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Review ArticleDelivery of Nucleic Acids and Nanomaterials by Cell-PenetratingPeptides Opportunities and Challenges

Yue-Wern Huang1 Han-Jung Lee2 Larry M Tolliver1 and Robert S Aronstam1

1Department of Biological Sciences Missouri University of Science and Technology Rolla MO 65409-1120 USA2Department of Natural Resources and Environmental Studies National Dong Hwa University Hualien 97401 Taiwan

Correspondence should be addressed to Yue-Wern Huang huangymstedu

Received 24 July 2014 Revised 18 September 2014 Accepted 23 September 2014

Academic Editor Pradeep Kumar

Copyright copy 2015 Yue-Wern Huang et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Many viral and nonviral systems have been developed to aid delivery of biologically active molecules into cells Among these cell-penetrating peptides (CPPs) have received increasing attention in the past two decades for biomedical applications In this reviewwe focus on opportunities and challenges associated with CPP delivery of nucleic acids and nanomaterials We first describe thenature of versatile CPPs and their interactions with various types of cargoes We then discuss in vivo and in vitro delivery of nucleicacids and nanomaterials by CPPs Studies on the mechanisms of cellular entry and limitations in the methods used are detailed

1 Introduction

11 Cell-Penetrating Peptides The plasma membrane playsessential roles in selective permeability osmotic balancecompartmentalization and cellular uptake Small polarmole-cules such as ions amino acids and sugars enter cells throughspecific carriers and channels in the membrane Largermacromolecules such as proteins DNAs and RNAs are gen-erally unable to use this mode of entry Consequently deliv-ery tools have been developed to facilitate cellular uptakeof large molecules for basic research and biomedical appli-cations (Figure 1) These include mechanical and electricaltransfection techniques such as microinjection bioballis-tics hydrodynamic force ultrasonic nebulization electro-poration chemicalbiochemical methods such as calciumphosphate coprecipitation membrane fusion catalyzed byartificial lipids peptidesproteins dendrimers adenovirus-associated virus vectors and lentiviral vectors [1] Some ofthese methods are suitable for in vitro or in vivo use whileothers are suitable for both These delivery methods canalso be categorized as involving viral or nonviral carriersystems Due to safety reasons nonviral delivery methodssuch as peptide- and lipid-based systems have receivedmore attention over the past 20 years than viral methods

Advantages of nonviral systems are ease and flexibility ofassembly minimal toxicity and low levels of immunogenicityand insertional mutagenesis

Among nonviral delivery methods cell-penetrating pep-tides (CPPs) have become increasingly popularThe first CPPwas discovered by two independent groups and is comprisedof a protein transduction domain (PTD) derived from thetransactivator of transcription (Tat) of the human immun-odeficiency virus type 1 (HIV-1) [2 3] This domain containseleven amino acids (YGRKKRRQRRR) that are responsiblefor cellular entry of Tat [4] Later a variety of CPPs are derivedfrom natural chimeric and synthetic sources (Table 1) [5 6]In general CPPs are (1) less than 30 amino acids (2) rich inarginine and lysine (3) positively charged or amphipathic (4)easy to prepare and (5) nontoxic [7]

In general the efficiency of a CPP in mediating cellularuptake is a function of its total electric charge and aminoacid sequence insofar as these properties determine its 3-D structures and potential interactions with membranesmolecules [8ndash10] In particular secondary amphipathicity isa critical determinant of cellular uptake [9 11ndash13] Data basesand predictive simulationmodels are available for identifyingbiomimetic cell-penetrating peptides based upon an array ofprotein characteristics [14ndash16]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 834079 16 pageshttpdxdoiorg1011552015834079

2 BioMed Research International

ProteinDNARNA

Cargo

Into the nucleus

To the cytoplasm

Cell-penetrating peptides

(cell membrane as a selective barrier)

+ + ++

++++

+ + ++

++++

+ + ++

++++

Cell-penetrating

peptides

CPP-tagged

cargo

Figure 1 Cell-penetrating peptides as a tool to deliver biologically active molecules

Table 1 A variety of cell-penetrating peptides mentioned in this paper

CPP Amino acid sequence ReferencesViral or natural CPPs

HIV Tat YGRKKRRQRRR [31ndash35]HIV Rev TRQARRNRRRRWRERQR [35]FHV coat RRRRNRTRRNRRRVR [35]HSV-1 protein VP22 DAATATRGRSAASRPTERPRAPARSASRPRRPVD [34]Penetratin RQIKIWFQNRRMKWKK [31 33 36 37]EB1 (penetratin analog) LIRLWSHLIHIWFQNRRLKWKKK [31]MPG GALFLGFLGAAGSTMGAWSQPKKKRKV [31 34 38ndash40]

PolyargininesPR9 FFLIPKGRRRRRRRRR [41ndash44]SR9 RRRRRRRRR [31 34 41 42 45]IR9 GLFEAIEGFIENGWEGMIDGWYGRRRRRRRRR [46ndash48]HR9 CHHHHHRRRRRRRRRHHHHHC [41ndash44 46]

Engineered CPPsTransportan CLIKKALAALAKLNIKLLYGASNLTWG [31 36]CADY GLWRALWRLLRSLWRLLWRA [31 49]C6 RLLRLLLRLWRRLLRLLR [13]C6M1 RLWRLLWRLWRRLWRLLR [13]PF20 (and variants see [9]) LLKLLKKLLKLLKKLLKLL [9]NAP KALKLKLALALLAKLKLA [9]Steryl-NAP Stearyl-KALKLKLALALLAKLKLA [9]POD GGG[ARKKAAKA]4 [25]

BioMed Research International 3

O

OH OO

O

O

O

OSHO

HO

N

O

OO

OO

O

OHS

N

O

O

O

O OH

SN

(2times)

n

n

O

NH

NH

HN NH2

O

O O

NH

NH

HN NH2

Cargo Bissulfosuccinimidyl suberate (BS3)CPP

(polyarginine) CPP-linked cargo

++

(a)

O

O

O

O

OH

N

N NN

N

N N

C

C

HNH

HNHN

NHNH

NH2

NH2

nn

CH3

CH3

CH3

CH3

CH3

CH3

+

Cargo Carbodiimide (EDC) CPP(polyarginine)

+

CPP-linked cargo

(b)

++NH2

NH2

NH2

O

O

O

OO

OO

O

O

O

O

O

O

OO

O

O

HO

HO

HS

N

N

N

N

NH

S

S

S

OH

Sulfo-SMCC Cysteine-modified CPP CPP-linked cargoCargo

(c)

+O O

O

O

O O

O

O

O O

O

O

O

OOH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NHNH

NH

NH

H2N NH

NH

H2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2NH2

O O

O

O

O O

O

O

O O

O

O

OH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NH

NH

NH

NH

H2N NH

NHH2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2

NH2

Streptavidin-taggedcargo

Biotinylated CPP (Tat PTD) CPP-linked cargo

(d)

Figure 2 Reaction scheme for linking CPPs to cargoesThe cargoes can be linked to the CPPs through a covalent linkage method such as (a)bissulfosuccinimidyl suberate (b) carbodiimide or (c) Sulfo-SMCC with a cysteine-modified CPP or through a noncovalent method suchas (d) biotin-streptavidin interaction

12 Versatile CPPs andTheir Interactions with Cargoes CPPshave been used as carriers of DNA RNA protein nano-materials and pharmaceuticals Association between CPPand cargo can be either covalent or noncovalent Covalentinteractions have been achieved by sulfosuccinimidyl suber-ate linkage carbodiimide conjugation and thiol-amine cou-pling Noncovalent interactions include biotin-streptavidininteractions electrostatic interactions and metal-affinityinteractions [18ndash20] (Figure 2) Covalent strategies have beenused to conjugate antibody fragments drugs and fluorescentlabels Covalent linking ensures strong association between

CPP and cargo and high transduction efficiency Howeverthe covalent-linking procedure may be labor-intensive time-consuming and costly The yield of the CPP-cargo covalentcomplex also decreases during separation from the unboundCPPs and cargoes Further to achieve covalent linkingcargoes are chemically modified which may compromisefunctionality Schwarze et al first demonstrated the deliveryof CPP-fusion proteins into various tissues in mice [21]Subsequently others showed that CPPs can carry covalentlylinked nucleic acids and nanomaterials into cells of a varietycell lines [22ndash24] Johnson et al used the cell-penetrating


peptide POD (peptide for ocular delivery) to deliver POD-GFP fusion protein to retina cornea and skin [25] Changet al first described a CPP-mediated covalent protein trans-duction in plants [26]

The advantages of noncovalent binding between CPP andcargo are ease of use ease of production versatility withrespect to cargo composition and preservation of cargo func-tionality [27] Noncovalent strategies have been used todeliver siRNA plasmids and splice correcting oligonucleoti-desNoncovalent bondingwas applied to the delivery of greenfluorescent protein (GFP) collagen and insulin into mouseskin tissues [28 29]We and others have successfully used thenoncovalent delivery in several representative organisms ofprokaryotes and unicellular yeasts including cyanobacteriabacteria archaea algae fungi and yeasts However nonco-valent delivery was not successful in multicellular fungi andgreen algae [30]

2 CPPs Delivery of Nucleic Acids

21 siRNA Delivery RNA interference (RNAi) is an evolu-tionarily conservedmechanism of gene expression regulationin animals and plants [84] Endogenous pre-microRNAs(pre-miRNAs) are synthesized and processed in the nucleusand then transported to cytoplasmThe pre-miRNA is short-ened and processed in the cytoplasm by an RNAse III enzyme(Dicer) to become mature microRNA A multienzyme com-plex that includes argonaute 2 and theRNA-induced silencingcomplex (RISC) binds to the microRNA and eliminates onestrand This activated complex then binds to an mRNAstrand that possesses a complementary sequence therebyinactivating its expression [85ndash87] Utilizing this principlesynthetic RNA molecules (small interfering RNA siRNA) of20ndash25 base pairs in length have been developed tomanipulatethe expression of specific genes (Figure 3) This techniquerepresents a new treatmentmodality in cancer infectious dis-eases and genetic disorders Currently there aremore than 20siRNAs undergoing clinical trials in various stages [88] Thebiggest challenge of this technique is delivery of siRNA acrossthe cytoplasmic membrane Carriers are needed to overcomethis barrier and CPPs represent an obvious attractive meansfor siRNA internalization

The CPPs transportan penetratin amphipathic peptidesand polyarginine have been extensively used to covalentlyor noncovalently deliver siRNA into animal and plant cells[31ndash33 36ndash40 45 49 89 90] Target gene products ofsiRNAs include Luciferase SOD1 EGFP p38 MAP kinaseCDK9 VEGF p53 and Oct-34 Stable noncovalent CPP andsiRNA complexes can be formed by CPPsiRNA electrostaticinteractions Alternately CPPs can be covalently linked tosiRNA duplexes through disulfide bond formation in whichCPPs containing N-terminal cysteines are conjugated tosiRNAmoleculeswith a 51015840-thiolmodified siRNA sense strand[31 34] It is critical to purify the CPPsiRNA complex inorder to investigate transduction efficiency of the covalentCPP-siRNA complexes Otherwise it is difficult to discernthe contribution of noncovalentCPP-siRNAcomplexes to thetransduction response

Potential drawbacks of direct conjugation of cationicCPPs with anionic siRNAs are charge neutralization inacti-vation of the CPP and aggregationprecipitation which maylimit siRNA entry into the cells [33 91 92] Eguchi andDowdy invented an elegant design that fused a Tat PTD witha double-stranded RNA-binding domain (dsRBD) [93] Thisdesign allowed siRNA to bind to dsRBD while leaving PTDto induce cellular uptake in primary and transformed cellsThis tactic was applied in a mouse model of glioblastomato deliver two siRNAs for simultaneous silencing of EGFRand Akt2 The result was selective destruction of tumor cellsand improved longevity of cancerousmice [94] Clearly CPP-mediated siRNA delivery has a promising future in diseasetreatment

22 DNA Delivery The delivery of functional exogenousDNA into organisms is important for transgenic research andgene therapy Most studies have focused on CPP-mediateddelivery into mammalian cells although our research teamhas demonstrated that CPPs can deliver biologically activemolecules into a variety of species including rotifers [46]cyanobacteria [95] insects [41] plants [96] and parame-cium [42] Internalization of CPP-mediated DNA transduc-tion involves a combination of pathways including classicalendocytosis caveolin- and clathrin-dependent endocytosismacropinocytosis and direct membrane penetration [46 9697] Various strategies have been developed to enhance trans-duction efficiency For instance stearylation of arginine-richCPPs drastically increases transduction efficiency of plasmidDNA [35 98] while hemagglutinin-2 (HA2) analogues orchemicals such as chloroquine and polyethylenimine (PEI)enhance transduction efficiency by catalyzing cargo releasefrom endosomes (see Section 43)

There have been attempts to deliver biologically activemolecules into the nucleus [99] Molecules can enter thenucleus from the cytoplasm by either passive diffusion oractive transport mechanisms Small molecules less than10 nm in diameter or 50ndash60 kDa in size can diffuse directlythrough nuclear pore complexes Most protein moleculesare transported by energy-dependent transport mechanismsinitiated by nuclear localization signals (NLS) These signalsare recognized by importin family proteins that mediate thetransport across the nuclear envelope with the participationof Ran proteins [100] An N-stearylated NLS was found toimprove CPP-mediated transfection activity by overcomingcell membrane and nuclear pore barriers [98] In contrastwe have found that constructs with NLS tag interact with theHA2 sequence thereby limiting delivery The detailed reasonof this NLS interference remained to be elucidated [78]

In addition to penetrating cytoplasmic membrane andnucleus we demonstrated that a CPP-piggyBac transposase(CPP-PBase) plasmid system could accomplish both proteintransduction and transposition [101] The system was able tosynchronously deliver covalently linked PBase and noncova-lently linked cis plasmid into human cells This one-plasmidldquotransposoductionrdquo has tremendous potential for safe andefficient cell line transformation gene therapy and functionalgenomics


O

O

O

O

OO

O

O

O

O

O

O

OH

OH

NHNH

NHNH

NHNH

N HN H

N HN H

N H

H2 N

H2 N

H2 N

H2 N

H2 N

H2 N

NH N

HN

HN

HN

H NH

NH

NH

NH

NH

NH

NH

NH

2

NH

2N

H2

shRNA

CPP (HIV Tat)

(a)

Dicer (dsRBD)

(b)

RISC

siRNA-boundRISC

mRNA

(c) (d) (e)

Figure 3 Simplified conceptual diagram (not drawn proportionally in size) of exogenous siRNA-mediated gene silencing (a) The siRNA(usually small hairpin RNA shRNA) can be modified to covalently interact with CPPs and then be transported through the cell membrane(b) shRNA binds to the double-strand RNA binding domain (dsRBD) of the enzyme Dicer and then is processed (c) The processed RNA isincorporated into the RNA-induced silencing complex (RISC)The passenger strand RNA is degraded (d)The guide strand RNA along withthe RISC binds to a complementary sequence of a targeted mRNA (e) The targeted mRNA is degraded and translation disrupted

3 CPPs Delivery of Nanomaterials

The improved sensitivity resolution and versatility of fluo-rescent microscopy and the discovery of fluorescent proteinshave revolutionized imaging in basic science and biomedicalapplications [102 103] These fluorescent proteins have beenextensively used for visualizing and tracking molecules indynamic cellular processesTheymay also be useful in diseasediagnosis and therapeutic planning Recently Nguyen et aladvanced the possibility of utilizing fluorescent proteinsto improve surgical precision [104] However the broademission spectra of current organic fluorophores impedemultiplex imaging while photobleaching limits their use inlong-term imaging [105 106] Furthermore cell autofluores-cence in the visible spectrum and a need of probes that emitin the near-infrared (NIR) region drive the need to developnew imaging probes

Nanomaterials are materials that have at least one dimen-sion in the range of 1ndash100 nm The development of nanoma-terials has revolutionizedmany industries such as computingand semiconductor optics energy and cosmetics [107] Sem-iconductor nanocrystals (aka quantum dots QDs) possess

high optical extinction coefficient a narrow range of emissionwavelength exceptional resistance to photo- and chemicaldegradation and high quantum yield [108 109] Theseproperties make QDs particularly attractive for long-termobservation of molecules in live cells and multiplex imagingas well as tumor targeting and diagnostics in vivo Howeverinorganic QDs are not permeable to cytoplasmic membraneand agglomerate easily Thus surface modifications of QDssuch as complexing with polyethylene glycol are requiredto achieve stable suspension (Figure 4(a)) Even so QDs arepoorly taken up by cells (Figure 4(b)) Josephson et al firstreported increased uptake of iron oxide nanoparticles cova-lently conjugated with Tat-PTD [110] These Tat-iron oxidenanoparticle complexes were internalized into lymphocytesand yielded magnetic labeling of cellsThis technology opensup the possibility for simultaneous diagnosis and treatmentof diseases (ie theranostics) when drugs are included in theimaging system

Stroh et al successfully labeled primary bone marrowcells with Tat-QD micelles ex vivo and observed the recruit-ment of the labeled bone marrow-derived precursor cells tothe tumor vasculature [111] This methodology may advance


P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe

ZnS ZnSDSPE-PEG(2000)

PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)

Figure 4 (a) Synthesis of water-soluble carboxylated CdSeZnS quantum dots Upon addition of ZnS as a shell to protect Cd core thesurface wasmodified with 1 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000] (DSPE-PEG 2000) and12-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-2 PE)The amount and ratio of PEG2-PEand DSPE-PEG(200) determine suspension stability in water (b) Fluorescence of CdSeZnS quantum dot in live cells with (left) and without(right) nona-arginine after a 1-hour exposure [17]

our understanding of stem cell proliferation and differenti-ation Many other studies have investigated CPP-mediateddelivery of QDs into living cells for basic science and biomed-ical application purposes [17 24 43 44 46ndash48 112ndash117] Ingeneral cellular uptake of the CPPQD complexes includesclassical endocytosis macropinocytosis and direct mem-brane penetration Factors that influence uptake efficiency ofCPPQD complexes include the size and the overall surfacecharge of the complexes For instance our data suggest thatelectropositive charges of CPPQD complexes (measured aszeta-potential) increase higher transduction efficiency [118]

Although Cd-based QDs at nontoxic levels can be usefulin research applications [119] they are not ideal agents

for therapeutic purposes Biocompatible fluorescent nan-odiamonds represent an attractive alternative Defect center(color center) of nanodiamonds can be created by irradi-ation with a high power laser beam followed by thermalannealing at 800∘C [120] Nanodiamonds thus have beenmodified producing strong and stable fluorescence with nophotoblinking (within 1ms) and no photobleaching [121]We found that histidine-modified arginine-rich CPP (HR9)can facilitate the cellular uptake of these fluorescent nan-odiamonds (Figure 5) Collectively a combined use of CPPsand nanoscaled materials (with or without fluorescence)may greatly enhance payload and efficiency for imaging andtherapeutic uses


Table 2 Examples of cellular uptake markers and organelle markers for green fluorescent CdSeZnS quantum dots in live cell imaging

Marker Function Color (ExEm) Incubation time ConcFM4-64 Endocytosis marker Red (506750) 15min 2120583MAlexa Fluor 568-Transferrin Clathrin-dependent endocytosis marker Red (580630) 5min 25 120583gmLAlexa Fluor 568-Cholera toxin B Caveolae-dependent endocytosis marker Red (580630) 10min 5 120583gmLTexas red-Neural Dextran 70 Macropinocytosis marker Red (595615) 30min 5 120583MTMR-Dextran Early endosome marker Red (555580) 5min 10mgmLLyso Tracker Red DND 99 Lysosome marker Red (577590) 10min 05 120583MEEA1-Alexa Fluor 568 Early endosome marker Red (580630) 5min 10 120583gmLLamp-1-Alexa Fluor 568 Late endosome marker Red (578603) 5min 10 120583gmLMito Tracker Deep 633 Mitochondria marker Red (640662) 10min 1 120583MHoechst 33342 Nuclei marker Blue (352461) 30min 5 120583M

Figure 5 HR9 CPP facilitates cellular uptake of green fluorescentnanodiamonds

4 Mechanisms of CPP-MediatedCellular Uptake

41 Complementary Methods to Study Mechanisms of Cellu-lar Uptake Fixed cell imaging was utilized to study CPP-mediated cellular uptake However the fixation procedureintroduced artifacts and yielded inaccurate data Live cellimaging has supplanted fixed cell imaging and become apowerful tool to study dynamic cellular process in CPP-mediated uptake Coupled with cellular uptake markersand organelle markers the subcellular localization of theCPPcargo complex can be identified (Table 2) Figure 6presents a comprehensive workflow of experiments on cellu-lar uptake intracellular uptake and subcellular localizationThere are some discrepancies among publications regardingthe identity of CPP uptake mechanisms due to the limiteduse of cellular process inhibitors Although pharmacologicalinhibitors (Table 3) can be used to inhibit internalizationprocesses these inhibitors are not completely specific andmay suppress more than one cellular uptake pathway Forinstance cytochalasin D (CytoD) and N-ethylmaleimide

(NEM) inhibit both clathrin- and caveolin-mediated path-ways CytoD also inhibits macropinocytosis This makes itdifficult to evaluate the contributions of different pathways totransduction and complicates the analysis of CPP-mediateduptakemechanisms To overcome this problem we suggestedthat RNAi be used as a complementarymethod to thoroughlyelucidate CPP-mediated uptake mechanism For instance inour study with CPP-mediated cellular uptake of CdSeZnSquantum dots pharmacological inhibitors reduced cellularuptake of the noncovalent CPPQD complex Howeveruptake efficiency of the CPPQD complex was not reduced bysiRNAs introduced to knockdown clathrin HC and caveolin1 (Figure 7) It is also worth noting that although the effectivedose of an inhibitor may be specified by commercial vendorsor literature a pilot study should be conducted to optimizethe concentration of an inhibitor for a specific cell line sincetoo high concentration of an inhibitor may be toxic to cellsand compromise cellular processes

42 Diverse Cellular Uptake Routes Understanding themechanisms underlying CPP-mediated cellular uptake andsubcellular localization of the carrier system is needed toimprove transduction efficiency and cargo functionality Ourunderstanding of uptake is still incomplete Proposed routesof entry include direct membrane penetration and varioustypes of endocytic pathways Empirical modeling evidencefrom several studies supports a directmembrane penetrationInitially CPPs bind to the phosphate groups of the phospho-lipids on the bilayer surface As the concentrations of CPPson cell surface increase the lipid molecules rearrange Sidechains of arginines translocate through the distal layer andform a water pore Finally a few CPPs diffuse through thepore followed by pore closure [122ndash125]

Most studies of CPP-mediated cellular uptake of nucleicacids and QDs have focused on endocytosis Endocytosisis an active process whereby cells internalize extracellularmaterial through cytoplasmic membranes This process isrequired by certain cells to obtain essential nutrition andexcrete cellular waste At least 10 different types of endocyticpathways involving various molecules have been delineated[126] Studies of cellular uptake of CPPcargo complexes have


Cellular uptake

Endocytosis

Clathrin-dependentendocytosis

Delivery

568-transferrin

Deliverysystem + Alexa

568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors

Caveolae-dependentendocytosis


568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran

Macro-pinocytosisinhibitors

Delivery system

neutral dextran

Intracellular transport

Microtubuleinhibitors

Subcellular localization

LysoTracker orMitoTracker orHoechst 33342

Colocalization(epifluorescent microscope)

EEA1-Alexa 568 or

system + Alexa

EEA1-Alexa 568 orsystem +

Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568

Figure 6 Diagram illustrating a comprehensive workflow of experiments designed to characterize the cellular uptake intracellular uptakeand subcellular localization of CPPs and their cargoes

focused on three pathways clathrin-mediated endocytosiscaveolin-dependent endocytosis and macropinocytosis [1734 43 44 97 127 128] Future studies should consider otherendocytic pathways CLICGEEC IL2R120573 Arf6-dependentflotillin-dependent circular dorsal ruffles and entosis

In endocytosis CPPcargo complexes might initiallyinteract with heparan sulfate proteoglycans (a pool of anioniccharge on the cell surface) However Gump et al recentlyrevised the role of glycosaminoglycans in Tat PTD-mediatedinduction of macropinocytosis [129] They found that trans-duction occurs efficiently in the absence of glycosamino-glycans and sialic acid and that the removal of cell surfaceproteins totally abolishes transduction They suggested thatadditional cell surface protein(s) are necessary for Tat PTDtransduction More studies are needed to identify theseproteins are and appreciate their roles in CPP membranetransduction

Collectively current data suggest that the routes of thecellular uptake for CPPcargo complexes are diverse reflect-ing the varied chemical and physical natures of the CPPs andcargoes entry may simultaneously involve multiple routes

43 Release from Lysosomal Entrapment A particular prob-lem associated with most of the CPP delivery systems isentrapment in lysosome which may lead to cargo degrada-tion and thus loss of intended functionality Multiple strate-gies have been developed to circumvent this problem Onemethod is to add to the CPP a section of the hemagglutinin

(HA) sequence from the human influenza virus (Table 4) HAis composed of two subunits hemagglutinin-1 responsiblefor binding to cells and hemagglutinin-2 (HA2) responsiblefor lysosomal escape [130] The N-terminal domain of theHA2 subunit possesses 23 amino acids in a hydrophobicregion referred to as fusion peptide [131] This fusion peptidedomain is buried inside the HA trimer in its resting confor-mation Upon acidification in the lysosome an irreversibleconformational change of HA2 occurs exposing the fusionpeptide and allowing it to insert into lysosomal membranesSubsequently a fusion pore is in the membrane is formedleading to transport of lysosomal contents into the cytosol

The sequence of CPP-HA2 can be chemically synthesizedor the HA2 sequence can be inserted into a CPP-containingplasmid The advantages of using a peptide synthesizerto produce a CPP-HA2 sequence are high purity ease ofprogramming the sequence and flexibility of residue mod-ification as well as molecular conjugation Disadvantagesinclude limited length of the sequence loss of yield duringpurification process and possible loss of native configurationThe advantages of using a CPP-HA2 plasmid are low costtime saving for production and the flexibility to include otherdesired functional sequences such as imaging moleculesDisadvantages are low purity and considerable time andlabor investments In addition to HA2 and its analogues thesequences of CPPs can be modified for lysosomal escapeCollectively these HA2 analogues and sequence variationsof CPPs exhibit different degrees of enhanced transductionefficiency ranging from 02- to 7000-fold (Table 4) Factors


Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature

Inhibitenergy-depend

entend

ocytosis

4∘C

[17]

Hypertonicm

edium

Inhibitclathrin-dependent

endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin

AInhibitenergy-depend

entend

ocytosis

(Allmetabolicinhibitors)

(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo

lymerizationinhibitor)

15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a

ndcaveolae-dependent

endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]

N-Ethylmaleimide(NEM

)Inhibitclathrin-a

ndcaveolin-dependent

endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip

inInhibitcaveolae-depend

entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin

Inhibitcaveolae-depend

entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin

Inhibitreceptor-mediatedendo

cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)

Control Sucrose MDCChlorpromazine

lowast

lowast

(a)

Lipid raftcaveolea inhibition

0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125

Control Clathrin HC siRNA Caveolin-1 siRNA

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)

Figure 7 Comparisons of clathrin- and caveolin-dependent cellular uptake pathways using pharmacological inhibitors and RNAi technique[17]

influencing efficiency include the nature of CPPs and HA2types of cargoes and sequence orientation of CPP and HA2

In addition to fusogenic amino acid sequences chemicalssuch as chloroquine and polycationic polyethylenimine (PEI)are commonly used to promote lysosomal escape Chloro-quine a weak base can enter the cell and accumulate invesicular compartments following protonation At low con-centrations chloroquine inhibits endosome acidification andmaturation by preventing the accumulation of free protonsAs its concentration increases it starts to accumulate counte-rions to protons (eg chloride ion) in endosomes leading toendosomal swelling and rupture [132 133] Endosomal releaseby chloroquine enhances of transduction efficiency [78 134135] The secondary and tertiary amines of low molecularweight PEI can be protonated in the acidic environment ofthe endosome leading to endosomal swelling and rupturePEI has been used to deliver DNA plasmids with improvedtransduction efficiency [81 136ndash139]Thedrawback of the PEIpolymer is that it is not biodegradable and is highly chargedThus interaction of this polymer with genetic materials in thecell nucleus might alter gene expression [140ndash142]

5 Conclusions

CPPs are capable of carrying nucleic acids and nanoma-terials into cells CPPs can interact with cargoes in cova-lent or noncovalent manners Complementary tools suchas pharmacological inhibitors and siRNA are being usedto decipher mechanisms of cellular uptake Depending onthe physiochemical natures of the CPPcargo complex themechanism of cellular entry may include classical endocy-tosis macropinocytosis clathrin- and caveolon-dependentpathways and direct membrane penetration A variety ofchemical and molecular methods have been introduced toovercome lysosomal entrapment in order to achieve higherfunctional yields As studies continue to advance our under-standing about CPPs this delivery modality will find consid-erable usage in clinical setting and basic science research

Abbreviations

CPP Cell-penetrating peptideCDK9 Cyclin-dependent kinase 9dsRBD Double-stranded RNA binding domain


Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable


GFP Green fluorescent proteinEGFP Enhanced green fluorescent proteinHA HemagglutininMAP kinase Mitogen-activated protein kinaseNLS Nuclear Localization SignalPOD Peptide for ocular deliveryPTD Protein transduction domainPEI PolyethylenimineQD Quantum dotRISC RNA-induced silencing complexRNAi RNA interferenceRNAse RibonucleasesiRNA Small interfering RNASOD1 Superoxide dismutase 1VEGF Vascular endothelial growth factor

Conflict of Interests

The authors confirm no conflict of interests with the contentsin this paper

References

[1] D Lou andW M Saltzman ldquoSynthetic DNA delivery systemsrdquoNature Biotechnology vol 18 no 1 pp 33ndash37 2000

[2] M Green and P M Loewenstein ldquoAutonomous functionaldomains of chemically synthesized human immunodeficiencyvirus tat trans-activator proteinrdquo Cell vol 55 no 6 pp 1179ndash1188 1988

[3] A D Frankel and C O Pabo ldquoCellular uptake of the tat proteinfrom human immunodeficiency virusrdquo Cell vol 55 no 6 pp1189ndash1193 1988

[4] E Vives P Brodin and B Lebleu ldquoA truncated HIV-1 Tatprotein basic domain rapidly translocates through the plasmamembrane and accumulates in the cell nucleusrdquoThe Journal ofBiological Chemistry vol 272 no 25 pp 16010ndash16017 1997

[5] K M Wagstaff and D A Jans ldquoProtein transduction cell pen-etrating peptides and their therapeutic applicationsrdquo CurrentMedicinal Chemistry vol 13 no 12 pp 1371ndash1387 2006

[6] A Graslund F Madani S Lindberg U Langel S Futaki andA Graslund ldquoMechanisms of cellular uptake of cell-penetratingpeptidesrdquo Journal of Biophysics vol 2011 Article ID 414729 10pages 2011

[7] J Regberg A Srimanee and U Langel ldquoApplications of cell-penetrating peptides for tumor targeting and future cancertherapiesrdquo Pharmaceuticals vol 5 no 9 pp 991ndash1007 2012

[8] E D Karagiannis A M Urbanska G Sahay et al ldquoRationaldesign of a biomimetic cell penetrating peptide libraryrdquo ACSNano vol 7 no 10 pp 8616ndash8626 2013

[9] J Regberg A Srimanee M Erlandsson et al ldquoRational designof a series of novel amphipathic cell-penetrating peptidesrdquoInternational Journal of Pharmaceutics vol 464 no 1-2 pp 111ndash116 2014

[10] A T Jones and E J Sayers ldquoCell entry of cell penetratingpeptides tales of tails wagging dogsrdquo Journal of ControlledRelease vol 161 no 2 pp 582ndash591 2012

[11] M Hallbrink K Kilk A Elmquist et al ldquoPrediction of cell-penetrating peptidesrdquo International Journal of Peptide ResearchandTherapeutics vol 11 no 4 pp 249ndash259 2005

[12] J Oehlke A Scheller BWiesner et al ldquoCellular uptake of an 120572-helical amphipathic model peptide with the potential to deliverpolar compounds into the cell interior non-endocyticallyrdquoBiochimica et Biophysica ActamdashBiomembranes vol 1414 no 1-2pp 127ndash139 1998

[13] M Jafari D N Karunaratne C M Sweeting and P ChenldquoModification of a designed amphipathic cell-penetrating pep-tide and its effect on solubility secondary structure and uptakeefficiencyrdquo Biochemistry vol 52 no 20 pp 3428ndash3435 2013

[14] G E Tusnady Z Dosztanyi and I Simon ldquoTransmembraneproteins in the Protein Data Bank identification and classifi-cationrdquo Bioinformatics vol 20 no 17 pp 2964ndash2972 2004

[15] R A Laskowski E G Hutchinson A D Michie A CWallaceM L Jones and J M Thornton ldquoPDBsum a Web-baseddatabase of summaries and analyses of all PDB structuresrdquoTrends in Biochemical Sciences vol 22 no 12 pp 488ndash490 1997

[16] M A Lomize A L Lomize I D Pogozheva and H I Mos-berg ldquoOPM orientations of proteins in membranes databaserdquoBioinformatics vol 22 no 5 pp 623ndash625 2006

[17] Y Xu B R Liu H-J Lee et al ldquoNona-arginine facilitatesdelivery of quantum dots into cells via multiple pathwaysrdquoJournal of Biomedicine and Biotechnology vol 2010 Article ID948543 11 pages 2010

[18] N Nitin L Laconte W J Rhee and G Bao ldquoTat peptide iscapable of importing large nanoparticles across nuclear mem-brane in digitonin permeabilized cellsrdquo Annals of BiomedicalEngineering vol 37 no 10 pp 2018ndash2027 2009

[19] Y Wei N R Jana S J Tan and J Y Ying ldquoSurface coatingdirected cellular delivery of TAT-functionalized quantum dotsrdquoBioconjugate Chemistry vol 20 no 9 pp 1752ndash1758 2009

[20] K Takayama A Tadokoro S Pujals I Nakase E Giralt and SFutaki ldquoNovel system to achieve one-pot modification of cargomolecules with oligoarginine vectors for intracellular deliveryrdquoBioconjugate Chemistry vol 20 no 2 pp 249ndash257 2009

[21] S R Schwarze A Ho A Vocero-Akbani and S F DowdyldquoIn vivo protein transduction delivery of a biologically activeprotein into the mouserdquo Science vol 285 no 5433 pp 1569ndash1572 1999

[22] E L Snyder and S F Dowdy ldquoCell penetrating peptides in drugdeliveryrdquo Pharmaceutical Research vol 21 no 3 pp 389ndash3932004

[23] X Nan P A Sims P Chen and X Sunney Xie ldquoObservationof individual microtubule motor steps in living cells withendocytosed quantum dotsrdquo The Journal of Physical ChemistryB vol 109 no 51 pp 24220ndash24224 2005

[24] B C Lagerholm M Wang L A Ernst et al ldquoMulticolorcoding of cells with cationic peptide coated quantum dotsrdquoNano Letters vol 4 no 10 pp 2019ndash2022 2004

[25] L N Johnson S M Cashman S P Read and R Kumar-SinghldquoCell penetrating peptide POD mediates delivery of recombi-nant proteins to retina cornea and skinrdquo Vision Research vol50 no 7 pp 686ndash697 2010

[26] M Chang J-C Chou C-P Chen B R Liu and H-J Lee ldquoNoncovalent protein transduction in plant cells bymacropinocytosisrdquo New Phytologist vol 174 no 1 pp 46ndash562007

[27] M Chang H Yue-Wern R S Aronstam and H-J LeeldquoCellular delivery of noncovalently-associated macromoleculesby cell-penetrating peptidesrdquo Current Pharmaceutical Biotech-nology vol 15 no 3 pp 267ndash275 2014


[28] Y-WHouM-H Chan H-R Hsu et al ldquoTransdermal deliveryof proteinsmediated by non-covalently associated arginine-richintracellular delivery peptidesrdquo Experimental Dermatology vol16 no 12 pp 999ndash1006 2007

[29] Y-H Wang C-P Chen M-H Chan et al ldquoArginine-richintracellular delivery peptides noncovalently transport proteininto living cellsrdquo Biochemical and Biophysical Research Commu-nications vol 346 no 3 pp 758ndash767 2006

[30] B R Liu J-C Chou and H-J Lee ldquoCell membrane diversityin noncovalent protein transductionrdquo Journal of MembraneBiology vol 222 no 1 pp 1ndash15 2008

[31] A Eguchi and S F Dowdy ldquosiRNA delivery using peptidetransduction domainsrdquo Trends in Pharmacological Sciences vol30 no 7 pp 341ndash345 2009

[32] Y-L Chiu A Ali C-Y Chu H Cao and T M RanaldquoVisualizing a correlation between siRNA localization cellularuptake and RNAi in living cellsrdquo Chemistry and Biology vol 11no 8 pp 1165ndash1175 2004

[33] J J Turner S Jones M M Fabani G Ivanova A A Arzu-manov and M J Gait ldquoRNA targeting with peptide conjugatesof oligonucleotides siRNA and PNArdquo Blood Cells Moleculesand Diseases vol 38 no 1 pp 1ndash7 2007

[34] B R Meade and S F Dowdy ldquoExogenous siRNA deliveryusing peptide transduction domainscell penetrating peptidesrdquoAdvanced Drug Delivery Reviews vol 59 no 2-3 pp 134ndash1402007

[35] S Futaki W Ohashi T Suzuki et al ldquoStearylated arginine-rich peptides a new class of transfection systemsrdquo BioconjugateChemistry vol 12 no 6 pp 1005ndash1011 2001

[36] A Muratovska and M R Eccles ldquoConjugate for efficientdelivery of short interfering RNA (siRNA) into mammaliancellsrdquo FEBS Letters vol 558 no 1ndash3 pp 63ndash68 2004

[37] T J Davidson S Harel V A Arboleda et al ldquoHighly efficientsmall interfering RNAdelivery to primarymammalian neuronsinduces microRNA-like effects before mRNA degradationrdquoJournal of Neuroscience vol 24 no 45 pp 10040ndash10046 2004

[38] F Simeoni M C Morris F Heitz and G Divita ldquoInsightinto the mechanism of the peptide-based gene delivery systemMPG implications for delivery of siRNA intomammalian cellsrdquoNucleic Acids Research vol 31 no 11 pp 2717ndash2724 2003

[39] P Lundberg S El-Andaloussi T Sutlu H Johansson and ULangel ldquoDelivery of short interferingRNAusing endosomolyticcell-penetrating peptidesrdquoThe FASEB Journal vol 21 no 11 pp2664ndash2671 2007

[40] S Veldhoen S D Laufer A Trampe and T Restle ldquoCellulardelivery of small interfering RNA by a non-covalently attachedcell-penetrating peptide quantitative analysis of uptake andbiological effectrdquo Nucleic Acids Research vol 34 no 22 pp6561ndash6573 2006

[41] Y J Chen B R Liu Y H Dai et al ldquoA gene delivery systemfor insect cells mediated by arginine-rich cell-penetrating pep-tidesrdquo Gene vol 493 no 2 pp 201ndash210 2012

[42] Y-HDai B R LiuH-J Chiang andH-J Lee ldquoGene transportand expression by arginine-rich cell-penetrating peptides inParameciumrdquo Gene vol 489 no 2 pp 89ndash97 2011

[43] B R Liu Y-W Huang J G Winiarz H-J Chiang and H-J Lee ldquoIntracellular delivery of quantum dots mediated bya histidine- and arginine-rich HR9 cell-penetrating peptidethrough the direct membrane translocation mechanismrdquo Bio-materials vol 32 no 13 pp 3520ndash3537 2011

[44] B R Liu S-Y Lo C-C Liu et al ldquoEndocytic trafficking ofnanoparticles delivered by cell-penetrating peptides comprisedof nona-arginine and a penetration accelerating sequencerdquoPLoS ONE vol 8 no 6 Article ID e67100 2013

[45] Y-HWang Y-W Hou and H-J Lee ldquoAn intracellular deliverymethod for siRNA by an arginine-rich peptiderdquo Journal ofBiochemical and Biophysical Methods vol 70 no 4 pp 579ndash586 2007

[46] B R Liu J-S Liou Y-J Chen Y-W Huang and H-JLee ldquoDelivery of nucleic acids proteins and nanoparticlesby arginine-rich cell-penetrating peptides in rotifers rdquo MarineBiotechnology vol 15 no 5 pp 584ndash595 2013

[47] B R Liu J-S Liou Y-W Huang R S Aronstam and H-JLee ldquoIntracellular delivery of nanoparticles and DNAs by IR9cell-penetrating peptidesrdquo PLoS ONE vol 8 no 5 Article IDe64205 2013

[48] B R Liu J G Winiarz J S Moon et al ldquoSynthesis charac-terization and applications of carboxylated and polyethylene-glycolated bifunctionalized InPZnS quantum dots in cellularinternalization mediated by cell-penetrating peptidesrdquo Colloidsand Surfaces B Biointerfaces vol 111 pp 162ndash170 2013

[49] L CrombezGAldrian-Herrada KKonate et al ldquoAnewpotentsecondary amphipathic cell-penetrating peptide for siRNAdelivery into mammalian cellsrdquo Molecular Therapy vol 17 no1 pp 95ndash103 2009

[50] X Wu X Zhao L Baylor S Kaushal E Eisenberg andL E Greene ldquoClathrin exchange during clathrin-mediatedendocytosisrdquo Journal of Cell Biology vol 155 no 2 pp 291ndash3002001

[51] F Johansson M Sommarin and C Larsson ldquoFusicoccinactivates the plasma membrane H+-ATPase by a mechanisminvolving the C-terminal inhibitory domainrdquo Plant Cell vol 5no 3 pp 321ndash327 1993

[52] T G Hammond F O Goda G L Navar et al ldquoMembranepotential mediates H+-ATPase dependence of ldquodegradativepathwayrdquo endosomal fusionrdquo Journal of Membrane Biology vol162 no 2 pp 157ndash167 1998

[53] R Fuchs S Schmid and I Mellman ldquoA possible role forNa+K+-ATPase in regulating ATP-dependent endosome acid-ificationrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 86 no 2 pp 539ndash543 1989

[54] T Suzuki S Futaki M Niwa S Tanaka K Ueda and YSugiura ldquoPossible existence of common internalization mecha-nisms among arginine-rich peptidesrdquo The Journal of BiologicalChemistry vol 277 no 4 pp 2437ndash2443 2002

[55] D A Richards S O Rizzoli and W J Betz ldquoEffects ofwortmannin and latrunculin A on slow endocytosis at the frogneuromuscular junctionrdquo Journal of Physiology vol 557 no 1pp 77ndash91 2004

[56] M Stahlhut and B van Deurs ldquoIdentification of filaminas a novel ligand for caveolin-1 evidence for the organiza-tion of caveolin-1-associated membrane domains by the actincytoskeletonrdquoMolecular Biology of the Cell vol 11 no 1 pp 325ndash337 2000

[57] T A Gottlieb I E Ivanov M Adesnik and D D SabatinildquoActin microfilaments play a critical role in endocytosis at theapical but not the basolateral surface of polarized epithelialcellsrdquo Journal of Cell Biology vol 120 no 3 pp 695ndash710 1993

[58] M R JackmanW Shurety J A Ellis and J P Luzio ldquoInhibitionof apical but not basolateral endocytosis of ricin and folate inCaco-2 cells by cytochalasin Drdquo Journal of Cell Science vol 107part 9 pp 2547ndash2556 1994


[59] J E Schnitzer J Liu and P Oh ldquoEndothelial caveolae havethe molecular transport machinery for vesicle budding dock-ing and fusion including VAMP NSF SNAP annexins andGTPasesrdquo The Journal of Biological Chemistry vol 270 no 24pp 14399ndash14404 1995

[60] L Rodriguez C J Stirling and P G Woodman ldquoMultiple N-ethylmaleimide-sensitive components are required for endoso-mal vesicle fusionrdquoMolecular Biology of the Cell vol 5 no 7 pp773ndash783 1994

[61] J E Schnitzer J Allard and P Oh ldquoNEM inhibits transcytosisendocytosis and capillary permeability implication of caveolaefusion in endotheliardquo American Journal of PhysiologymdashHeartand Circulatory Physiology vol 268 no 1 part 2 pp H48ndashH551995

[62] A Subtil I Gaidarov K Kobylarz M A Lampson J H Keenand T EMcgraw ldquoAcute cholesterol depletion inhibits clathrin-coated pit buddingrdquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 12 pp 6775ndash6780 1999

[63] J E Schnitzer P Oh E Pinney and J Allard ldquoFilipin-sensitive caveolae-mediated transport in endothelium reducedtranscytosis scavenger endocytosis and capillary permeabilityof select macromoleculesrdquo Journal of Cell Biology vol 127 no 5pp 1217ndash1232 1994

[64] N ArakiM T Johnson and J A Swanson ldquoA role for phospho-inositide 3-kinase in the completion of macropinocytosis andphagocytosis by macrophagesrdquo Journal of Cell Biology vol 135no 5 pp 1249ndash1260 1996

[65] B R Liu J F Li S W Lu et al ldquoCellular internalizationof quantum dots noncovalently conjugated with arginine-richcell-penetrating peptidesrdquo Journal of Nanoscience and Nan-otechnology vol 10 no 10 pp 6534ndash6543 2010

[66] M M Fretz G A Koning E Mastrobattista W Jiskoot andG Storm ldquoOVCAR-3 cells internalize TAT-peptide modifiedliposomes by endocytosisrdquo Biochimica et Biophysica ActamdashBiomembranes vol 1665 no 1-2 pp 48ndash56 2004

[67] A Gonzalez-Noriega J H Grubb V Talkad and W SSly ldquoChloroquine inhibits lysosomal enzyme pinocytosis andenhances lysosomal enzyme secretion by impairing receptorrecyclingrdquo Journal of Cell Biology vol 85 no 3 pp 839ndash8521980

[68] M A Wolfert and L W Seymour ldquoChloroquine and amphi-pathic peptide helices show synergistic transfection in vitrordquoGene Therapy vol 5 no 3 pp 409ndash414 1998

[69] P DrsquoArcy Hart and M R Young ldquoAmmonium chloride aninhibitor of phagosome-lysosome fusion in macrophages con-currently induces phagosome-endosome fusion and opens anovel pathway studies of a pathogenic mycobacterium and anonpathogenic yeastrdquo Journal of Experimental Medicine vol174 no 4 pp 881ndash889 1991

[70] H Michiue K Tomizawa F-Y Wei et al ldquoThe NH2terminus

of influenza virus hemagglutinin-2 subunit peptides enhancesthe antitumor potency of polyarginine-mediated p53 proteintransductionrdquoThe Journal of Biological Chemistry vol 280 no9 pp 8285ndash8289 2005

[71] J S Wadia R V Stan and S F Dowdy ldquoTransducible TAT-HAfusogenic peptide enhances escape of TAT-fusion proteins afterlipid raft macropinocytosisrdquo Nature Medicine vol 10 no 3 pp310ndash315 2004

[72] T Yoshikawa T Sugita Y Mukai et al ldquoOrganelle-targeteddelivery of biological macromolecules using the protein trans-duction domain potential applications for Peptide aptamer

delivery into the nucleusrdquo Journal ofMolecular Biology vol 380no 5 pp 777ndash782 2008

[73] A B Gustafsson M R Sayen S D Williams M T Crow andR A Gottlieb ldquoTAT protein transduction into isolated per-fused hearts TAT-apoptosis repressor with caspase recruitmentdomain is cardioprotectiverdquoCirculation vol 106 no 6 pp 735ndash739 2002

[74] I Navarro-Quiroga J Gonzalez-Barrios F Barron-Moreno VGonzalez-Bernal D B Martinez-Arguelles and D Martinez-Fong ldquoImproved neurotensin-vector-mediated gene transfer bythe coupling of hemagglutinin HA2 fusogenic peptide and Vp1SV40 nuclear localization signalrdquoMolecular Brain Research vol105 no 1-2 pp 86ndash97 2002

[75] S H Chin Lee R Jefferies PWatt et al ldquoIn vitro analysis of theTAT protein transduction domain as a drug delivery vehicle inprotozoan parasitesrdquo Experimental Parasitology vol 118 no 3pp 303ndash307 2008

[76] T Sugita T Yoshikawa Y Mukai et al ldquoImproved cytosolictranslocation and tumor-killing activity of Tat-shepherdin con-jugates mediated by co-treatment with Tat-fused endosome-disruptive HA2 peptiderdquo Biochemical and Biophysical ResearchCommunications vol 363 no 4 pp 1027ndash1032 2007

[77] S Oliveira I van Rooy O Kranenburg G Storm and RM Schiffelers ldquoFusogenic peptides enhance endosomal escapeimproving siRNA-induced silencing of oncogenesrdquo Interna-tional Journal of Pharmaceutics vol 331 no 2 pp 211ndash214 2007

[78] J S Liou B R Liu A L Martin Y W Huang H J Chiangand H J Lee ldquoProtein transduction in human cells is enhancedby cell-penetrating peptides fused with an endosomolytic HA2sequencerdquo Peptides vol 37 no 2 pp 273ndash284 2012

[79] C Plank B Oberhauser K Mechtler C Koch and E WagnerldquoThe influence of endosome-disruptive peptides on gene trans-fer using synthetic virus-like gene transfer systemsrdquoThe Journalof Biological Chemistry vol 269 no 17 pp 12918ndash12924 1994

[80] I Neundorf R Rennert J Hoyer et al ldquoFusion of a shortHA2-derived peptide sequence to cell-penetrating peptidesimproves cytosolic uptake but enhances cytotoxic activityrdquoPharmaceuticals vol 2 no 2 pp 49ndash65 2009

[81] S L Lo and S Wang ldquoAn endosomolytic Tat peptide producedby incorporation of histidine and cysteine residues as a nonviralvector for DNA transfectionrdquo Biomaterials vol 29 no 15 pp2408ndash2414 2008

[82] K Takayama I Nakase H Michiue et al ldquoEnhanced intracel-lular delivery using arginine-rich peptides by the addition ofpenetration accelerating sequences (Pas)rdquo Journal of ControlledRelease vol 138 no 2 pp 128ndash133 2009

[83] S Kobayashi I Nakase N Kawabata et al ldquoCytosolic targetingof macromolecules using a pH-dependent fusogenic peptide incombination with cationic liposomesrdquo Bioconjugate Chemistryvol 20 no 5 pp 953ndash959 2009

[84] A Fire S Xu M K Montgomery S A Kostas S E Driver andC CMello ldquoPotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegansrdquo Nature vol 391 no6669 pp 806ndash811 1998

[85] L Peters andGMeister ldquoArgonaute proteinsmediators of RNAsilencingrdquoMolecular Cell vol 26 no 5 pp 611ndash623 2007

[86] C Wostenberg J W Lary D Sahu et al ldquoThe role of humanDicer-dsRBD in processing small regulatory RNAsrdquo PLoSONEvol 7 no 12 Article ID e51829 2012

[87] A de Fougerolles H-P Vornlocher J Maraganore and JLieberman ldquoInterfering with disease a progress report on


siRNA-based therapeuticsrdquoNature ReviewsDrugDiscovery vol6 no 6 pp 443ndash453 2007

[88] R Kanasty J R Dorkin A Vegas and D Anderson ldquoDeliverymaterials for siRNA therapeuticsrdquo Nature Materials vol 12 no11 pp 967ndash977 2013

[89] N Unnamalai B G Kang and W S Lee ldquoCationic oligopep-tide-mediated delivery of dsRNA for post-transcriptional genesilencing in plant cellsrdquo FEBS Letters vol 566 no 1ndash3 pp 307ndash310 2004

[90] D Zeineddine E Papadimou K Chebli et al ldquoOct-34 dosedependently regulates specification of embryonic stem cellstoward a cardiac lineage and early heart developmentrdquo Devel-opmental Cell vol 11 no 4 pp 535ndash546 2006

[91] B R Meade and S F Dowdy ldquoEnhancing the cellular uptakeof siRNA duplexes following noncovalent packaging with pro-tein transduction domain peptidesrdquo Advanced Drug DeliveryReviews vol 60 no 4-5 pp 530ndash536 2008

[92] A Eguchi B R Meade Y-C Chang et al ldquoEfficient siRNAdelivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion proteinrdquo Nature Biotechnologyvol 27 no 6 pp 567ndash571 2009

[93] A Eguchi and S F Dowdy ldquoEfficient siRNA delivery by novelPTD-DRBD fusion proteinsrdquo Cell Cycle vol 9 no 3 pp 424ndash425 2010

[94] HMichiue A EguchiM Scadeng and S FDowdy ldquoInductionof in vivo synthetic lethal RNAi responses to treat glioblastomardquoCancer Biology ampTherapy vol 8 no 23 pp 2306ndash2313 2009

[95] B R Liu Y-W Huang and H-J Lee ldquoMechanistic studies ofintracellular delivery of proteins by cell-penetrating peptides incyanobacteriardquo BMCMicrobiology vol 13 no 1 article 57 2013

[96] S-W Lu J-W Hu B R Liu et al ldquoArginine-rich intracellulardelivery peptides synchronously deliver covalently and nonco-valently linked proteins into plant cellsrdquo Journal of Agriculturaland Food Chemistry vol 58 no 4 pp 2288ndash2294 2010

[97] J M Gump and S F Dowdy ldquoTAT transduction the molecularmechanism and therapeutic prospectsrdquo Trends in MolecularMedicine vol 13 no 10 pp 443ndash448 2007

[98] H-Y Wang J-X Chen Y-X Sun et al ldquoConstruction of cellpenetrating peptide vectorswithN-terminal stearylated nuclearlocalization signal for targeted delivery of DNA into the cellnucleirdquo Journal of Controlled Release vol 155 no 1 pp 26ndash332011

[99] K H Bremner L W Seymour A Logan and M L ReadldquoFactors influencing the ability of nuclear localization sequencepeptides to enhance nonviral gene delivery rdquo BioconjugateChemistry vol 15 no 1 pp 152ndash161 2004

[100] Q Ding L Zhao H Guo and A C Zheng ldquoThe nucleocyto-plasmic transport of viral proteinsrdquoVirologica Sinica vol 25 no2 pp 79ndash85 2010

[101] C-Y Lee J-F Li J-S Liou Y-C Charng Y-W Huang andH-J Lee ldquoA gene delivery system for human cells mediatedby both a cell-penetrating peptide and a piggyBac transposaserdquoBiomaterials vol 32 no 26 pp 6264ndash6276 2011

[102] O Shimomura F H Johnson and Y Saiga ldquoExtractionpurification and properties of aequorin a bioluminescent pro-tein from the luminous hydromedusan Aequoreardquo Journal ofcellular and comparative physiology vol 59 pp 223ndash239 1962

[103] R Y Tsien ldquoThe green fluorescent proteinrdquo Annual Review ofBiochemistry vol 67 pp 509ndash544 1998

[104] Q T Nguyen E S Olson T A Aguilera et al ldquoSurgerywith molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves

survivalrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 107 no 9 pp 4317ndash4322 2010

[105] A P Alivisatos W Gu and C Larabell ldquoQuantum dots ascellular probesrdquo Annual Review of Biomedical Engineering vol7 pp 55ndash76 2005

[106] X Michalet F F Pinaud L A Bentolila et al ldquoQuantum dotsfor live cells in vivo imaging and diagnosticsrdquo Science vol 307no 5709 pp 538ndash544 2005

[107] EPA Office of Research and Development NanomaterialResearch Strategy EPA Office of Research and DevelopmentWashington DC USA 2009

[108] F Chen and D Gerion ldquoFluorescent CdSeZnS nanocrystal-peptide conjugates for long-term nontoxic imaging and nucleartargeting in living cellsrdquo Nano Letters vol 4 no 10 pp 1827ndash1832 2004

[109] M A Reed J N Randall R J Aggarwal R J Matyi T MMoore and A E Wetsel ldquoObservation of discrete electronicstates in a zero-dimensional semiconductor nanostructurerdquoPhysical Review Letters vol 60 no 6 pp 535ndash537 1988

[110] L Josephson C-H Tung A Moore and R WeisslederldquoHigh-efficiency intracellular magnetic labeling with novelsuperparamagnetic-tat peptide conjugatesrdquo BioconjugateChemistry vol 10 no 2 pp 186ndash191 1999

[111] M Stroh J P Zimmer D G Duda et al ldquoQuantum dotsspectrally distinguish multiple species within the tumor milieuin vivordquo Nature Medicine vol 11 no 6 pp 678ndash682 2005

[112] Y-W Huang H-J Lee B R Liu H-J Chiang and C-HWu ldquoCellular internalization of quantum dotsrdquo Methods inMolecular Biology vol 991 pp 249ndash259 2013

[113] B R Liu Y-W Huang H-J Chiang and H-J Lee ldquoCell-penetrating peptide-functionalized quantum dots for intracel-lular deliveryrdquo Journal of Nanoscience and Nanotechnology vol10 no 12 pp 7897ndash7905 2010

[114] B R Liu H J Chiang Y W Huang M H Chan H HChen and H J Lee ldquoCellular internalization of quantumdots mediated by cell-penetrating peptidesrdquo PharmaceuticalNanotechnology vol 1 no 2 pp 151ndash161 2013

[115] E B Voura J K Jaiswal HMattoussi and SM Simon ldquoTrack-ing metastatic tumor cell extravasation with quantum dotnanocrystals and fluorescence emission-scanning microscopyrdquoNature Medicine vol 10 no 9 pp 993ndash998 2004

[116] J B Delehanty I L Medintz T Pons F M Brunel P E Daw-son and H Mattoussi ldquoSelf-assembled quantum dot-peptidebioconjugates for selective intracellular deliveryrdquo BioconjugateChemistry vol 17 no 4 pp 920ndash927 2006

[117] G Ruan A Agrawal A I Marcus and S Nie ldquoImaging andtracking of tat peptide-conjugated quantum dots in living cellsnew insights into nanoparticle uptake intracellular transportand vesicle sheddingrdquo Journal of the American Chemical Societyvol 129 no 47 pp 14759ndash14766 2007

[118] B R Liu M-H Chan H-H Chen S-Y Lo Y-W Huangand H-J Lee ldquoEffects of surface charge and particle sizeof cell-penetrating peptidenanoparticle complexes on cellularinternalizationrdquo inCell Membrane I Mandraccia andG SlavinEds pp 43ndash57 Nova Science 2013

[119] G Von Maltzahn D-H Min Y Zhang et al ldquoNanoparticleself-assembly directed by antagonistic kinase and phosphataseactivitiesrdquo Advanced Materials vol 19 no 21 pp 3579ndash35832007


[120] T-L Wee Y-W Mau C-Y Fang H-L Hsu C-C Han andH-C Chang ldquoPreparation and characterization of green flu-orescent nanodiamonds for biological applicationsrdquo Diamondand Related Materials vol 18 no 2-3 pp 567ndash573 2009

[121] Y-R Chang H-Y Lee K Chen et al ldquoMass productionand dynamic imaging of fluorescent nanodiamondsrdquo NatureNanotechnology vol 3 no 5 pp 284ndash288 2008

[122] M Reuter C Schwieger A Meister G Karlsson and A BlumeldquoPoly-l-lysines and poly-l-arginines induce leakage of nega-tively charged phospholipid vesicles and translocate throughthe lipid bilayer upon electrostatic binding to the membranerdquoBiophysical Chemistry vol 144 no 1-2 pp 27ndash37 2009

[123] N Schmidt AMishra G H Lai andG C LWong ldquoArginine-rich cell-penetrating peptidesrdquo FEBS Letters vol 584 no 9 pp1806ndash1813 2010

[124] G Ter-Avetisyan G Tunnemann D Nowak et al ldquoCell entryof arginine-rich peptides is independent of endocytosisrdquo TheJournal of Biological Chemistry vol 284 no 6 pp 3370ndash33782009

[125] H D Herce A E Garcia J Litt et al ldquoArginine-rich peptidesdestabilize the plasma membrane consistent with a pore for-mation translocation mechanism of cell-penetrating peptidesrdquoBiophysical Journal vol 97 no 7 pp 1917ndash1925 2009

[126] G J Doherty and H T McMahon ldquoMechanisms of endocyto-sisrdquo Annual Review of Biochemistry vol 78 pp 857ndash902 2009

[127] A van den Berg and S F Dowdy ldquoProtein transduction domaindelivery of therapeutic macromoleculesrdquo Current Opinion inBiotechnology vol 22 no 6 pp 888ndash893 2011

[128] M C Shin J Zhang K A Min et al ldquoCell-penetratingpeptides achievements and challenges in application for cancertreatmentrdquo Journal of Biomedical Materials Research Part A vol102 no 2 pp 575ndash587 2014

[129] J M Gump R K June and S F Dowdy ldquoRevised roleof glycosaminoglycans in TAT protein transduction domain-mediated cellular transductionrdquoThe Journal of Biological Chem-istry vol 285 no 2 pp 1500ndash1507 2010

[130] J J Skehel and M D Waterfield ldquoStudies on the primarystructure of the influenza virus hemagglutininrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 72 no 1 pp 93ndash97 1975

[131] S AWharton S RMartin RWH Ruigrok J J Skehel andDC Wiley ldquoMembrane fusion by peptide analogues of influenzavirus haemagglutininrdquo Journal of General Virology vol 69 no8 pp 1847ndash1857 1988

[132] P Erbacher AC RocheMMonsigny andPMidoux ldquoPutativerole of chloroquine in gene transfer into a human hepatoma cellline by DNAlactosylated polylysine complexesrdquo ExperimentalCell Research vol 225 no 1 pp 186ndash194 1996

[133] I Mellman R Fuchs and A Helenius ldquoAcidification of theendocytic and exocytic pathwaysrdquo Annual Review of Biochem-istry vol 55 pp 663ndash700 1986

[134] I Nakase S Kobayashi and S Futaki ldquoEndosome-disruptivepeptides for improving cytosolic delivery of bioactive macro-moleculesrdquo Biopolymers vol 94 no 6 pp 763ndash770 2010

[135] S Yang D J Coles A Esposito D J Mitchell I Toth andR F Minchin ldquoCellular uptake of self-assembled cationicpeptide-DNA complexes multifunctional role of the enhancerchloroquinerdquo Journal of Controlled Release vol 135 no 2 pp159ndash165 2009

[136] J S Suk J Suh K Choy S K Lai J Fu and J HanesldquoGene delivery to differentiated neurotypic cells with RGD

and HIV Tat peptide functionalized polymeric nanoparticlesrdquoBiomaterials vol 27 no 29 pp 5143ndash5150 2006

[137] S R Sirsi R C Schray X Guan et al ldquoFunctionalized PEG-PEI copolymers complexed to exon-skipping oligonucleotidesimprove dystrophin expression in mdx micerdquo Human GeneTherapy vol 19 no 8 pp 795ndash806 2008

[138] E Kleemann M Neu N Jekel et al ldquoNano-carriers forDNA delivery to the lung based upon a TAT-derived peptidecovalently coupled to PEG-PEIrdquo Journal of Controlled Releasevol 109 no 1ndash3 pp 299ndash316 2005

[139] J-P Behr B Demeneix J-P Loeffler and J Perez-Mutul ldquoEffi-cient gene transfer into mammalian primary endocrine cellswith lipopolyamine-coated DNArdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 86 no18 pp 6982ndash6986 1989

[140] W T Godbey K KWu and A GMikos ldquoPoly(ethylenimine)-mediated gene delivery affects endothelial cell function andviabilityrdquo Biomaterials vol 22 no 5 pp 471ndash480 2001

[141] W T Godbey K K Wu and A G Mikos ldquoSize mattersmolecular weight affects the efficiency of poly(ethylenimine)as a gene delivery vehiclerdquo Journal of Biomedical MaterialsResearch vol 45 no 3 pp 268ndash275 1999

[142] W T Godbey K K Wu and A G Mikos ldquoTracking theintracellular path of poly(ethylenimine)DNA complexes forgene deliveryrdquo Proceedings of National Academy of Sciences ofthe United States of America vol 96 no 9 pp 5177ndash5181 1999

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


ProteinDNARNA

Cargo

Into the nucleus

To the cytoplasm

Cell-penetrating peptides

(cell membrane as a selective barrier)

+ + ++

++++

+ + ++

++++

+ + ++

++++

Cell-penetrating

peptides

CPP-tagged

cargo

Figure 1 Cell-penetrating peptides as a tool to deliver biologically active molecules

Table 1 A variety of cell-penetrating peptides mentioned in this paper

CPP Amino acid sequence ReferencesViral or natural CPPs

HIV Tat YGRKKRRQRRR [31ndash35]HIV Rev TRQARRNRRRRWRERQR [35]FHV coat RRRRNRTRRNRRRVR [35]HSV-1 protein VP22 DAATATRGRSAASRPTERPRAPARSASRPRRPVD [34]Penetratin RQIKIWFQNRRMKWKK [31 33 36 37]EB1 (penetratin analog) LIRLWSHLIHIWFQNRRLKWKKK [31]MPG GALFLGFLGAAGSTMGAWSQPKKKRKV [31 34 38ndash40]

PolyargininesPR9 FFLIPKGRRRRRRRRR [41ndash44]SR9 RRRRRRRRR [31 34 41 42 45]IR9 GLFEAIEGFIENGWEGMIDGWYGRRRRRRRRR [46ndash48]HR9 CHHHHHRRRRRRRRRHHHHHC [41ndash44 46]

Engineered CPPsTransportan CLIKKALAALAKLNIKLLYGASNLTWG [31 36]CADY GLWRALWRLLRSLWRLLWRA [31 49]C6 RLLRLLLRLWRRLLRLLR [13]C6M1 RLWRLLWRLWRRLWRLLR [13]PF20 (and variants see [9]) LLKLLKKLLKLLKKLLKLL [9]NAP KALKLKLALALLAKLKLA [9]Steryl-NAP Stearyl-KALKLKLALALLAKLKLA [9]POD GGG[ARKKAAKA]4 [25]


O

OH OO

O

O

O

OSHO

HO

N

O

OO

OO

O

OHS

N

O

O

O

O OH

SN

(2times)

n

n

O

NH

NH

HN NH2

O

O O

NH

NH

HN NH2



++

(a)

O

O

O

O

OH

N

N NN

N

N N

C

C

HNH

HNHN

NHNH

NH2

NH2

nn

CH3

CH3

CH3

CH3

CH3

CH3

+


+

CPP-linked cargo

(b)

++NH2

NH2

NH2

O

O

O

OO

OO

O

O

O

O

O

O

OO

O

O

HO

HO

HS

N

N

N

N

NH

S

S

S

OH


(c)

+O O

O

O

O O

O

O

O O

O

O

O

OOH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NHNH

NH

NH

H2N NH

NH

H2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2NH2

O O

O

O

O O

O

O

O O

O

O

OH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NH

NH

NH

NH

H2N NH

NHH2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2

NH2



(d)















O

O

O

O

OO

O

O

O

O

O

O

OH

OH

NHNH

NHNH

NHNH

N HN H

N HN H

N H

H2 N

H2 N

H2 N

H2 N

H2 N

H2 N

NH N

HN

HN

HN

H NH

NH

NH

NH

NH

NH

NH

NH

2

NH

2N

H2

shRNA

CPP (HIV Tat)

(a)

Dicer (dsRBD)

(b)

RISC

siRNA-boundRISC

mRNA

(c) (d) (e)








P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe


PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)















Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O

OH OO

O

O

O

OSHO

HO

N

O

OO

OO

O

OHS

N

O

O

O

O OH

SN

(2times)

n

n

O

NH

NH

HN NH2

O

O O

NH

NH

HN NH2



++

(a)

O

O

O

O

OH

N

N NN

N

N N

C

C

HNH

HNHN

NHNH

NH2

NH2

nn

CH3

CH3

CH3

CH3

CH3

CH3

+


+

CPP-linked cargo

(b)

++NH2

NH2

NH2

O

O

O

OO

OO

O

O

O

O

O

O

OO

O

O

HO

HO

HS

N

N

N

N

NH

S

S

S

OH


(c)

+O O

O

O

O O

O

O

O O

O

O

O

OOH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NHNH

NH

NH

H2N NH

NH

H2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2NH2

O O

O

O

O O

O

O

O O

O

O

OH

OHNH

NH

NH

NH

NH

NH

NH N

HNH

NH

NH

H2N

H2N

NH

NH

NH

NH

H2N NH

NHH2N NH

NH

H2N NH

NH

H2NNH

NH

NH2

NH2

NH2



(d)















O

O

O

O

OO

O

O

O

O

O

O

OH

OH

NHNH

NHNH

NHNH

N HN H

N HN H

N H

H2 N

H2 N

H2 N

H2 N

H2 N

H2 N

NH N

HN

HN

HN

H NH

NH

NH

NH

NH

NH

NH

NH

2

NH

2N

H2

shRNA

CPP (HIV Tat)

(a)

Dicer (dsRBD)

(b)

RISC

siRNA-boundRISC

mRNA

(c) (d) (e)








P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe


PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)















Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














O

O

O

O

OO

O

O

O

O

O

O

OH

OH

NHNH

NHNH

NHNH

N HN H

N HN H

N H

H2 N

H2 N

H2 N

H2 N

H2 N

H2 N

NH N

HN

HN

HN

H NH

NH

NH

NH

NH

NH

NH

NH

2

NH

2N

H2

shRNA

CPP (HIV Tat)

(a)

Dicer (dsRBD)

(b)

RISC

siRNA-boundRISC

mRNA

(c) (d) (e)








P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe


PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)















Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O

O

O

O

OO

O

O

O

O

O

O

OH

OH

NHNH

NHNH

NHNH

N HN H

N HN H

N H

H2 N

H2 N

H2 N

H2 N

H2 N

H2 N

NH N

HN

HN

HN

H NH

NH

NH

NH

NH

NH

NH

NH

2

NH

2N

H2

shRNA

CPP (HIV Tat)

(a)

Dicer (dsRBD)

(b)

RISC

siRNA-boundRISC

mRNA

(c) (d) (e)








P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe


PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)















Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




P

P

P

PPPP

P

P

P

P

P

P

PP P P

P

P

PP

P

P

P

PP P P

P

P

P

P

P

P

PPPP

P

P

OOO O O

OH

NH+4

HN

POminus

OO

O O OOH

NH+4HN

POminus

O

OO

OO

OH

NH+4

HN

P Ominus

OO

OO

O

OH N

H+ 4

HN

P Ominus

OO

OO

OO

H NH

+ 4

HN

P Ominus

O

OO

OO

O

H

NH+4

HNP O

minus

O

OO

OO

OH

NH+4

HNPOminus

OO O

OO

OH

NH+4

HNPOminus

O OOOO

O H

NH+4

HNPOminus

O

OO

OOOH

NH+4

HN

POminus

OO

OO

OO

H

NH+

4

HNPOminus

O

OO

OO

O

H

NH+4

HN

POminus

OO

OO

OO H

NH +4 HN

PO minus

OOO

OO

OO

OH

NH+4

HN POminus

OH

OO

OO

O

OO

OHN

H+4

H N

POminus

OH

O OO

O OOO

OH

NH+4

HNPOminus

OHO

O

O

OO

OO

OH

NH+4

HN

P Ominus

OH

OO

O

OO

OO OH

NH+4

HN

POminus

OH

CdSe CdSe


PEG-2-PE

(OCH2 CH

2 )45 OCH

3

(OCH2 CH

2 )45 OCH

3

(OCH2CH2)45

(OCH2CH

2 )45OCH3

(OCH2CH2

)45OCH3

(OCH 2CH 2

) 45OCH 3

(OCH 2

CH 2) 45

OCH 3

(OCH

2CH

2) 45O

CH3

(OCH

2CH

2) 4

5O

CH3

(OCH

2CH2)45

OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH2)45OCH3

(OCH2CH

2)45OCH

3

OO

OO

O

OHN

H+4

H N

POminus

(OCH

2 CH2 )

45 O

CH3

OO

OO

OO

HNH

+4

H N

POminus

(OCH

2CH2)45O

CH3

(OCH2CH2)45

(OCH

2 CH2 )45

(OCH2CH2

)45

(OCH

2CH

2) 45

(a)

(b)















Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials













Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Cellular uptake

Endocytosis


Delivery

568-transferrin


568-transferrin

lowastDelivery

FM4-64

Energyinhibition

Clathrininhibitors



568

Caveolaeinhibitors


568-CTB

Delivery

FM4-64

Macropinocytosis

Delivery system

neutral dextran


Delivery system

neutral dextran






EEA1-Alexa 568 or

system + Alexa


Delivery system +

Delivery system +

Delivery system +

system +

+ Texas red-

+ Texas red-

Lamp-1-Alexa 568

Lamp-1-Alexa 568









Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Table3Ph

armacologicalandph

ysicalinhibitorsof

cellu

laru

ptakep

rocess

Inhibitor

Mechanism

Working

cond

ition

Ref

Lowtemperature


entend

ocytosis

4∘C

[17]

Hypertonicm

edium


endo

cytosis

(Diss

ociatecla

thrin

lattice)

02ndash045

Msucrose

[1750]

Potassium

depletion


endo

cytosis

(Diss

ociatecla

thrin

lattice)

50DMEM

w1mM

ouabain

[50]

Fusic

occin

Endo

cytosis

inhibitor

(H+ -AT

Pase

activ

ator)

10120583M

[51]

Valin

omycinnigericin


entend

ocytosis

(Na+K

+AT

Pase

mod

ulatorK

+selectiveion

opho

re)

2120583M

[5253]

Sodium

azidesodium

fluorideantim

ycin


entend

ocytosis


(015

15

mM2120583gmL)

[17]

Okadaicacid

Endo

cytosis

andautoph

agyinhibitor

15120583M

[426]

Nocod

azole


endo

cytosis

(Cause

microtubu

ledepo

lymerization)

10ndash25120583

M[1754]

Latrun

culin

AInhibitm

icropino

cytosis

(F-actin

depo


15120583M

[55]

Cytochalasin

D(C

ytD)

Inhibitm

acropino

cytosis

clathrin-a


endo

cytosis

(InhibitF

-actin

rearrang

ement)

1ndash30120583M

[56ndash

58]


)Inhibitclathrin-a


endo

cytosis

(Inhibitend

osom

alfusio

nenergy

metabolism

)01ndash3m

M[59ndash

61]

Methyl-120573

-cyclodetritrin

(M120573CD

)Inhibitclathrin-a


endo

cytosis

(Depleteor

sequ

esterc

holeste

rol)

2mM

[62]

Filip


entend

ocytosis

(Inhibitlipid

raft

cholesterolbinding

)5120583

gmL

[63]

Nystatin


entend

ocytosis

(Inhibitlipid

raft

sequ

esterc

holeste

rol)

5120583gmL

[63]

Wortm

annin


cytosis

(InhibitP

I-3kinase)

100n

M[64]

5-(N

-Ethyl-N

-isop

ropyl)-am

ilorid

e(EIPA

)Inhibitm

icropino

cytosis

(InhibitN

a+H

+exchanger)

100120583

M[65]

Dextran

sulfate

Inhibitb

inding

ofCP

Pto

cellmem

brane

5120583gmL

[66]

DMSO

ethanol

Dire

ctmem

branetranslocatio

nenhancer

101

[43]

Oleicacidlimon

enePE

GAlldirectmem

branetranslocatio

nenhancers

5[2843]

Chloroqu

ine

Lysosomotropica

gent

(Cause

vesic

ular

lysis

)100amp25120583M

[6768]

Ammon

ium

chlorid

eInhibitthe

fusio

nof

lysosomes

with

endo

somes

10mM

[6769]


Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Clathrin inhibition

0

25

50

75

100

125M

ean

fluor

esce

nce (

o

f con

trol

)


lowast

lowast

(a)


0

25

50

75

100

125

Control

lowastlowast

Mea

n flu

ores

cenc

e (

of c

ontr

ol)

FilipinNystatin

(b)

siRNA knockdown

0

25

50

75

100

125


Mea

n flu

ores

cenc

e (

of c

ontr

ol)

(c)




5 Conclusions


Abbreviations



Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Table4HA2analoguesa

ndothersequencev

ariatio

nsin

CPPs

toovercomelysosom

alentrapment

Nam

eAminoacid

sequ

ence

aa

Structuralorder

Cargo

Purity

Efficiency

Ref

HA2analogues

GLF

EAIEGFIEN

GWEG

MID

GWYG

23pH

A2-p53-R9

p53

NA

5x[70]

GDIM

GEW

GNEIFG

AIAGFL

G20

pTat-C

repTat-H

A2

Nocargo

NA

2ndash6x

[71]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

PM10

NA

05ndash1x

[72]

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

pTat-H

A-hA

RCor

pTat-H

A-120573-gal

120573-galA

RCNA

NA

[73]

GLF

EAIAEF

IEGGWEG

LIEG

CAKK

K25

HA2-NT

(NT=neurotensin

)NT

gt90

22

[74]

GLF

GAIAGFIEN

GQWGMID

G20

HA2-Tat

FPNA

NA

[75]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23HA2-Tat

Shepherdin

gt90

3x[76]

GLF

EAIEGFIEN

GWEG

MID

GWYG

CGLF

EAIEGFIEN

GWEG

MID

GWYG

C(dim

eric)

48EG

FRsiR

NALFdiNF-7

EGFR

siRNA

NA

gt2x

[77]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23CP

P-HA2

mCh

erry

NA

80ndash9

0x[78]

GLF

EAIEGFIEN

GWEG

MID

GWYG

23GALA

-INF

Luciferase

NAgt1000x

[79]

GLL

EALA

ELLE

11HA2-CP

PFP

gt95

2x[80]

Sequ

ence

varia

tions

inCP

Ps10H

CHHHHHRK

KRRQ

RRRR

HHHHHC

22C-

5H-Tat-5H-C

Luciferase

gt98gt7000x

[81]

HR9

CHHHHHRR

RRRR

RRRH

HHHHC

21C-

5H-R9-5H

-CDNAs

FPsQ

Ds

87

20x

[43]

PasR8

FFLIPK

GRR

RRRR

RRGC

17Pas-CP

PAlexa

488

NA

18x

[82]

PR9

FFLIPK

GRR

RRRR

RRR

16Pas-CP

PDNAsFP

sQDs

99

475x

[44]

GALA

WEA

ALA

EALA

EALA

EHLA

EALA

EALE

ALA

A30

GALA

-EGFP

FITC

NA

68x

[83]

NA=

notavailable





References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







References































































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






































































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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