEG comodified liposomal system (C-TAT-SL). Adapted from Ref.41 with permission. 128x126mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 30 of 38

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Fig.2: Amidization of TAT’s primary amines to succinyl amides and their acid-triggered hydrolysis. Adapted from Ref.50 with permission. 282x188mm (72 x 72 DPI)

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.3: Schematic illustration of colon-specific delivery with activatable CPP-PNA (After the reductive cleavage of azobenzene bond, a self-immolative moiety releases the intact lysine residue on the CPP backbone). Adapted from Ref. 53 with permission. 162x102mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 32 of 38

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Fig.4: Surface charge of TH-Lip was shifted from negative to positive under tumor microenvironment where the pH value declined, and it was internalized into tumor cells efficiently via instant electrostatic attraction between protonated TH peptide and cellular membrane and then the subsequent endocytosis. Adapted from Ref.73 with permission. 270x200mm (96 x 96 DPI)

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 5: Surface HA could be degradated by HAase in HAase-rich tumor milieu and exposed peptide R6H4, where it could be applied for enhanced cell penetration to mildly acidic tumor microenvironment for improved cellular uptake. Upon internalization, R6H4 could facilitate liposomal endosomal/lysosomal escape. Adapted from Ref.80 with permission. 146x101mm (220 x 220 DPI)

ACS Paragon Plus Environment

Page 34 of 38

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Fig.6: Liposomes modified with R8-RGD could specifically bind to integrin avβ3 receptors expressed on the brain capillary endothelial cells and transport across the BBB through a synergetic effect of both R8 and RGD. Adapted from Ref.99 with permission. 167x119mm (96 x 96 DPI)

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.7: pHLIP peptides exhibit three distinct states. In State I, pHLIPs are soluble and unstructured in aqueous solution. In State II, pHLIPs bind reversibly to the outer leaflet of membrane bilayers, remaining largely unstructured at physiological pH. In acidic extracellular environments a pHLIP inserts its C-terminus across the membrane to form State III, a stable transmembranea-helix. Adapted from Ref. 136 with permission. 227x94mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 36 of 38

Page 37 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

Fig.8: Schematic presentation of pHLIP/PEG coated liposomes (A) and their interaction with cellular membranes (B). Adapted from Ref. 135 with permission. 115x109mm (96 x 96 DPI)

ACS Paragon Plus Environment

Molecular Pharmaceutics

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig.9: Multistep Binding and Penetration Mechanism of iRGD. Adapted from Ref. 139 with permission. 107x101mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 38 of 38