ripl peptide (iplvvplrrrrrrrrc)-conjugated liposomes for enhanced intracellular drug delivery to...

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

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European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier .com/locate /e jpb

Research paper

RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhancedintracellular drug delivery to hepsin-expressing cancer cells

http://dx.doi.org/10.1016/j.ejpb.2014.03.0160939-6411/� 2014 Elsevier B.V. All rights reserved.

Abbreviations: Hpn, hepsin; IPL, IPLVVPLC, Hepsin specific sequence; R8,RRRRRRRRC, Octa arginines sequence; RIPL, IPLVVPLRRRRRRRRC, Hepsin specificand cell penetrating sequence; RIPL-Lipo, RIPL peptide-conjugated liposomes; CL,conventional liposomes; CLSM, confocal laser scanning microscopy; CPHP, cellpenetrating homing peptide; CPP, cell penetrating peptide; D.W., distilled water;DSPE-PEG2000-mal, distearoyl phosphatidyl ethanolamine-polyethylene glycol-maleimide; DTNB, 5,50-dithio-bis(2-nitrobenzoic acid); EE, entrapment efficiency;FITC-dextran, fluorescein dextran isothiocyanate; Fmoc SPPS, 9-fluorenylmethyl-oxycarbonyl solid phase peptide synthesis; MFI, mean fluorescence intensity; MMP,matrix metalloproteases; MW, molecular weight; pArg, polyarginine; PBS, phos-phate buffer saline; PC, phosphatidylcholine; PDI, polydispersity index; RIPL-FITC,fluorescence-tagged RIPL peptide; uPa, Urokinase plasminogen activator.⇑ Corresponding author. College of Pharmacy, Chung-Ang University, 221

Heuksuk-dong, Dongjak-gu, Seoul 156-756, Republic of Korea. Tel.: +82 2 8205609; fax: +82 2 826 3781.

E-mail address: [email protected] (Y.W. Choi).

Please cite this article in press as: M.H. Kang et al., RIPL peptide (IPLVVPLRRRRRRRRC)-conjugated liposomes for enhanced intracellular drug delihepsin-expressing cancer cells, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.03.016

Min Hyung Kang a, Min Jung Park a, Hyun Joon Yoo a, Kwon Yie hyuk a, Sang Gon Lee a, Sung Rae Kim a,Dong Woo Yeom a, Myung Joo Kang b, Young Wook Choi a,⇑a College of Pharmacy, Chung-Ang University, Seoul, Republic of Koreab College of Pharmacy, Dankook University, Cheonan-Si, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 December 2013Accepted in revised form 11 March 2014Available online xxxx

Keywords:LiposomeCell penetrating/homing peptideIntracellular deliveryPolyarginineIPLTargetingHepsin

Background: To facilitate selective drug delivery to hepsin (Hpn)-expressing cancer cells, the RIPL peptide(IPLVVPLRRRRRRRRC; 16mer; 2.1 kDa) was synthesized as a novel cell penetrating/homing peptide(CPHP) and conjugated to a liposomal carrier.Methods: RIPL peptide-conjugated liposomes (RIPL-Lipo) were prepared by conjugating RIPL peptides tomaleimide-derivatized liposomal vesicles via the thiol-maleimide reaction. Vesicle size and zeta potentialwere examined using a Zetasizer. Intracellular uptake specificity of the RIPL peptide, or RIPL-Lipo, wasassessed by measuring mean fluorescence intensity (MFI) after treatment with a fluorescent markerin various cell lines: SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaT for Hpn(�).FITC-dextran was used as a model compound. Selective translocational behavior of RIPL-Lipo to LNCaPcells was visualized by fluorescence microscopy and confocal laser scanning microscopy. Cytotoxicitiesof the RIPL peptide and RIPL-Lipo were evaluated by WST-1 assay.Results: RIPL peptides exhibited significant Hpn-selectivity. RIPL-Lipo systems were of positively chargednanodispersion (165 nm in average; 6–24 mV depending on RIPL conjugation ratio). RIPL-Lipo with theconjugation of 2300 peptide molecules revealed the greatest MFI in all cell lines tested. Cellular uptakeof RIPL-Lipo increased by 20- to 70-fold in Hpn(+) cells, and 5- to 7-fold in Hpn(�) cells, compared tothe uptake of FITC-dextran. Cytosolic internalization of RIPL-Lipo was time-dependent: bound instantly;internalized within 30 min; distributed throughout the cytoplasm after 1 h. Cytotoxicities of RIPL peptide(up to 50 lM) and RIPL-Lipo (up to 10%) were minor (cell viability >90%) in LNCaP and HaCaT cells.Conclusion: By employing a novel CPHP, the RIPL-Lipo system was successfully developed forHpn-specific drug delivery.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

The poor selectivity of chemotherapeutic agents for specifictarget sites or cells is one of the major obstacles in chemotherapy[1–3]. Surface-functionalized delivery systems including liposomalnanocarrier have been widely applied to target specific cancer cellsand efficiently transfer a cargo to cytoplasm through diverse mech-anisms [4–8]. Active targeting can be accomplished by molecularrecognition of the diseased cells by various specific moleculesoverexpressed at the site of disease via ligand-receptors orantigen–antibody interactions [9], e.g., folate receptor as a targetprotein to distinguish ovarian and cervical cancer cells fromnormal cells [8,10]. Prostate-specific membrane antigen (PSMA)has been widely used for prostate cancer targeting [11–13],

very to

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2 M.H. Kang et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

however, hepsin has recently been addressed as a biomarker todetect early prostate cancer [14].

Hepsin (Hpn) belongs to the hepsin/TMPRSS/enteropeptidasesubfamily within the class of type II transmembrane serine prote-ases [15]. This extracellular protease has been detected at signifi-cant levels in many different types of mammalian cells, includinghuman hepatoma cells (HepG2), peripheral nerve cells (PC12),and prostate cancer cells (LNCaP) [16]. Notably, Hpn is consistentlyup-regulated in cancer cells, but is either absent or expressed atvery low levels in normal prostate and benign prostatic hypertro-phy [14]. Based on this difference, IPLVVPL (IPL) has been intro-duced as an Hpn-specific peptide possessing both high affinityand high selectivity for Hpn.

The employment of cell penetrating peptides (CPPs) is a usefulapproach for enhanced cytosolic drug delivery [17]. Among CPPs,polyarginine (pArg) peptides have been widely used in studiesaiming to enhance the cellular uptake of various drugs in vitroand in vivo. For example, the octaArg (R8)-doxorubicin conjugateand R8 functionalized liposomes effectively suppressed tumor pro-liferation without any significant weight loss in mice [18–20], andpArg-conjugated cationic liposomes exhibited effective intracellu-lar delivery of small interfering RNA [21,22]. Nevertheless, theapplication of CPPs for enhanced intracellular drug delivery hasbeen limited, because the majority of known CPPs are non-selec-tive for specific cells or tissue. Many attempts to assign selectivityto CPPs by the conjugation of a homing peptide have been made.For instance, by the conjugation of PEGA (CPGPEGAGC; a breastvasculature-specific peptide) with pVEC (LLIILRRRIRKQAHAHSK; aCPP), PEGA-pVEC showed both cell-specific and cell-penetratingproperties [23]. Attachment of DV3 (LGASWHRPDKG; CXC chemo-kine receptor 4 ligand) to TAT peptide (GRKKRRQRRRPQ; a CPP)resulted in an enhancement of tumor cell killing compared withtreatment with non-targeted parenteral peptides [24]. These chi-meric peptides are called ‘‘cell penetrating homing peptides’’(CPHP) and have been successfully used for the targeted deliveryof drug molecules, nanoparticles, and liposomes to various tissuesincluding tumors [25].

Therefore, in the present study, RIPL peptide(IPLVVPLRRRRRRRRC) was synthesized as a novel CPHP and conju-gated to a liposomal carrier to facilitate selective drug delivery.Conformational and physical properties of different types of RIPLpeptide-conjugated liposomes were characterized in terms of theextent of surface modification, vesicle size and zeta potential.Using the various cell lines including LNCaP, SK-OV-3, and MCF-7as Hpn-expressing cells and DU-145, PC-3, and HaCaT asHpn-non-expressing cells, intracellular uptake behaviors of RIPLpeptide-conjugated liposomes containing fluorescence-labeledmacromolecules as a model probe were observed by fluorescencemicroscopy, confocal laser scanning microscopy (CLSM), and flowcytometry. Cytotoxicities of the RIPL peptide and the peptide-conjugated liposomal carriers were also evaluated.

2. Materials and methods

2.1. Materials

Soya phosphatidylcholine (PC) and distearoyl phosphatidylethanolamine–polyethylene glycol–maleimide (DSPE-PEG2000-mal)were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Tween80, cystein hydrochloride anhydrous, 5,50-dithio-bis(2-nitrobenzoicacid) (DTNB) and fluorescein dextran isothiocyanate (FITC-dextran,4 kDa) were purchased from Sigma (St. Louis, MO, USA). RIPL pep-tides (IPLVVPLRRRRRRRRC, 16mer), octa-arginine (R8; RRRRRRRRC),and IPL (IPLVVPLC) were synthesized by Peptron Co. (Daejeon,Korea). The polyethersulfone ultrafiltration membrane was

Please cite this article in press as: M.H. Kang et al., RIPL peptide (IPLVVPLRRRRhepsin-expressing cancer cells, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.o

purchased from Millipore (Billerica, MA, USA). Phosphate buffer sal-ine (PBS; pH 7.4, 10x) and cell culture materials including RoswellPark Memorial Institute medium (RPMI) 1640 medium, fetal bovineserum, penicillin–streptomycin, and trypsin-EDTA (0.25%) wereobtained from Invitrogen (Carlsbad, CA, USA). NUNC CC2 chamberslides were purchased from Nalgene Nunc International (Rochester,NY, USA). Human prostate cancer cell lines (LNCaP, DU145 and PC3),an ovarian carcinoma cell line (SK-OV-3), a breast cancer cell line(MCF-7), and a human keratinocyte cell line (HaCaT) were pur-chased from the Korean Cell Line Bank (Seoul, Korea). All otherchemicals and reagents purchased from commercial sources wereof analytical or cell culture grade.

2.2. Synthesis of RIPL peptide

Peptides were synthesized by Fmoc SPPS (9-fluorenylmethyl-oxycarbonyl solid phase peptide synthesis) and purified by reversephase HPLC. Amino acid units were coupled one by one from theC-terminal using an automated peptide synthesizer (ASP48S, PeptronInc.). S-trityl-L-cysteine-2-chlorotrityl resin was used to attach thefirst amino acid of the C-terminal to a resin. All the amino acids usedin the peptide synthesis were those protected by trityl, t-butyloxy-carbonyl, t-butyl, and the like, whereby the N-terminal is protectedby Fmoc, and residues are all removed in acid. As a couplingreagent, 2-(1H-benzotriazol-1-y1)-1, 1, 3, 3-tetramethyluroniurnhexafluorophosphate/hydroxyl-benzotriazole/N-methylmorpholinewas used. An elution in preparative HPLC (Shimadzu, Kyoto, Japan)purification was carried out on a Vydac Everest C18 column(250 � 22 mm; 10 lm) with a water–acetonitrile linear gradient(3–40% v/v of acetonitrile) containing 0.1% (v/v) trifluoroacetic acid.Molecular weights of the purified peptide were confirmed using LC/MS (Agilent HP1100 series). The settings for the LC/MS, operated inthe electrospray positive ion mode, with a liquid chromatographyflow of 0.4 mL/min were as follows: drying gas flow, 8 L/min;nebulizer pressure, 35 psig; nebulizer temperature, 350 �C;capillary voltage, 4.0 kV.

2.3. Structure prediction and model construction of RIPL peptide

Secondary structure prediction was carried out to determine thestructural significance of targeting sequences using PSIPRED, whichis based on a dictionary of protein secondary structure (DSSP) [26].Three-dimensional models of the RIPL peptide were constructed byMODELLER for the selection of the best model with the highestconfidence score. The above-mentioned tools were accessedthrough the Local Meta-Threading Server (http://zhanglab.ccmb.med.umich.edu/LOMETS/) [27]. The structure of the complexbetween the RIPL peptide and Hpn is predicted and described byZDOCK, a protein-docking algorithm (http://zlab.umassmed.edu/zdock/) [28].

2.4. Preparation of RIPL-conjugated liposomes

The RIPL-conjugated liposomes (RIPL-Lipo) were prepared byconjugating RIPL peptides to maleimide-derivatized liposomalvesicles via a thiol-maleimide reaction as previously reported[29]. All liposomal vesicles were prepared by the thin film hydra-tion method. First, PC and Tween80 were dissolved at a molar ratioof 9:1 in chloroform–methanol mixture (1:1) in a round bottomflask. After adding DSPE-PEG2000-maleimide at 0.2–4.0% of the totalmolar concentration, the organic solvent was removed by rotaryvacuum evaporation at 35 �C, above the phase transition tempera-ture of the phospholipid and Tween80, and solvent traces wereremoved under nitrogen gas streaming. Thin lipid film was thenhydrated with 1 mL distilled water containing 10 mg/mL ofFITC-dextran. The total molar concentration of the liposomal

RRRRC)-conjugated liposomes for enhanced intracellular drug delivery torg/10.1016/j.ejpb.2014.03.016

http://zhanglab.ccmb.med.umich.edu/LOMETS/

http://zhanglab.ccmb.med.umich.edu/LOMETS/

http://zlab.umassmed.edu/zdock/

http://zlab.umassmed.edu/zdock/


M.H. Kang et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 3

constituents was 7.16 mM initially. Prepared liposomal solutionwas extruded on Mini-Extruder with 20 passes through a 200 nmpolyethersulfone membrane for homogenous size distributionand efficient entrapment. Finally, the RIPL peptide solution wasadded to the maleimide-derivatized liposomal solution andallowed to react for 12 h at room temperature. RIPL-Lipo werepurified from un-reacted RIPL peptide and un-entrapped FITC-dex-tran using a cellulose ester dialysis membrane (100 kDa MWCO)against distilled water for 48 h. After dialysis, liposomal stock solu-tions were prepared by replenishing the purified liposomes withdistilled water to 5 mL, finally resulted in total molar concentrationof the liposomal constituents as 1.432 mM. Based on the composi-tion ratio of conjugated RIPL peptide on liposomal surface as 0.1,0.35, 1.0, and 2.0 mole ratio, the liposomal stock solutions weredesignated as RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, andRIPL(2.0)-Lipo, respectively. For comparison, conventional lipo-somes (CL) were separately prepared with PC and Tween80 asdescribed above, excluding the addition of DSPE-PEG2000-malei-mide and RIPL peptide. And empty liposomes were separately pre-pared by the same procedure except for hydration with distilledwater under the absence of FITC-dextran.

2.5. Conformational characterization of RIPL-Lipo

The number of external maleimide groups and the conjugationrate of RIPL peptides on maleimide-derivatized liposomes werecalculated indirectly by determining the amount of unreacted cys-teine in an Ellman’s reaction [4]. To block the unreacted maleimideresidue, maleimide-derivatized liposomes were incubated at roomtemperature for 30 min with a three-fold molar amount of cysteinehydrochloride anhydrous. A known amount of DTNB (0.1 mg/mL)was added to react with the unreacted cysteine, leading to the for-mation of a cysteine-TNB (5-thio-2-nitrobenzoic acid) adduct tocause the concomitant release of an equivalent of free TNB. Theamount of liberated TNB was analyzed by HPLC to estimate theamount of cysteine used for adduct formation under the assump-tion that every external maleimide was blocked stoichiometricallyby a cysteine addition. The conjugation rate of RIPL peptides onmaleimide-derivatized liposomes was calculated as describedabove by determining unreacted maleimide of RIPL-Lipo after dial-ysis. The HPLC systems consisted of a pump (W2690/5, Waters,USA), UV detector (W2489, k = 412 nm, Waters, USA), a data sta-tion (Empower3, Waters, USA), and a C18 column (Shiseido, Japan)at the flow rate of 1.0 mL/min. The mobile phase was composed ofa mixture of methanol and 10 mM ammonium formate solution(5:95 v/v).

2.6. Size and zeta potential of RIPL-Lipo

Liposomal stock solutions (10 lL) were diluted to 800 lL of1 mM KCl solution and were examined for size distribution andPDI (polydispersity index) using a dynamic light scattering particlesize analyzer (Zetasizer Nano-ZS; Marlvern Instrument, Worcester-shire, UK) equipped with a 50 mV laser at a scattering angle of 90�.Zeta potential measurements were taken with disposable capillarycells and the M3-PALS measurement technology, built into theZetasizer system. All measurements were carried out in triplicateunder ambient conditions.

2.7. Determination of FITC-dextran and encapsulation efficiency

The concentration of FITC-dextran in liposomal stock solutionswas measured by HPLC with fluorescence detection at an excita-tion wavelength of 485 nm and emission wavelength of 520 nm.Chromatography was carried out on a C18 column (Shiseido, Japan)with methanol–10 mM phosphate buffer (5:95 v/v) at the flow rate


of 1.0 mL/min. The FITC-dextran peak was separated with a reten-tion time of 1.3 min. Prior to the assay, liposomal samples weresubjected to pre-treatment procedure by the addition of 2% tri-ton-X and sonication to break up the vesicle entirely. Separately,entrapment efficiency (EE) of FITC-dextran was determined bycentrifugation. Aliquot of unpurified solution of RIPL-Lipo wasdiluted 4000 times with distilled water and centrifuged (12,000g,30 min), and the amount of FITC-dextran in the supernatant([FITC]supernatant) was determined by HPLC. EE was calculated bythe following equation:

EEð%Þ ¼½FITC�initial � ½FITC�supernatant

½FITC�initial� 100

where [FITC]initial is the amount of FITC-dextran initially added inthe film hydration process.

2.8. Cell culture

All cell lines used in this study, including human prostate can-cer cells (LNCaP, DU145 and PC3), ovarian carcinoma cells (SK-OV-3), breast cancer cells (MCF-7), and human keratinocyte cells(HaCaT), were grown in RPMI 1640 medium supplemented with10% (v/v) heat-inactivated fetal bovine serum, 100 units/ml peni-cillin G, and 100 lg/mL streptomycin. Cultures were maintainedat 37 �C in a humidified 5% CO2 incubator. The cells were subcul-tured every 2–4 days and were used for experiments at passages5–20.

2.9. In vitro cell uptake specificity of RIPL peptide

Cell specificity and cell penetration efficiency of the synthesizedpeptides were estimated by determining the mean fluorescenceintensity (MFI) of FITC or FITC-dextran via flow cytometry (FAC-SCalibur; Becton Dickinson, New Jersey, USA). To analyze the cellspecificity of the RIPL peptide, various cell lines including LNCaP,SK-OV-3, MCF-7, DU145, PC3 and HaCaT were seeded in growthmedia at a density of 1 � 106 per well in a 6-well plate. After reach-ing 70–80% confluence, the cells were incubated with 1 lM RIPLpeptide-conjugated FITC (RIPL-FITC) in RPMI medium for 2 h at37 �C, washed with PBS three times, and subjected to flow cytom-etry for the quantification of MFI values via acquisition of 10,000events per histogram. To evaluate the cell penetration efficiency,LNCaP cells were co-incubated with FITC-dextran (28 lg/mL) and3 lM of R8, IPL, or RIPL peptides for 2 h at 37 �C. Cells were thenharvested and washed with PBS, followed by the measurement ofMFI values as described above.

2.10. In vitro cell uptake study of RIPL-Lipo

The uptake of liposomes into cultured cells was examined bydetermining the MFI of the probe using a FACS, and visualized bymonitoring the cell association using a fluorescence microscopeand CLSM. Briefly, LNCaP, SK-OV-3, MCF-7, DU145, PC3, and HaCaTcells were seeded in growth media at a density of 1 � 106 cells perwell into NUNC CC2 chamber slides. After reaching 70–80% conflu-ence, the cells were incubated for 2 h at 37 �C in RPMI media(2 mL) containing liposomal stock solutions (0.1 mL) of CL andRIPL-Lipo, in which the concentration of FITC-dextran was 28 lg/mL. In order to measure MFI values by flow cytometry, the cellswere treated as previously described. For microscopic observation,the cells were visualized using a fluorescence microscope under200�magnification (Motic, Beijing, China). Separately, LNCaP cellstreated as described above were washed three times with PBS,mounted onto slides without fixation process, and the fluorescenceof the probes delivered to the cells was monitored using a Zeiss




LSM 510 Meta confocal microscope with Z-sectioning mode under400� magnification (Carl Zeiss, Oberkochen, Germany). Confocalimage of live cells was obtained with different chamber slides atpredetermined time points of 10 min, 30 min, 1 h, and 2 h. Z stackswere created at 1 lm intervals throughout the 20 lm of the sec-tions with a guard region of 2 lm excluded from top and bottomof the Z stack.

2.11. Cytotoxicity assessment

The cytotoxicities of RIPL peptides and RIPL-Lipo were evalu-ated in LNCaP and HaCaT cells by WST-1 assay as previouslyreported [4,30]. Based on the cleavage of WST-1 into formazanby mitochondrial dehydrogenases in viable cells, a colorimetricassay for quantifying cell proliferation and cell viability was carriedout. LNCaP and HaCaT cells were seeded in growth medium at adensity of 1 � 104 per well into a 96-well plate. After reaching70–80% confluence, the cells were incubated at 37 �C for 2 h inRPMI medium containing RIPL peptide or empty RIPL-Lipo at dif-ferent concentrations. In positive and negative controls, methanoland distilled water (D.W.) were used to replace the sample treat-ment, respectively. Cells were then incubated with WST-1 reagentat 37 �C for 2 h, and the absorbance of WST formazan dye was mea-sured at 450 nm using a microplate reader. Cell viability was calcu-lated as the percentage of viable cells relative to the untreatedsample.

2.12. Statistical analysis

Values were processed using Microsoft Excel 2010 software andpresented as mean ± standard deviation (n = 3). Statistical signifi-cance was determined by Student’s t-test and considered to be sig-nificant at P < 0.05.

3. Results

3.1. Synthesis and identification of peptides

R8, IPL, RIPL peptide, and RIPL-FITC were synthesized by FmocSPPS using automated peptide synthesizer and purified with pre-parative HPLC. The molecular weight (MW) of a synthesized pep-tide was calculated by first, summing every MW of all aminoacids in the relevant peptide sequence, then, by subtracting fromthat sum the number of peptide bonds multiplied by the MW ofwater, since the formation of peptide bonds is always accompaniedby the loss of one water molecule. The molecular ion peaks of R8,IPL, RIPL peptide, and RIPL-FITC were measured as 1370, 853, 2102,and 2619, respectively. As listed in Table 1, calculated MWs of allpeptides closely corresponded to the MWs of the purified peptidesobserved by LC/MS analysis.

3.2. Cell uptake specificity of RIPL peptide

Cellular uptake specificity of the RIPL peptide by Hpn-express-ing cells was assessed by measuring MFI after various cell lines(SK-OV-3, MCF-7, and LNCaP for Hpn(+); DU145, PC3, and HaCaTfor Hpn(�)) were treated with fluorescent macromolecules.FITC-dextran was used as a model compound because it is mini-mally transported through cell membranes. As shown in Fig. 1,MFI values of FITC-dextran alone were less than those of RIPL-FITCin all tested cell lines. The fluorescent marker itself was poorlytransported into the cells, regardless of cell type. In comparison,the uptake of RIPL-FITC was remarkably high. Significant differ-ences, at P < 0.05, were found in all Hpn(+) cell lines, but not inHpn(�) cell lines. Particularly in the LNCaP cell line, the uptake


of RIPL-FITC was 8.3-fold greater than that of FITC-dextran, sug-gesting a selective interaction of the RIPL peptide with the specificcell line. For further evaluation how the RIPL peptide enhances theuptake of FITC-dextran, comparative uptake studies in LNCaP cellwere performed with equivalent peptides (R8, IPL, RIPL) or in theabsence of a peptide (Control). As depicted in Fig. 2, when com-pared to the control, R8 and RIPL significantly increased the MFIvalues, but IPL did not. The MFI value representing R8 uptakewas greater than that of RIPL, possibly due to the reduced molefraction of pArg in the RIPL peptide. These results suggested thatpArg and IPL played important roles in the enhancement of cellpenetration and cell homing function, respectively.

3.3. Characteristics of RIPL-Lipo

RIPL-Lipo systems were successfully prepared with PC, Tween80, DSPE-PEG2000-mal, and RIPL peptides, and characterized forphysical and conformational properties (Table 2). We investigatedthe physical characteristics of liposomal nanocarrier systems interms of vesicular size, polydispersity index, zeta potential, andEE. The average size of all systems was observed to be about160 nm by dynamic light scattering. Low polydispersity indices,below 0.07, indicated a narrow and homogenous size distribution.Compared to the slight negative charge of CL, all RIPL-Lipo systemswere positively charged. RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo,and RIPL(2.0)-Lipo showed zeta potentials of about 6, 16, 24, and27 mV, respectively, indicating the proportions of liposome to theamount of attached RIPL peptide. This peptide contains positivelycharged amino acid (Arg) molecules in the sequence. The concen-tration of FITC-dextran in liposomal stock solutions was measuredas 0.56 ± 0.02 mg/mL for all RIPL-Lipo. Conjugation of RIPL peptidesto liposomal surfaces did not affect EE, revealing about 28% ofinitially added amount of FITC-dextran on average for allformulations.

Conformational aspects of RIPL-Lipo were characterized bydetermining the external amounts of maleimide groups and boundpeptide molecules. The molar amount of external maleimide groupsacting as a substrate for peptide conjugation was determined byEllman’s reaction. The molar amount of external maleimide[Mal]external, was calculated as [Cys]initial � [TNB]detected, where[Cys]initial is the molar amount of cysteine initially added and[TNB]detected is the molar amount of TNB detected by HPLC. Ellman’sassay revealed that approximately 51 ± 2.3% of the initially addedDSPE-PEG-mal was oriented externally at the liposomal surface,regardless of the amount of DSPE-PEG-mal added (0.2–4.0%). Thenumber of external maleimide groups per vesicle was then calcu-lated as follows: [N]vesicle � [Mal]external/[Lipid]total, where [N]vesicle

is the number of lipid molecules that formed a vesicle, [Mal]external

is the molar amount of external maleimide as described above,and [Lipid]total is the molar amount of total lipids initially added.[N]vesicle was estimated by the following formula [31]: 4pr2 � 2/A,where r and A refer to the radius of the liposomes (80 nm) andthe cross-sectional area of the PC head group (0.72 nm2), respec-tively. From these equations, we calculated that the vesicle wasformed by approximately 223,000 lipid molecules. The extent ofRIPL peptide conjugation on maleimide-derivatized liposomeswas demonstrated by determining the amount of unreacted malei-mide after dialysis of the product. The conjugation reaction wascompleted voluntarily, yielding no detectable, residual TNB. As aresult, RIPL peptide molecules located on the external surface ofthe vesicle proportionally increased with the amount of peptideadded: approximately 230, 800, 2300, and 4550 peptide moleculesper vesicle for RIPL(0.1)-Lipo, RIPL(0.3)-Lipo, RIPL-Lipo, andRIPL(2.0)-Lipo, respectively.



Table 2Physical and conformational characteristics of liposomal nanocarriers.

Formulations Composition (mol ratio) Physical characteristi

PC Tween80 DSPE-PEG-Mal Peptide Size (nm) PDI

CL 90 10 – – 160.3 ± 5.4 0.038RIPL(0.1)-Lipo 89.8 10 0.2 0.1 162.7 ± 4.3 0.048RIPL(0.3)-Lipo 89.4 9.9 0.7 0.35 164.5 ± 2.4 0.052RIPL-Lipo 88.2 9.8 2.0 1.0 164.2 ± 2.7 0.065RIPL(2.0)-Lipo 86.4 9.6 4.0 2.0 165.9 ± 3.9 0.070

* Values calculated as described in the text.a Zeta potential.b Entrapped efficiency for FITC-dextran.

Fig. 3. Effect of the number of conjugated RIPL peptides per vesicle on the cellularuptake of RIPL-Lipo. LNCaP cells were treated with RIPL(0.1)-Lipo, RIPL(0.3)-Lipo,RIPL-Lipo, and RIPL(2.0)-Lipo containing equivalent FITC-dextran (28 lg/mL).Values represent mean ± S.D. (n = 3).

Table 1Sequence and molecular weight of peptides used.

Abbreviation Sequence Calculated MW Observed MW Description

R8 RRRRRRRRC 1369.83 1370 Known as cell penetrating sequence [36]IPL IPLVVPLC 852.51 853 Known as hepsin-specific sequence [14]RIPL IPLVVPLRRRRRRRRC 2101.32 2102 A novel peptide for cell penetrating and hepsin targetingRIPL-FITC IPLVVPLRRRRRRRRCK-FITC 2618.80 2619 Used as a fluorescent maker by RIPL peptide binding

Fig. 1. Cell specificity of RIPL peptide in Hpn(+) and Hpn(�) cell lines. Various cellswere incubated with 1 lM RIPL-FITC or FITC-dextran alone at 37 �C and pH 7.4 for2 h. Mean fluorescence intensity (MFI) was measured as cellular uptake by flowcytometry. Values represent mean ± S.D. (n = 3). Statistical analysis was performedusing the Student’s t-test (*P < 0.05 versus paired group; **P < 0.005 versus pairedgroup).

Fig. 2. Effect of admixed peptides for FITC-dextran uptake in LNCaP cell. Cells weretreated with FITC-dextran (6.2 lM) and different peptides (3 lM) for 2 h, whilecontrol was treated with FITC-dextran alone. Values represent mean ± S.D. (n = 3).Statistical significances were compared using the Student’s t-test (*P < 0.05 versuspaired group; **P < 0.005 versus paired group).



3.4. Selection of the optimized RIPL-Lipo system

Surface modification of the vesicular nanocarrier could be a cru-cial factor for cell interaction. Therefore, when preparing the fourRIPL-Lipo systems, it was necessary to screen the RIPL-Lipo con-structs for further experiments. We analyzed the effect of the con-jugated RIPL peptide ratios on the cellular uptake of RIPL-Lipo, bymeasuring MFI after treatment in LNCaP cell line. As shown inFig. 3, cellular uptake was dependent on the number of RIPL pep-tides. MFI values increased proportionally up to 2300 moleculesof the conjugated peptides, but, afterward, the value plateaued.Therefore, RIPL-Lipo, which has 2275 peptide molecules, wasselected and used thereafter as the optimized system.

3.5. In vitro cellular uptake evaluation of RIPL-Lipo

Cellular uptake efficiency of RIPL-Lipo was assessed by flowcytometry in various cell lines such as LNCaP, DU145, and PC3for human prostate cancer, MCF-7 for breast cancer, SK-OV-3 forovarian carcinoma, and HaCaT as a reference. Cells were treatedwith different samples of FITC-dextran alone, FITC-dextran-loadedCL or RIPL-Lipo. As shown in Fig. 4 (upper panel), a greater shift of

cs Conformational characteristics*

ZPa (mV) EEb (%) Total maleimides/vesicle Peptide molecules/vesicle

�2.4 ± 3.2 29.3 ± 2.7 – –6.1 ± 0.8 28.4 ± 1.3 446 227

16.2 ± 1.1 29.0 ± 1.7 1589 81024.2 ± 2.7 28.3 ± 2.1 4460 227427.2 ± 0.9 27.7 ± 2.4 8920 4549



Fig. 4. Flow cytometry results for FITC-dextran uptake in Hpn(+) and Hpn(�) cell lines incubated for 2 h. Upper panel shows the treatment effect: untreated cells (black); cellstreated with FITC-dextran alone (red), FITC-dextran loaded CL (green), and FITC-dextran loaded RIPL-Lipo (blue). Lower panel indicates the relative ratio of MFI values indifferent treatments: (a) FITC-dextran loaded CL versus FITC-dextran alone; (b) FITC-dextran loaded RIPL-Lipo versus FITC-dextran alone; (c) FITC-dextran loaded RIPL-Lipoversus FITC-dextran loaded CL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


the FI graph was observed after RIPL-Lipo treatment, whereas littleto no shift was observed after treatments with CL or FITC-dextranalone. The MFI values of RIPL-Lipo were counted in the order of


SK-OV-3 (419) > LNCaP (214) > MCF-7 (153) > PC3 (87) > DU145(33) > HaCaT (21), indicating selective binding and cellular uptakeof RIPL-Lipo due to cell penetrating and homing function of the



Fig. 5. Fluorescence microscopy images of various cell lines incubated with RIPL-Lipo at 37 �C for 2 h. Concentration of FITC-dextran was 28 lg/mL. Scale bar indicates100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


conjugated RIPL peptide. In terms of cell specificity, the relativeratio of MFI values in different treatments was calculated for fur-ther analysis (lower panel in Fig. 4). Compared to FITC-dextranalone, CL revealed less than 3-fold increase in all cell types tested,whereas RIPL-Lipo showed 5- to 7-fold increase in Hpn(�) cells(DU145, PC3, and HaCaT) and approximately 20- to 70-foldincrease in Hpn(+) cells (SK-OV-3, MCF-7, and LNCaP). Likewise,in comparison with CL, RIPL-Lipo increased the MFI values by asmuch as 2- to 5-fold in Hpn(�) cells and 9- to 26-fold in Hpn(+)cells. Therefore, in summary, cellular uptake of the macromoleculewas increased by liposomal formulation, and the uptake was fur-ther enhanced by RIPL peptide modification of the liposomal sur-face. Interestingly, Hpn-specific intracellular delivery was feasiblewith RIPL-Lipo.

3.6. Selective binding and internalization of RIPL-Lipo to Hpn(+) cell

To better understand the selectivity and translocational behav-ior of RIPL-Lipo, it was visualized by fluorescence microscopy andCLSM. Hpn-favoring RIPL-Lipo was added to a suspension ofHpn(+) or Hpn(�) cells and microscopic monitoring took place atappropriate time points. First, in the fluorescence microscopicobservation (Fig. 5), the addition of RIPL-Lipo revealed a cell-typedependency: prominent green spots were visible in Hpn(+) cells,whereas only weak or faint marks were visible in Hpn(�) cells. Thisobservation was consistent with the results of flow cytometry anal-ysis, indicating RIPL-Lipo selectively binds to and interacts withHpn-expressing cells. In the case of CL, regardless of cell type, fluo-rescence spots were invisible (data not shown). Meanwhile, the


translocational behavior of RIPL-Lipo in LNCaP cells was furtherinvestigated by CLSM with orthogonal views of Z stacks (Fig. 6).Immediately after the treatment (10 min), green fluorescence withmild intensity in XY plane was observed in the vicinity of cell mem-branes, indicating selective binding of RIPL-Lipo to the extracellularmembrane of Hpn(+) cells. In comparison, the intensity in Z direc-tions (XZ and YZ planes) was very weak or negligible. After30 min, green spots with moderate intensity were observed in theinterior compartment of the cell, exhibiting evidence of internaliza-tion of RIPL-Lipo. After 1 h, in all orthogonal planes, green spotsbecame intensified and were distributed throughout the cell struc-ture. This intense response was maintained for 2 h after the treat-ment. Cytosolic internalization of RIPL-Lipo was time-dependent.

3.7. Cytotoxicity of RIPL peptide and RIPL-Lipo

The toxicities of RIPL peptide and RIPL-Lipo were examinedusing LNCaP and HaCaT cells. The cells were treated with varyingamounts of peptide or liposomes, and cell viability was examinedby WST-1 assay. Cell viability in the untreated group was deemedto be 100%. As shown in Fig. 7A, toxicity was minor (cell viability>90%) with the addition of up to 50 lM of RIPL peptide in bothcells. However, cell viability decreased to less than 70% at concen-trations above 100 lM for LNCaP or 500 lM for HaCaT cells. Addi-tionally, as depicted in Fig. 7B, RIPL-Lipo revealed no toxicity whenadded in concentrations less than 10% (cell viability >90%) in bothcell lines, though cell viability decreased as the concentrationincreased over 10%. However, the concentration range used in thisexperiment was very low: less than 5 lM or 5% for RIPL peptide



Fig. 6. Confocal images with orthogonal views of LNCaP cells incubated with RIPL-Lipo at 37 �C at pH 7.4. Concentration of FITC-dextran was 28 lg/mL. The positions of thesection plane were shown by colored lines; XY plane (blue), XZ plane (green), YZ plane (red). Scale bar indicates 50 lm. (A) Immediately after the treatment, a small amount ofRIPL-Lipo strongly bound to extracellular membrane because of Hpn selectivity; (B) after 30 min, RIPL-Lipo were translocated into cells owing to endocytosis and cellpenetration; (C) after 1 h, florescence intensity of RIPL-Lipo in cytosol was increased overall; (D) after 2 h, intense fluorescence was maintained. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)


and RIPL-Lipo, respectively. Cytotoxicity of CL was negligible inboth cell types (data not shown). Therefore, we suggest thatRIPL-Lipo did not cause any significant toxicity in either cell type.

4. Discussion

Various approaches for selective ligand tagging of molecularcargo and drug carriers have been tested to enhance the cellularuptake specificity of therapeutic drugs and/or imaging agents. Inparticular, much attention has been paid to the use of cell-specificand cell-penetrating peptides or proteins, which may result in sig-nificant increases in drug concentration at target site or cell, there-fore dramatically reducing unwanted side effects [32]. Given thatthe cell membrane is a major biological barrier for the delivery oftherapeutic drugs and particulate carriers [32], successful employ-ment of efficient intracellular target moieties, which selectivelybind to cell surface receptors and promote selective uptake ofattached cargos, is a point of focus in emerging medicine, espe-cially for cancer chemotherapy. In previous research, CPPs havebeen successfully employed for enhanced intracellular drug deliv-ery [4,33,34]. CPP-conjugated nanoparticles could be accumulatedin cancer tissues by the enhanced permeability and retention effecttermed ‘‘passive targeting.’’ Then, the nanoparticles would beinternalized into cancer cells by CPP-mediated translocation. How-ever, because of their non-selectivity, the application of CPPs hasbeen very limited. In recent years, several CPHPs have been suc-cessfully employed for targeted drug delivery. For example,ErbB2-binding peptide (homing sequence), TAT-derived peptide


(CPP), and STAT3BP (signal transducers and activators of transcrip-tion proteins) were serially connected, demonstrating selectiveinhibition of the growth of ErbB2-overexpressing cells in vitroand a greater reduction in tumor growth in xenograft modelsin vivo [35]. In the present study, we synthesized a novel CPHP,which was named RIPL peptide (16mer; 2.1 kDa), and developeda RIPL-Lipo system for Hpn-specific drug targeting with enhancedcellular uptake.

RIPL peptide was successfully synthesized by Fmoc SPSS, anautomated peptide synthesizer, and its secondary and tertiarystructures were verified with relevant software programs. Thisnovel CPHP carries two essential domains of IPL for targetingaction and R8 for cell penetrating action. IPL analogues were ran-domly selected by phage display in Hpn-transfected PC3 cells,due to a shared homology in secondary structure of proteinsequences [14]. To investigate structural similarity between RIPLpeptide and IPL analogues, we developed a prediction for the sec-ondary structure of the RIPL peptide through use of the PSIPREDmethod, in which secondary structure is designated as C (coil), E(strand), and H (helix). The sequence of IPL analogues has a con-nection of coil and strand structure (CCEE) in common (Table 3).In the RIPL peptide, the C-terminus of the IPL peptide was con-nected to the R8 peptide: Despite the pArg linkage, this commonsecondary structure was retained; therefore, its Hpn targetingproperty would not be hampered. We demonstrated that fluores-cence-tagged RIPL peptide (RIPL-FITC) exhibited greater cellularuptake in Hpn(+) cells than the uptake of FITC-dextran alone(Fig. 1), indicating the retention of Hpn affinity, regardless of R8linkage.



Fig. 7. Cytotoxicities of RIPL peptide (A) and empty RIPL-Lipo (B) in LNCaP andHaCaT cells by WST-1 assay. For positive and negative controls, methanol (MeOH)and distilled water (D.W.) were used to replace the sample treatment, respectively.The bars represent the concentration range used in this study. Data are mean ± SD(n = 3).

Table 3Secondary structure of peptides predicted by PSIPRED method.

Peptide sequence Secondary structurea

IPL analoguesIPLVVPL CCEECCCIPLWVPL CCEECCCIPLVLVPL CCEEEECCIPLVVPLGGSCK CCEEEECCCCCC

RIPL peptideIPLVVPLRRRRRRRRC CCEEEHHHHHHHHCCC

a Represented by C (coil), E (strand), and H (helix).


Cell penetration efficiency of Arg-rich peptides is related to thenumber of Args in peptide sequence. When the number of Args wasincreased by 16, R8 peptides exhibited optimal translocation intomouse macrophage RAW 264.7 cells [36]. In this regard, the RIPLpeptide was considered to include the optimal number of Args.The cell penetrating capability of the RIPL peptide could be esti-mated with a CPP prediction method [37], in which the value inter-vals of CPPs are described using a set of five descriptors (Z1–Z5)that represent a composite of the physical characteristics of theamino acids. Among the five descriptors, the most relevant areZ1 (lipophilicity), Z2 (steric bulk), and Z3 (polarity). The valueintervals for efficient CPPs range as follows: Z1 ranges from�1.25 to 1.92; Z2 ranges from �1.22 to 1.29; Z3 ranges from�0.5 to �1.94. The numerical calculation of average Z values foramino acids constituting RIPL was obtained as 0.48 (Z1), 0.73(Z2), and �2.5 (Z3): lipophilicity and steric bulk were satisfactory,but polarity was not. Nevertheless, the inclusion of this novelpeptide allowed for significantly better cell penetration than the


control or IPL (Fig. 2). In comparison with R8, however, cell-penetrating efficiency of the RIPL peptide was somewhat reduced.This reduction was commonly presented in artificial CPHP, possi-bly due to a decreased molar fraction of relevant peptides in theconjugate. For instance, the cell penetration efficiency of pVEC-PEGA (LLIILRRRIRKQAHAHSK-CPGPEGAGC), a combination of pVEC(CPP) and PEGA (aminopeptidase binding peptide), was 0.25-foldless than that of the constituting peptides [23]. Moreover, insteadof PEGA, when a linear breast tumor homing peptide (CREKA)was conjugated to pVEC, the efficiency was decreased to one-thirdof the constituting peptides [38]. These observations may be attrib-uted to the reduced molar fraction of positively charged aminoacids like Arg and lysine. In addition, because the cell surface isnegatively charged, a reduction in positivity could reduce the elec-trostatic interaction between peptide and cell, thereby decreasingthe cell-penetrating effect [39].

Cellular binding and the internalization of ligand-modified lip-osomes may depend on the extent of conjugation of the targetingligand and CPPs to the liposomal surface [34,40]. For the successfuldevelopment of RIPL-Lipo, the architecture of RIPL conjugation tothe liposomal surface should be configured in linear or brush con-formation aspects: When the distance (D) between PEG moleculeson the nanoparticle surface is shorter than Rf, which defines theFlory diameter of the space occupied by a PEG molecule, the lateralpressure between the overcrowded PEG linkers will force theextension of the PEG chains into a linear conformation. This exten-sion of ligand moieties away from the surface into more linear con-formations contributes to the interaction between ligand andtarget molecule [2,41]. The minimal number of PEG linkers neededfor a linear conformation (nPEG) was calculated as follows:nPEG = 4pr2/D2, where r refers to the radius of liposomes and Dvalue is equal to the Rf of the linker molecules. The RIPL-Lipo sys-tem was measured to be 80 nm in radius and used PEG2000, whoseRf value is approximately 5.6 nm in solution, as a linker [2]. There-fore, the nPEG needed in order to adopt a linear conformation wascalculated to be about 2500. In this study, as shown in Fig. 3, cel-lular uptake of RIPL-Lipo was maximized at the conjugation of2300 peptide molecules. This result is in agreement with the theo-retical calculation, since RIPL peptide was conjugated to PEG link-ers. In addition, the number of conjugated CPPs on the liposomalsurface for optimal system efficiency is dependent on the type oftarget ligand, liposomal size, and cell type. In previous studies,the translocation of liposomes modified with the TAT peptide orpenetratin was proportional to the number of peptide moleculesattached to the liposomal surface [34]. The penetratin-conjugatedliposomal system showed strong cell-association and internaliza-tion at the conjugation level of 120 peptide molecules [42]. In con-trast, the HVGGSSV peptide, a tumor vasculature-targeting peptideselected by phage display as an IPL peptide without cell penetrat-ing property, was attached at a level of 2% of total lipid (about 1700peptide molecules) to enhance tumor accumulation [43]. To thebest of our knowledge, our study is the first documented attemptto optimize the extent of CPHP conjugation on a liposomal surface.

As illustrated in Fig. 8B, we propose that RIPL-Lipo might beendocytosed after selective binding to Hpn(+) cells and temporar-ily entrapped in endosomes, later escaping into the cytosol. RIPL-Lipo may selectively bind to the cell surface due to Hpn recognitionby IPL domain, then, it may undergo proteolytic cleavage by serineprotease exposing the R8 peptide and triggering cell penetration. Ithas been generally recognized that arginine-rich, peptide-conju-gated cargos are translocated by endocytosis [44–47], even thoughthe cellular uptake mechanism of arginine-rich peptides wasthought to undergo non-endocytic translocation [48]. Therefore,liposomes carrying cleaved pArg may be translocated by theendocytic pathway, which leads to lysosomal delivery andsubsequent degradation or escape to cytosol compartment. Similar



Fig. 8. Schematic diagram describing the tertiary structures of the RIPL peptide and Hpn (A) and uptake mechanism and intracellular pathways of RIPL-Lipo in Hpn(+) andHpn(�) cells (B). RIPL-Lipo selectively bound to Hpn, therefore, less interacted with Hpn(�) cells. Protease cleaved polyarginine of RIPL sequence. The uptake of liposomes byendocytosis would cause temporary entrapment in endosome, however, liposomes could escape into cytosol or be degraded by lysozyme. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)


mechanisms for translocation of the iRGD peptide (CRGDKGPDC)-modified liposomes were reported: The RGD motif of the iRGDpeptide recognizes the vb3/avb5 integrins on tumor endothelialcells; iRGD is then cleaved by proteases to expose the crypticCendR element, RGDK/R, at the C-terminus, and eventually theCendR element mediates binding to neuropilin-1 to induce vascu-lar and tissue penetration [49]. There are some arguments as towhether or not the endosomally-entrapped liposomes could avoidlysosomal breakage and travel to cytosol while remaining intact[17]. In the case of RIPL-Lipo, due to CPP functioning of R8, the lip-osomes could escape from the endosome being delivered to cyto-sol. It was evident that pH-sensitive formulations, which weredesigned to hide a Tat moiety at physiological pH but expose theCPP moiety in an acidic endosomal environment, could translocatethrough the endosomal membrane, passing by lysosomal degrada-tion, and subsequently increasing the cytotoxicity of cancer cells[50,51]. Our observation with CLSM also supported this behavior,as shown in Fig. 6, resulting in the scattered dot-like fluorescencethroughout the cytoplasm even after 2 h.

Cancer-associated proteases including Hpn, matrix metallopro-teases (MMP), and urokinase plasminogen activator (uPa) havebeen focused on targeted drug delivery: MMP was used to selec-tively activate a CPP moiety with MMP-sensitive linker in tumorsites [52]; uPa was applied to uPa-sensitive polymer-caged lipo-somes using a protease function [53]. However, there has been lit-tle application of Hpn protease in drug delivery. We firstlydemonstrated that Hpn protease functionality could be appliedto developing strategies of drug targeting. Instead of PSMA, Hpncould be used as a diagnostic tool for prostate cancer, in whichmetastasis accompanies Hpn overexpression [54]. By the applica-tion of the RIPL peptide and RIPL-Lipo, we also proved that Hpnwas a selectable target molecule for breast and ovarian cancercells. In particular, RIPL-Lipo would be a promising tool for Hpn-selective targeting for various drugs covering cell membraneimpermeable macromolecules, protein drugs, diagnostics, andchemotherapeutics.

5. Conclusion

In the present study, we synthesized a novel CPHP, which wehave named RIPL peptide (16mer; 2.1 kDa), and successfully devel-oped a RIPL-Lipo system for Hpn-specific drug delivery, revealingenhanced selectivity and cellular uptake compared to conventional


liposomes. The cellular uptake of RIPL-Lipo was maximized withthe conjugation of 2300 peptide molecules. Cytosolic internaliza-tion of RIPL-Lipo was time-dependent, and the uptake mechanismand intracellular pathways related to RIPL-Lipo have been postu-lated. In the future, the RIPL-Lipo system is a promising tool thatcould be extensively used for the therapy and/or diagnosis ofHpn-related cancers.

Acknowledgements

This research was supported by Basic Science Research Programthrough the National Research F (NRF) funded by Ministry of Edu-cation, Science and Technology (2011-0009876).

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