International Journal of Pharmaceutics 510 (2016) 394–405

Pharmaceutical Nanotechnology

Brain tumor-targeted therapy by systemic delivery of siRNA withTransferrin receptor-mediated core-shell nanoparticles

Lin Weia,b, Xi-Ying Guob, Ting Yangb, Min-Zhi Yub, Da-Wei Chena,**, Jian-Cheng Wangb,*aDepartment of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, ChinabBeijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, State Key Laboratory of Natural and Biomimetic Drugs, School ofPharmaceutical Sciences, Peking University, Beijing, 38 Xueyuan Road, 100191, China

A R T I C L E I N F O

Article history:Received 12 February 2016Received in revised form 26 June 2016Accepted 28 June 2016Available online 29 June 2016

Keywords:siRNAGene therapyGliomaCore-shell nanoparticleTargeted delivery

A B S T R A C T

Treatment of brain tumor remains a great challenge worldwide. Development of a stable, safe, andeffective siRNA delivery system which is able to cross the impermeable blood-brain barrier (BBB) andtarget glioma cells is necessary. This study aims to investigate the therapeutic effects of intravenousadministration of T7 peptide modified core-shell nanoparticles (named T7-LPC/siRNA NPs) on braintumors. Layer-by-layer assembling of protamine/chondroitin sulfate/siRNA/cationic liposomes followedby T7 peptide modification has been carried out in order to obtain a targeted siRNA delivery system. Invitro cellular uptake experiments demonstrated a higher intracellular fluorescence intensity of siRNA inbrain microvascular endothelial cells (BMVECs) and U87 glioma cells when treated with T7-LPC/siRNANPs compared with PEG-LPC/siRNA NPs. In the co-culture model of BMVECs and U87 cells, a significantdown-regulation of EGFR protein expression occurred in the U87 glioma cells after treatment with theT7-LPC/siEGFR NPs. Moreover, the T7-LPC/siRNA NPs had an advantage in penetrating into a deep regionof the tumor spheroid compared with PEG-LPC/siRNA NPs. In vivo imaging revealed that T7-LPC/siRNANPs accumulated more specifically in brain tumor tissues than the non-targeted NPs. Also, in vivo tumortherapy experiments demonstrated that the longest survival period along with the greatestdownregulation of EGFR expression in tumor tissues was observed in mice with an intracranial U87glioma treated with T7-LPC/siEGFR NPs compared with mice receiving other formulations. Therefore, webelieve that these transferrin receptor-mediated core-shell nanoparticles are an important potentialsiRNA delivery system for brain tumor-targeted therapy.

ã 2016 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

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1. Introduction

Glioma is one of the most aggressive and lethal types of cancer.Owing to the high rates of growth, invasiveness and infiltration,patients with gliomas have an extremely poor prognosis: the 5 yearsurvival rate is less than 10% (Gao et al., 2014; Li et al., 2014a;Zanotto-Filho et al., 2013). The special pathological and physiolog-ical characteristics make glioma treatment very difficult. Forchemotherapy, the non-targeted nature of drugs and the physio-logical barriers, including the blood-brain barrier (BBB), haslimited its efficacy (Garanti et al., 2016; Qi et al., 2014). Thedevelopment of anti-cancer drugs able to cross the BBB and

* Corresponding author at: Department of Pharmaceutics, School of Pharmaceu-tical Sciences, Peking University, Xueyuan Road 38, Beijing, 100191, China.** Corresponding author.

E-mail addresses: chendawei@syphu.edu.cn (D.-W. Chen),wang-jc@bjmu.edu.cn (J.-C. Wang).

http://dx.doi.org/10.1016/j.ijpharm.2016.06.1270378-5173/ã 2016 Elsevier B.V. All rights reserved.

specifically target cancer cells would be a promising approach forglioma treatment.

In order to meet this challenge, many attempts have focused onnanoparticle drug delivery systems which could make a significantcontribution to increase the permeability and retention (EPR)effect and cross the blood-brain barrier (Kang et al., 2014; Resnieret al., 2013). However, because of a lack of specific targeting, it isdifficult for the nanoparticles to accumulate in brain tumorsmerely based on their nano-size. Thus, various targeted strategieshave been applied in the field of nanocarriers to overcome the BBBand target the glioma (Mei et al., 2014; Zhang et al., 2014). Amongthese strategies, a receptor-mediated drug delivery system is oneof the most promising strategies (Chirio et al., 2014; Hao et al.,2013). Transferrin receptors (TfR) are highly expressed in bothbrain microvascular endothelial cells (BMVECs) and brain gliomacells (Kang et al., 2015; Srimanee et al., 2016). A seven-peptide(sequenced HAIYPRH, T7) screened by a phage display system has ahigher affinity for TfR, with a Kd of �10 nM. In recent years, T7

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peptide has been used as a ligand in targeted drug delivery systemsfor transferrin receptor-mediated glioma therapy (Han et al., 2011;Youn et al., 2014). Zong and co-workers have developed dual-targeting doxorubicin liposomes conjugated with transferrin (T7)and cell-penetrating peptide (TAT) (DOX-T7-TAT-LIP) for trans-porting drugs across the BBB and targeting brain glioma (Zonget al., 2014b). Jiang and co-workers have designed glioma-targeteddendrimer nanoparticles (DGL-PEG-T7) for the delivery of DNA invitro and in vivo (Kuang et al., 2013).

In our previous study, we developed T7 peptide modified core-shell cationic nanoparticles [T7 peptide-liposome-protamine-chondroitin sulfate/siRNA nanoparticle (T7-LPC/siRNA NP)] con-taining EGFR siRNA, which are dramatically internalized in MCF-7cells and exhibited significant cell growth inhibition against MCF-7cells in vitro (Yang et al., 2011). From the promising siRNA silencingeffect resulting from T7-LPC/siRNA NP, it is expected that thesesiRNA-loaded nanoparticles could also make a contribution totransferrin receptor-mediated glioma therapy. T7-LPC/siRNA NPpossesses its own merits in terms of its nanostructure and materialcomposition. Firstly, T7-LPC/siRNA NP is a typical core-shell typenanoparticle which exhibits high structural stability and drugprotection ability (Hadinoto et al., 2013; Tseng et al., 2009).Secondly, the materials (lipids, protamine, chondroitin sulfate)used in this siRNA delivery system are biocompatible (Hathcockand Shao, 2007).

The aim of this study was to investigate the targetedtherapeutic efficacy of T7-LPC/siRNA NP in mice bearing a glioma(Scheme 1). In the co-culture model of brain microvascularendothelial cells (BMVECs) and U87 glioma cells, the penetrationacross the BBB and the down-regulation of specific protein wereinvestigated. A tumor spheroid model was used to predict thepenetration in tumor tissue and the inhibitory effects on tumorgrowth for T7-LPC/siRNA NP. In vivo imaging and in vivo tumorgrowth inhibition confirmed the efficient distribution andtherapeutic efficacy of the transferrin receptor-mediated core-shell siRNA-loaded nanoparticles.

Scheme 1. Schematic illustration of the preparation and biological effects of T7-LPC/sassembling of protamine/chondroitin sulfate/siRNA/cationic liposomes followed by T7 pwere observed from the mice bearing intracranial U87 glioma after intravenous admin

2. Materials and methods

2.1. Materials

Cholesterol, dioleoyl-phosphatidylethanol-amine (DOPE) anddioleoyl-trimethylammonium-propane (DOTAP) were obtainedfrom Avanti Polar Lipids, Inc. (Alabaster, AL, USA); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine with covalently linkedpolyethylene glycol of molecular weight 2000 (DSPE-PEG2000)and 1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine-n-[poly-(ethyleneglycol)]-hydroxy succinamide, PEG2000] (DSPE-PEG2000-NHS) were obtained from NOF Co. (Tokyo, Japan); The peptideHAIYPRH (named T7 peptide) was obtained from ShanghaiC-Strong Co., Ltd. (Shanghai, China). Chondroitin sulfate (CS)was obtained from Shandong Yibao Biological Product Co., Ltd.(Shandong, China); Protamine was obtained from Sigma-Aldrich(St. Louis, MO); OPTI-MEM was obtained from Invitrogen (NY,USA); Agarose was obtained from GENE COMPANY (Hong Kong,China).

Anti-EGFR siRNA, named siEGFR (sense strand: 50-AGG AAUUAA GAG AAG CAA CAU dTdT-30; antisense strand: 50-AUG UUGCUU CUC UUA AUU CCU dTdT-30), negative control scrambledsiRNA named siNC (sense strand: 50-UUC UCC GAA CGU GUC ACGUTT-30; antisense strand: 50-ACG UGA CAC GUU CGG AGA ATT-30)and fluorescein-labeled siRNA (50 end of the sense strand, namedFAM-siRNA and Cy5-siRNA) were synthesized by Ribobio Co. Ltd(Suzhou, China).

2.2. Cell culture and animals

U87 cells (human glioblastoma cells) were obtained from theInstitute of Materia Medica, Chinese Academy of Medical Scienceand Peking Union Medical College (Beijing, China). Cells werecultured in MEM supplemented with 1% non-essential aminoacids, 10% FBS (PAN, GER), 100 IU/mL penicillin and 100 m g/mLstreptomycin. BMVECs (murine brain microvascular endothelialcells) were kindly provided by Dr. Xiao-yan Liu, Peking University.Cells were cultured in DMEM supplemented with 20% FBS (GIBCO,

iRNA NPs. The targeted siRNA delivery system is obtained from the layer-by-layereptide modification. The best downregulation of EGFR expression in tumor tissuesistration of T7-LPC/siEGFR NPs.

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USA), 100 IU/mL penicillin, 100 m g/mL streptomycin, 40 U/mlheparin, and 100 mg/mL endothelial cell growth factor. All cellswere maintained at 37 �C in a humidified atmosphere containing5% CO2. Cells for all experiments were in the logarithmic phase ofgrowth.

Male nu/nu mice (20 � 25 g) were purchased from Vital RiverLaboratory Animal Center (Beijing, China) and were raised in aspecific pathogen-free (SPF) environment. All care and handling ofthe animals were in compliance with the approved guidelines ofthe Institutional Authority for Laboratory Animal Care of PekingUniversity.

2.3. Preparation and characterization of T7-LPC/siRNA nanoparticles

The core-shell type T7-LPC/siRNA nanoparticles were preparedby layer-by-layer assembling according to the methods reported ina previous study (Feng et al., 2014). Briefly, DOTAP, DOPE, andcholesterol (1:1:1, n/n/n) were dissolved in dichloromethane andevaporated under vacuum to prepare a thin film followed byultrasonic dispersion to obtain cationic liposomes. The negativelycharged nanoparticle core was a ternary complex composedof siRNA, chondroitin sulfate (CS) and protamine. Then, a solutionof protamine was added to the mixture of siRNA and CS(siRNA/CS = 1/1, w/w) to obtain a negatively charged ternarycomplex by electrostatic interaction. The weight ratio of prot-amine/(siRNA + CS) in the nanoparticle core was 1/1.2 (w/w). Theresulting nanoparticle core was then allowed to stand at roomtemperature for 10 min, then coated with the cationic lipid shell(lipids/siRNA = 2000/1, n/n) and incubated at room temperature for10 min. Finally, PEG-DSPE or T7-PEG-DSPE (the detailed synthesisand characterization is seen in Supplementary Information) wasadded to the nanoparticle suspension for PEGylation. The molarratio of PEG in total lipids was 5%. After incubation at 55 �C for15 min, non-targeted PEG-LPC/siRNA NPs or targeted T7-LPC/siRNA NPs were obtained. The particle size and zeta potential of thedifferent nanoparticles were measured at 25 �C using dynamiclight scattering (MalvernZetasizer nano ZS, Malvern, UK).

2.4. Cellular uptake

Confocal laser scanning microscopy (CLSM) was used to observethe intracellular distribution of siRNA. LPC/siRNA NPs, PEG-LPC/siRNA NPs, and T7-LPC/siRNA NPs, containing Cy5-siRNA, wereprepared as described above. BMVECs and U87 cells wereseparately seeded on glass-bottom dishes at a density of 2 �105

cells per dish and incubated for 24 h. Then, the culture medium wasdiscarded and followed by two washes with PBS. The Cy5-siRNAnanoparticles were dissolved in 2 mL fresh OPTI-MEM and treatedwith the cells. The final concentration of Cy5-siRNA was 100 nM.After 4 h incubation, the cells were washed three times with PBSand fixed with 4% paraformaldehyde for 10 min at room tempera-ture. Then, the cell nucleus and F-actin were individually stainedwith Hoechst 33258 and Rhodamine labeled phalloidin 10 min atroom temperature. Finally, images of the cells were recorded usinga Leica TCS SP8 confocal fluorescence microscope (Leica Micro-systems, Heidelberg, Germany).

Flow cytometry analysis was used to quantify the intracellularfluorescence intensity of FAM-siRNA after cellular uptake. BMVECsand U87 cells were separately seeded on glass-bottomed dishes ata density of 3 � 105 cells per dish and incubated for 24 h. The FAM-siRNA encapsulated nanoparticles were incubated with the cellsseparately for 4 h. The final concentration of FAM-siRNA was100 nM. After incubation, the cells were harvested and washedthree times with pre-cooled PBS solution. The fluorescenceintensity of FAM-siRNA per 10,000 cells was measured using FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA, USA).

2.5. Studies of in vitro gene silencing

The EGFR mRNA levels in U87 cells were measured by qRT-PCR(quantitative real-time polymerase chain reaction). The cells weretreated with siEGFR, T7-LPC/siNC NPs, PEG-LPC/siEGFR NPs, andT7-LPC/siEGFR NPs for 24 h. The final concentration of siEGFR usedin the experiment was 100 nM. Total RNA was extracted usingTRNzol reagent according to the manufacturer’s protocol. The RNAwas transcribed into the first-strand cDNA using a reversetranscription kit (Promega, Wisconsin, USA). Next, the cDNA wassubjected to qRT-PCR analysis, EGFR and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using GoTaq1 qPCR MasterMix of Promega. GADPH was amplified as an endogenousreference. The analysis was performed by a real-time PCR detectionsystem (IQ5 Bio-Rad, USA) and the relative gene expression wasquantified by the DCt method using the Optical System Softwareversion 2.0 (IQ5 Bio-Rad, USA).

2.6. Studies on the penetration across the BBB in the co-culture cellmodel

To evaluate the BBB penetration ability of the siRNA formu-lations, a co-culture model of BMVECs and U87 cells wasestablished. BMVECs were seeded at a density of 1 �105

cells/cm2 on a 2% gelatin coated upper-well of a 12-insert cell(Corning, NY, USA, 0.4 mm pore size, 12 mm diameter). After about8 days’ culture, the cell monolayer integrity was monitored by atransendothelial electrical resistance (TEER) instrument (MillicellERS-2, Inc., Millipore, USA). Only a cell monolayer with a TEERexceeding 200 Vcm2 was selected for this experiment (Raymondet al., 2016; Wang et al., 2015b; Yang et al., 2013). In addition, theculture medium was added to the outer slot of the incubator in aslightly lower quantity than that of the inner slot to observe thechange between the bilateral liquid surface of the outside andinside slots. If a different liquid level was maintained over 4 h, wecan also infer that the cell monolayer formed a tight junction(Wang et al., 2015a; Xie et al., 2004; Yang et al., 2013). U87 cellswere seeded at a density of 2 � 105 cells/per cell on the bottom of a12-insert cell. After co-incubation for 24 h, the model could be usedfor experiments.

CLSM was used to compare the penetration velocity of thesiRNA formulations in the co-culture model of BMVECs and U87cells. LPC/siRNA NPs, PEG-LPC/siRNA NPs, and T7-LPC/siRNA NPs,containing Cy5-siRNA, were added to the upper-well of a 12-insertcell (shown in Fig. 4A). Then, the U87 cells on the bottom of the cellwere visualized immediately by CLSM after adding samples, andphotos were recorded every 5 min until 1 h.

To assess the EGFR protein down-regulation ability of the siRNAformulations after penetrating the BBB model, an EGFR enzyme-linked immunosorbent assay (ELISA) was applied. LPC/siEGFR NPs,PEG-LPC/siEGFR NPs and T7-LPC/siEGFR NPs were added to theapical compartment of the BBB model. The final concentration ofsiEGFR used in the experiment was 100 nM. After 24 h incubation,the EGFR protein in the U87 cells was extracted by RIPA reagentand analyzed using a human EGFR ELISA kit (Ray-Biotech, USA)according to the manufacturer’s instructions.

2.7. Studies of the penetration ability in the tumor spheroid model

To prepare three-dimensional tumor spheroids, U87 cells wereseeded at a density of 5 �103 cells per well in 96-well plates coatedwith 50 mL of 2% low melting point agarose. Then, 5 days after thecells were seeded, the tumor spheroids were treated with PEG-LPC/siRNA and T7-LPC/siRNA NPs, containing Cy5-siRNA, andincubated at 37 �C. Then, the spheroids were rinsed with cold PBS.Finally, the spheroids were imaged under the CLSM.

Fig. 1. (A) Size distribution and (B) zeta potential of LPC/siRNA NPs, PEG-LPC/siRNA NPs and T7-LPC/siRNA NPs. Data were shown as mean � SD (n = 3). **P < 0.01, * P < 0.05.

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To assess the growth inhibition of the siRNA nanoparticlesagainst the U87 spheroid, T7-LPC/siNC, T7-LPC/siEGFR, and PEG-LPC/siEGFR NPs were incubated with the U87 spheroids. The finalconcentration of siRNA was 100 nM. Growth inhibition wasmonitored by measuring the size of the U87 glioma spheroidsusing an inverted microscope 5, 6, 7, 8 and 9 days after thespheroids were seeded. The major (dmax) and minor (dmin)diameters of each spheroid were measured, and the spheroidvolume was calculated using the following formula: V = (p �max

�min)/6 (Li et al., 2014b; Zong et al., 2014a) The glioma spheroidvolume ratio was estimated from the following formula: R = (Vdayi/Vday0) � 100%, where Vdayi was the U87 glioma spheroid volume atthe ith day after applying the drug, and Vday0 was the U87 gliomaspheroid volume prior to administration. The resulting data wereexpressed as the mean values � SD (n = 3).

2.8. In vivo distribution of siRNA-loaded nanoparticles

Glioma-bearing nu/nu mice were prepared by intracranialinjection (striatum, 1.8 mm right lateral to the bregma and 3 mm ofdepth) of 1 �105 U87 cells suspended in 5 mL serum-free mediainto male nude mice with a body weight of 20–25 g (Gu et al., 2013;Huang et al., 2011; Zong et al., 2014a). More than 95% of the micerecovered from the surgery. At the 18th day after implantation,glioma-bearing mice were injected intravenously with DiR labeledPEG-LPC/siRNA and T7-LPC/siRNA NPs containing Cy5.5-siRNA,through the tail vein. The final concentration of Cy5.5-siRNA was2 mg/Kg, and DiR was 500 mg/Kg. Then, 1, 3, 6, 12, 24, and 48 h afteradministration, the mice were anesthetized and visualized by aCambridge Research & Instrumentation in vivo imaging system(CRi, MA, USA). Then, mice were sacrificed at predetermined times,and the glioma-bearing brains were excised carefully to observethe siRNA accumulation.

2.9. In vivo glioma therapy

Nu/nu mice bearing intracranial U87 glioma model wasestablished as described in Section 2.8. The mice were randomlydivided into 4 groups (11 animals per group). Ten days after tumorinoculation, the mice were given (1) 5% glucose, (2) T7-LPC/siNCNPs, (3) PEG-LPC/siEGFR NPs, (4) T7-LPC/siEGFR NPs via tail veininjection at an siRNA dose of 1 mg/kg given every other day (a totalfor 4 doses). Eight mice randomly chosen in each group were usedfor monitoring survival. The survival time was calculated from day0 (tumor inoculation) to the day of death. Kaplane Meier survivalcurves were plotted for each group. Also, the expression level ofEGFR in brain tissue was measured using an ELISA kit (Ray-Biotech,USA). The tumor tissue of the remaining mice in each group wasextracted and analyzed using a human EGFR ELISA kit according tothe manufacturer’s instructions (as described in Section 2.6).

2.10 In vivo safety evaluationHemogram analysis was performed to evaluate the in vivo

safety properties of the nanoparticles. During tumor treatment, themice were randomly chosen for collection of blood from the orbitand measurement of the changes in hematopoiesis and immune-related indexes on the 4th, 7th, and 10th day after treatment began.

2.10. Statistical analysis

The results were expressed as mean � standard deviation. Forstatistical analysis between two groups, Student’s t-test forindependent means was applied. Comparisons between multiplegroups were made by one-way analysis of variance (ANOVA)followed by an LSD multiple comparison test.

3. Results and discussion

3.1. Characterization of PEGylated LPC/siRNA NPs

The average hydrodynamic diameters of the different LPC/siRNA, PEG-LPC/siRNA, T7-LPC/siRNA NPs were 91.56 � 5.13 nm,89.23 � 0.95 nm, and 83.82 � 4.07 nm, respectively. After DSPE-PEG or T7-PEG-DSPE insertion (Detailed synthesis and characteri-zation given in Supplementary Information), the size of thenanoparticles decreased slightly (Fig. 1A). The particle sizes of thetwo PEGylated LPC/siRNA NPs all had a narrow size distribution(polydispersity index <0.2). The zeta potentials of LPC/siRNA, PEG-LPC/siRNA, and T7-LPC/siRNA NPs were 43.84 � 2.57 mV,34.13 � 1.60 mV, and 33.82 � 1.05 mV, respectively. According tothe results, PEGylated LPC/siRNA NPs possessed a lower positivecharge than unmodified LPC/siRNA, which was caused by theshield provided by the hydrophilic PEG chain (Fig. 1B). PEG formeda hydration layer around the lipid surface of LPC/siRNA NPs. Thesurface charge was shielded by the hydration layer whichexpanded the shear plane. Thus, the zeta potential of particleswas reduced (Wang et al., 2010).

3.2. Cellular uptake

The affinity of T7-LPC/siRNA NPs for monolayer BMVECs andU87 cells was qualitatively and quantitatively analyzed by confocallaser scanning microscopy (CLSM) and flow cytometry (FCM),respectively. BMVEC is the main component of the BBB and U87cells are a subspecies of brain glioma cells.

As shown in Fig. 2A and B, significant intracellular fluorescencewas found in both BMVECs and U87 cells treated with T7-LPC/Cy5-siRNA NPs, while a lower fluorescence was found in cells treatedwith PEG-LPC/Cy5-siRNA NPs. Also, the CLSM photos of LPC/Cy5-siRNA NPs showed clear shrinkage of the cells and clearcytomorphosis.

Fig. 2. Cell uptake and intracellular distribution of siRNA complexes in BMVECs and U87 cells. (A) CLSM images of BMVECs and (B) U87 cells transfected with differentnanoparticles containing Cy5-siRNA (red). The Rhodamine labeled phallacidin (green) was used to show the cytoskeleton and Hoechst 33258 (blue) for nucleus. Intracellularfluorescence intensity of BMVECs(C and D) and U87 cells (E and F) analyzed by flow cytometry after treated with different FAM-siRNA formulations. The final concentration ofsiRNA is 100 n M. Data were shown as mean � SD (n = 3). **P < 0.01, * P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

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FAM-siRNA was used as a fluorescent probe to monitor theinternalization of nanoparticles in BMVECs and U87 cells. Based onthe quantized results of flow cytometry shown in Fig. 2D and F, thefluorescence ratio of T7-LPC/FAM-siRNA NPs and PEG-LPC/FAM-siRNA NPs was 550.35 � 75.06% and 328.37 � 13.02% in BMVECscompared with the negative control. In U87 cells, the fluorescenceratio of T7-LPC/FAM-siRNA NPs and PEG-LPC/FAM-siRNA NPs was522.27 � 28.61% and 228.18 � 11.75%. Thus, in both BMVECs andU87 cells, a higher intracellular fluorescence intensity of FAM-siRNA was found in cells treated with T7-LPC/FAM-siRNA NPs thanPEG-LPC/FAM-siRNA NPs, which was consistent with the qualita-tive results observed from the CLSM images in Fig. 2A and B. Afterconjugation with T7 peptide, the nanoparticles clearly accumulat-ed in the cells, indicating that T7 peptide modification couldpromote the cellular uptake of T7-LPC/siRNA NPs through areceptor-mediated pathway due to the special affinity fortransferrin receptors overexpressed in BMVECs and U87 cells.These results suggested that the targeting nanoparticle have thepotential to cross the BBB and access the glioma cells in vivo.During the experiment, it was found that, under the sametreatment conditions, the cells treated with LPC/FAM-siRNA NPs(without PEGylation) were relatively more difficult to collect thanT7-LPC/siRNA NPs and PEG-LPC/siRNA NPs (with PEGylation).Combined with the cytomorphosis in the CLSM images (Fig. 2A andB), cytomorphosis always occurs on the change in cell surfacestructure or a decrease in the physiological activity of the cells. Inaddition, the results of the MTT assay (Fig. S2C), showed that LPC/siNC NPs were clearly cytotoxic. These phenomena were probablydue to the fact that LPC/siRNA NPs possessed a high unshielded netcharge which could destroy the surface structure and physiologicalactivity of the cells. Thus, the treated cells were not able to tightlyadhere to the flask anymore and disappeared or were disruptedduring the experiment. This was suggested that with the T7peptide-mediated active target function T7-LPC/siRNA NPs couldbe efficiently internalized in BMVECs and U87 cells. Moreover,without PEG modification, LPC/siRNA NPs are too toxic for directadministration to cells. Therefore, PEGylation is necessary toimprove the safety of the cationic carriers.

3.3. In vitro EGFR gene silencing effects

The EGFR gene silencing effect in monolayer U87 cells wasassessed using qRT-PCR. Based on the results shown in Fig. 3, the

Fig. 3. EGFR mRNA determined by qRT-PCR after treatment with variousformulations in U87 cells. Data were shown as mean � SD (n = 3). **P < 0.01, *P < 0.05.

rank of EGFR mRNA silencing ratios was T7-LPC/siEGFR > PEG-LPC/siEGFR > siEGFR > T7-LPC/siNC. EGFR mRNA levels were maximallysuppressed after the U87 cells were treated with T7-LPC/siEGFRNPs compared with other formulations. Also, T7-LPC/siNCproduced almost no EGFR knockdown, which confirmed that thesilencing efficacy was caused by T7-LPC/siEGFR NPs. In addition,we noticed that the PEG-LPC/siEGFR NPs also reduced genetranscription. This may because, at a monolayer cell level, the PEG-LPC/siEGFR NPs could enter the glioma cells because of their nano-size and positive charge. It has been reported that many PEG-modified gene delivery systems enter into cells through micro-pinocytosis, and then silence the relevant genes (Lühmann et al.,2008; Nakase et al., 2007; Walsh et al., 2006). This suggests that areceptor-mediated targeting delivery system could help increasethe cellular up-take of siRNA and, thus, promote its gene silencingefficacy.

3.4. Penetration across the BBB

To predict the ability of T7-LPC/siRNA NPs to cross the BBB,simulating in vivo conditions, a co-culture model of BMVECs/U87cells was established. Fig. 4A shows the co-culture BBB model.BMVECs, the main component of the BBB, can form tight junctionsto hamper the vast majority of molecules from being transportedacross the BBB into brain parenchyma.

Cellular uptake of different Cy5-siRNA NPs in U87 cells wasinvestigated by CLSM. The transport in the BBB model of siRNA NPswas studied from 0 min to 60 min. As seen in Fig. 4B, the BBBcrossing velocity of the NPs was in order T7-LPC/siRNA > PEG-LPC/siRNA � LPC/siRNA. As shown in the results, even at 20 min, Cy5-siRNA fluorescence could be detected in U87 cells, which suggestedthat T7-LPC/siRNA NPs cross the BBB model rapidly. At each point,the red fluorescence signals of Cy5-siRNA in T7-LPC/siRNA NPswere significantly stronger than those in LPC/siRNA NPs and PEG-LPC/siRNA NPs, while there was no significant difference betweenthe untargeted nanoparticles, indicating that T7-modified nano-particles could promote BBB crossing and cellular uptake through areceptor-mediated pathway due to the special affinity fortransferrin receptors overexpressed in BMVECs and U87 cells.The LPC/siRNA NPs were clearly cytotoxic, and could destroy thesurface structure and physiological activity, and reduce thetransport capacity of the cells. Thus, the injured BMVECs werehardly involved in the transportation of LPC/siRNA NPs.

Furthermore, the transfection efficiency of the NPs wasevaluated in the co-culture model of BMVEC/U87 cells, and wasquantitatively analyzed using an ELISA kit. After treatment withLPC/siEGFR NPs, PEG-LPC/siEGFR NPs or T7-LPC/siEGFR NPs, thecells treated with T7-LPC/siEGFR NPs exhibited the highest proteindown-regulation in U87 cells (Fig. 4C). These results are consistentwith the CLSM results (Fig. 4B) and indeed support our hypothesisthat the constructed gene delivery system could cross the BBB todown-regulate the target RNA.

These results suggested that T7-LPC/siRNA NPs are able to crossthe BBB and target glioma cells in vivo.

3.5. Tumor spheroid penetration

An in vitro U87 glioma tumor spheroid culture model wasapplied to evaluate the solid tumor penetration ability of nano-particles. A major barrier in vivo reducing the therapeutic efficacyof siRNA nanoparticles is the restriction of interstitial transportwhich blocks the diffusion of siRNA nanoparticles into the targetsolid tumor tissue. However, the monolayer cell model could notstimulate such a physiological feature and, thus, could notaccurately reflect the accumulation effect in vivo. Thus, it isnecessary to carry out further studies of the penetrative behavior

Fig. 4. Schematic for the co-culture model of BMVECs and U87 cells and an assay for BBB penetrate. (A) Co-culture model of BMVECs and U87 cells and (B) CLSM images of U87cells transfected with different nanoparticles, after crossing the co-culture BBB model, containing Cy5-siRNA (red) and Hoechst 33258 (blue) for nucleus. (C) EGFR proteinexpression of U87 cells after various formulations treatment determined by ELISA. Data were shown as mean � SD (n = 3). **P < 0.01, * P < 0.05, N.S. means no significance. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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of the NPs on three dimension tumor spheroids which is closer tothe in vivo microenvironment of solid tumors.

Fig. 5A shows the penetration ability of T7-LPC/Cy5-siRNA NPsand PEG-LPC/Cy5-siRNA NPs into glioma U87 tumor spheroidsviewed by CLSM. After incubation with the NPs, images werecaptured at different layers of a spheroid from the top to themiddle. Furthermore, the semi-quantitative intensity in U87 tumorspheroids was calculated from the fluorescence intensity of T7-LPC/siRNA NPs and PEG-LPC/siRNA NPs (Fig. 5B). After a 4 hincubation, the penetration of T7-LPC/siRNA NPs and PEG-LPC/siRNA NPs was restricted to the outer few cell layers, and thespheroid cores were all dark with no obvious signals. However, T7-LPC/siRNA NPs exhibited stronger fluorescence than PEG-LPC/siRNA NPs. After a 6 h treatment, deeper Cy5-siRNA penetration forT7-LPC/siRNA NPs was found. The Cy5-siRNA signal spread fromthe periphery toward the core of the spheroids and clearly coveredthe entire globe. The fluorescence signals of Cy5-siRNA in PEG-LPC/siRNA NPs markedly increased on the periphery of the tumorspheroids while there was little change in the center. Thisobservation suggested that the T7-LPC/siRNA NPs had an advan-tage in penetrating into a deep region of the tumor spheroid.

3.6. Growth inhibition of tumor spheroid

The tumor spheroid inhibition of the targeting siRNA deliverysystem was evaluated using U87 glioma spheroids which imitatethe solid tumor in vivo. Fig. 5C displays the inhibitory effect on

glioma U87 tumor spheroids after treatment with different siEGFRNPs. According to the results, the relative volumes of the sphericaltumor were in the following order: T7-LPC/siEGFR < PEG-LPC/siEGFR < T7-LPC/siNC < PBS. For siEGFR formulations, T7-LPC/siEGFR NPs exhibited stronger spheroid growth inhibition thanPEG-LPC/siEGFR NPs, indicating that U87 glioma spheroids weremore sensitive to T7-LPC/siRNA NPs. Clear spheroid growth wasobserved for T7-LPC/siNC NPs and PBS treatment. T7 peptidemodification of the nanoparticles was responsible for theseoutcomes. Since the tumor spheroid could imitate the microenvi-ronment of in vivo glioma solid tumor, the clear tumor spheroidinhibitory effect of T7-LPC/siRNA indicated a favorable therapeuticeffect in vivo.

3.7. In vivo imaging of glioma-bearing nude mice

In order to evaluate the safety and effectiveness of a nanoscaledelivery system, drug accumulation in tumor tissue and othermajor organs should be investigated. The ability of T7-LPC/siRNANPs to cross the BBB and deliver the payload to brain tumors wasdemonstrated by in vivo real time observation after systemicadministration. For these studies, we used double labeled siRNAnanoparticles. DiR (lex = 748 nm, lex = 780 nm) was used as a near-infrared fluorescence probe to be inserted into the lipid shell of thenanoparticles. Cy5.5-siRNA(lex = 673 nm, lex = 692 nm) wasentrapped in the core of the nanoparticles. Thus, during the

Fig. 5. CLSM images of U87 glioma spheroids incubation with different nanoparticles containing Cy5-siRNA (red). The Hoechst 33258 (blue) labeled nucleus. (B) Semi-quantitative intensity of inner region at different sections of U87 tumor spheroids. (C) Inhibitory effect of various formulations to U87 glioma spheroids. The data arepresented as the means � SD (n = 3). **P < 0.01, * P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of thisarticle.)

L. Wei et al. / International Journal of Pharmaceutics 510 (2016) 394–405 401

observation process, the fluorescence of DiR and Cy5.5-siRNAcould simultaneously reflect the location of the vectors and siRNA.

In Fig. 6A, weak fluorescence signals of DiR and Cy5.5-siRNAwere observed in the brain, mostly in the liver and kidney. Thissuggested that T7-LPC/siRNA NPs could penetrate the BBB andaccumulate in brain of the mice. As the glioma volume is relativelysmaller compared with the liver, a dramatic difference inaccumulation of fluorescence substance in the brain and othertissues was observed. Moreover, the clear fluorescence in the liver

and kidney may be caused by the reticular epithelial system (RES)that captured the nanoparticles. In addition, the fluorescence ofDiR and Cy5.5-siRNA showed a familiar distribution during thewhole observation period, indicating that the vectors remainedstable in the in vivo circulation and there was no disruption, whichfavored siRNA protection and delivery.

Next, the observation parameters were optimized to monitorthe fluorescence signal changes in the brain (Fig. 6B). In addition,the brains of the mice were excised after the animals were

Fig. 6. In vivo distribution of nanoparticles was observed by in vivo imaging system. The nude mice bearing U87 glioma tumor were given by intravenous injection via tail vein.(A) the whole body imaging of the mice in vivo, (B) the brain of the mice in vivo imaging and (C) ex vivo imaging of brain excised from nu/nu nude mice.

402 L. Wei et al. / International Journal of Pharmaceutics 510 (2016) 394–405

Fig. 7. (A) Kaplane Meier survival curves of gliom-bearing mice treated with different formulations at day 10, 13, 16 and 19 post glioma cells inoculation. The dose of siEGFRwas 1 mg/kg. (B) EGFR protein expression levels determined by ELISA in tumors. Data were shown as mean � SD (n = 3). **P < 0.01, * P < 0.05, N.S. means no significance.

L. Wei et al. / International Journal of Pharmaceutics 510 (2016) 394–405 403

sacrificed and imaged at predetermined times (Fig. 6C). As shownin Fig. 6B, the fluorescence signals of both DiR and Cy5.5-siRNA inthe T7-LPC/siRNA group displayed in brain 1 h after injection,became strongest after 3 and lasted until 48 h. However, negligiblefluorescence signals were detected in the mouse brains at anypoint after intravenous injection of PEG-LPC/Cy5.5-siRNA NPs invivo and ex vivo. It was suggested that T7 modification couldpromote increased tumor accumulation by transferrin receptor-mediated targeting delivery, and PEGylation could prolong thecirculation time of the nanoparticles in blood to increase the BBBbinding efficacy. In addition, the in vivo distribution results of PEG-LPC/siRNA also demonstrated the integrity of the blood-brainbarrier.

The in vivo image results showed that the T7-LPC/siRNA NPsefficiently crossed the BBB and exhibited good tumor targetingability.

Fig. 8. Hematological indicators including RBC, PLT, LYM, GRN

3.8. In vivo anti-glioma efficacy

For further investigation of the potential of T7-LPC/siEGFR NPsin anticancer therapy in vivo, the Kaplane Meier survival curve ofU87 glioma-bearing mice was used (Fig. 7A). The ultimate goal foran siRNA delivery system is to achieve optimal therapeutic efficacywith an acceptable safety profiles during in vivo applications. Theclinical gene therapeutic benefits are mainly determined based onthe quality of life and prolonged survival time of cancer patients.According to the results, in the T7-LPC/siEGFR NPs group, thebeginning and the ending of mouse death was later than in anyother groups. The mean survival time of the T7-LPC/siEGFR NPs(37 days) group was longer than that of the other groups treatedwith 5% glucose (31 days), PEG-LPC/siEGFR NPs (33 days) or T7-LPC/siNC NPs (33 days), and this was mainly attributed to the target

, and HGB. Data are presented as the mean � SD (n = 4).

404 L. Wei et al. / International Journal of Pharmaceutics 510 (2016) 394–405

systemic delivery of T7-LPC/siRNA NPs and the anti-glioma activityof siEGFR.

To further investigate the anti-tumor mechanism, EGFRexpression in glioma was determined using ELISA analysis.According to Fig. 7B, the EGFR protein level of 5% glucose, T7-LPC/siNC, PEG-LPC/siEGFR, T7-LPC/siEGFR was 25.51 �3.00,25.55 � 5.49, 19.36 � 4.23, 12.53 � 3.96 (pg/mg protein), respec-tively. T7-LPC/siEGFR NPs exhibited the highest EGFR proteindown-regulation effect of in vivo glioma compared with the otherformulations. The EGFR level of the 5% glucose and T7-LPC/siNCgroup was compared while the PEG-LPC/siEGFR group showedonly a slight down-regulation effect. These results suggested thatT7-LPC/siEGFR has a greater glioma-targeting ability and exhibitsits tumor inhibition effects by down-regulating EGFR expression ina sequence-specific manner. Therefore, T7-LPC/siEGFR NPs showgreat potential for glioma therapy in an in vivo pharmacodynamicevaluation.

3.9. In vivo safety evaluation

Considering that siRNA and cationic nanoparticles afterintravenous injection may cause immune responses and resultin systemic toxicity, it is necessary to evaluate the biological safetywhen intending to develop a cationic siRNA delivery system. Thechanges in hematopoiesis and immune-related indexes of mouseblood were used to estimate the in vivo toxic effects. Red blood cell(RBC), hemoglobin (HGB), and platelet (PLT) are indexes ofhematopoiesis. Lymphatic cell (LYM), and neutrophil granulocyte(GRN) are indexes of the immune system. The results (Fig. 8)showed that the positively charged nanoparticles produced nosignificant effects on these indexes compared with 5% glucose,suggesting that these nanoparticles do not easily evoke hemolysisor immune reactions. Thus, the positively charged nanoparticlesexhibited a low in vivo toxic effect.

4. Conclusion

In the present study, it was demonstrated that T7-LPC/siRNANPs could effectively deliver siEGFR into U87 glioma cells throughTfR-mediated internalization and inhibit tumor proliferation byinducing EGFR down-regulation. In vivo experiments furthershowed that T7-LPC/siEGFR NPs could exhibit a higher accumula-tion in the brain tumor site and a higher therapeutic efficacycompared with non-targeted PEG-LPC/siEGFR NPs. All the resultssuggested that the T7-LPC/siEGFR would be a potential deliverysystem for glioma targeting therapy in vivo.

Acknowledgments

The authors acknowledge financial support from the Ministry ofScience and Technology of China (Grant No. 2013CB932501), theNational Natural Science Foundation of China (Grant No.81273455, 81473158), and Programs from Ministry of Educationof China (NCET-11-0014 and BMU20110263).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.ijpharm.2016.06.127.

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