Cyclic Cell-Penetrating Peptides As Efficient Intracellular Drug

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Review

Cyclic Cell-Penetrating Peptides As Efficient Intracellular Drug Delivery Tools Shang Eun Park, Muhammad Imran Sajid, Keykavous Parang, and Rakesh Kumar Tiwari Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00633 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Molecular Pharmaceutics

Cyclic Cell-Penetrating Peptides As Efficient Intracellular Drug Delivery Tools Shang Eun Park†, Muhammad Imran Sajid†,‡, Keykavous Parang†*, and Rakesh Kumar Tiwari†* †Center

for Targeted Drug Delivery, Department of Biomedical and Pharmaceutical Sciences,

Chapman University School of Pharmacy, Harry and Diane Rinker Health Science Campus, Irvine, California 92618, United States ‡Faculty

of Pharmacy, University of Central Punjab, Lahore 54000, Pakistan

Keywords: Amphiphilic, Cyclic Peptide, Cell-Penetrating Peptides, Cyclic Cell-Penetrating Peptides, Cell Impermeability, Stability, Delivery

ACS Paragon Plus Environment

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ABSTRACT

Cyclic cell-penetrating peptides are relatively a newer class of peptides that have a huge potential for the intracellular delivery of therapeutic agents aimed at treating challenging ailments like multidrug resistance bacterial diseases, cancer, and HIV infection. Although, cell-penetrating peptides (CPPs) have been extensively explored as intracellular delivery vehicles, however, they have some inherent limitations like poor stability, endosomal entrapment, toxicity, and suboptimal cell penetration. Owing to their favorable properties that avoid these limitations, cyclic CPPs can provide a good alternative to linear CPPs. Several reviews have been published in the last decade that cover CPPs and cyclic peptides independently. To the best of our knowledge, this is one of the first reviews that covers cyclic CPPs comprehensively in the light of studies published so far. In this review, we have detailed examples of cyclic CPPs, their structures, cyclization strategies followed by a detailed account of their advantages over their linear counterparts. A hot area in cyclic CPPs is the exploration of cell-penetration mechanisms; this review highlights this topic in detail. Finally, we will review the applications of cyclic CPPs, followed by conclusions and future prospects.

1.

Introduction Biological membranes pose a major obstacle in the permeation of therapeutic agents to the target

sites within the cells due to their lipophilic nature. Nevertheless, many useful therapeutic agents are either hydrophilic or macromolecules that cannot adequately reach the target site intracellularly.1 Several approaches have been adopted for the delivery of macromolecules and hydrophilic therapeutic moieties, such as membrane perturbation methods and viral transfections, however, these approaches possess intrinsic limitations of immunogenicity, higher toxicity, and


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poor yield.2, 3 Cell-penetrating peptides (CPPs) have emerged as a powerful delivery system for the intracellular delivery of membrane-impermeable therapeutic agents.4 CPPs are short oligopeptides consisting of 5 to 30 amino acids that have the potential to load various cargos of interest as well as the ability to cross the biological membranes.1 Owing to their ability to efficiently permeate across plasma membranes, they have been extensively investigated as molecular vehicles to transport several therapeutic and imaging agents into the cells.5,

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However, there are some limitations associated with CPPs originating due to their functional and structural features that limit their usefulness as drug delivery tools, such as poor stability that prevent them from reaching to the target site in intact and biologically active form.7 In addition, most of the conventional CPPs have low cell specificity, limiting their direct application to the target cells to achieve the desired effect.8 To overcome these problems, several research groups have designed and synthesized a number of cyclic peptides that provide superior cell-penetrating ability with improved physicochemical characteristics to avoid the limitations posed by the linear CPPs. Recently, several studies have reported the advantages of cyclization in improving the physicochemical characteristic, stability, and effectiveness of peptides as drug delivery tools. Cyclic cell-penetrating peptides (cyclic CPPs) is a new area that warrants further exploration. Although there are several studies that employed cyclic CPPs in the various biomedical applications and explored their structure-activity relationship, however, to the best of our knowledge, there is no current review that covers the applications of cyclic CPPs. While submitting this manuscript, another review was published which provided some mechanistic understanding of cyclic peptides9 however, it did not provide their applications in the drug delivery. Recently, numerous reviews addressed the cyclic peptides10-12 and cell-penetrating


3


peptides1,

13, 14

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separately. The purpose of this review is to address cyclic CPPs and their

applications. We will present the examples of cyclic CPPs used in various biomedical applications followed by advantages of cyclic CPPs compared to their linear counterparts. Also, we will focus on the cyclization strategies employed in recent studies. The hot area in cyclic CPPs is the exploration of cell-penetration mechanisms and their applications in drug delivery; this review highlights this topic in detail. Finally, we will review the applications of cyclic CPPs followed by conclusions and future prospects.

2.

Classes of CPPs CPPs can be classified either on the basis of their origin or according to their physicochemical

properties or their conformation. Schematic illustration of their classification is presented below. As per origin, CPPs can be classified as protein-derived, chimeric, and synthetic CPPs,15 while on the basis of physicochemical properties they can be classified as cationic, hydrophobic, and amphipathic CPPs (Figure 1).16 A database called CPPsite 2.0 is currently available that contains entries of about 1700 CPPs and provides comprehensive information of their 3D structures. This database is also useful for looking up the sources of information, their types, and variety of cargo molecules, e.g., proteins, nucleic acids, and nanoparticles that have been examined for the intracellular delivery using these peptides.17 Cyclic CPPs are a newer class of CPPs that have not been reviewed extensively; this review focuses on their various aspects.


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Figure 1. A schematic diagram illustrating the types of CPPs.

3.

Examples of cyclic CPPs Cyclic CPPs are derived from different methods. Many are synthesized in the laboratory while

others are purified from natural products and characterized. Several of them have been shown to improve the cell-penetrating ability from their linear counterparts. A number of chemical modifications were employed to improve stability and cell permeability. Various research groups have synthesized or discovered cyclic CPPs. Parang and co-workers were one of the pioneers to report the application of cyclic CPPs containing alternatively charged and hydrophobic amino acids as drug delivery tools in 2011.18 Several cyclic peptides containing L-amino acids were designed and synthesized to investigate their cell-penetrating properties and to examine their role as molecular transporters. Nischan et al. modified one of the first thoroughly investigated CPPs, TAT peptide which was derived from the viral protein for its ability to transduce into the cells,19 into cyclic form (cTAT, N3-PEG2-[K(rRGrKkRr)E]), and found out that cyclic TAT peptide has a


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considerably higher ability than its linear counterpart to deliver proteins (GFP) into the cell.20 (See Figure 2 for the structures of above-mentioned peptides) Around the same time, Cascales et al. discovered and characterized disulfide-rich cyclic CPPs derived from plants. They also examined the mechanism of their cellular transport and found convincing results for their cell-penetrating ability, leading them to conclude that this new class of peptides can be used as a delivery vehicle for potential drug candidates.21 Later, D’Souza et al. did more structural modifications to these peptides and examined their cell-penetrating properties, suggesting that these disulfide-rich cyclic peptides can act as cyclic CPPs.22 Traboulsi et al. investigated the effect of structural modifications on the cell-penetrating ability and proteolytic stability of macrocyclic peptides.23 Qian et al. designed and synthesized eighteen cyclic peptides, containing arginine and L-2-naphthylalanine, and found them to be a powerful tool for intracellular delivery of drugs and as a chemical probe.24, 25 Structures of these peptides are shown in Figure 3. Also, Ichimizu et al. designed cyclic cell-penetrating albumin derivative and explored its role as a versatile nanovehicle for intracellular delivery of therapeutic agents.26 Buckton and McAlpine alkylated side chain of hydrophilic cyclic CPPs and modified L-lysine to D-lysine and showed improved cell-permeability.27 Table 1 shows the names and sequences of numerous reported cyclic CPPs. (see sections 6 and 7 for further details) Synthetically engineered cyclic peptides have been rigorously investigated, and numerous cyclic CPPS were proven to be efficient molecular transporters for enhancing the efficacy of existing chemotherapeutic, antiviral and antibacterial agents.28-38 For instance, Shirazi et al. used cyclic peptide [W(RW)4K] containing arginine and tryptophan residues and conjugated it with doxorubicin through a ß-alanine linker. They found out that this cyclic CPP can be employed as an intracellular drug delivery tool.29 Thereafter, Darwish et al. inserted cysteine on the cyclic


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peptide to generate [C(WR)4K] to attach doxorubicin through disulfide bridge and found high nuclear uptake and localization of drug moiety in the nucleus.35 Also, Shirazi et al. designed linear and cyclic peptides containing tryptophan and lysine. Here, the change of arginine residue to lysine was made to see the differential effect of cationic nature in the cyclic CPPs. Furthermore, they prepared gold nanoparticles using these cyclic CPPs and loaded fluorescently labeled anti-HIV drugs [emtricitabine (FTC) and lamivudine (3TC)]. The comparative flow cytometry studies clearly indicated that cellular uptake of these drugs was significantly higher in the presence of cyclic [KW]5-capped gold nanoparticles than that of linear (KW)5 gold nanoparticles, linear (KW)5 peptide, and cyclic [KW]4 peptides (see Section 7 for further details).32 Adding to the previous work, Shirazi et al. synthesized several linear and cyclic peptides containing cysteine and arginine residues like linear (CR)3, linear (CR)4, linear (CR)5, cyclic [CR]4, and cyclic [CR]5 to investigate their ability to deliver sample cargo drugs intracellularly and to understand the role of cyclization and amino acid residues to see improvement in the cellpenetrability and found cyclic [CR]4 significantly enhanced cellular uptake of cell-impermeable phosphopeptides (F´-GpYEEI) in the presence of endocytic inhibitors and sodium azide in lymphoblastic leukemia cell line (CCRF-CEM).38 Also, Shirazi et al. prepared tryptophan and histidine-containing linear (WH)5 and its cyclic decapeptides [WH]5 to investigate the intracellular delivery of cell-impermeable phosphopeptides and the anti-HIV drug, emtricitabine, using this peptide.36 In another effort, El-Sayed et al. reported the synthesis of cyclic [HR]4 peptides as a molecular transporter, and it was discovered that this peptide could double the permeability of cellimpermeable fluorescence-labeled phosphopeptide.37 Also, Oh et al. synthesized two bicyclic peptides containing tryptophan and arginine residues namely [W5G]‐(triazole)‐[KR5] and FITC[W5E]‐(β‐Ala)‐[KR5] (Figure 4). These bicyclic peptides significantly improved the cellular


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delivery of cargo, and confocal microscopy revealed that the peptides were localized both in cytosol and nucleus. The results from the study suggest these peptides can act as a new class of cyclic CPP and cellular delivery tools.33

NH

H2N HN

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18

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8

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Figure 2. Cationic and Amphiphilic Cyclic Peptides.

H2N

HN

O

NH

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HN

N

O

NH

[WH]5

H2N

N

O N H

36

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N H

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[CR]438

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[HR]437

[W(RW)4K]29

NH

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H 2N

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OH H2N

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NH H2N

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cTAT peptide20

NH

SH

NH

N

O

NH

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NH

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O

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NH

SH

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NH2 HN

N3

NH

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NH

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NH

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O

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[WK]532

NH O

N

O

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NH

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NH

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HN

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2

NH

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Figure 3. Cationic and Amphiphilic Cyclic Peptides. *r denotes D-arginine.


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H 2N

NH

H 2N

H 2N

NH

NH

O

O HN O

H 2N N

NH HN

O O

NH

O

HN O

N+

NH

NH

O

O

NH

NH

NH2 N H

O

HN O

HN HN

HN

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O

NH

NH

O

NH2

N H H 2N

H N

O

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NH NH

H N

HN

HN

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NH2

O

NH

O

HN O

HN

NH2

HN

NH

O

NH

NH

N H

[FRRRRQ]24

O

NH

O N H

[FRRRRAAAAA]Rho25 H2N O HO

HN

O

O

O

N

NH O

O

HN

S

NH

O

O N

O

O

NH

C

P I

S

NH2

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OH

NH

N

S

S S

S

S I

C

A

G

P

C H

D

S

D

L K K CH R R

MCoTI-II*21, 22, 53

SFTI-1*21, 22

R

T

K

G

O O

S

S D G G V

G

HC Y

NH OH

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Kalata B1*21

Figure 4. Amphiphilic peptides, disulfide-rich polycyclic peptides, and L-2-naphthylalanine containing CCPPs. * Dithio bridge is internalized in the cyclic structure ᶲ is L-2-naphthylalanine Each Amino acid is abbreviated as one letter Table 1. Names and sequence of CCPPs. Type

Name

Sequence

Reference

Amphipathic

[WR]3

[WRWRWR]

18

Amphipathic

[WR]4

[WRWRWRWR]

18

Amphipathic

[WR]5

[WRWRWRWRWR]

18

Amphipathic

FITC-[W4R3K]

FITC-[WWWWRRRK]

18

Amphipathic

FITC-[W5R4K]

FITC-[WWWWWRRRRK]

18


10

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Amphipathic

kalata B1

[GLPVCGETCVGGTCNTPGCTCSWPVCTR 21 N]

Amphipathic

SFTI-1

[GRCTKbSIPPICFPD]

Amphipathic

MCoTI-II

[SGSDGGVCPKILKKCRRDSDCPGACICRG 39, 21, 22 NGYCG]

Cationic

cTAT

N3-PEG2-[K(rRGrKkRr)E]

20

Cationic

FITC-Cyc-R4

FITC-βA-[KRRRRE]-NH2

23

Cationic

FITC-Cyc-R6

FITC-βA-[KRRRRRRE]-NH2

23

Cationic

FITC-Cyc-R7

FITC-βA-[KRRRRRRRE]-NH2

23

Cationic

FITC-[R8]

FITC-βA-[KRRRRRRRRE]-NH2

23

Cationic

FITC-[R9]

FITC-βA-[KRRRRRRRRRE]-NH2

23

Cationic

FITC-[r7]

FITC-βA-[KrrrrrrrE]-NH2

23

Cationic

FITC-[r8]

FITC-βA-[KrrrrrrrrE]-NH2

23

Cationic

FITC-[r9]

FITC-βA-[KrrrrrrrrrE]-NH2

23

Cationic

FITC-[(L,D)-R6]

FITC-βA-[KRrRrRrE]-NH2

23

Cationic

FITC-[(L,D)-R7]

FITC-βA-[KrRrRrRrE]-NH2

23

Cationic

FITC-[(L,D)-R8]

FITC-βA-[KRrRrRrRrE]-NH2

23

Cationic

FITC-Bicyc-0-[R6] FITC-βA-[ERRRK]-[KRRRE]-NH2

23

Cationic

FITC-Bicyc-1-[R6] FITC-βA-[ERRRK]-βA-[KRRRE]-NH2

23

Cationic

FITC-Bicyc-2-[R6] FITC-βA-[ERRRK]-(βA)2-[KRRRE]-NH2

23

Cationic

FITC-Bicyc-3-[R6] FITC-βA-[ERRRK]-(βA)3-[KRRRE]-NH2

23

Cationic

FITC-(R1)-[R5]

FITC-βA-R-[KRRRRRRE]-NH2

23

Cationic

FITC-(R2)-[R5]

FITC-βA-RR-[KRRRRRE]-NH2

23

Cationic

FITC-(R3)-[R3]

FITC-βA-RRRR-[KRRRE]-NH2

23

Cationic

FITC-(R4)-[R3]

FITC-βA-RRR-[KRRRRE]-NH2

23

Amphipathic

[FΦRRRRQ]

[FΦaRRRRQ]

24

Amphipathic

[FΦR4-A5]Rho

[FΦaRRRRAAAAA]Rho

25

21, 22


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Cationic

[r12]

GSSG[CrrrrrrrrrrrrC]

26

Amphipathic

[W(RW)4K]

[WRWRWRWRK]

29

Amphipathic

FITC-[WK]5

FITC-βA-[WKWKWKWKWK]

32

Amphipathic

[WK]5

[WKWKWKWKWK]

32

Amphipathic

FITC-Bicyc[KW4E]-(β-Ala)[KR5]

FITC-[KWWWWE]-(β-Ala)-[KRRRRR]

33

Amphipathic

[W5G]-(triazole)- [WWWWWG]-(triazole)-[KRRRRR] [KR5]

33

Amphipathic

[C(WR)4K]

[CRWRWKWRWR]

35

Amphiphatic

[WH]5

[WHWHWHWHWH]

36

Cationic

[HR]4

[HRHRHRHR]

37

Amphipathic

[C(WR)4K]

[CRWRWKWRWR]

35

Cationic

[CR]4

[CRCRCRCR]

38

Amphipathic

[WR]6

[WRWRWRWRWRWR]

40

Amphipathic

[WR]7

[WRWRWRWRWRWRWRWR]

40

Amphipathic

[WR]8

[WRWRWRWRWRWRWRWRWR]

40

Amphipathic

[WR]9

[WRWRWRWRWRWRWRWRWRWR]

40

Amphipathic

FITC-[W9R8K]

FITC-[WWWWWWWWWRRRRRRRRK]

40

a

Φ is L-2-naphthylalanine

b Lys

residues were labeled with Alexa-488.

r = D-arginine, Cyc = cyclic. Bicyc = Bicyclic, FITC = Fluorescein isothiocyanate

4. Advantages of cyclic CPPs compared to their linear counterparts According to the several reports described above and numerous other studies performed on cyclic CPPs, it is believed that they offer convincing advantages compared to their linear counterparts.41-43 Cyclic CPPs possess one or more advantages over their liner counterparts:


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1) They possess significantly increased cell permeability compared to their linear counterparts,4 2) Cyclic CPPs have generally higher resistant to proteolysis,24, 44-46 3) Some cyclic CPPS exhibit efficient endosomal escape,47 and some show endosomalindependent uptake,18 4) Improved higher affinity of the cyclic peptides to the target receptor is observed compared to the linear form,6, 48 5) A few cyclic CPPs have nuclear targeting property.18

1) Cyclic CPPs possess significantly increased cell permeability compared to their linear counterparts Permeability properties of cyclic CPPs compared to linear CPPs has been explored in numerous studies, and it appears from the data reported so far that cyclization of the peptides confers greater penetrability. 18, 49, 50 Also, charge on the amino acid residues and the nature of amino acids also influence the cellular uptake of cyclic CPPs (see section 6 for more details). For instance, in an important study, Lättig-Tünnemann et al. explored the cellular uptake properties of arginine-rich CPPs (Figure 2) and surprisingly found that the cyclic form of TAT was able to permeate into cells on an average of 15 min earlier, accumulated into cells in larger amounts and exhibited enhanced transduction kinetics. Although, both cyclic TAT and linear TAT have similar charges, the investigators attributed the higher uptake efficiency to the cyclization that caused guanidinium groups of arginine to move to distant positions that resulted in better interaction of the peptide with the cell surface.51 Following up the work on cyclic TAT, Nischan et al. employed cyclic TAT peptide as a delivery tool for a green fluorescent protein (GFP). Although this conjugation was tedious using a small peptide as a linker, however, by employing oligo-ethylene spacer, this


13


Page 14 of 49

conjugation was successfully accomplished by copper-catalyzed bio-orthogonal azide-alkyne cycloaddition in which azide group was attached to cyclic TAT peptide, and Lhomopropargylglycine was tethered to GFP at its N-terminus using genetic engineering. In their experiments, they demonstrated that GFP conjugated to cyclic TAT peptide could be efficiently reached not only into the cytoplasm but also into the nucleus of the living cells 15 minutes earlier than its linear form.51 Interestingly, however, unmodified GFP did not translocate into the cell at all even at 30-fold higher concentration than the GFP conjugated to cyclic TAT. As a comparison, GFP was conjugated with linear Tat peptide result in only 1% uptake efficiency at 150 mM concentration whereas using the same concentration; cyclic Tat-GFP complex was able to reach the cytoplasm of virtually all cell.20 This study demonstrated the higher cellular uptake efficiency of cyclic peptides compared to their linear form, and this cyclization strategy can be employed for the delivery of various potential drug candidates. The superior penetrability of cyclic CPPs was repeatedly confirmed by the studies of amphiphilic cell-penetrating peptides. For instance, Mandal et al. discovered that cyclic peptides rich in arginine and hydrophobic tryptophan residues possess significantly more cell permeability compared to linear CPPs.18 Mandal et al. demonstrated enhanced delivery of fluorescently labeled negatively-charged phosphopeptides, nucleoside reverse transcriptase inhibitor anti-HIV drugs, and doxorubicin in the presence of [WR]5 and [WR]4. In their experiments, the confocal microscopy data confirmed that cyclization was crucial for increasing cellular permeability.18 In another similar work on tryptophan and arginine-containing peptide, Shirazi et.al demonstrated significantly higher cellular uptake of cyclic [WR]4 compared to linear (WR)4.30 This study continued to develop another set of peptides in which they replaced tryptophan with lysine and synthesized cyclic [WK]5 and linear (WK)5 capped gold nanoparticles to investigate the


14

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internalization at different locations in the cells. The results revealed significantly higher nuclear localization of cyclic [WK]5 compared to corresponding linear (WK)5.32 Comparable results were obtained when tryptophan and histidine-containing cyclic [WH]5 and linear (WH)5 peptides were tested for cellular uptake.36 On the same lines, the studies of cysteine- and arginine-rich cyclic CPPs demonstrated both cyclization strategy and the number of amino acid residues significantly impact their transporting ability of the cargo.38 Later, El-Sayed et al. designed homochiral cyclic CPPs containing arginine and histidine and investigated them as molecular transporters. The results revealed that cyclic [HR]4 compared to linear (HR)4 showed better permeability.37 Along with the studies of cysteine- and arginine-rich cyclic CPPs, the results of these studies also revealed not only cyclization but also the combination of amino acid residues is important in determining the degree of cellular internalization. Most surprisingly, amphiphilic nature of cyclic CPPs, i.e., having an alternate segment of the positively charged residues such as histidine, arginine or lysine and hydrophobic residues such as tryptophan, phenylalanine, and naphthylalanine are very important in making the cyclic peptide a cell-penetrating peptide. Pei group adopted a slightly different approach by designing macrocyclic arginine-rich CPPs with one to three hydrophobic residues inside the ring system. These peptides were expected to not only facilitate target binding but also influence membrane transport. They tested the transport efficiency of short peptide motifs [FΦRRRRQ] cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) and found out that these transporter motifs showed 2 to 5 fold higher cellular internalization than that of linear nona-arginine (R9).52 Traboulsi et al. investigated macrocyclic cell-penetrating peptides (Figure 4) to determine the structure-activity relationship and found out that site of cyclization, ring size, and stereochemistry of arginine residues all contribute to the cell uptake properties of cyclic CPPs. This observation is consistent with the


15


Page 16 of 49

earlier evidence, as described above, that presence of the positive or the negative charges on the cyclic ring is an important factor influencing the cellular uptake.23 They also found out that the introduction of a second ring, (i.e., bicyclic peptide: FITC-βA-[ERRRK]-(βA)n-[KRRRE]-NH2, n= 0 – 3) in the peptide structure does not significantly influence the cellular uptake. However, the introduction of arginine residues in the structure affects the cellular uptake (Figure 4). For instance, by tethering endo- and exo-cyclic arginine in the peptide structure, they found out hepta-arginine to be as much effective in its uptake by the HeLa cell lines as was its linear nona-arginine form. Amongst different assortment of combinations of a linear and cyclic peptide, a peptide containing three exocyclic and four endocyclic arginine residue, 6-FITC-R3-[R4EK] (Fig. 4), was found to induce maximum cellular uptake.23 A year later, Oh et al. tried to improve the cell penetrability by designing bicyclic amphiphilic peptides namely [W5G]-(triazole)-[KR5] and FITC-[KW4E]-(β-Ala)[KR5]. The results of the study demonstrated improved cellular uptake. For instance, delivery of fluorescein (F´) labeled phosphopeptide increased by ~7 to ~19 folds in human ovarian adenocarcinoma cell lines. It is still too early to state whether the bicyclic nature of the peptides affects the cell permeability or not as there may be some other factors that lead to conflicting results described above. More studies are needed to reach a definite conclusion.33 In addition to the work performed on synthetic cyclic CPPs, reports from plant-derived natural cyclic CPPs (MCoTI-II, SFTI-1, Kalata B1, Figure 5) also signifies the importance of cyclization in improving cell permeability. For instance, MCoTI-II, a macrocyclic peptide rich in disulfide backbone, was reported to penetrate cells as determined by fixed cell imaging.39 These findings were further confirmed by two more studies using live cell imaging.21,

53

Elaborating on this,

Cascales et al. confirmed that both T- and M- class cyclotides and SFTI-1 were able to penetrate


16

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the cells efficiently, and they coined the term cyclic cell-penetrating peptides to nominate these novel peptides.21 The observation of having an alternate segment of the positively charged residues and hydrophobic residues as critical key in making the cyclic peptide a cell-penetrating peptide was further corroborated by Ermondi et al. They synthesized three polar cyclic hexapeptides ([ANGGAW], [ADGGAW], and [AKGGAW] (Figure 6)), with different amino acid residue on the second amino acid to give different charges at physiologic pH. They found out that the permeability of positively-charged peptide was significantly higher than the anionic and neutral derivatives.54 Qian in his thesis work also showed that cyclic CPPs entered into the cell at significantly higher concentrations as compared to canonical arginine rich linear CPPs.4 Dougherty et al. and White et al. reached to the same conclusion while investigating macrocyclic peptides that cyclization and distribution of the charges on the side chains influences the cellular uptake properties.49, 50


17


NH

H 2N

=6-FITC, 6-FAM

NH

H 2N

N H

NH

HN

O H N

HN

OH

O NH

O

H N

HN

NH

6-FITC-Bicyc-0-R6 H 2N

O NH

O

H 2N

O

O

H N

O

HN

NH2

HN

H N

O

O

NH

6-FITC-R4-[R3EK]

H N O

O

HN

NH2

NH

NH2

HN

HN

NH

H 2N

H N

N H

H N

O

NH

NH

HN

NH2

NH

O

O

O

HN HN

O

NH

NH

HN

O

NH

H N

O

NH2

NH NH2

O

NH

O

O

H N

NH

NH H N

NH

23

NH2

HN

NH

NH

N H

O

HN

O O

H 2N

23

HN

O

O HN

NH

O

O

O

NH2

HN

NH

NH2

HN

H 2N

HN

H N

HN

HN H N

NH

NH

NH

NH

O

O

HN H N

H N

N H

NH NH

H 2N

NH

H N

O

NH2

H 2N

NH2 NH

O

H N

N H

NH

NH

NH

NH

O

HN

O

O

H 2N

HN

O

HN

H 2N

NH

O

H N

H N O

NH2 NH

NH

O

O

HN

O

H 2N

H N

HN

NH

O

O

O

O

HO

O

H N

NH

HN

H 2N

NH

NH

H 2N

Page 18 of 49

NH2

NH

O

O

NH

N H O

H 2N

O

H 2N

6-FITC-R3-[R4EK]23

6-FITC-Bicyc-1-R623

NH

NH

H 2N NH

H 2N NH H 2N

NH

H N

NH

O

NH

HN

H N

O

O

HN H N

O NH

H N

O

O

N H HN

NH

NH

NH O

H N

H N

NH HN

O O

NH

H 2N

NH2

NH2

NH

O

O

O

O

NH

NH

NH

H N

N H

HN

NH2

NH

HN NH

O

NH2

HN O

NH

O

O

O H 2N

6-FITC-Bicyc-2-R623

NH

N H

H N

H 2N

O

H 2N

NH

O

O

O H N

N H

O O

O

H 2N

H 2N

NH

HN

HN

HN

NH

H 2N

NH

O

6-FITC-R2-[R5EK]23

NH

N H

HN

NH2

HN HN NH2

HN H 2N

NH

NH

H 2N

HN H 2N

NH

NH NH H 2N

NH

H N O

O NH

O

NH

HN O HN H 2N

H N

O

O

NH

HN

O

O

H N

H N O

NH

HN

HN

O

HN

NH

O

H N

NH

O

O O

HN

H N

N H

NH

NH2 NH

H 2N NH

NH

NH

NH

NH

NH2 H2N

H N

H N

H N

HN

O

O

O

NH

O

NH

O NH HN

O

O

O

O

O H 2N

6-FITC-Bicyc-3-R623

NH

O

H 2N

O

NH2 N H

6-FITC-R-[R6EK]

23

O

N H

N H

NH2 NH

HN

HN HN

HN

NH

HN

NH2

NH2

Figure 5. Fluorescent-tagged mono and bicyclic CPPs.


18

Page 19 of 49 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


HN

NH2

HN

H N

HN

O

O

O

HN

H2N

HN

N H

6-FAM-[W5R4K]18 HN

O

NH

N H

NH

NH

NH

NH

H2N

NH HN

HN H N

H N

HN

HN

HN

O

NH

O

O

O

HN O

HN N H

NH O

O NH

O N H

HN

HN

O

O N H

H N

HN

O

O

NH

O HN

HN

N N N

O

NH

O

O HN

NH

N H

HN

O O

H2N

NH

HN

NH

HN NH2

HN

HN

NH

NH2

H N

HN

N H

O

HN

H N O

NH H2N

HN

O O

O N H

NH2

NH

O

O

O N H

O

HN

NH NH

O

NH

HN

H N

NH O

O

HN

NH2

HN

NH O

O HN

NH

NH2

HN

HN

HN

H N

O

O

HN

H2N

[W5G]-(triazole)-[KR5]33

O

NH2

HN

H N

H N

N H

N H

NH NH

HN

O

O

NH2

NH

NH

NH

NH

O

NH2

NH

HN

HN

H N

HN

O O

NH2

H N

N H

NH

O

HN

HN

NH

NH

O

HN O

6-FAM-[W9R8K]39

NH2

NH

N H

NH

O

HN

NH

H2N

6-FAM-[WK]532

HN

H2N

NH

N H

O

HN

H N

HN

O

O

HN

H N

H2N

NH

H N

O

O O

O

O

H N

NH

H N

O

O

HN NH2

O

HN NH

H2N

O

H N

NH NH

NH

NH

NH

H2N

NH

NH2

NH2

H N

O O

O

HN

H2N

NH NH

HN

NH

NH HN

O O

N H

H2N

NH

NH

O

O

O N H

HN

O

O

O

O

6-FAM-[W4R3K]18

OH

HN

NH

NH

HN

O

H N

HN

HN O

O

HN

H N

H N

O

O

O

HN

NH2

N H

H N

HN

O

HO

HN

NH

=6-FITC, 6-FAM

NH

HN

NH

HN

NH2

NH

H2N

6-FAM-[KW4E]-(ß-Ala)-[KR5]33

Figure 6. Fluorescent-tagged mono and bicyclic CPPs.


19


NH

H2N

H N

H N

H2N

NH NH

NH

H2N

NH

NH

NH

H2N H N

H N

HN O O

O

O

HO

H2N

NH

=6-FITC, 6-FAM

Page 20 of 49

NH2 O

O

NH

H N

H N

O

NH

O

O

NH

NH

OH

HN

NH

NH

O

O

N H

O

O

HN

N H

O HN

NH

HN

6-FITC-[R4]23

O

O O

HN

H2N

6-FITC-[R6]23

NH

NH

NH2

O

NH HN

N H H2N

NH2

HN

O

O

O

NH2

HN HN NH2 HN

H2N

H2N

NH H2N

H2N H N

HN

H N

H N

NH

O

O O

NH

O

HN

O

6-FITC-[R7]23

NH2

H2N NH

O

HN

O

N H

NH

NH

NH2

O

HN

HN

H2N

6-FITC-[R8]

HN

23

N H

H N HN

H N

O

NH2 O

O

H N

O

NH

N H

O

O

HN

NH2

HN NH

O

HN

H2N

O

O

O

HN

NH2

6-FITC-[R9]

23

O

H2N

HN

O

HN HN HN

HN

NH2 NH2

HN

O

HN

NH2

O

O O

NH HN

NH2

O O

H2N

HN

O

HN

L.D.

O

NH2

NH

NH2 NH

HN

NH2

HN

HN HN

HN

NH

NH

N H

O

HN

Figure 7. Fluorescent-tagged CCPPs. r is D- amino acids;

N H

NH

HN NH2

NH2

NH O

6-FITC-[r9]*23

NH

N H

NH O

NH

NH2

HN

NH

HN HN

H2N

H N O

O

O

NH

N H

O

NH

HN

O

NH

HN

NH

N H

O

N H

NH

HN

O

6-FITC-L,D[R8]23

NH2

NH

HN

H N

H N

O

NH O

H2N NH

H N

NH

N H

NH O

O

NH NH

NH

H N

O

NH

H2N

NH

H2N

O

O

NH2 HN

NH2

H2N

NH

HN

H N

H N

HN

H2N NH

NH2

HN

HN

NH

NH

6-FITC-[r8]*

HN

N H

O

23

HN

NH NH

NH

NH

O

O

HN

H2N

HN

NH

NH2

HN NH

O

NH2

H2N

NH H2N

H2N

NH

O

O HN

NH2

HN HN

NH

NH

O

O HN HN

NH2

N H

NH

N H

O

NH2

NH

O

NH

NH

O

O

HN

H2N

NH

NH2

HN NH

O

NH2

HN

NH

NH2 O

O

O

NH

O

H2N

NH

O

O

HN

H N

H N

NH

NH O HN

O

HN

N H

NH

N H

O

NH NH

O

NH

HN

O

N H

NH2 O

H2N

H N

NH

O

O

NH

NH

NH

N H

O

HN

H N

H N NH

H N

NH2

NH

H2N

H2N

H N

O

O

H2N

NH

NH NH2

O

O

H2N

NH2

HN

HN

NH

H2N

NH

O

6-FITC-L,D[R7]23

HN

O

NH

NH

NH

N H

O

HN

H2N

6-FITC-[r7]*23

NH

N H

NH2

NH2

H2N

H2N

H N

H N

O

NH

NH

H N

O

HN

NH

HN

HN HN

HN

H2N

NH

HN

NH

HN

NH

NH

N H

H2N

NH2 HN

O

O

HN

NH O

O

NH2

NH

O

NH2

NH

HN

23

O

N H

NH

O

HN

O O

NH2

NH O

O NH

NH

N H

O

HN H N

H N

NH

O

HN

N H

H2N

O

O

NH

NH

NH

NH2 N H

NH

NH2

HN

O O

NH

O

O HN

H N

NH H N

O

O

HN H N

H2N

NH

H2N NH

N H

H N

NH

N H

O

O

6-FITC-L,D[R6]

NH

NH

NH

H2N

NH

H2N

NH

NH

H2N

NH

NH

NH

NH2

is a mixture of L and D forms of

amino acids.


20

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2) Cyclic CPPs have generally higher resistant to proteolysis. Enhancing CPPs rigidity by cyclization is an emerging concept in the design of cell-penetrating peptides51, 55 and several studies have pointed out that cyclization of CPPs accounts for resistance to proteolysis.24, 44-46 Qian et al. designed and synthesized a series of cFΦR4 analogs by modifying the stereochemistry and/or peptide sequence and investigated their cellular uptake, using unnatural amino acids and changing their sequence lengths, and they found out that cyclic peptides rich in arginine residues, cFΦR4 peptide analogs, [FΦRRRRQ], where Φ stands for L-2-naphthylalanine (Figure 3) possess excellent proteolytic stability as determined by the pharmacokinetic studies performed on mice24. Heitz et al. investigated the role of cyclic backbone in conferring stability to peptide framework, and they found out that the stability of the cyclic peptides is largely associated with the cysteine knot framework and suggested that cyclization predominantly reduces the susceptibility of the peptides to thermal unfolding.56 It is suggested that cyclization reduces proteolysis due to the removal of reactive C- and N-termini and by providing shielding components against the proteolytic enzymes.57

3) Some cyclic CPPs exhibit efficient endosomal escape, and some show endosomal-independent uptake In addition to enhanced intracellular uptake and resistance to proteolysis, some studies performed on cyclic CPPs mention their endosomal escape property. For instance, Mandal et al. reported in their mechanistic studies that fluorescently labeled F-[W5R4K] was internalized into the nuclei of the cells rapidly without the need of endosome-derived vesicles. This endosomeindependent cellular uptake is a great advantage for those CPPs that require endosomal escape to exit out of the resulting endosome.18 In the same report mentioned above by Qian et al., it was


21


Page 22 of 49

shown that [FΦRRRRQ] (Figure 3) binds directly to cell membrane phospholipids, permeates the cells dominantly via endocytosis, and escape endosomes efficiently. These demonstrated that the efficiency of cellular uptake is directly correlated with their binding affinity to the cell membrane phospholipids and endosomal escape is dependent upon their binding to the endosomal membrane. The cyclic CPPs escaped endosomes by binding to the endosomal membrane that subsequently causes induction of CPP-enriched lipid domains to bud off as small vesicles.24 It was hypothesized that this smooth endosomal escape is caused by the stabilizing interaction of these peptides with the curvature of membranes resulting in budding off of the vesicles. This demonstrates efficient endosomal escape by the cyclic CPPs. However, the precise mechanisms remain elusive. It is also postulated that the same attributes that cause intracellular uptake could also be important to promote endosomal vesical disruption and eventually cause passive diffusion. One good example of this is the stiffening of the membrane due to interaction peptide containing positively charged residues leading to the formation of pH gradients and increasing in the concentration of vesicles content.15,

58

Recently, and while submitting this review, Dougherty et al. published a

comprehensive account addressing the penetrating mechanism of cyclic peptides. For, more details, readers are encouraged to follow the cited reference.59 It has also been reported that a peptide derived from the endosomal escape sequence of influenza virus haemagglutinin protein (HA) can enhance the cellular uptake and/or cytosolic delivery of cargo.

For

instance,

HA2

fusion

peptide

with

the

sequence

of

“GLFGAIAGFIENGWEGMIDGWYG” discovered from HA2 subunit of the influenza virus X31 strain was identified to promote the fusion of a peptide to the cell membrane and its internalization into the cell.60 Alfredo et al. has reviewed these endosomal escape domains in detail.61 It was found that these membrane-active peptides have been successfully used for the delivery of siRNA,


22

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oligonucleotides, plasmid DNA, lipofectamine, and fluorescently-labeled dextran. However, the major concern for their use of a successful tool for efficient cellular internalization is the wellreported toxicity. For instance, HA2E5-TAT was found to be toxic to HeLa cells even at a concentration more than ~5 μM.62 Several cyclic CPPs, on the other hand are relatively safe (See section 7).

4) Improved higher affinity of the cyclic peptides to the target receptor is observed compared to the linear form Another advantage of cyclic CPPs compared to the linear form is their improved higher affinity to the target receptor. For instance, using genetic engineering, it is possible to insert cysteine residues at the terminal ends of a known peptide sequence [r12] (GSSG[CrrrrrrrrrrrrC]) resulting in the formation of cyclic peptide within a phage protein due to the formation of a disulfide bridge (Figure 2).26 This constrained conformation significantly enhanced the affinity of the peptide to its target receptor compared to its linear counterpart. These reports indicate that some of the cyclic CPPs possess increased affinity of the peptide ligand to its membrane receptor, a property attributed to cyclization.48 5) A few cyclic CPPs have nuclear targeting property Another important property of cyclic CPPs is their ability to reach and deliver cargos to the nucleus. For instance, Mandal et al. reported cyclic peptides containing tryptophan and arginine to have nuclear targeting and non-covalent molecular transport properties.18 later, Shirazi et al reported the design of a novel nuclear-targeting drug delivery system containing gold nanoparticles and cyclic peptides containing lysine and tryptophan residues.32 Based on these studies, further investigated is required to explore the potential of cyclic CPPs as a nuclear targeting tool.


23


5.

Page 24 of 49

Cyclization Strategies While exploring studies, we came across two popular cyclization strategies viz. cyclization by

bridging two cysteine side chains and cyclization via formation of amide bonds between Nterminus alpha amine and C-terminus alpha carboxyl functional groups. There are several cyclic peptides cyclized in amide formation of lysine and aspartic acid or glutamic acid side chain or using a staple peptide, but for the purpose of cyclic CPPs, these methods are not covered in this text. Pfaff et al. in 1994 reported the cyclization of peptides by making amide bond between the C-terminus alpha carboxyl group and N-terminus alpha amine group.63 Cyclization by bridging the cysteine side chains present at the either N-terminal and C-terminal ends of the peptides has been reported by Hart et al. and later by De Groot et al..64, 65 Using disulfide bridge for the cyclization is more popular in the synthesis of cyclic CPPs compared to classical amide bond cyclization. Disulfide bridge cyclization is advantageous because of its easiness of cyclization, reversibility in the reductive environment that adds to the peptide’s conformational definition. Lättig-Tünnemann et al. reported that cyclization performed via a disulfide bridge technique on TAT peptide and arginine-rich peptides caused efficient uptake kinetic compared to their respective linear forms.51 While in another study, Qian et al. employed the same cyclization strategy using disulfide bridging technique by the introduction of cysteine residues at the ends, and they found out that the CCPPs produced this way were efficiently taken up by HeLa cell lines and exhibited endosomal escape.45 There are other cyclization strategies that have been employed to develop cyclic peptide as well. For a detailed discussion on this topic, the readers can consult the cited following reviews.66, 67


24

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6.

Transport mechanism of Cyclic CPPs There are several hypotheses for the mechanism of translocation of CPPs. Although many

scientists came up with these hypotheses including “A proton sponge” hypothesis,59,68, 69membrane fusion mechanism,70, 71 transient pores mechanism,71 local disruption of endosomal complex and leaky endosomal membrane fusion,72-74 the exact mechanism for the translocation of CPPs remain elusive due to numerous kinds of different cyclic CPPs. Very recently, a new review provided more compressive mechanistic data for CPPs.9 According to the literature, the proposed main routes of entry of CPPs into the cells are direct penetration, diffusion as well as endocytosis. Endocytosis is a broader term and includes both phagocytosis and pinocytosis.75,

76

Although,

initial translocation studies performed on CPPs proposed that the direct penetration is the main route of entry of CPPs, however, with more studies published in this area, it is believed that endocytosis is the predominant mechanism for the penetration of CPPs. Nonetheless, it is not yet absolutely clear, which mechanism is responsible, and it is most likely that different mechanisms participate for internalization of CPPs when employing various experimental conditions.76 Few studies have been conducted on understanding the translocation mechanism of cyclic CPPs and this area needs further studies seeing the conflicting results reported so far. Banerjee et al. investigated the intracellular transport mechanism of arginine-rich cyclic peptides [WR]4 and [WG(triazole-KR-NH2)]3 and found out that the charges on peptide from environment, their conformational flexibility, and the hydrophilic-lipophilic balance (HLB), all play important roles in determining their molecular transporter and self-aggregating properties. Their findings were supported by the results of molecular dynamics simulation.77 Also, the cellular uptake performed on linear (WH)5 and cyclic [WH]5 (Figure 2) demonstrated that presence of endocytosis inhibitors, such as chlorpromazine, 5-(N-ethyl-N-isopropyl)-amiloride (EIA), methyl-β-


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cyclodextrin, and chloroquine, did not significantly affect the internalization ability of cyclic peptide [WH]5,36 a finding which suggests that endocytosis may not be the predominant mechanism of intracellular transport for tryptophan and histidine-containing cyclic CPPs. For instance, in one study, the cellular uptake of the fluorescence-labeled [W5R4K] (Figure 5) was found to be concentration and time-dependent.18 The uptake was observed even after 5 min. Rapid internalization and localization of FITC-[W5R4K] to the nucleus in SK-OV-3 cells were also observed at 4 C and in the presence of sodium azide, to see if the cellular uptake was energy-dependent. In the presence of different endocytic inhibitors, such as methyl β-cyclodextrin, chlorpromazine, chloroquine, and nystatin, F´-[W5R4K] did not show any significant difference in cellular uptake in CCRF-CEM cells. Therefore, Mandal et al. concluded the endocytosis mechanism of F´-[W5R4K] was neither clathrin-mediated, caveolae-mediated endocytosis, nor micropinocytosis.18 These results suggested that the cellular uptake for this peptide does not require endosome-derived vesicles. Similarly, Duchardt et al. investigated the transport mechanism of cationic CPPs and ruled out endocytosis as a mode of their cellular uptake. They found out the cellular uptake was dependent upon the peptide’s concentration as well as inhibition of endocytosis.78 Later, Lättig-Tünnemann et al., while exploring the cellular uptake of cyclic Tat and arginine-rich CPPs, discovered that in addition to endocytosis, these peptides traverse biological membranes in a non-endocytic mode.51 In another study, Shirazi et al. explored the cellular uptake mechanism of peptide-capped gold nanoparticles (linear F′-(KW)5-AuNPs and cyclic F′-[KW]5-AuNPs) in the presence of several endocytosis inhibitors including nystatin (50 μg/mL), chloroquine (100 μM), chlorpromazine (30 μM), methyl-β-cyclodextrin (2.5 mM), and 5-(N-ethyl-N-isopropyl)-amiloride (EIA, 50 μM). The data from the study illustrate that cellular uptake of the cyclic CPP capped AuNPs is not affected significantly in the presence of these inhibitors, suggesting that the uptake is not mediated


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exclusively by clathrin- or caveolae-mediated endocytosis and micropinocytosis. It was speculated that surface decoration of AuNPs by c[KW]5 and l(KW)5 may improve the interaction of tryptophan and lysine residues with negatively charged phospholipids and hydrophobic amino acids in the plasma membrane that could be the major driving force for cellular internalization. This hydrophobic interaction can potentially disrupt the outer phospholipid monolayer followed by the peptide internalization and increased intracellular uptake of the cargo.32 Adding on to the mechanistic studies, Hanna et al. investigated whether cellular uptake of cyclic CPPs was dependent upon endocytosis. Fluorescently labeled peptide F′-[W9R8K] (Figure 5) was examined for the cellular uptake studies using CCRF-CEM cells in the presence of endocytosis inhibitors such as nystatin chlorpromazine, chloroquine, EIA, and methyl-β-cyclodextrin. The results of the study demonstrated that the uptake of the peptide was reduced in the presence of chloroquine, but it was remarkably decreased in the presence of chlorpromazine, EIA, nystatin, and methyl-β-cyclodextrin suggesting that cellular uptake may be dependent on clathrin or caveolin-mediated endocytosis as well as phagocytosis. However, the endocytosis inhibitors did not completely block the cellular uptake of the cargo that points out for the possibility of other pathways involved in the delivery of F′-[W9R8K]. With all that said, uptake of this peptide is not completely dependent on endocytosis, and possibly other mechanisms of entry are involved as well. Furthermore, ATP depletion method was used to determine whether the cellular uptake was energy dependent, for which sodium azide was used. It is pertinent to mention here that sodium azide is used to block ATP synthesis as it inhibits oxidative phosphorylation. Cellular uptake studies performed on CCRF-CEM cells with F′-[W9R8K] revealed that cellular uptake was reduced by 1.8-fold in the presence of sodium azide. Significant cellular uptake, however, was still observed suggesting that different mechanisms contribute towards peptide’s internalization.40 The


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cellular internalization of the peptide was significantly reduced by 5.6 fold when incubated at 4 °C suggesting that the peptide’s cellular uptake is significantly affected by temperature.40 Similarly, Shirazi et al. tried to investigate the molecular transport mechanism of cyclic CPP, [CR]4, and reached to the same conclusion that several mechanisms participate in the internalization of cyclic CPPs. For instance, a mixture of fluorescently labeled cell impermeable phosphopeptide, F´GpYEEI, and [CR]4 was employed for the mechanistic studies in the presence of endocytosis inhibitors. It was observed that cellular uptake of F´-GpYEEI was not significantly affected in the presence of chlorpromazine and chloroquine, however, the uptake was reduced by 20% in the presence of methyl-β-cyclodextrin and by 19% in the presence of sodium azide. Therefore, it can be interpreted that ATP depletion can account for internalization. Methyl-β-cyclodextrin is responsible for removing cholesterol out of the plasma membrane that disrupts the clathrinmediated endocytosis. Thus, it can be inferred that ATP depletion and clathrin-mediated endocytosis are the potential pathways contributing partially to the cellular uptake of these cyclic CPPs.38 Contrary to the observation of Hana et al. 40, they observed that the decreasing incubation temperature to 4 °C did not block the uptake of the cargo38. In contrast to the above reports, Qian et al. investigated the internalization mechanistic using model lipid vesicles, pharmacologic inhibitors and gene mutations that affect the internalization steps, indicating that cyclic CPPs translocate into cells via endocytosis.4, 52 Later, Oh et al. studied the mechanism of cellular uptake of a fluorescently labeled bicyclic peptide F´-[KW4E]-(𝛽-Ala)[KR5] in the presence of endocytosis inhibitors and found out that main pathway for the cellular internalization was mediated by clathrin-and caveolin-dependent endocytosis.33 In the light of mechanistic studies conducted by our research group as well as by other researchers, it can be reasonably argued that multiple mechanisms operate during the translocation


28

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of cyclic CPPs. Macropinocytosis, clathrin-mediated endocytosis, and caveolae/lipid-raftmediated endocytosis do contribute towards their internalization into cells. However, the sequence of amino acid in cyclic CPPs, nature of the peptide, concentration, temperature as well as the period of incubation are other important factors in the cellular uptake of cyclic CPPs.

7.

Biomedical Applications of cyclic CPPs Cyclic CPPs owe desirable properties like cell-penetration, proteolytic stability, safety, ability

to conjugate with potential therapeutic and imaging agents, and early endosomal escape. Thus, they have been used in numerous drug delivery applications. The summary of biomedical applications of the cyclic CPPs is presented in Table 2. We have summarized below some of the highlights of these applications. 1) Intracellular delivery of cargo including drug, protein, phosphopeptide, and bioactive molecules,20, 30-33, 37-39 2) Cyclic CPPs can target nucleus,18 3) Cyclic CPPs are used as antibacterial or bacteriostatic agents,79 4) Cyclic CPPs deliver siRNA in cancer cell lines,28, 80-83 5) Cyclic CPP is a promising scaffold for nanoparticle or small bioactive molecule design,26, 31, 32, 84, 85

6) Cyclic CPPs can silence kinase protein, such as KSP and JAK2 in cancer,82

siRNA-mediated gene silencing is an innovative area in cancer therapeutics and has been rigorously investigated; however, the efficient intracellular delivery of siRNA remains a daunting task. Several reports garner hope in cyclic CPP’s potential in siRNA delivery. For instance, Shirazi et al. studied the cyclic peptide-capped gold nanoparticles for enhanced delivery of siRNAs. In this study, the authors tested [WR]5-AuNPs to deliver small interfering RNA molecules in human


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cervix adenocarcinoma (HeLa) cells and observed 2 to 3.8 fold increase in siRNA delivery when conjugated with cyclic peptide-based nanoparticles compared to incubation of siRNA alone after 24 h. Cytotoxicity studies performed on these conjugates showed their comparative safety versus other carrier systems such as lipofectamine.86 Similarly, Welch et al. reported the cytosolic delivery of short interfering RNAs (siRNAs) using disulfide-constrained cyclic peptides (Ac[C(FKFE)2C]G-NH2 and Ac-[C(WR)4C]G-NH2, (Figure 6) in both in vitro experiments as well in vivo to mouse lungs, and they revealed the potential of these cyclic CPPs to design efficient siRNA delivery agents, which are important for perturbing the expression of disease-related genes.80 For instance, they used human H441 cells and found that D-Ac-C(FKFE)2CG-NH2 caused ~30% knockdown of TTF-1 gene, and the L-Ac-C(FKFE)2CG-NH2 caused ~40% knockdown of TTF-1. Hung et al. tried to explore the potential of arginine and lysine-containing (R5K2 peptide) difatty acyl conjugates in siRNA delivery. They synthesized several difatty acyl conjugates and found that C16- and C18-conjugated cyclic CPPs showed significant silencing against kinesin spindle protein (KSP) and janus kinase 2 (JAK2) proteins which was further enhanced in the presence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).87 Very recently, Saghar et al. also reported the efficient intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468 using several tryptophan- and arginine-containing cyclic CPPs. Among the studied peptides, [WR]5 appeared to be most significant in the delivery of siRNA and generated silencing of KSP and JAK2 in MDA-MB-231 cells in the presence of the peptides and DOPE.82 Cyclic CPPs possess great potential as a molecular transporter and has an ability to solve the challenge of transporting macromolecules and other hydrophilic therapeutic moieties. Several studies suggest their usefulness in improving the effectiveness of existing anti-cancer and anti-


30

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microbial agents. Parang and co-workers have also rigorously evaluated the applications of several CCPPs as molecular transporters.32, 37, 38, 40 For instance, [WK]5 was successfully shown to be an effective delivery vehicle for model anti-HIV drugs.32 [WH]5 was employed for the efficient delivery of a cell-impermeable cargo.36 [CR]4 demonstrated enhanced cellular uptake of negatively charged phosphopeptide,38 and [HR]4 was shown to be an effective molecular transporter.37 Previously, Parang and co-workers reported that cyclic [WR]5 and similar tryptophan and arginine containing CCPPs could self-assemble into nanostructure and potentially can be employed for the stabilization of protein biomolecules and silver nanoparticles.83 Also, potentiating the activity of doxorubicin in drug-resistant cancer cells was examined by Shirazi et al.29 They found out that doxorubicin conjugated with a cyclic peptide containing arginine and tryptophan residues, [W(RW)4], resulted in a significant increase of antiproliferative activity. They observed increased trafficking of the drug to the nucleus and decreased efflux of doxorubicin-cyclic CPP conjugate in comparison to control free doxorubicin.29 Later, Shirazi et al. investigated the application of cyclic [WR]5 to improve the intracellular uptake of curcumin, a rigorously investigated therapeutic agent for multiple conditions. They used both the peptide-curcumin conjugate as well as the peptide/curcumin physical mixture for the cellular uptake studies. Flow cytometry studies showed that cellular internalization was enhanced by 5.7 folds when used as peptide/curcumin physical mixture compared to the incubation of curcumin alone after 3 h in the human leukemia cell line (CCRF-CEM), whereas, peptide-curcumin conjugate resulted in 4-fold increase in cellular uptake. Furthermore, antiproliferative activity of curcumin was increased by 20% using [WR]5/curcumin physical mixture compared to ~13% increase with [WR]5-curcumin conjugate after incubation in CCRF-CEM cells after 72 h.28 Thereafter, Darwish et al. conjugated curcumin and doxorubicin with cyclic peptide [C(WR)4K2(β-A)] to improve the solubility of curcumin and enhance the


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antiproliferative activity of doxorubicin. The data from the study suggest the selectivity of peptide conjugates towards the different cancerous cell lines e.g., CCRF-CEM and SKOV-3, without toxicity to normal human embryonic kidney cell line (HEK-293).88 These results suggest the potential of cyclic CPPs as potential cargo delivery tools for therapeutic agents. Shirazi et al. also designed and synthesized cyclic peptide-based selenium nanoparticles and tested them as a drug transporter system. In this study, the authors noted a significant improvement in the delivery of various anticancer drugs, such as gemcitabine, clofarabine, doxorubicin, etoposide, irinotecan, paclitaxel, fludarabine, epirubicin, dasatinib, and camptothecin when conjugated with [W5R4C]−SeNPs against human leukemia (CCRF-CEM) cells and SK-OV-3 cells. They concluded that this cyclic CPP-based drug delivery system could be successfully used as a nanosized delivery tool for anticancer and other negatively charged therapeutic moieties.31 The second mitochondria-derived activator of caspase (Smac) is a pro-apoptotic mitochondrial protein released during the apoptosis. It has been observed that seven N-terminal amino acid sequence (AVPIAQK) of Smac (SmacN7) possess intrinsic anticancer property. However, this heptapeptide is cell impermeable as well as unstable in the cellular environment. Melek et al. designed several polyarginine containing cyclic peptides by incorporating SmacN7 sequence and found that cyclization helped in improving the cellular uptake as well as stability of Smac linear heptapeptide. Interestingly, this cyclization strategy also resulted in the enhanced anticancer ability of SmacN7 when tried in multiple myeloma tumor cells.89 Observing the cell penetrating ability of oligocyclic peptide toxins, namely maurocalcine and crotamine, that exhibit the similar property as that of CPPs, Wallbrecher et al. designed a bicyclic cell-penetrating peptide and showed that presence of arginine residues either in the cyclic ring or as a part of the side chain significantly enhanced the cell-penetrating ability of the cyclic peptides.


32

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They examined the cellular uptake of this peptide in the HeLa cell lines using flow cytometry and confocal microscopy and reported that these bicyclic peptides were able to serve as a potential scaffold for the intracellular delivery of small molecules.90 Recently, Ichimizu et al. designed palmitoyl-cyclic-[r12] (Figure 2) and functionalized it with human serum albumin (HAS) to investigate the effect of this cyclic peptide in improving the cell-penetrating ability of HSA. The effect of resulting CPP-HAS conjugate was tested in enhancing the pharmacological activity of three model drugs: paclitaxel, doxorubicin, and thioredoxin fusion protein. The results of the experiments demonstrated that palmitoyl-cyclic-(D-R)12/HAS system could act as a versatile nano drug delivery system for a wide variety of pharmaceutical applications.26 Although various studies point to the ability of cyclic CPPs as a delivery tool for small molecules, Nischan et al. examined the potential of a cyclic CPP as a delivery tool for full-length protein. In their experiment, they employed the cyclic form of well-known CPP termed TAT, cTAT, as a delivery agent for green fluorescent protein (GFP) and found out that cyclic-CPP–GFP conjugates are efficiently internalized into the living cells with immediate bioavailability in the cytosol as well as nucleus, which expands the application of cyclic CPPs for the delivery of fulllength proteins into the living cells.20 Inspired by a natural product FR235222 (Figure 6), Hilário et al. synthesized a cyclic tetrapeptide and post functionalized it with cargo model, a bioactive and fluorescent triazole aminocoumarin, to test its cytosolic penetration and cytotoxicity. This study successfully demonstrated the ability of this CCPP to deliver small bioactive molecules not only for cytosolic but also for nuclear delivery.84 Oh et al. synthesized several amphiphilic cyclic peptides and their analogs and reported their potent antibacterial activities against multidrug-resistant pathogens. Among the cyclic peptides


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studied, cyclic peptide [R4W4] exhibited the most potent antibacterial activity against MRSA with the MIC of 2.67 μg/mL. Four arginine and four tryptophan amino acids were found to have an antimicrobial activity after diverse modifications in the cyclic ring using other cationic and hydrophobic amino acids. However, interestingly, the four alternate arginine and tryptophan residues in cyclic [WR]4 makes them cell penetrating without antimicrobial property. They found fluorescence-labeled-[R4W4] peptide penetrate cellular membrane which could be used for delivery of non-permeable antibiotics. Therefore, to show the proof of molecular transporter property of this antibacterial peptide, they used this cyclic peptide in combination with tetracycline and demonstrated a 4 to 8-fold increase in bactericidal activity against MRSA compared to tetracycline alone. Authors suggested that combination therapy of antibiotic with the cyclic CPPs has the potential to effectively inhibit multidrug-resistant strains.34


34

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H N

HN

NH

O O

HN

O

O

O

HN

NH

N H

O

HN

NH

O O

O

O

H N

HN

NH

O

N H

H 2N

NH

O O

NH

O

H N

HN

O

O

NH

NH

O

O

NH

N H

HO O

NH2

O

[ANGGAW]52

[AKGGAW]52

[ADGGAW]52

O

O

N

N

O HN

O

NH

HN

O

O N H

O

O

OH

O

NH

N H

OH

O

Compound 1# 85

FR235222# 85 NH H 2N

HO NH2

NH HN H N

HN

O

HN NH H 2N

H N

OH

O

HN

NH

O

HN

HN

O

O

NH

S O

S

O O

HN O

O

O

HN NH

O NH2

N H

HN

NH

HO

O

NH

O

S

O HN

NH2

HN

O

S O

HN

OH

O

HN O

O

NH2

N H

OH

NH

N H

O

H 2N

HN HN

O

O

NH

O

NH

N H

O

NH2

Ac-C(WR)4CG-NH280

Ac-C(FKFE)2CG-NH280

Figure 8. Examples of cyclic CPPs; #Natural product and its synthetically modified tetracyclopeptide.


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Table 2. Summary of Biomedical Applications of cyclic CPPs. Peptide

Sequence

Application

[WR]5

[WRWRWRWRWR]

To deliver a small interfering RNA molecule 28, 82, 86 (siRNA) in human cervix adenocarcinoma (HeLa) cells and triple negative breast cancer cell lines. Also, to improve the intracellular uptake of curcumin

Cyclic Ac-CaFKFEFKFECaG-NH2 For functional delivery of siRNAs Ac−C(FKFE )2CG−NH2 peptides

Reference

80

[R5K2]

[RRRRKRK]

siRNA delivery and silencing of kinesin spindle 87 protein (KSP) and janus kinase 2 (JAK2) proteins in human breast cancer cel lines (MDA-MB-231 and MDA-MB-435)

[R4W4]

[RRRRWWWW]

As an antibacterial peptide against multidrug- 34 resistant pathogen

Roquefortine [W-dehydroHf]

Bacteriostatic activity over gram-positive bacteria, 85 inhibition of cytochrome P450 3A

[WK]5

[WKWKWKWKWK]

As a cellular nano drug delivery system utilizing 32 anti-HIV drugs (emtricitabine (FTC) and lamivudine (3TC) as model drugs

[WH]5

[WHWHWHWHWH]

To enhance the delivery of cell-impermeable cargo 36 like phosphopeptides and the anti-HIV drug emtricitabine.

[CR]4

[CRCRCRCR]

As a molecular carrier to enhance the cellular 38 uptake of e negatively charged phosphopeptide (F´GpYEEI) and fluorescence-labeled lamivudine (F´3TC)

[HR]4

[HRHRHRHR]

As a molecular transporter to improve the delivery 37 of cell-impermeable cargo

[W5G](triazole)[KR5]

[WWWWWG]-(triazole)[KRRRRR]

As cellular delivery agents

33


36

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[W5E]-(β- [WWWWWE]-(β-A)Ala)-[KR5] [KRRRRR]]

As cellular delivery agents

[W(RW)4K]- [WRWRWRWRWK]-βAf βA

To enhance the antiproliferative activity of 29 doxorubicin towards drug-resistant cancer cell lines.

[C(WR)4K2( [CRWRWK(βA)fWRWR] βA)]

To improve the solubility of curcumin and enhance 88 the antiproliferative activity of doxorubicin

[WR]4 [WR]5

As a nuclear targeting molecular transporter

and [WRWRWRWR]

33

18

and [WRWRWRWRWR]

[W5R4C]

[WRWRWRWRWC]b

To improve antiproliferative activities of several 31 anticancer drugs, such as doxorubicin, gemcitabine, clofarabine, etoposide, camptothecin, irinotecan, epirubicin, fludarabine, dasatinib, and paclitaxel in the presence of [W5R4C]-capped selenium nanoparticles

Cyclic TAT N3-PEG2-[K(rRGrKkRr)E] For delivery of a protein into the cell peptidec

20

Modular [Alkylated lithium enolate- For efficient cytosolic and nuclear delivery of small 84 tetrapropargyl bromide- bioactive molecules. cyclopeptide homoallylglycinefluorochrome] Bicyclic cell- FITC-C6dAs a promising scaffold for the design of small 90 penetrating ACSGSGSGCGSGSGSCGe bioactive molecules peptide Palmitoylpolyarginine peptides

C16-[r12]

A versatile nanovehicle for intracellular delivery of 26 small bioactive molecules

a Disulfide

bond between cysteines; b Peptide name not consistent with the peptide sequence; c Sequence listed in Table 1.; d C6 refers to Aminohexanoic acids, C16 refers to Palmitoyl acid; e Cysteines form a disulfide bridge with T3-scaffold; f ß-Alanine is on the side chain of Lysine residue

8.

Conclusion and further prospects


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The efficient intracellular delivery of bioactive molecules can assist the treatment of several challenging diseases, such as cancer, neurodegenerative diseases, bacterial resistance, and HIV. However, the impermeability of the cell membrane is a major challenge in achieving this goal. CPPs appeared as a promising tool for overcoming this challenge, but due to some unfavorable properties like proteolytic degradations, decreased stability in the biological fluids, endosomal trapping, and toxicity issues, no compound has reached yet to the clinical trial stage. On the other hand, cyclization of the linear peptides confers some favorable properties and avoid the limitation posed by the linear CPPs. Furthermore, many endogenous peptides in nature are cyclic and have important physiological functions.79 Another observation is that many cyclic peptides, such as daptomycin, polymixine B, vancomycin, gramicidin C, and cyclosporin reached the market as drugs to cure several diseases.91 These evidence engender hope in a new class of compounds termed as cyclic CPPs. As described above, several studies point to the importance of cyclic CPPs as efficient delivery tools for not only small bioactive therapeutic agents but also for the effective cellular uptake of full-length proteins. Although, numerous studies conducted on cyclic CPPs provide convincing results as delineated in this review, yet many more studies are needed to understand the exact mechanism of each penetration categories and to realize their full potential as a successful new drug delivery system. Macropinocytosis, clathrin-mediated endocytosis, and caveolae/lipid-raft-mediated endocytosis can be possible categories, yet this is not all inclusive as many factors will influence the degree of cellular uptake of cyclic CPPs. Areas which needs further exploration in this field are the assessment of pharmacokinetic properties and testing these cyclic CPPs in in vivo models. Although, in vitro data suggest the safety of various cyclic CPPS, however, sufficient in vivo data is required for the translational application of these peptide-based therapeutic agents. Now the pharmaceutical industry is actively involved in the


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investigation and realization of the potential of CCPPs. It is believed that further exploration into this new yet powerful area of research can lead to a significant breakthrough in cancer therapy as well as other intractable diseases.

AUTHOR INFORMATION Corresponding Author

*Email: [email protected]; Tel.: +1-714-516-5489 (KP); [email protected]; Tel.: +1714-516-5483 (RKT) Author Contributions

The manuscript was written through the contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors greatly acknowledge the financial support for this research from the Chapman University School of Pharmacy


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Cyclic Cell-Penetrating Peptides As Efficient Intracellular Drug Delivery Tools Shang Eun Park, Muhammad Imran Sajid, Keykavous Parang*, and Rakesh Kumar Tiwari*

Cyclic Cell-Penetrating Peptide

Intracellular Drug Delivery


Cyclic Cell-Penetrating Peptides As Efficient Intracellular Drug

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