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Biomaterials 77 (2016) 149e163

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

A surface charge-switchable and folate modified system for co-delivery of proapoptosis peptide and p53 plasmid in cancer therapy

Si Chen, Lei Rong, Qi Lei, Peng-Xi Cao, Si-Yong Qin, Di-Wei Zheng, Hui-Zhen Jia,Jing-Yi Zhu, Si-Xue Cheng, Ren-Xi Zhuo, Xian-Zheng Zhang*

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan, 430072, PR China

a r t i c l e i n f o

Article history:Received 23 July 2015Received in revised form2 November 2015Accepted 6 November 2015Available online 10 November 2015

Keywords:Detachable complexesCharge-switchableTumor targetingCo-delivery systemCancer therapy

* Corresponding author.E-mail address: xz-zhang@whu.edu.cn (X.-Z. Zhan

http://dx.doi.org/10.1016/j.biomaterials.2015.11.0130142-9612/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

To improve the tumor therapeutic efficiency and reduce undesirable side effects, ternary FK/p53/PEG-PLL(DA) complexes with a detachable surface shielding layer were designed. The FK/p53/PEG-PLL(DA)complexes were fabricated by coating the folate incorporated positively charged FK/p53 complexeswith charge-switchable PEG-shield (PEG-PLL(DA)) through electrostatic interaction. At the physiologicalpH 7.4 in the bloodstream, PEG-PLL(DA) could extend the circulating time by shielding the positivelycharged FK/p53 complexes. After the accumulation of the FK/p53/PEG-PLL(DA) complexes in tumor sites,tumor-acidity-triggered charge switch led to the detachment of PEG-PLL(DA) from the FK/p53 com-plexes, and resulted in efficient tumor cell entry by folate-mediated uptake and electrostatic attraction.Stimulated by the high content glutathione (GSH) in cytoplasm, the cleavage of disulfide bond resulted inthe liberation of proapoptosis peptide C-KLA(TPP) and the p53 gene, which exerted the combined tumortherapy by regulating both intrinsic and extrinsic apoptotic pathways. Both in vitro and in vivo studiesconfirmed that the ternary detachable complexes FK/p53/PEG-PLL(DA) could enhance antitumor efficacyand reduce adverse effects to normal cells. These findings indicate that the tumor-triggered decom-plexation of FK/p53/PEG-PLL(DA) supplies a useful strategy for targeting delivery of different therapeuticagents in synergetic anticancer therapy.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

As a serious threat to human health, cancers are characterizedby the abnormal apoptosis of tumor cells, which grants them theability to survive in harsh environments like hypoxia and malnu-trition, and escape programmed cell death [1,2]. Therefore,apoptosis regulation as a new strategy for cancer therapy hasattracted increasing research attention, and promising results havebeen reported in recent years [3]. For example, Wang et al.demonstrated that the apoptosis regulation peptide AVPIcombiningwith the tumor suppress p53 gene could promote tumorcell apoptosis and overcome multidrug resistance [4]. Veryrecently, we used a dual-functional polypeptide xPolyR8-KLA(TPP)(RK) to deliver proapoptotic peptide C-KLA(TPP) and p53 gene intotumor cells simultaneously. This dual-functional polypeptide couldinduce efficient cells apoptosis through regulating intrinsic

g).

“mitochondria” apoptosis pathway by C-KLA(TPP) and extrinsic“death” receptors signaling apoptotic pathway by p53 [5]. Althoughthe apoptosis regulation agents could promote tumor cell apoptosisefficiently, their application was still limited by many drawbacks,such as rapid clearance from the bloodstream [6,7], non-specificcellular uptake [8,9], and potential toxicity to normal tissues [10].These drawbacks led to the limited antitumor efficacy and adverseeffects to normal tissues. To overcome these disadvantages, variousfunctional components were introduced to the delivery system ofthe apoptosis regulation agents, such as biocompatible polymers toprolong the circulation time in bloodstream, targeting moieties toenhance their accumulation at tumor sites, and cell penetratingcomponents to promote cellular internalization, etc. [11].

It is well known that positively charged carriers would lead tosevere aggregation by binding to serum proteins, which is one ofmain causes for the rapid clearance from circulation, thereforereduce the antitumor efficacy and limit their applications in vivo[12,13]. Negatively charged or neutral polymers can be employed asa protective shell to shield the positively charged surface andprolong the circulation time in blood [14]. For example,

S. Chen et al. / Biomaterials 77 (2016) 149e163150

poly(ethylene glycol) (PEG), a neutral polymer, was widely used toprolong the circulation time in bloodstream. However, compared tothe positively charged carriers, the negatively or neutrally chargedsurfaces reduce the cell entry efficiency due to the inefficientinteraction between the cell membrane and the particles, and leadto the decreased antitumor efficacy. To address this issue, theprotective shell should be detached once the therapeutic agentsarrived at the desired sites to facilitate the cell uptake of the agents.

Taking advantage of the slight pH difference between extracel-lular acidity of tumor sites (pHe ~ 6.8) and normal tissues (pH ~ 7.4),charge-switchable systems have been developed for the prolongedcirculation and improved cell uptake by the tumormicroenvironment-triggered shell detachment. These systemscould stay stable in bloodstream and avoid the non-special ab-sorption by serum proteins. After the systems accumulated in tu-mor sites, the low pH triggered ionization and the detachment ofprotective moiety result in positive surface charge, which facilitatethe intracellular endocytosis [15].

In this study, we fabricated ternary FK/p53/PEG-PLL(DA) com-plexes with tumor environmental detachable surface shielding toimprove the antitumor efficacy and reduce undesirable side effects.First, a tumor targeted polypeptide (xPolyR8(FA)-KLA(TPP), FK) wassynthesized by cross-linking folate (FA) incorporated cell pene-trating peptide FA-CR8C (CR8C(FA)) and triphenylphosphonium(TPP) incorporated proapoptosis peptide TPP-KK(KLAKLAK)2C (C-KLA(TPP)). Then the positively charged polypeptide FK compressedp53 plasmid to form FK/p53 complexes. Finally, a switchable shield,poly(ethylene glycol)-block-2,3-dimethylmaleic anhydride (DA)-modified poly(L-lysine) (PEG-PLL(DA)), was coated on the surface ofFK/p53 through electrostatic interaction to form the ternary com-plexes FK/p53/PEG-PLL(DA). At the physiological pH 7.4, due to thecharge shielding effect of PEG-PLL(DA), FK/p53/PEG-PLL(DA) couldavoid the non-special absorption by serum proteins and extend thecirculating time. Once the FK/p53/PEG-PLL(DA) complexes accu-mulated in acidic extracellular tumor tissue, the amide bonds werehydrolyzed, leading to the charge repulsive removal of the PEG-PLL(DA) shell and explosion of the folate decorated positivelycharged FK/p53 complexes. Thereafter, the positively charged FK/p53 complexes could be internalized by tumor cells effectively dueto the folate receptor-mediated cell uptake and electrostaticattraction [16]. After internalization, the FK/p53 complexes weredegraded in cytoplasm under the stimulation of glutathione (GSH)and further released C-KLA(TPP) and p53 plasmid to induce cellapoptosis by regulating both intrinsic and extrinsic apoptoticpathways [5]. Both in vitro and in vivo studies demonstrated thatthe FK/p53/PEG-PLL(DA) complexes could enhance antitumor ef-ficacy and reduce the side effects effectively.

2. Materials and methods

2.1. Materials

Rink Amide-AM resin (100e200 mesh, loading: 0.7 mmol/g), 2-Chlorotrityl chloride resin (100e200 mesh, loading: 1.20 mmol/g),N-fluorenyl-9-methoxycarbonyl (Fmoc) protected D-amino acids(Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH and Fmoc-Leu-OH) and L-amino acids (Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH), benzylox-ycarbonyl protected L-lysine (H-Lys(Z)-OH), O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) were obtained from GL Biochem. Ltd.(Shanghai, China). N,N-dimethylformamide (DMF), diisopropylethyl-amine (DIEA) and trifluoroacetic acid (TFA) were acquired fromShanghai Chemical Co. (Shanghai, China) and distilled prior to use.Piperidine, thioanisole, phenol, ethanedithiol (EDT), dichloromethane(DCM), methanol, anhydrous ether, dimethylsulfoxide (DMSO), and

1,4-dithiothreitol (DTT)were provided by Shanghai Chemical ReagentCo. (Shanghai, China). Amino-monomethoxypoly(ethylene glyco)(PEG-NH2, Mw 2000), triphosgene, 2,3-dimethylmaleic anhydride(DA) and folate (FA) were obtained from Aladdin Reagent Co. Ltd.(Shanghai, China). Triphenylphosphonium (TPP) was obtained fromAdamas Reagent Co. Ltd. (Shanghai, China). All other solvents andreagents were analytical grade, provided by Sinopharm ChemicalReagent Co. Ltd. (Shanghai, China), and used directly.

GelRed™ was provided by Biotium (California, USA). QIAfilter™plasmid purification Giga Kit was obtained from Qiagen (Hilden,Germany). Fetal bovine serum (FBS), penicillin-streptomycin,Minimum Essential Medium (MEM), Dulbecco's Modified Eagle'sMedium (DMEM), phosphate buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), JC-1 (5,50,6,60-tetrachloro-1,10,3,3’-tetraethylbenzimidazolycarbocya-nine iodide) fluorescent dye, Hoechst 33342 and YOYO-1 iodidewere obtained from Lonza Group Ltd. (Basel, Switzerland). MicroBCA protein assay kit was provided by Thermo Fisher Scientific Inc.(Rockford, USA). Polyethyleneimine (PEI) (25 kDa) was obtainedfrom SigmaeAldrich Co. LLC. (Missouri, USA).

2.2. Synthesis of CR8C, CR8C(FA) and C-KLA(TPP)

CR8C (Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys) andCR8C(FA) (Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys-FA) weresynthesized manually on 2-Chlorotrityl chloride resin (1.20 mmol/g), and C-KLA(TPP) (TPP-Lys-Lys-Lys-Leu-Ala-Lys-Leu-Ala-Lys-Lys-Leu-Ala-Lys-Leu-Ala-Lys-Cys) was synthesized manually on RinkAmide-AM resin (0.7 mmol/g), using standard Fmoc-based solid-phase synthesis technique [17,18]. The TPP and FAwere attached tothe peptide as normal amino acid. Peptides were cleaved from theresin in a cleavage cocktail for 1.5 h. The collected cleavage mixturewere concentrated and precipitated in ether. The precipitate wascollected and lyophilized before further use. The purity of all theproducts was assayed by analytical high performance liquid chro-matography (HPLC), and the corresponding purity was proved to beabove 90%. Themolecular weight of C-KLA(TPP), CR8C and CR8C(FA)was measured by electrospray ionization mass spectrometry (ESI-MS). CR8C: calculated 1474.79, found 738.8 (Mþ 2H)2þ. CR8C(FA):calculated 1895.97, found 949.0 (Mþ 2H)2þ and 633.1 (Mþ 3H)3þ.C-KLA(TPP): calculated 2224.43, found 1112.6 (Mþ 2H)2þ.

2.3. Synthesis and characterization of disulfide linked polypeptides

xPolyR8-KLA(TPP) (RK) polypeptide was synthesized by oxida-tive polymerization of CR8C and C-KLA(TPP) at a molar ratio of 3:1in 4ml phosphate-buffered saline (PBS) containing 30% DMSO (v/v)for 96 h at room temperature [19,20]. xPolyR8(FA)-KLA(TPP) (FK)polypeptide was obtained by oxidation of CR8C(FA) and C-KLA(TPP)at a molar ratio of 3:1 using the same technique as for polypeptideRK. The products acquired were purified by dialysis using regen-erated cellulose membrane (MWCO: 3500 Da). The molecularweight and polydispersity (Mw/Mn) of RK and FKweremeasured bysize-exclusion chromatography and multi angle laser light scat-tering (SEC-MALLS). And the Mws of RK and FK were measured tobe 8700 and 8500, respectively. Themolar ratio of CR8C:C-KLA(TPP)in RK and CR8C(FA):C-KLA(TPP) in FK was determined by protonNMR analysis. The molar ratio was found to be 3:1 in both RK andFK. Proton NMR (300 MHz, D2O, d, ppm) of RK: 0.7e1.0 (-CH3),1.2e2.0 (eCH2CH2CH2eCH2-, eCH2CH2eCH2-), 2.6e3.3(eCH2eNH2, eCH2-NH-), 4.0e4.5 (eCHeNH-), 7.5e8.0 (-C6H5).Proton NMR (D2O, d, ppm) of FK: 0.7e1.0 (-CH3), 1.2e2.0(eCH2CH2CH2eCH-, eCH2CH2eCH-), 2.6e3.3 (eCH2eNH2, eCH2-NH-), 4.0e4.5 (eCHeNH-), 7.5e8.0 (-C6H5, eC6H4eNH,eC6H2N4OeNH2).

S. Chen et al. / Biomaterials 77 (2016) 149e163 151

2.4. Synthesis of benzyloxycarbonyl-L-lysine-N-carboxyanhydride(Lys(Z)-NCA)

H-Lys(Z)-OH (7.0 g) was dissolved in dry THF (75 ml) and thesolution was stirred at 50 �C under a steady flow of N2. Then asolution of triphosgene (3.0 g) in dry THF (10 ml) was addeddropwise to the solution and stirred for another 2.5 h. Thereafter,the solution was concentrated and precipitated in excess n-hexane.Finally, Lys(Z)-NCA was obtained by recrystallizing twice in THF/n-hexane and dried under vacuum [21]. Proton NMR spectra ofLys(Z)-NCAwas recorded on a Varian Unity 300 MHz spectrometer.Proton NMR (dimethyl sulfoxide-d6 (DMSO-d6), d, ppm): 1.20e1.75(eCH2CH2CH2eCH2-), 2.99 (eCH2eNH-), 4.43 (eCHeNH-), 5.00(eCH2eC6H5), 7.27e7.36 (-C6H5), 9.09 (eNHeC¼O-).

2.5. Synthesis of monomethoxypoly(ethylene glycol)-poly(L-lysine)(PEG-PLL)

PEG-PLL(Z) was synthesized by ring-opening polymerization(ROP) of Lys(Z)-NCA (2.0 g) initiated by PEG-NH2 (0.2 g) in DMF(15ml) under a steady flow of N2 at 40 �C for 72 h. Then themixturewas concentrated and precipitated in ether to obtain PEG-PLL(Z).Proton NMR spectra of PEG-PLL(Z) was recorded on a VarianUnity 300 MHz spectrometer. Proton NMR (DMSO-d6, d, ppm):1.20e1.75 (eCH2CH2CH2eCH2-), 2.95 (eCH2eNH-), 3.50(eCH2CH2eO-), 4.10 (eCHeNH-), 5.08 (eCH2eC6H5), 7.10e7.40(-C6H5).

Thereafter, PEG-PLL(Z) (1.0 g) was dissolved in TFA (10 ml) in anice bath, then HBr/HAc (5 ml) was added dropwise and the mixturewas stirred for 1.5 h. Afterward, the mixture was concentrated andprecipitated in ether. The precipitate was dissolved in DMF (10 ml)and the solution was dialyzed against distilled water for 72 h usinga regenerated cellulose membrane (MWCO: 3500 Da), and finallylyophilized to obtain PEG-PLL [22]. Proton NMR spectra of PEG-PLLwas recorded on a Varian Unity 300 MHz spectrometer. ProtonNMR (D2O, d, ppm): 1.20e1.75 (eCH2CH2CH2eCH2-), 2.95(eCH2eNH-), 3.50 (eCH2CH2eO-), 4.10 (eCHeNH-).

2.6. Synthesis of poly(ethylene glycol)-blocked-2,3-dimethylmaleicanhydride-modified poly(L-lysine) (PEG-PLL(DA))

0.2 g of PEG-PLL were dissolved in phosphate buffer (pH 8.0),and 0.4 mg of DA were added [23]. 24 h later, the solution wasdialyzed against distilled water (the pH valuewas adjusted to 8.0 by0.1 M NaOH solution) using a regenerated cellulose membrane(MWCO: 3500 Da), and finally lyophilized to obtain PEG-PLL(DA).Proton NMR spectra of PEG-PLL(DA) was recorded on a VarianUnity 300MHz spectrometer. Proton NMR (D2O, d, ppm): 1.20e1.75(eCH2CH2CH2eCH2-), 3.10 (eCH2eNH2), 3.50 (eCH2CH2eO-), 4.10(eCHeNH-)

2.7. Monitor the hydrolysis of PEG-PLL(DA) at pH 6.8 by protonNMR

The PEG-PLL(DA) was dispersed in D2O/DCl (pH ¼ 6.8) andincubated for certain timescales (0, 5, 30, and 60 min), afterwardthe samples were characterized by proton NMR using a VarianUnity 300 MHz spectrometer.

2.8. Cell culture and amplification of plasmid DNA (pDNA)

Human cervix adenocarcinoma (HeLa) cells and mouse embryofibroblast (NIH3T3) cells were used in this study. HeLa and NIH3T3cells were cultured in DMEM and MEM (containing 1% antibiotics(penicillin-streptomycin, 10000 U/ml)) with 10% FBS, respectively.

The DNA plasmids p53 and pGL-3 were used in this study. Redfluorescent protein-tagged p53 expression plasmid and luciferasereporter gene plasmid (pGL-3) were amplified and purified byE. coli JM109 [24,25]. pDNA was dissolved in TriseEDTA (TE) buffersolution at a concentration of 200 ng/ml, and stored at �20 �C forfurther use.

2.9. Preparation of polypeptide/pDNA and polypeptide/pDNA/PEG-PLL(DA) complexes

The complexes of polypeptide/pDNA at different weight ratios(wpolypeptide/wpDNA) were prepared by adding pDNA (1 mg) intoappropriate 1 mg/ml polypeptide solutions at corresponding vol-ume, and then diluted to 100 ml with PBS aqueous solution (10 mM,pH 7.4) and vortexed for 5 s. Finally the complexes were incubatedat 37 �C for 30 min and used immediately [26,27].

Polypeptide/pDNA/PEG-PLL(DA) complexes were prepared byadding PEG-PLL(DA) solution to polypeptide/pDNA complexes. Af-ter incubation at 37 �C for 15 min, polypeptide/pDNA/PEG-PLL(DA)complexes were obtained and used immediately [28].

2.10. Agarose gel retardation assay

Polypeptide/pDNA complexes (wpolypeptide/wpDNA ranging from0.5 to 7) and FK/pDNA/PEG-PLL(DA) complexes (wpolypeptide/wpDNA ¼ 2, wPEGePLL(DA)/wpolypeptide ranging from 0 to 4) werediluted to 8 ml with 10 mM PBS (pH 7.4). After incubated at 37 �C for30 min, the complexes were electrophoresed on the agarose gelcontaining GelRed™ (w/v: 0.7%) for 60 min at 80 V with Tris-acetate (TAE) running buffer [29]. Naked pDNA was used as acontrol.

2.11. Particle size and zeta potential measurements

The particle size and the zeta potential of polypeptide/pGL-3complexes (wpolypeptide/wpGL-3 ¼ 5, 10, 15, 20, 30 and 40) andpolypeptide/pGL-3/PEG-PLL(DA) complexes (wpolypeptide/wpGL-

3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) at pH 7.4 were tested byNano-ZS ZEN3600 (Malvern Instruments) at 25 �C [30]. And thezeta potentials of polypeptide/pGL-3/PEG-PLL(DA) (wpolypeptide/wpGL-3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) at various incubationtimes were measured in PBS (10 mM) at pH 6.8 or 7.4, respectively.Briefly, all complexes were prepared with PBS (10 mM, pH 7.4) in100 ml at first. The zeta potentials at pH 7.4 were measured atvarious incubation timescales after the prepared complexes dilutedto 1 ml with PBS (10 mM, pH 7.4). For pH 6.8, the prepared com-plexes were diluted to 1 ml with PBS (10 mM, pH 6.8), and the pHvalue of the complexes solution was adjusted to 6.8 by adding10 mM HCl solution, and the zeta potentials at pH 6.8 weremeasured at various incubation timescales.

The particle size and zeta potential of FK/pGL-3 complexes(wpolypeptide/wpGL-3 ¼ 20) at various GSH concentrations (0, 1, 2, 5,10 and 20 mM) at pH 7.4 were also measured.

2.12. Transmission electron microscopy (TEM)

After the FK/pGL-3 and FK/pGL-3/PEG-PLL(DA) complexes(wpolypeptide/wpGL-3 ¼ 20) were incubated at pH 6.8 or 7.4 for30 min, their morphologies were observed by TEM (JEM-2100) atan acceleration voltage of 100 kV. The FK/pGL-3 or FK/pGL-3/PEG-PLL(DA) solutions were dripped to copper grid, stained withphosphotungstic acid solution, and dried under infrared lightbefore detection [31].

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2.13. BSA adsorption

2 mg/ml bovine serum albumin (BSA) solutions (1 ml) wasadded to polypeptides, polypeptide/pGL-3 (wpolypeptide/wpGL-

3 ¼ 20) or polypeptide/pGL-3/PEG-PLL(DA) (wpolypeptide/wpGL-

3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) complexes solution, andshaken at 37 �C for 30 min. Then the samples were centrifuged at8000 rpm and the supernatants were collected to determine theBSA concentration using a UV/Vis spectrophotometer (Lambda 35,PerkinElmer Inc., USA) at 280 nm. The amount of BSA adsorbed wasdefined as [BSA]adsorbed¼ ([BSA]t e [BSA]s)/w, where [BSA]t was thetotal amount (mg) of BSA presented in the mixture initially, [BSA]srepresented the amount (mg) of BSA in the supernatant, and wwasthe total amount (mg) of the polypeptide, polypeptide/pGL-3complexes or polypeptide/pGL-3/PEG-PLL(DA) complexes inmixture [32]. The particle sizes of the samples after incubated withBSA solution (final concentration 1 mg/ml) at 37 �C for 30 minwerealso measured.

2.14. Western blotting analysis of the folate receptor expression

HeLa cells and NIH3T3 cells were seeded in 6-well platesrespectively, and incubated for 24 h. After that the cells were lysedin 50 ml RIPA buffer and resuspended in 50 ml 2� SDS sample buffercontaining 1% b-mercaptoethanol. The samples were boiled for5 min and separated on a 10% SDS-PAGE afterward. Then, theproteins were transferred to a PVDF membrane (Millipore), andblocked for 1 h in PBS solution containing 5% skim milk. The folatereceptors were identified by incubating the membranes withpolyclonal antibodies against Folate Receptors (1:100; Santa Cruz)at 4 �C overnight and subsequently treated with the secondaryantibody HRP-labeled goat anti-rabbit IgG (1:3000 dilution, SantaCruz Biotechnology) for 1 h. Specific proteins were detected byenhanced chem-iluminescence (ECL, Pierce). GAPDH antibody(1:10000 dilution, Epitomics) was used as protein loading control.

2.15. Cell uptake characterization by confocal microscopy and flowcytometry

Cell uptake behaviors of RK/pGL-3 and FK/pGL-3 (wpolypeptide/wpGL-3 ¼ 20) in HeLa and NIH3T3 cells at pH 7.4 were observed byconfocal laser scanning microscope (CLSM) (C1eSi, Nikon, Japan).pDNA (1 mg) was incubated with YOYO-1 (2.5 ml, 10 � 10�6 M) for15 min at 37 �C before 1 mg/ml polypeptide (20 ml) were added. Thenthe complexes were diluted to 100 ml with 10 mM PBS (pH 7.4) andincubated at 37 �C for another 30 min. Finally, the complexes weresupplemented to 1 ml by medium with 10% FBS and added to theHeLa cells or NIH3T3 cells. After 1 h incubation, the cells werewashed thrice by PBS, and the cell nuclei were labeled by Hoechst33342. The fluorescence images were acquired by CLSM [33].

The quantitative study of cellular uptake behaviors ofpolypeptide/pGL-3 complexes at pH 7.4 was analyzed by flowcytometry (BD FACSAria™ III, USA). pGL-3 was pre-stained withYOYO-1. HeLa and NIH3T3 cells were incubated with polypeptide/pGL-3 complexes (wpolypeptide/wpGL-3 ¼ 20) for 1 h. Thereafter, thecells were dissociated with 0.25% trypsin, collected, re-suspendedin PBS, filtrated, and estimated by flow cytometry [33].

Cell uptake characterization of RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) (wpolypeptide/wpGL-3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) in HeLa and NIH3T3 cells were also observedusing CLSM. The pGL-3 of RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) were labeled by YOYO-1, then the complexes weresupplemented to 1 ml by medium (pH 6.8 or 7.4) with 10% FBS, andincubated with HeLa cells or NIH3T3 cells for 1 h, the CLSM datawere acquired as described above.

The quantitative study of RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) internalization was carried out by using flow cytom-etry. pGL-3 in RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA)was stained with YOYO-1. HeLa and NIH3T3 cells were incubatedwith RK/pGL-3/PEG-PLL(DA) or FK/pGL-3/PEG-PLL(DA) at either pH7.4 or 6.8 for 1 h. Then, the flow cytometry data were acquired asdescribed above.

For the pDNA release evaluation of FK/pGL-3/PEG-PLL(DA)complexes in vitro, pGL-3 plasmid was stained with YOYO-1,while the C-KLA(TPP) moiety of FK was pre-labeled by Rhoda-mine B (Rh B). HeLa cells were incubated with FK/pGl-3/PEG-PLL(DA) complexes (wpolypeptide/wpGL-3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) at pH 6.8 for 1 h, then the medium was replacedwith fresh medium containing 10% FBS. 0 h or 10 h later, the fluo-rescence image was acquired by CLSM.

For JC-1 assay, HeLa cells were incubated with FK/pGL-3/PEG-PLL(DA) complexes (wpolypeptide/wpGL-3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) at pH 6.8 for 1 h, then incubated with freshmedium containing 10% FBS for 0 h, 12 h, 24 h and 48 h. Finally, themitochondria were labeled by JC-1 for 30 min, and the fluorescenceimage was acquired by CLSM.

2.16. In vitro transfection

The transfection efficiency of RK/pGL-3 and FK/pGL-3 atdifferent wpolypeptide/wpGL-3 ratios ranging from 5 to 40 wasassessed in HeLa and NIH3T3 cells, and PEI/pGL-3 (wPEI/wpGL-

3 ¼ 1.3) was used as the positive control. 24 h after the cells wereseeded in 24-plate, polypeptide/pGL-3 complexes at differentwpolypeptide/wpGL-3 ratios were added into the plates and co-incubated for 1 h. After that, the complexes containing mediumwere replaced with fresh medium and the cells were incubated foranother 48 h. The luciferase expression was assessed by the lucif-erase assay system according to the manufacturer's protocol. Therelative light unit (RLU) was analyzed through chemiluminometer(Lumat LB9507, EG&G Berthold, Germany) and the total proteinconcentration was measured by BCA protein assay kit, and thetransfection efficiency was demonstrated as RLU/mg protein [34].

The transfection efficiency of the RK/pGL-3/PEG-PLL(DA) andFK/pGL-3/PEG-PLL(DA) complexes in HeLa and NIH3T3 cells wasalso measured. When the cells were ready for use, the polypeptide/pGL-3/PEG-PLL(DA) complexes with 1 mg pGL-3 and a particularamount of polypeptide (wPEGePLL(DA)/wpolypeptide ¼ 1.5, wpolypeptide/wpGL-3 ¼ 5, 10, 15, 20, 30 and 40, respectively) in 1 ml medium (pH7.4 or 6.8) with 10% FBS were added and incubated with cells for1 h, then the transfection data were acquired using the same pro-cedure as described above.

2.17. In vitro p53 protein assay

The transfection efficiency of p53 DNA mediated by RK/p53, FK/p53, RK/p53/PEG-PLL(DA) and FK/p53/PEG-PLL(DA) (wPEGePLL(DA)/wpolypeptide ¼ 1.5) complexes at wpolypeptide/wp53 ratio of 20 wasevaluated in HeLa cells with CLSM. When the cells were ready foruse, RK/p53, FK/p53, RK/p53/PEG-PLL(DA) or FK/p53/PEG-PLL(DA)with 1 mg p53 DNA in 1 ml medium (pH 6.8) with 10% FBS wereadded and incubated with HeLa cells for 1 h. Then, the mediumwasreplaced with DMEM containing 10% FBS, and the cells wereincubated for another 48 h. The fluorescence images were acquiredby CLSM [35].

2.18. Cytotoxicity assay in vitro

The cytotoxicity of the polypeptides in HeLa and NIH3T3 cellswas evaluated via MTT assay. The cells at a density of 6000 cells/

S. Chen et al. / Biomaterials 77 (2016) 149e163 153

well were seeded in 96-well plate and incubated for 24 h. After that,the polypeptides at different concentrations were added andincubated with cells for 48 h. Afterward the medium was replacedwith 200 ml fresh medium with 10% FBS. Then 20 ml MTT solutionwas added, and the cells were incubated for another 4 h. Finally, themedium was removed, and 200 ml DMSO was added. The absor-bance was measured by microplate reader (Model 550, Bio-Rad,USA) at 570 nm. The relative cell viability was defined as follows:cell viability (%) ¼ (OD570samples-OD570blank/OD570control-OD570blank) � 100. The OD570control was recorded in the absence ofpolypeptides and OD570samples was recorded in the presence ofpolypeptides [36].

Cytotoxicity of polypeptide/pGL-3 and polypeptide/p53 com-plexes against HeLa and NIH3T3 cells were also tested using MTTassay. When the cells were ready for use, polypeptide/pDNA com-plexes at various concentrations of polypeptides with 1 mg pDNAwere added. After 1 h, the media was replaced with fresh mediumcontaining 10% FBS, and the cells were incubated for another 48 hbefore cytotoxicity data acquired using the same procedure asdescribed above.

Cytotoxicity of RK/pDNA/PEG-PLL(DA) and FK/pDNA/PEG-PLL(DA) complexes against HeLa and NIH3T3 cells was also testedviaMTTassay. 24 h after the cells were seeded in 96-well plates, RK/pDNA/PEG-PLL(DA) and FK/pDNA/PEG-PLL(DA) complexes with aparticular concentration of polypeptides containing 1 mg pGL-3 orp53 (wPEGePLL(DA)/wpolypeptide ¼ 1.5) in 100 ml medium (pH 7.4 or6.8) with 10% FBS were added. 1 h later, the medium was replacedwith fresh mediumwith 10% FBS at pH 7.4. After further incubationfor 48 h, the data of cell viability were acquired as described above.

2.19. Western blot

After the cells seeded in 6-well plate at a density of 2� 104 cells/well for 24 h, the polypeptides, polypeptide/pGL-3 complexes(wpolypeptide/wp53 ¼ 20) or polypeptide/pGL-3/PEG-PLL(DA) com-plexes (wPEGePLL(DA)/wpolypeptide ¼ 1.5, wpolypeptide/wp53 ¼ 20) with20 mg polypeptide in 1 ml mediumwere added and incubated withHeLa cells at pH 6.8 for 1 h. Afterward the medium was replacedwith fresh DMEM containing 10% FBS, and the cells were incubatedfor another 48 h. After which, cells were washed with PBS thriceand lysed with 50 ml RIPA buffer. Then the lysates were re-suspended in 50 ml 2 � SDS sample buffer containing 1% b-mer-captoethanol, boiled for 5 min and separated on a 10% SDS-PAGE.After electrophoresis, the proteins were transferred to PVDFmembranes. The membranes were blocked for 1 h in TBST con-taining 5% skim milk, and incubated with the primary antibodyrabbit monoclonal anti-caspase-3 antibody (1:1000 dilution, CellSignaling Technology), rabbit monoclonal anti-cytochrome c anti-body (1:500 dilution, Boster) or rabbit anti-human p53 (1:1000dilution, Abcam) at 4 �C overnight. After which, the membraneswere incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG, 1:10000 dilution, Santa Cruz Biotechnology), and thespecific proteins were tested by enhanced chemiluminescence(ECL, Pierce). GAPDH antibody (1:10000 dilution, Epitomics) wasused as protein loading control [37].

2.20. In vivo study

Female BALB/c mice (6 weeks old) were obtained from Zhon-gnan Hospital (Wuhan, China) and used as the animal model forex vivo imaging. And the transplantable murine hepatoma22 (H22)model was chose in this study. To establish H22 tumor-bearingmouse model, H22 cells were intraperitoneally injected to aBALB/c mouse, and ascites containing H22 cells were collected fromthe peritoneal cavity of the mouse after 6 days. Then 100 ml of the

ascites containing H22 cells (1 � 107/ml) were subcutaneouslyinoculated to the BALB/c mice at the right flank. The mice weresupplied with sufficient water and food and until the tumor wereready for use, then the mice were intravenously injected with RK,FK, FK/p53 or FK/p53/PEG-PLL(DA) (wPEGePLL(DA)/wpolypeptide ¼ 1.5),respectively. The C-KLA(TPP) moiety in the samples were pre-labeled by RhB, and the doses of polypeptide and p53 were 100and 5 mg per mouse respectively. 6 h after injection, the mice weresacrificed, the organs (heart, liver, spleen, lung, and kidney) as wellas the solid tumor tissues were excised and washed with PBS. Af-terward, the organs and the tumor tissues were imaged using theMaestro in vivo Imaging System (Cambridge Research&Instrumentation, Inc., USA) at excitation and emission wave-lengths of 550 and 580 nm, respectively [38,39].

Tumor suppression study and side effects were also investigatedin this study. After the tumor volume reached around 150 mm3, themice were randomly divided into five groups (six mice per group),and treated with PBS, RK, FK, FK/p53 or FK/p53/PEG-PLL(DA)(wPEGePLL(DA)/wpolypeptide ¼ 1.5) for each group, respectively. Thesamples were intravenous injected in the 1st day, 3rd day and 5thday. The doses of polypeptide and p53 were 300 and 15 mg permouse respectively. Tumor growth was monitored using a caliper.The tumor volume was calculated as: V ¼ 0.5 � (tumorlength) � (tumor width)2. Relative tumor volume was defined as V/V0, and V0 was the tumor volume prior to treatment. Body weightchange (%) defined as W/W0, and W0 was the weight of mice whenthe treatment was initiated. The mice were sacrificed after the tu-mor volume and body weight monitored for 14 days. Then, theorgans and the solid tumor tissues were excised for histologicalexamination by standard hematoxylin and eosin (H&E) staining[40].

3. Results and discussion

3.1. Polymer synthesis and characterization

In this study, PEG-PLL(Z) was obtained from the ROP of Lys(Z)-NCA using PEG-NH2 (Mw 2000) as an initiator, and then PEG-PLLwas obtained by deprotection of PEG-PLL(Z) using HBr/HAc. Thesuccessful synthesis of PEG-PLL(Z) and PEG-PLL was validated byproton NMR spectroscopy. The degree of polymerization of PEG-PLL(Z) and PEG-PLL was both found to be 28 by proton NMRspectrometry (Fig. S2BeC). The molecular weight of PEG-PLL wasdetermined by proton NMR spectrometry to be 6000, and thisresult agreed well with the data (Mw ¼ 6200 g/mol) calculatedaccording to SEM-MALLS. Subsequently, PEG-PLL was modifiedwith DA to obtained final polymer PEG-PLL(DA). The successfulsynthesis of PEG-PLL(DA) was also confirmed by proton NMRspectroscopy (Fig. S2D).

To verify the acidity-triggered hydrolysis of the amide bond ofPEG-PLL(DA), the proton NMR spectra of PEG-PLL(DA) at differenttime scales (0, 5, 30, 60min) after incubated at pH 6.8 was recorded.As shown in Fig. S3, with the increasing of incubation time, the peaka decreased and the peak b increased. The decreased integral ratioof a to b represented the transformation of the carboxyl groups inDA to amino groups, which indicated the acidity-triggered hydro-lysis of the amide bonds.

3.2. Characterization of complexes

The ability to compress pDNA and form stable complexes was aprerequisite of gene vectors [41,42]. After the complexes wereformed, the complexes could protect pDNA against enzymaticdegradation [32]. The pDNA condensing capabilities of RK and FKwere tested by agarose gel electrophoresis assay. As shown in

Fig. 1. (A) Zeta potential changes of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) complexes exposed at pH 7.4 or 6.8 for different time periods. (B) BSA pro-tein adsorption by polypeptide, polypeptide/pGL-3 complexes or polypeptide/pGL-3/PEG-PLL(DA) complexes. Data are shown as the mean ± SD (n ¼ 3).

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Fig. S4, with the increasing amount of polypeptide, the free pDNAwas decreased and the mobility of pDNA was reduced. The com-plete pDNA retardationwas achieved at the wpolypeptide/wpDNA ratioof 1 for RK and 1.6 for FK, respectively. These results showed that RKand FK were able to condense pDNA effectively at low wpolypeptide/wpDNA ratios, and the ability to compress pDNA of FK was slightlyweaker than that of RK, which might be ascribed to the chargedensity reduction of the polypeptide after the introduction offolate.

The introduction of the anionic PEG-PLL(DA) may vary the sta-bility of the polypeptide/pDNA complexes. To verify the stability ofthe polypeptide/pDNA complexes after coated with PEG-PLL(DA),the agarose gel electrophoresis assay was applied to investigatethe compaction of pDNA. As shown in Fig. S4C, FK/pDNA/PEG-PLL(DA) did not release pDNA with the addition of PEG-PLL(DA).This result indicated that the polypeptide/pDNA complexes couldmaintain their stability after coated with PEG-PLL(DA).

To further evaluate the pDNA compressing capability of poly-peptide, the hydrodynamic size and zeta potential of thepolypeptide/pGL-3 complexes at various wpolypeptide/wpGL-3 ratiosranging from 5 to 40 were measured in aqueous solution. As shownin Fig. S5A, the hydrodynamic size of RK/pGL-3 and FK/pGL-3complexes showed a similar trend, i.e., with the increasing of thewpolypeptide/wpGL-3 ratios, the particle size decreased at first andincreased thereafter. RK can condense pGL-3 into smaller particlesthan FK at the same weight ratio. The minimum particle size of RK/pGL-3 complexes was around 76 nm, while the minimum particlesize of FK/pGL-3 complexes was around 133 nm. These resultssuggested that RK could compress pDNA more efficiently than FK.Meanwhile, the zeta potentials of RK/pGL-3 and FK/pGL-3 com-plexes were both above 15 mV. Moreover, the zeta potential of FK/pGL-3 was slightly lower than that of RK/pGL-3, which may beattributed to the reduction of charge density after folate attach-ment on the polypeptide FK. This slight zeta potential differentialcould also lead to the weaker pDNA condensing capability of FKcompared to RK as mentioned above.

To further investigate the stability of polypeptide/pDNA com-plexes after coated with PEG-PLL(DA), the particle size and zetapotential of polypeptide/pGL-3/PEG-PLL(DA) complexes at variouswpolypeptide/wpGL-3 ratios ranging from 5 to 40 (wPEGePLL(DA)/wpolypeptide ¼ 1.5) at pH 7.4 were measured. As shown in Fig. S5Aand S5B, the hydrodynamic size of polypeptide/pGL-3 complexeswas increased after coated with PEG-PLL(DA). And the positivelycharged polypeptide/pGL-3 complexes (Fig. S5A) were changedinto negatively charged particles (Fig. S5B). These results suggestedthat the PEG-PLL(DA) shell was coated on polypeptide/pDNAcomplexes successfully, which shielded the positive charge of thepolypeptide/pGL-3 complexes.

DA-conjugated amines were stable at physiological conditions(pH ~ 7.4), while the amide bonds would promptly degrade underthe extracellular acidity of tumor sites (pHe ~ 6.8) [43,44]. Toidentify the charge shielding effect of PEG-PLL(DA), the zeta po-tential of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA)(wpolypeptide/wpGL-3 ¼ 20, wPEGePLL(DA)/wpolypeptide ¼ 1.5) com-plexes were monitored at simulated physiological conditions (pH7.4) and extracellular acidic tumor sites (pHe 6.8). As shown inFig. 1A, at pH 6.8, the initially negatively charged FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) complexes quickly changedinto positively charged within 10 min, and reached a plateauaround 15 mV after 20 min incubation, while both of these twocomplexes kept negatively charged at pH 7.4 even after 100 minincubation. This acid dependent zeta potential change was mainlyattributed to the fact that the rapid hydrolysis of amide bondsunder the pHe led to the detachment of the charge switching shellPEG-PLL(DA) and exposure of the positively charged surface of the

complexes (as illustrated in Scheme 1D). This charge switch capa-bility of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) com-plexes indicated that they possessed the negatively charged surfacein physiological conditions, such as blood streams, to avoid un-wanted aggregation. Their surface charge changed into positivewhen arrived at tumor sites to facilitate the tumor cell entry.

The size and shape of FK/pGL-3 and FK/pGL-3/PEG-PLL(DA)complexes were further verified by TEM. After FK/pGL-3 and FK/pGL-3/PEG-PLL(DA) complexes incubated at pH 6.8 or 7.4 for30 min, the morphology was observed by TEM respectively. Asshown in Fig. S6, most of the FK/pGL-3 and FK/pGL-3/PEG-PLL(DA)complexes exhibited compact and spherical morphology. Afterincubated at pH 7.4 for 30 min, the size of FK/pGL-3 complexes wasaround 20 nm, and the size of FK/pGL-3/PEG-PLL(DA) was around50 nm. The size of FK/pGL-3/PEG-PLL(DA) complexes was biggerthan that of FK/pGL-3 complexes indicated that the PEG-PLL(DA)shell was coated on FK/pGL-3 complexes successfully. But afterincubated at pH 6.8 for 30 min, the size of FK/pGL-3/PEG-PLL(DA)complexes decreased to around 20 nm which was similar to thatof FK/pGL-3 complexes. The reduced size of FK/pGL-3/PEG-PLL(DA)complexes was mainly attributed to the fact that fast hydrolysis ofamide bonds under the extracellular acidic pH led to the chargeswitch and electrostatically removal of PEG-PLL(DA). All the resultsimplied that PEG-PLL(DA) could be coated on FK/pGL-3 complexesto form FK/pGL-3/PEG-PLL(DA) complexes in physiological condi-tions and would be removed at acidic extracellular tumor sites to

Scheme 1. (A) Chemical structures of C-KLA(TPP), CR8C(FA), PEG-PLL and PEG-PLL(DA). (B) Schematic illustration of the formation of reductive polypeptide FK. (C) Schematicillustration of charge-switchable PEG-PLL(DA). (D) Illustration of the preparation of FK/p53/PEG-PLL(DA) complex with a PEG-PLL(DA) shell. The FK/p53 complex coated with thePEG-PLL(DA) shell to form the detachable FK/p53/PEG-PLL(DA) complex. At the extracellular acidity of tumor sites (pHe), the detachable FK/p53/PEG-PLL(DA) complex removed thePEG-PLL(DA) shell and exposed the FK/p53 complex. (E) Illustration of the stealth property and accumulated in tumor to promote cell uptake of detachable FK/p53/PEG-PLL(DA)complexes. The FK/p53/PEG-PLL(DA) complexes minimized nonspecific interactions with serum components under physiological conditions and exposed the positively charged FK/p53 complexes by removed the shell at the tumor site to promote cell internalization. (F) Illustration of FK/p53 complexes mediated cancer therapy. I: FK/p53 complexes attached totumor cells via electrostatic adherence and folate binding; II: FK/p53 complexes internalized by the tumor cells; III: FK/p53 complexes endosomal escaped to cytoplasm; IV: FK/p53complexes degraded by GSH-triggered to release of the p53 and C-KLA; V: p53 imported to the nucleus; VI: mitochondria destroyed by C-KLA.

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expose the FK/pGL-3 complexes which could facilitate the tumorcell entry.

To further investigate whether the FK/pGL-3 complexes coulddissociate in a reductive environment, the hydrodynamic size andzeta potential of the FK/pGL-3 complexes (wpolypeptide/wpGL-3 ¼ 20)

in the presence of GSH at different concentrations were alsomeasured. As shown in Fig. S5C, the hydrodynamic size of thecomplexes showed a GSH concentration dependent manner. Withthe increase of GSH concentration, the zeta potential reduced from16 mV to �3 mV, while the hydrodynamic size of the complexes

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increased from 167 nm to 636 nm. These significant changes of zetapotential and hydrodynamic size were attributed to the cleavage ofdisulfide bond in the polypeptide, which resulted in the loose anddegradation of FK/pGL-3 complexes. These results implied that theFK/pDNA complexes could dissociate in the reductive environment,leading to the release of pDNA and C-KLA(TPP).

3.3. BSA adsorption

In order to demonstrate whether the PEG-PLL(DA) coating couldreduce the non-specific interactions of positively charged com-plexes with serum proteins, BSA was used as a model protein tosimulate the non-specific protein adsorption at simulated physio-logical conditions (pH 7.4). As shown in Fig. 1B, the capability toinhibit BSA adsorption of RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) complexes was much more strong than that of freepolypeptide and polypeptide/pGL-3 complexes. The strongest in-hibition capability against BSA adsorption of polypeptide/pGL-3/PEG-PLL(DA) complexes was mainly attributed to the fact thatPEG-PLL(DA) shielded the positive charge of the complexes anddiminished the adsorption of BSA proteins. And the particle size ofthe samples after incubated with BSAwas also measured. As shownin Fig. S5D, among all the samples, complexes with PEG-PLL(DA)coating demonstrated the smallest particle size, which indicatedthe excellent stability in blood. All the results suggested thatcoating with PEG-PLL(DA) on the positively charged complexes inphysiological conditions could reduce the aggregation and enhancethe stability in the blood circulation, and extend the blood circu-lation time of complexes potentially.

3.4. Cell uptake characterization by confocal microscopy and flowcytometry

In order to estimate whether the folate moiety could enhancecellular internalization of positively charged complexes, the celluptake of RK/pGL-3 and FK/pGL-3 complexes (wpolypeptide/wpGL-

3¼ 20) in the folate-receptor over-expressed Hela cells (Fig. S7) andfolate receptor negative NIH3T3 cells (Fig. S7) were assessed byCLSM. As shown in Fig. 2AeB, after cultured with cells for 1 h, thefluorescence intensity of FK/pGL-3 was slightly stronger than thatof RK/pGL-3 in Hela cells, while the trend in NIH3T3 cells wasreversal. These differences were attributed to the different mech-anisms of cellular internalization in the two cell lines. FK/pGL-3complexes were mainly internalized into HeLa via receptor-mediated internalization, while RK/pGL-3 complexes were proneto electrostatic interaction-mediated pathway. Meanwhile, bothkinds of complexes entered into NIH3T3 cells via electrostaticinteraction-mediated internalization and RK/pGL-3 complexeswith higher zeta potential exhibited better capacity of internali-zation. To further demonstrate the cellular uptake behaviors ofpolypeptide/pGL-3 complexes, the cell uptake of polypeptide/pGL-3 complexes was also determined quantitatively by flow cytometry.As shown in Fig. 2 GeH, the cell uptake of folate incorporated FK/pGL-3 was approximately 1.5-fold higher than that of RK/pGL-3in Hela cells. The more effective internalization of FK/pGL-3 com-plexes was mainly due to fact that the folate ligands increase thefolate-mediated uptake in folate receptor over-expressed cells.These results implied that the folate functionalization couldenhance the ability of internalization in targeted tumor cells. InNIH3T3 cells, the cell uptake of RK/pGL-3 was higher as comparedwith FK/pGL-3, the less internalization of FK/pGL-3 in NIH3T3 cellsmay due to the fact that the folate attachment reduced the chargedensity of complexes and decreased the driving force of cell uptake.

To identify the role of the tumor-acidity-triggered PEG-PLL(DA)played in the internalization, RK/pGL-3/PEG-PLL(DA) and FK/pGL-

3/PEG-PLL(DA) were compared at simulated physiological condi-tions (pH 7.4) and extracellular acidic tumor sites (pHe 6.8) by CLSMand flow cytometry. As shown in Fig. 2AeF, at pH 7.4, in both Helaand NIH3T3 cells, FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) complexes showed limited internalization compared toFK/pGL-3 and RK/pGL-3 complexes. The limited internalization ofpolypeptide/pGL-3/PEG-PLL(DA) complexes was attributed to thefact that PEG-PLL(DA) shielded the positively charge of complexesand reduced the driving force of cell uptake at pH 7.4. The resultimplied that charge shielding effect of PEG-PLL(DA) could reducethe non-specific cellular uptake in physiological conditions andnormal tissues. Moreover, much stronger fluorescence intensity ofRK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) was found incytoplasm at pH 6.8, indicating more efficient uptake occurred atpH 6.8 than that at pH 7.4 in both HeLa and NIH3T3 cells. Thisuptake improvement was due to electrostatically removal of PEG-PLL(DA) and exposure of the positively charged complexes whichwas favorable for the cell uptake. These results indicated that thepolypeptide/pGL-3/PEG-PLL(DA) complexes could remove the PEG-PLL(DA) shell to expose the positively charged polypeptide/pGL-3complexes which restored the ability of cell entry when arrivedat tumor sites. As shown in Fig. 2IeJ, FK/pGL-3/PEG-PLL(DA) andRK/pGL-3/PEG-PLL(DA) complexes showed limited internalizationat pH 7.4 in both Hela and NIH3T3 cells. However, at pH 6.8, the celluptake levels of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA)were approximately 3.5-fold and 2.5-fold higher than that at pH 7.4in Hela cells. While in NIH3T3 cells, the cell uptake of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) was approximately 2-fold and 3-fold higher than that at pH 7.4, respectively. These re-sults were similar to that of CLSM, and the difference of cell uptakelevels at different pHs may due to the charge shielding effect ofPEG-PLL(DA) at pH 7.4 which minimized the uptake, while theremoval of charge switched PEG-PLL(DA) at pH 6.8 led to moreefficient cell uptake through electrostatic interaction. Meanwhile,the fluorescence intensity of FK/pGL-3/PEG-PLL(DA) was 1.5-foldhigher than that of RK/pGL-3/PEG-PLL(DA) at pH 6.8 in Hela cells.The different internalization of two complexes in HeLa cells wasdue to the fact that folate moiety enhanced the ability of targetedcell entry after PEG-PLL(DA) removal.

Z-stack CLSM was also used to further assess the cellular dis-tribution of polypeptide/pGL-3/PEG-PLL(DA) complexes afterinternalized by tumor cells. As shown in Fig. S8, the green fluo-rescence (pGL-3) was distributed all over the cells. This resultclearly demonstrated the excellent cellular internalization ability ofpolypeptide/pDNA/PEG-PLL(DA) complexes at pH 6.8.

All the cell uptake analyses confirmed that the FK/pDNA/PEG-PLL(DA) complexes could reduce the cell uptake in physiologicalconditions and normal tissues by the charge shielding effect of PEG-PLL(DA). After FK/pDNA/PEG-PLL(DA) arrived at tumor sites, thecomplexes removed the PEG-PLL(DA) shell and exposed the folateincorporated positively charged FK/pDNA complexes to enhancethe cellular internalization of folate receptor over-expressed tumorcells.

To investigate whether the exposed FK/pDNA complexes coulddissociate in cytoplasm, the release of pDNA and C-KLA(TPP) fromFK/pGL-3/PEG-PLL(DA) complexes at pH 6.8 in Hela cells wasassessed by CLSM. The C-KLA(TPP) moiety of FK was pre-labeled byRh B (red), while pGL-3was stainedwith YOYO-1 (green). As shownin Fig. S9A, the green and red fluorescence overlapped very well togive a yellow color in Fig. S9A3. The highly overlap of green and redfluorescence was due to the efficient binding of FK with pGL-3. 10 hlater (Fig. S9B), although some green and red fluorescencewere stilloverlapped, most of them were split from each other, which indi-cated that pGL-3 and C-KLA(TPP) could be released from thecomplexes efficiently (Fig. S9B3e4). These results confirmed that

Fig. 2. Cell uptake study in Hela (A, C, E, G, I) and NIH3T3 cells (B, D, F, H, J). CLSM images treated with polypeptide/pGL-3 complexes (A, B) or polypeptide/pGL-3/PEG-PLL(DA)complexes at pH 7.4 (C, D) or 6.8 (E, F) for 1 h. Scale bars: 20 mm. Flow cytometry of polypeptide/pGL-3 complexes (G, H) or polypeptide/pGL-3/PEG-PLL(DA) complexes (I, J)after incubated with cells for 1 h (*p < 0.05 as compared with the data of blank, *p < 0.01 as compared with the data of blank).

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after internalized by HeLa cells for 10 h, the disulfide bond of the FKwas cleaved under the reductive environment of cytoplasm,resulting in the successful release of C-KLA(TPP) and gene.

The JC-1 assay were also performed against HeLa cells to furtherverify that the FK/pDNA/PEG-PLL(DA) complexes could result in thedisruption of mitochondria after internalized by tumor cells. Asshown in Fig. 3, with the increasing incubation time, the J-mono-mer green fluorescence increased accompanied with the decreaseof J-aggregates red fluorescence. This fluorescence change wasascribed to the mitochondrial dysfunction of the HeLa cells, whichwas caused by the fact that the released C-KLA(TPP) moiety couldspecifically disrupt the mitochondrial membrane [45]. Therefore,this result demonstrated that the FK/pDNA/PEG-PLL(DA)

complexes could disrupt mitochondria effectively after internalizedby tumor cells.

3.5. In vitro transfection

The transfection efficiency of FK/pGL-3 and RK/pGL-3 com-plexes was evaluated in Hela and NIH3T3 cells. As shown inFig. 4AeB, in HeLa cells, the transfection efficiencies of FK/pGL-3and RK/pGL-3 were both increased initially and then reached astable plateau, and finally decreased slightly. The transfection ef-ficiency of FK/pGL-3 was slightly higher than that of RK/pGL-3 inHeLa cells, the higher transfection efficiency of FK/pGL-3 mainlydue to the fact that folate incorporated FK/pGL-3 complexes could

Fig. 3. CLSM images with JC-1 assay of Hela cells with FK/pGL-3/PEG-PLL(DA) com-plexes at pH 6.8 for 0 h (A), 12 h (B), 24 h (C) and 48 h (D). Scale bars: 20 mm.

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internalize more effectively as compared with RK/pGL-3 in folatereceptor over-expressed cells. In NIH3T3 cells, the transfection ef-ficiency of FK/pGL-3 was lower than RK/pGL-3, and the lower

Fig. 4. Luciferase expression mediated in HeLa cells (A, C) and NIH3T3 cells (B, D) by the polypH 6.8 (filled with diagonal lines) and pH 7.4 (blank). Data are shown as mean ± SD (n ¼ 4) (of PEI/pGL-3).

transfection efficiency of FK/pGL-3 was attributed to less uptake ofFK/pGL-3 than RK/pGL-3 in folate receptor negative cells. The resultimplied that compared with RK/pDNA, the folate incorporated FK/pDNA exhibited enhanced transfection efficiency in targeted tumorcells and decreased transfection efficiency in normal cells, whichagreed well with the data of cell uptake.

To identify the effect of the tumor-acidity-triggered PEG-PLL(DA) in transfection, the transfection efficiency of FK/pGL-3/PEG-PLL(DA) and RK/pGL-3/PEG-PLL(DA) at simulated physiolog-ical conditions (pH 7.4) and extracellular acidic tumor sites (pHe6.8) were evaluated. As shown in Fig. 4CeD, FK/pGL-3/PEG-PLL(DA)and RK/pGL-3/PEG-PLL(DA) complexes showed limited trans-fection efficiency at pH 7.4, the limited transfection efficiency ofpolypeptide/pGL-3/PEG-PLL(DA) was mainly attributed to the factthat the shielding effect of PEG-PLL(DA) at pH 7.4 reduced the celluptake. At pH 6.8 the transfection efficiency of polypeptide/pGL-3/PEG-PLL(DA) became much higher which even comparable to PEI/pGL-3 in both Hela and NIH3T3 cells. The high transfection effi-ciency of polypeptide/pGL-3/PEG-PLL(DA) at pH 6.8 mainly due tothe fact that the exposed positively charged complexes enhancedthe cell uptake and promoted the transfection efficiency. The resultwas consistent with the data of cell uptake and indicated that PEG-PLL(DA) could reduce the transfection efficiency of complexes atsimulated physiological conditions while enhance the transfectionefficiency at extracellular acidic tumor sites. Moreover, FK/pGL-3/PEG-PLL(DA) exhibited slightly higher transfection efficiency thanRK/pGL-3/PEG-PLL(DA) in HeLa cells, and much lower transfectionefficiency than RK/pGL-3/PEG-PLL(DA) in NIH3T3 cells at pH 6.8.This result indicated that the folate moiety enhanced the trans-fection efficiency in targeted cells after the removal of PEG-PLL(DA)shell.

3.6. In vitro p53 protein assay

In order to visualize the transfection of the complexes at pH 6.8directly by CLSM, the red fluorescent protein-tagged p53 DNA wasused to prepare the complexes. As shown in Fig. S10, the

peptide/pGL-3 complexes (A, B) or polypeptide/pGL-3/PEG-PLL(DA) complexes (C, D) at*p < 0.05 as compared with the data of PEI/pGL-3, *p < 0.01 as compared with the data

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fluorescence images of the cells treated by FK/p53 and FK/p53/PEG-PLL(DA) exhibited more and stronger red fluorescence, whichindicated more efficient transfection as compared with both RK/p53 and RK/p53/PEG-PLL(DA) in HeLa cells at pH 6.8. These resultsagreed well with the transfection assay of pGL-3, and also validatedthat the introduction of targeting agent could improve the trans-fection efficiency of positively charged complexes in targeted cells.

3.7. Cytotoxicity assay in vitro

To investigate whether the enhanced cellular internalization bythe folate modification could lead to increased anticancer activity,cell cytotoxicity assay was performed in the folate-receptor over-expressed Hela cells and folate receptor negative NIH3T3 cells. As

Fig. 5. Cell viability of polypeptide (A, B), polypeptide/pDNA complexes (C, D) and polypeptid(A, C, E, G) and NIH3T3 cells (B, D, F, H) for 48 h. Data are shown as mean ± SD (n ¼ 4).

displayed in Fig. 5AeB, concentration-dependent toxicity towardscells was found in both RK and FK. The toxicity was attributed to thefact that C-KLA(TPP) moiety of polypeptide could specificallydisrupt themitochondrial membrane and result in cell death. It wasalso found that RK exhibited high cytotoxicity in both HeLa cellsand NIH3T3 cells due to its excellent cellular internalization ability.In addition, the toxicity of FK was much higher than that of RK inHeLa cells, the higher toxicity of FK was due to the fact that folate-modified enhanced the cell uptake in folate receptor over-expressed tumor cells and result in more tumor cell death. Incontrast, lower toxicity of FK was found in NIH3T3 cells, which wasattributed to the fact that folate attachment reduced the chargedensity of complexes and led to less internalization in normal cells.The purpose of co-delivery of p53 and C-KLA(TPP) was to achieve

e/pDNA/PEG-PLL(DA) complexes at pH 7.4 (E, F) or 6.8 (G, H) incubated with HeLa cells

Fig. 6. Fluorescent images of excised organs detected at 6 h post-injection with (A) RK,(B) FK, (C) FK/p53 or (D) FK/p53/PEG-PLL(DA).

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synergistic effect. The anticancer activity of polypeptide/p53 com-plexes was further evaluated, and polypeptide/pGL-3 was used ascontrol. As shown in Fig. 5CeD, FK/p53 and RK/p53 exhibitedmuchhigher cytotoxicity than that of FK/pGL-3 or RK/pGL-3. The highercytotoxicity of polypeptide/p53 complexes was attributed to theco-delivery of p53 gene and C-KLA(TPP), while the p53 gene sup-pressed tumor cell growth through the extrinsic “death” receptorssignaling pathway. C-KLA(TPP) could trigger mitochondria-basedprogrammed cell death by regulated the intrinsic “mitochondria”pathway. This result indicated that co-delivery p53 and proa-poptosis peptide C-KLA(TPP) could induce significantly enhancedcell inhibition. Moreover, the cytotoxicity of FK/p53 was muchhigher than that of RK/p53 in HeLa cells. The higher cytotoxicity ofFK/p53 was attributed to the fact that FK/p53 led to more effectiveinternalization of targeted cells and resulted in more targeted tu-mor cell inhibition. However, in NIH3T3 cells, FK/p53 showed lowercytotoxicity than RK/p53 because the uptake of FK/p53 in NIH3T3cells was less than RK/p53, which results in less cell death. Clearly,compare with RK/p53, the folate incorporated FK/p53 increasedanticancer activity in targeted cells and reduced the side effects ofnormal cells.

In order to evaluate the effect of charge-switchable PEG-PLL(DA)of anticancer activity, the cytotoxicity of RK/p53/PEG-PLL(DA) andFK/p53/PEG-PLL(DA) was assessed in HeLa and NIH3T3 cells atsimulated physiological conditions (pH 7.4) and extracellular acidictumor sites (pHe 6.8). For comparison, the cytotoxicity of RK/pGL-3/PEG-PLL(DA) and FK/pGL-3/PEG-PLL(DA) was also evaluated. Asshown in Fig. 5EeH, RK/p53/PEG-PLL(DA) and FK/p53/PEG-PLL(DA)show lower cytotoxicity than RK/p53 or FK/p53 at pH 7.4. Thelimited cytotoxicity of polypeptide/p53/PEG-PLL(DA) was attrib-uted to the low cellular internalization which caused by the chargeshielding effect of PEG-PLL(DA) at pH 7.4. In addition, RK/p53/PEG-PLL(DA) and FK/p53/PEG-PLL(DA) exhibited higher cytotoxicity atpH 6.8 than pH 7.4 in both HeLa and NIH3T3 cells, which wasprobably due to the removal of charge switched PEG-PLL(DA) andexposure of the positively charged complexes at pH 6.8 whichfacilitated the cell uptake and resulted inmore cell death. As shownin Fig. 5GeH, RK/p53/PEG-PLL(DA) showed higher cytotoxicity to-ward NIH3T3 cells than that of HeLa cells at pH 6.8. The highertoxicity was attributed to the excellent cellular internalizationability of RK/p53/PEG-PLL(DA) at pH 6.8 and the lower tolerabilityof NIH3T3 cells. Furthermore, compared to RK/p53/PEG-PLL(DA),FK/p53/PEG-PLL(DA) showed lower cytotoxicity toward NIH3T3cells, and higher cytotoxicity against HeLa cells at pH 6.8. The dif-ference of cytotoxicity in different cells was due to the fact thatsynergic effect of folate bind and electrostatic interaction enhancedthe cell uptake in targeted cells. These results demonstrated thatthe enhanced cellular internalization by the folate modified andcharge-switchable not only increased anticancer activity, but alsoreduced the side effects of normal cells.

3.8. Western blot analysis

In order to further validate the synergistic effect of C-KLA(TPP)and p53, the expression of the apoptosis associated proteins,caspase-activating proteins (caspase-3), cytochrome c and p53, wasexamined by western blot analysis. As shown in Fig. S11, at pH 6.8,polypeptide/p53 complexes showed a much higher level of p53protein expression than that of polypeptide. Moreover, comparedto polypeptide and polypeptide/p53, polypeptide/p53/PEG-PLL(DA) not only induced much higher p53 expression and morecytochrome c release, but also induced more caspase-3. The highexpression of p53, cytochrome c and caspase-3 by polypeptide/p53/PEG-PLL(DA) might due to the fact that the PEG-PLL(DA)shield reduced the non-specific interactions of complexes with

serum proteins to some extent even in pH 6.8 and resulted in morecell uptake. Furthermore, due to the targeting property of folic acidmoiety in the FK peptide, the amounts of cytochrome c, caspase-3and p53 protein expressed in HeLa cells mediated by FK/p53/PEG-PLL(DA) were slightly higher than that by RK/p53/PEG-PLL(DA).These results indicated that FK/p53/PEG-PLL(DA) could promotethe release of cytochrome c and the expression of caspase-3 andp53 effectivelywhich could block the anti-apoptotic effect of cancercell, and thus lead to cell apoptosis effectively.

3.9. The fluorescence image studies

To investigate the biodistribution of RK, FK, FK/p53 and FK/p53/PEG-PLL(DA), all samples were labeled by RhB, ex vivo RhB fluo-rescence imaging of the organs (heart, liver, spleen, lung, kidney)and tumors in H22 tumor-bearing mice was observed. As shown inFig. 6, 6 h after injection, compared with other samples, the FK/p53/PEG-PLL(DA) treated mice hold the weakest RhB signal in bothkidney and liver. Moreover, strong fluorescence signal was onlyfound in tumor tissues for FK/p53/PEG-PLL(DA) treated mice, whilenegligible signals were found in tumor tissues from mice treatedwith other samples. The effective accumulation in tumor sites ofFK/p53/PEG-PLL(DA) was attributed to the excellent stability dur-ing the blood circulation, reduced clearance by the reticuloendo-thelial system, EPR effect and the targeting ability of folate. Theseresults indicated that FK/p53/PEG-PLL(DA) could accumulate intotumor effectively and reduce the uptake by liver and kidney, whichcould lead to high antitumor efficiency with low side effects.

3.10. Tumor suppression study and side effects evaluation

To assess the in vivo antitumor efficacy of RK, FK, FK/p53 and FK/p53/PEG-PLL(DA), BALB/c mice bearing H22 xenograft tumor wereused as animal model. As shown in Fig. 7A, on the 14th day, therelative tumor volume of RK, FK, FK/p53 and FK/p53/PEG-PLL(DA)were 15.4, 7.3, 4.6 and 1.5, respectively, while that of PBS reached29.0. The smaller relative tumor volume included the more effec-tive growth inhibition, and the tumors of all treated groupsexhibited growth inhibition compared to the negative control.Moreover, FK/p53/PEG-PLL(DA) exhibited the highest efficiency oftumor growth inhibition compared to all the samples (Fig. 7A, C andD), The high antitumor activity of FK/p53/PEG-PLL(DA) may be onaccount of the charge shielding effect of PEG-PLL(DA) prolongedblood circulation time and reduced clearance by the reticuloen-dothelial system. When the complexes arrived at tumor sites, the

Fig. 7. Tumor suppression study in vivo via intravenous injection once every other day in the first five days (black arrows). (A) Relative tumor volume after post-treatment indifferent groups, the statistical significance of FK/P53/PEG-PLL(DA) compared to all other groups (*p < 0.001 versus PBS, #p < 0.001 versus RK, *p < 0.001 versus FK and þp < 0.05versus FK/p53) as determined by a Student's-test; (B) body weight changes of the mice with different groups; (C) images of the mice were taken on the 14th day after treatment, theblack dotted line circle indicate the tumors; (D) images of the tumors at the 14th day post-treatment. (E) H&E staining images of organs and tumor tissues which were sacrificed atthe 14th day after treatment of various groups. I: PBS, II: RK, III: FK, IV: FK/p53, V: FK/p53/PEG-PLL(DA).

S. Chen et al. / Biomaterials 77 (2016) 149e163 161

removal of PEG-PLL(DA) and exposure of the folate decoratedpositively charged FK/p53 could result in the effective accumula-tion in tumor sites. After internalized by tumor cells, FK/p53 wasdegraded in cytoplasm under the stimulation of GSH which furtherreleased p53 gene and C-KLA(TPP) moiety for the efficient cellapoptosis by regulating both extrinsic and intrinsic apoptoticpathways. The antitumor activity of FK/p53 was lower than FK/p53/PEG-PLL(DA) due to the fact that positively charged FK/p53 withoutPEG-PLL(DA) shell may lead to aggregation in blood circulation andresult in less accumulation in tumor sites. Due to the co-delivery C-

KLA(TPP) and p53, the antitumor activity of FK/p53was higher thanFK and RK. All the results of tumor suppression study demonstratedthat the surface charge-switchable and folate incorporated co-delivery system FK/p53/PEG-PLL(DA) could inhibit tumor growtheffectively.

The mouse body weight was monitored throughout the thera-peutic period to evaluate the side effects of all groups. In Fig. 7B, thebody weights of all groups showed similar smooth body weightfluctuations suggesting no indication of systemic toxicity versusnegative control.

S. Chen et al. / Biomaterials 77 (2016) 149e163162

To further evaluate the antitumor efficacy and side effects, theorgans and tumor of treated mice were separated for H&E stainingat 14th day. As shown in Fig. 7E, comparedwith negative control, alltreated groups were no obvious degenerations, abnormalities orlesions found in the major organs. These results were indicated thelow systemic toxicity for all groups and agreed well with the data ofbody weight change. Moreover, the amount of dead tumor cellstreated by RK, FK, FK/p53 and FK/p53/PEG-PLL(DA) successivelyincreased, and the FK/p53/PEG-PLL(DA) treated group had largestnecrosis area compared with the other groups. The result indicatedthat FK/p53/PEG-PLL(DA) could lead tomore tumor cells death thanother treated group, which was consistent with the data of tumorsuppression study. All the results validated that the FK/p53/PEG-PLL(DA) complexes could enhance antitumor efficacy and reducethe side effects effectively.

4. Conclusions

In this study, we designed and fabricated a surface charge-switchable and folate modified co-delivery system FK/p53/PEG-PLL(DA) to improve the antitumor efficacy and reduce undesir-able side effects which result in effective cancer therapy. This sys-tem combined multiple functions, such as the excellent stability inthe blood circulation, targeting ability of tumor sites and the abilityof effective internalization by targeted cells, displayed great po-tential for co-delivery proapoptosis peptide C-KLA(TPP) and p53gene via intravenous injection in the cancer therapy. At the physi-ological conditions, FK/p53/PEG-PLL(DA) complexes could extendthe circulating time due to the charge shielding effect of PEG-PLL(DA) in the bloodstream. At tumor sites with extracellularacidity, the removal of PEG-PLL(DA) led to the exposure of folateincorporated positively charged FK/p53 to facilitate the targetedtumor cell entry. FK/p53/PEG-PLL(DA) complexes exhibited highaccumulation in tumor sites, superior efficiency of tumor growthinhibition and low toxicity of normal tissue in vivo. The ternary co-delivery system FK/p53/PEG-PLL(DA) with the detachable surfacelayer supplies a useful strategy for synergetic anticancer therapy.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (51125014, 51233003, 21474077 and51533006).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2015.11.013.

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