Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136
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Colloids and Surfaces B: Biointerfaces
jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb
elective transfection with osmotically active sorbitol modified PEIanoparticles for enhanced anti-cancer gene therapy
im Cuc Thi Nguyena,f,1, Muthunarayanan Muthiahb,f,1, Mohammad Ariful Islamc,. Santhosh Kalashb, Chong-Su Choc, Hansoo Parke, Il-Kwon Leed, Hyeoung-Joon Kimd,
n-Kyu Parkb,f,∗, Kyung A. Choa,f,∗∗
Department of Biochemistry, Chonnam National University Medical School, Gwangju 501-746, South KoreaDepartment of Biomedical Science and Chonnam National University Medical School, Gwangju 501-746, South KoreaDepartment of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South KoreaDepartment of Hematology-Oncology, Chonnam National University Hwasun Hospital, Hwasun, Jeollanamdo 519-763, South KoreaSchool of Integrative Engineering, Chung-Ang University, Dongjak-gu, Seoul 156-756, South KoreaBK21 PLUS Center for Creative Biomedical Scientists at Chonnam National University, Chonnam National University Medical School, Gwangju 501-746,outh Korea
r t i c l e i n f o
rticle history:eceived 3 February 2014eceived in revised form 1 May 2014ccepted 2 May 2014vailable online 14 May 2014
Polysorbitol-mediated transporter (PSMT) has been previously shown to achieve high transfection effi-ciency with minimal cytotoxicity. Polysorbitol backbone possesses osmotic properties and leads toenhanced cellular uptake. The PSMT/pDNA nanoparticles were prepared and the particle size, surfacecharge of the nanoparticles was determined for the study. PSMT delivers genes into cells by the caveolaemediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Trans-fection through the caveolae may help PSMT to selectively transfect cancer cells rather than normal cells.Transfection of the luciferase gene by PSMT was tested in various cell types including cancer cell lines, pri-mary cells, and immortalized cells. Luciferase transgene expression mediated by PSMT was remarkablyincreased in HeLa cells compared to expression using the control carrier Lipofectamine. Moreover, thetoxicity of PSMT was comparable to the control carrier (Lipofectamine) in the same cells. Selective trans-fection of cancer cells using PSMT was further confirmed by co-culture of cancer and normal cells, which
showed that transgene expression was pre-dominantly achieved in cancer cells. A functional p53 genewas also delivered into HeLa cells using PSMT and the selective transgene expression of p53 protein incancer cells was analyzed through western blotting and confocal microscopy. HeLa cells transfected withPSMT/p53 plasmid nanoparticles showed cellular damage and apoptosis, which was confirmed throughpropidium iodide staining.
Successful gene therapy depends on the efficient and safe deliv-ry of a therapeutic gene. The main obstacles to achieving efficientene delivery lie in selection of the gene carrier and its transfection
∗ Corresponding author at: Department of Biomedical Sciences, Chonnamational University Medical School, Gwangju 501-746, South Korea.el.: +82 61 379 8481; fax: +82 61 379 8455.∗∗ Corresponding author at: Department of Biochemistry, Chonnam National Uni-ersity Medical School, Gwangju 501-746, South Korea.
efficiency [1,2]. Viral gene carriers have enhanced transfectionefficiencies, but their associated toxicities limit their use as clinicalvectors [3,4]. Recently, the focus has turned towards non-viralcarriers, but such carriers still have to compete with viral carriers interms of transfection efficiency. The initial search for non-viral car-riers focused on the use of polylysine, while later studies examinedtransfection efficiencies achieved using polyethylenimine (PEI) [5].While PEI was efficient as a non-viral carrier, its non-degradablenature produced toxicity in cells [6]. PEI was thus chemically mod-ified to reduce its toxicity and increase biocompatibility. These
modifications included its conjugation with biocompatible poly-mers such as chitosan, PEG, and hyaluronic acid. Degradable unitswere also introduced into PEI for facilitating its degradation intosmaller units after entering cells [7,8]. To some extent, di-sulphide
K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136 127
S the spw normao
matefctt(iesWcPwmeiawrtu
cheme 1. The therapeutic gene expression in the cancer cells is enhanced due to
hich is found to have high expression in cancer cells compared to normal cells. The
f the PSMT/pDNA nanoparticle.
odified PEI (ssPEI) also reduced toxicity when it was formulateds a non-viral carrier for gene delivery. The ssPEI was stable inhe extracellular environment and reductive in the intracellularnvironment; these characteristics helped to deliver the gene andacilitated degradation of the carrier over time [9]. However, issuesoncerning accelerated cellular uptake and intracellular localiza-ion remained areas of concern. Recently, polysorbitol-mediatedransporter (PSMT), whose structure is based on sorbitol diacrylateSDA) and low molecular weight PEI (LMW PEI), was synthesizedn our group to facilitate cellular uptake of genetic material andnhance transfection efficiency in cells [10]. The PSMT transporterystem has a polysorbitol backbone possessing osmotic properties.
hen the carrier binds to the cells, the osmotic condition of theells are altered and the cellular uptake of the particle is enhanced.SMT interacts with the cell through the caveolae mediated path-ay. It was hypothesized that osmotically active PSMT gene carrieright be able to selectively transfect and treat caveolae over-
xpressing cancer cells with tumor suppressor gene, as describedn Scheme 1. Compared to the currently available transfectiongents and polymeric carriers, cytotoxicity was greatly reduced
ith PSMT. PSMT polymer was synthesized using Michael addition
eaction, so the low molecular PEI was conjugated alternativelyo SDA. This was due to the fact that only non-toxic lower molec-lar PEI was conjugated alternatively to hydrophilic SDA, and
ecific interaction of sorbitol component in the PSMT/pDNA to the caveolae proteinl cells have basal level of caveolae protein leading to reduced uptake and expression
degradability was assured with the ester linkage formed by SDAmodification. Transfection efficiency is an important and interest-ing phenomenon because it varies among different types of cellsaccording to their origin, proliferation status, cellular constituents,membrane architecture, and receptors present on the membrane[11,12]. Normal cells differ from cancer cells in their membranearchitecture, cellular uptakes, and various other characteristicswhich may influence transfection efficiency [13]. Our previousstudy demonstrated that the selective caveolae-dependent endo-cytic pathway played a significant role for increasing efficiency oftransfection by osmotic PSMT-mediated gene delivery [14]. Thecaveolae protein is over-expressed in most of cancer cells, while itis less pronounced in normal cells [15–18]. Based on such findings,we hypothesized that PSMT-based transfection of therapeuticgenes would be selectively enhanced in cancer cells compared tonormal cells. A comparison of PSMT mediated transfection in cancercells and normal cells of various origins would provide additionalinformation regarding the use of PSMT as a carrier for selectivegene delivery. We were therefore interested to examine whetherthe transfection efficiency of PSMT is consistent across different
cell lines, or selectively depending on cellular caveolae activation.
In this study, we have physico chemically characterized thePSMT/pDNA nanoparticles and tested both the capability of PSMTto transfect various cell types and its cytotoxicity to cells. Selective
ransfection using the carrier was confirmed by co-culture exper-ments performed after differential labeling of normal cells andancer cells. Finally, PSMT was utilized for cancer cell-specific ther-peutic gene delivery. Following intracellular delivery of p53 tumoruppressor gene/PSMT nanoparticles, morphological changes andhe apoptotic status of cells were assessed with respective experi-
ents.
. Materials and methods
.1. Reagents
Branched PEI (bPEI) 25 kDa and Poly-L lysine were purchasedrom Sigma-Aldrich (St. Louis, MO, USA). Plasmid gWiz-luc (Alde-ron, Madison, Wisconsin, USA) was transformed into competentscherichia coli DH5� cells by a heat shock method. The gWiz-lucas then propagated in bacterial cultures grown in Luria-Bertani
LB) media (BD, Franklin Lakes, New Jersey, USA) containing00 mg/mL kanamycin (Biosesang Inc., Korea) and extractednd purified using a mini DNA-spin kit (Intron Biotechnol Co.,orea). Lipofectamine 2000, Cell TraceTM Oregon Green, propid-
um iodide, and TOPO T-A cloning vector were purchased fromnvitrogen (Carlsbad, CA, USA). The CellTiter 96® AQueous Non-adioactive Cell Proliferation Assay (MTS assay) was purchased
rom Promega (Madison, WI, USA) and used as per the manufac-urer’s protocol. Glass coverslips were purchased from MarienfeldLauda-Königshofen, Germany). Plasmid pLentiH1.4-monomerRFPas purchased from Macrogen, (Seoul, Korea) and used for the
ver-expression of Red fluorescent protein (RFP) reporter in cells.rimary antibody against p53 protein (Santa Cruz, Dallas, Texas,SA) was used to confirm expression of p53 protein by westernlotting.
.1.1. Cell linesVarious cancer cell lines, including human cervical cancer
HeLa), BALB/c colon carcinoma (CT-26), C57BL/6 colon carcinomaMC38), and human breast adenocarcinoma cell line (MCF7) wereurchased from American Type Culture Collection (ATCC; Manas-as, VA, USA) and used in this study. Both primary and immortalizedells were used for comparisons of PSMT-mediated transfection.uman diploid fibroblasts (HDF) were isolated from human fore-
kins as previously described [19]. Stable clonal lines of humanrain microvessel endothelia cells (HEN7) were generated fromuman fetal telencephalon by using retroviral vector encodinguman telomerase reverse transcriptase and beta-galactosidase.rimary mouse peritoneal macrophages (M�) were isolated from57BL/6 mice as previously described [20].
.1.2. Synthesis of PSMTPSMT composed of Low Molecular Weight (LMW) branched PEI
nd Sorbitol Di-Acrylate (SDA) was synthesized via the Michaelddition reaction as previously reported [10]. In brief, LMWranched PEI and SDA were separately dissolved in a vial withnhydrous DMSO. Then, SDA was slowly dropped to PEI solutionn a three-neck reaction flask under dry nitrogen purge at a stoi-hiometric ratio of 1:1 of PEI to SDA. The reaction was performedt 80 ◦C for 24 h with magnetic stirring. Then, the reaction mixtureas dialyzed against distilled water (MWCO: 3500 Da) at 4 ◦C for
8 h and lyophilized. The final products were stored at −70 ◦C forhe steps.
.2. Preparation and physico chemical characterization of
SMT/plasmid DNA nanoparticles
The plasmid DNA binding ability of the PSMT was evaluatedy electrophoresis through a 1.2% agarose gel containing ethidium
B: Biointerfaces 119 (2014) 126–136
bromide intercalating dye. The nitrogen-to-phosphate (N/P) molarratio was varied by adding predetermined PSMT concentrations to afixed amount of the plasmid DNA. The PSMT and plasmid DNA solu-tions were mixed at N/P ratios from 5 to 20 and vortexed briefly.bPEI 25 kDa was taken as control. The complexes were kept at roomtemperature for 30 min for complete complexation before beingloaded into an agarose gel. Electrophoresis was carried out at 100 Vfor 50 min, and DNA retention was visualized under ultraviolet(UV) illumination. Particle sizes and zeta potentials were mea-sured using a Zetasizer Nano ZS (Malvern Instruments, Malvern,UK).
2.3. Cell culture and analysis of PSMT transfection efficiency
HDF, HeLa, CT-26, and MC-38 cells were cultured in Dulbecco’sModified Eagle’s Medium (DMEM) supplemented with 10% fetalbovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). M� and MCF7cells were cultured in RPMI-1640 supplemented with 10% FBS.HEN7 cells were cultured in endothelia basal medium (EGM-2 Bul-let Kit, Lonza, Switzerland).
Cells were seeded in 24-well culture plates in a 5% CO2 atmo-sphere at 37 ◦C. After reaching confluence, the cells were washed3 times with DPBS (Takara, Japan) and transfected with 1 �g ofplasmid DNA complexed with either Lipofectamine 2000 pre-pared according to the manufacturer’s instructions or PSMT atdifferent charge ratios in Opti-MEM solution for 4 h, followed bya 48 h post-incubation in culture medium containing 10% FBSat 37 ◦C in a 5% CO2 incubator. At 48 h post-transfection, thecells were lyzed in 1× lysis buffer (Promega, Madison, WI, USA).Transfection results were obtained by measuring the extent oftransgene expression in the lysate, as quantified by luciferaseactivity determined using the Luciferase Assay System (Promega,Madison, WI, USA). A microplate luminometer (Microlumat PlusLB96 V, Berthold Technologies, Germany) was set for a 3 s delaywith signal integration for 10 s. Luciferase activity was normal-ized to the amount of total protein in a sample. A calibrationcurve created using a bovine serum albumin standard wasused to measure protein concentration. Transfection activity wasexpressed as relative light units (RLU) per milligram of cellularprotein.
2.4. Cytotoxicity of PSMT/plasmid nanoparticles
HDF and HeLa were seeded in 96-well tissue culture plateswith DMEM medium containing 10% FBS. The cytotoxicity ofPSMT/pLuc NP was evaluated by determining cell viability aftera 24 h incubation in DMEM media containing 10% FBS with dif-ferent concentrations of PSMT. Numbers of viable cells weredetermined by measuring mitochondrial reductase activity usingthe tetrazolium-based colorimetric method (MTS assay, Promega)according to the manufacturer’s instructions.
2.5. Comparison of transgene expression by PSMT/plasmid NP intransformed cancer cells and normal cells.
HDF and HeLa cells were cultivated in DMEM at 37 ◦C underhumidified 5% CO2 for 3 days, and were then sub-cultured afterreaching confluence. Cells were seeded on Poly L-lysine coatedglass coverslips at a density of 1 × 104 cells per well in DMEMmedium containing 10% FBS for 24 h. The cells were then treatedwith PSMT/RFP reporter plasmid NP or PSMT-p53 NP for 4 h in opti-MEM (Gibco, NY, USA) and then transferred to DMEM medium
containing 10% FBS for further incubation. After 24 h, the cover-slips were washed with DPBS and fixed with 4% paraformaldehyde(PFA) for 10 min at room temperature (RT). The treated cells werewashed 3 times with DPBS and permeabilized with 0.05% Triton
-100 in DPBS for 10 min at RT. After washing, the samples wereounter-stained with DAPI, mounted on glass slides, and observedy confocal microscopy (Carl Zeiss Microimaging Inc. Oberkochen,ermany).
For the co-culture model, HeLa cells were first seeded onlass coverslips overnight and then co-seeded with Oregon Greenabeled-HDF on the same coverslips. After confirming the fullttachment of HDF, transfection experiments were performedsing either Lipofectamine/plasmid NP or PSMT/plasmid NP. Theamples were then fixed, permeabilized, and counter-stained withAPI at 16 h post-transfection as described above.
.6. Confirmation of transgene expression by western blottingfter treatment with PSMT/p53 tumor suppressor gene NP
HeLa cells were seeded on 6-well plates one day before transfec-ion with p53 plasmid. NP formed with either Lipofectamine/p53P or PSMT-p53 NP was delivered to HeLa cells and the intracellularroteins were collected at 24 h post-transfection by scrapping thereated cells in lysis buffer [50 mM Tris-HCl; 150 mM NaCl; 1 mMDTA, 60 mM Octyl-�-D-glucopyranoside, 1 mM phenylmethylsul-onyl fluoride protease inhibitor cocktail (1:500)], followed by briefonication for 10 s. The protein concentration of each sample waseasured using the Bradford assay. Protein samples were sepa-
ated by SDS-PAGE and then transferred to a PVDF membrane. Theembrane was blocked in 5% skim milk for 1 h at RT and incu-
ated over-night with anti-p53 primary antibody at 4 ◦C, followed
y incubation with peroxidase-conjugated anti-mouse secondaryntibody for an additional 1 h. After washing 3 times, the mem-ranes were developed using an enhanced chemiluminescenceetection kit.
ig. 1. Physico-chemical characterization of PSMT/pDNA nanoparticles. The complexatiossay. The particle size and surface charge of the PSMT/pDNA nanoparticles were measur
: Biointerfaces 119 (2014) 126–136 129
2.7. Detection of apoptosis induced by treatment with PSMT/p53plasmid NP
HeLa cells were seeded on Poly-L lysine coated coverslips ata density of 1 × 104 cells per well in DMEM medium containing10% FBS for 24 h. The cells were then transfected with either p53plasmid alone or PSMT/p53 plasmid NP (PSMT-p53 NP). At 24 hpost-transfection, the cells were stained with 500 mM PI in DMEMmedia supplemented with 10% FBS for 15 min in a 5% CO2 atmo-sphere at 37 ◦C for detection of apoptosis. After staining, the cellswere washed 3 times in DPBS, fixed with 4% PFA, permeabili-zed with 0.05% Triton X-100, and counter-stained with DAPI asdescribed above.
2.8. Statistical analysis
Quantitative data are expressed as means ± SD. The means werecompared using an independent samples t-test. P values less than0.05 were considered statistically significant.
3. Results and discussion
Hyperosmotic activity improves transfection capability byincreasing the cellular uptake of osmotically active gene carriers[21]. The PSMT transporter system has a polysorbitol backbonepossessing osmotic properties. As a result, the osmotically activecomponent in PSMT might also be responsible for the synergisti-
cally enhanced cellular uptake and transfection efficiency of PSMT.In our previous study, the transfection efficiency with PSMT wasanalyzed in human lung adenocarcinoma epithelial cells (A549),human lung bronchio-alveolar carcinoma cells (H322), and human
n between the PSMT carrier and the pDNA was analyzed by agarose gel retardationed by dynamic light scattering and Zeta potential analysis, respectively.
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Fig. 2. Transfection efficiency of PSMT for cancer and primary cells. Transfection efficiency of the PSMT carrier in different cell types was analyzed by the luciferase assay.H hargeL propet
coewf
3n
artst1N2R
3c
stadmvb(imewtclTf
complexes formed at N/P 20 and N/P 30. It was therefore evidentthat increasing the carrier concentration did not increase transfec-tion efficiency. The transfection efficiency of PSMT/pLuc NP was
Fig. 3. Transfection efficiency of PSMT in various cell lines (a). PSMT/pLuc NP wasprepared at N/P 15 and incubated with cells at 24 h after seeding. Transfection effi-ciency of PSMT/pLuc NP was analyzed in colon cancer cells (CT-26, MC38), breastcancer cells (MCF7), primary cells (HDF and M�), immortalized cells (HEN7), and
eLa cancer cells and HDF primary cells were transfected with PSMT/pLuc NP at cipofectamine/pLuc NP was used as a positive control to compare the transfection
he mean ± SD (error bars) of 3 independent experiments (*P < 0.05).
ervix epithelial carcinoma cells. All 3 of these cell lines hadriginated from the cancers and showed enhanced transfectionfficiency with a PSMT carrier. We therefore wanted to confirmhether the use of PSMT would be beneficial for enhancing trans-
ection with all types of cells of different lineages.
.1. Physico chemical characterization of PSMT/pDNAanoparticles
PSMT/pDNA nanoparticles (NP) were completely retarded in agarose gel at an N/P ratio of 15, whereas bPEI/pDNA NP wasetarded in the gel at an N/P ratio of 5 (Fig. 1a). The particle sizes ofhe PSMT/pDNA NP ranged from 100 to 200 nm (Fig. 1b, Left). Theurface charge of the PSMT/pDNA was measured by zeta poten-ial analysis and the charges were negative at the lower N/P ratio, but the transition to the positive charge was observed at the/P ratio 3. Surface charge of the PSMT/pDNA NP at the N/P ratio0–40 was within the range between +20 mV and +30 mV (Fig. 1b,ight).
.2. Efficiency of PSMT transfection in the co-culture of cancerells and normal cells
Two cell types representing cancer cells and normal cells wereelected for this study to test the effect of PSMT on transfec-ion efficiency in various cell types. HeLa cells were chosen as
representative cell line for transformed cells, and HDF (humaniploid fibroblasts) cells were selected as normal cells. To deter-ine transfection efficiency, luciferase plasmid was mixed with
arying concentrations of PSMT, and the complexes were incu-ated with HeLa and HDF cells to examine luciferase expressionFig. 2). PSMT mediated luciferase expression was significantlyncreased in HeLa cells when compared to luciferase expression
ediated by Lipofectamine. Lipofectamine and PSMT utilize differ-nt pathways for transfection. Lipofectamine is a cationic carrierhich was designed and made commercially available for efficient
ransfection. It is reported that the liposomes generally utilize the
lathrin mediated uptake pathway [22]. Lipofectamine’s cationicipid molecules are formulated with a neutral co-lipid (helper lipid).he DNA-containing liposomes (with positive charge on their sur-aces) can fuse with the negatively charged endosomal membrane
ratios (N/P) of 10, 15, 20, and 30, and luciferase activity was measured after 48 h.rties of PSMT. Control cells alone were used as negative controls. Results represent
of living cells, due to the neutral co-lipid mediating fusion of theliposome with the cell membrane, allowing nucleic acid to crossinto the cytoplasm and DNA contents to be available to the cell forreplication or expression. However, the results showed that trans-gene expression in HeLa cells with the PSMT carrier (3 × 106) wasapproximately 30% higher than that with Lipofectamine (2 × 106).PSMT/luciferase plasmid nanoparticles (NP) (PSMT/pLuc NP) com-plexed at different charge ratios (N/P) of 10, 15, 20, and 30 wereincubated with HeLa cancer cells, and transfection properties werecompared. The highest transfection efficiency was achieved at N/P15, where a lower amount of PSMT was used compared to the
cervical cancer cells (HeLa) after 48 h. Non-treated cells were used as a negative con-trol, and Lipofectamine/pLuc NP (Lipo/pLuc) was used as a positive control. Resultsrepresent the mean ± SD (error bars) of 3 independent experiments. Effect of endo-cytosis inhibitor (�-methyl cyclodextrin) on transfection efficiency of PSMT/DNAcomplexes in HeLa, CT-26 and MC 38 cells (b).
K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136 131
F MT wt ith DNp erime
spfdrtfuarraPtcftftaTitci
ig. 4. Cytotoxicity of PSMT on HeLa and HDF cells. Different concentrations of PSreatment to determine cell viability. Non-treated cells (control) or cells treated wositive control. Results represent the mean ± SD (error bars) of 3 independent exp
omewhat reduced at higher charge ratios of 20 and 30 when com-ared to N/P 10. N/P 15 was found to be the optimal charge ratioor transfection with PSMT, and higher concentrations producedecreased transfection accompanied by severe cytotoxicity. Theseesults suggest it may be feasible to use lower amounts of PSMTo enhance its biocompatibility. Finally, the optimal charge ratioor transfection varied depending on cell type. Interestingly, whensing PSMT as a carrier, levels of transfection in HDF cells remainedt low or negligible levels irrespective of the N/P charge, whichanged from 10 to 30. However, the commercial lipid based car-ier Lipofectamine produced considerable transfection in both HDFnd HeLa cells. This result suggests that the transfection property ofSMT is selective in cancer cells, but not in normal cells. Transfec-ion level varies between the cancer cell and normal cell. Primaryells and immune cells are hard to transfect cells, but the trans-ection of lipofectamine in HDF cells was comparatively enhancedhan PSMT transfection in those cells. Lipofectamine and PSMTollow a different pathway for cellular uptake which influences theransfection capabilities. Lipofectamine utilizes the clathrin medi-ted pathway and PSMT utilizes the caveolae mediated pathway.ransfection of the PSMT was based on the caveolae expression
n the cells. Some of the cancer cells have reduced caveolae pro-ein expression which in turn affected the transfection in thoseells. (Supplementary Fig. 2 and Fig. 3a). If PSMT were injectedn an in vivo tumor model, the enhanced expression should be
ere added to cells after 24 h of seeding. The MTS assay was performed 24 h afterA (DNA control) were used as negative controls, and Lipofectamine was used as ants.
predominantly found in the tumor region when compared to nor-mal regions, thus reducing unwanted off-target side effects byexpressing the therapeutic gene in an enhanced manner only inthe tumor region.
To further confirm the selective transfection property of PSMT,we conducted experiments using cancer cells and normal cells ofdifferent origins. Cancer cells including HeLa, BALB/c colon car-cinoma (CT-26), C57BL/6 colon carcinoma (MC38), and humanbreast adenocarcinoma cell line (MCF7) were chosen to test theconsistency of the PSMT carrier’s transporter properties. HDFs,immortalized cerebral endothelial cells (HEN7), and primarymouse peritoneal macrophage (M�) cells were selected to rep-resent normal cells. From the previous experiment using HeLaand HDF cells, we deduced that PSMT nanoparticles formed at thecharge ratio (N/P) of 15 should exhibit enhanced transfection prop-erties. In this new experiment, all cancer and normal cells weretransfected with PSMT at N/P 15, and their luciferase expressionwas compared. In comparison with previous studies [14], the trans-fection was found to be high in cancer cells, but the transfectionwas not compared to normal cells. We have compared with nor-mal cells, primary cells and cells of different origin in this study. In
agreement with the former experiment, an enhanced transfectionefficiency with PSMT was found with cancer cells, which possessa highly active caveolae-mediated pathway [19,23–25], but not innormal cells (Scheme 1) (Fig. 2). Among the cancer cell lines tested,
132 K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136
F F cellsN to visu
l7coTsssat
3
irdpimc
ig. 5. RFP reporter gene transfection into HeLa and HDF using PSMT. HeLa and HDP. Confocal image was taken 48 h post-transfection. Cells were stained with DAPI
uciferase expression was more pronounced only in HeLa and MCF- cells (Fig. 3), both of which originated from a human source. Wehecked the caveolae expression among the cancer cells, the cave-lae protein was found to be abundant in HeLa and MCF-7 cells.he caveolae expression is different within cancer cells, which ishown by us and also by other studies [26]. But more detailedtudy is needed to confirm these assessments, which is beyond thecope of this study. We have also blocked the endocytosis pathwaynd confirmed the reduction in both caveolae mediated uptake andransgene expression (Fig. 3b and supplementary Fig. 2).
.3. Cytotoxicity of PSMT in cells from different origins
The cytotoxicity of a carrier substance must be analyzed becauset may drastically reduce the viability of treated cells. If the car-ier itself is toxic, the effect of the therapeutic gene cannot beistinguished from effects of the carrier. Carrier toxicity may also
roduce unwanted side effects if it reaches non-targeted organs
n the body. PSMT carrier consists of PEI carrying numerous pri-ary amine groups with a highly positive charge. This positive
harge may rupture cellular membranes due to strong electrostatic
were seeded on coverslips for 1 day and then transfected with PSMT/RFP plasmidalize the nucleus.
interaction with the plasma membrane [27,28]. We selected HeLacancer cells and HDF normal cells to study the toxic effects of PSMTand Lipofectamine carriers. Different amounts of PSMT carrier,equivalent to the polymer concentrations used for nanoparticlesformed at N/P ratios of 10–20, were incubated with HeLa and HDFcells. One day after polymer treatment, toxicity was analyzed usingthe MTS assay, which determines the number of viable cells amongan entire population of cells. The viability of HeLa and primary cellstreated with Lipofectamine was reduced in comparison to PSMTtreated cells (Fig. 4). A minimal toxicity was observed with thecarrier and the cell viability of >80% was observed in cancer cellstreated with PSMT. HeLa cells treated with PSMT with an N/P of10–20 showed >60% viability. However, in primary cells, toxicityincreased with increasing concentrations of PSMT. While the carrierwas more toxic to primary cells than to cancer cells, PSMT was non-toxic at lower concentrations. Based on the results of transfectionefficiency and cell viability studies, the PSMT with N/P 15 demon-
strated the most enhanced transfection properties accompanied byreduced toxicity among the N/P ratios tested. The PSMT/plasmid NPformed at N/P 15 was used in further studies for comparisons withthe Lipofectamine/plasmid complex.
K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136 133
Fig. 6. Determination of selective transfection with PSMT NP in co-culture experiment. HeLa cells were seeded on coverslip overnight and Oregon Green labeled HDFc r plasp
tbhtsw2tilbtc
3d
gpstiTottT
ells were co-seeded. After the cells were fully attached to coverslips, RFP reporteost-transfection.
Toxicity of the carriers against normal cells can be reduced whenhe carrier is targeted to cancer cells. For targeting, the carrier has toe modified with targeting moieties like peptides, aptides, carbo-ydrates, etc. In the context of this study, it helps to understand thathe carrier does not mediate the cancer cell suppression and theuppression was due to the tumor suppressor gene P 53 deliveredith PSMT. In a previous study [14], the toxicity was tested against
93T normal cells and the carrier was not toxic to those cells. Sohe toxicity to normal cell line is varying between cell to cell andt may be partially due to their different origin and the expressionevel of survival related genes. HDF is a primary cell, which mighte more sensitive to the gene carrier. We are currently improvinghe carrier for achieving cyto-compatibility to all kinds of normalells.
.4. Transgene expression after PSMT mediated reporter geneelivery
Following quantitative confirmation of transfection efficiency,ene expression and visualization capabilities were analyzed torovide qualitative visualization of the effect of transgene expres-ion mediated by PSMT. We selected the RFP expressing plasmid forhis study. RFP plasmid was complexed with PSMT at N/P 15 andncubated with HeLa cancer cells and HDF primary cells (Fig. 5).ransgene expression of the red fluorescent protein was observed
nly in the transfected cancer cells. The primary cells were notransfected and were only visible due to DAPI staining; however,ransfection and expression of RFP were not detected in these cells.hese results demonstrate that transgene expression mediated by
mid was delivered into the cells using PSMT. Confocal images were taken at 16 h
PSMT is more specific in cancer cells than in normal cells. Enhancedgene transfer by osmotic PSMT is mediated through the selectivecaveolae endocytic pathway, which was previously demonstratedby an author of this manuscript [14]. The fact that cancer cells havemore caveolae than normal cells might be the primary reason forthis observation.
3.5. PSMT carriers selectively enhanced transfection in aco-culture study
In the previous experiment, the cells were treated with thePSMT/reporter plasmid complex while in an environment specificfor the cell type. We next investigated whether cancer cell-specificdelivery of PSMT would be maintained when both cell types (can-cer and normal types) were present in the same milieu. Therefore,we co-cultured HeLa cancer cells and HDF primary cells in the sameenvironment (Fig. 6). Primary cells were labeled with Oregon greenand the cancer cells were unlabeled. After labeling, the cells wereco-cultured and treated with PSMT/RFP plasmid NP. The expressionof Red fluorescent protein was observed only in the cancer cellsand not in primary cells (Fig. 6). This result indicates that cancercells were transfected with RFP and that primary cells labeled withOregon green had not been transfected. Therefore, we believe thatintracellular uptake and transgene expression with PSMT appearsto be selective for transformed cells and does not occur in primary
cells. If a therapeutic gene is delivered using PSMT, expression ofthe therapeutic gene should be more pronounced in the cancer cellsthan in normal cells, due to the caveolae mediated transfection ofPSMT in caveolae over-expressed cancer cells.
134 K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136
Fig. 7. Therapeutic transgene expression mediated by PSMT in cancer cells. HeLa cancer cells were treated with PSMT/p53 plasmid NP15 (PSMT/p53) and the expression ofp smid ac alizingp
3g
gpmambioccp
53 protein was confirmed by western blotting. Lipofectamine/p53 (Lipo/p53) plaontrols. (a) p53-mCherry plasmid was delivered with PSMT to cancer cells for visulasmid NP, and the confocal images were taken at 24 h post transfection.
.6. PSMT mediated delivery of functional p53 tumor suppressorene for cancer therapy
Successful reporter gene expression mediated by the PSMT sug-ests that a functional therapeutic gene could also be delivered. The53 tumor suppressor gene was selected as the functional plas-id to be delivered using PSMT as the carrier, and the resulting
nti-cancer effects were analyzed. A delivered therapeutic plas-id generally has to cross numerous internal and external barriers
efore reaching the target region. However, if the therapeutic genes delivered with a carrier molecule, the gene will be assisted in
vercoming most of the barriers and protected until it reaches theancer region. Functional plasmid p53 can induce apoptosis in theancer cells, and proliferation of cancer cells can be reduced if the53 gene is over-expressed. Thus, PSMT carrier was employed in
nd PSMT/Topo vector (PSMT/T-A vector) plasmid NP15 treated cells were used as therapeutic transgene expression. (b) Cells were treated with PSMT/p53-mcherry
this study to achieve effective delivery and expression of the p53gene. The functional plasmid p53 was delivered with PSMT car-rier and its expression in HeLa cells was confirmed by westernblotting (Fig. 7a). The expression of p53 protein was enhancedwhen delivered with PSMT compared to delivery using the freep53 plasmid. Free p53 plasmid DNA has a negative charge andits interaction with the negatively charged cell membrane will begreatly hindered. As a result, free p53 plasmid should not be eas-ily incorporated by cells. The PSMT mediated expression of p53was comparable to p53 expression mediated by Lipofectamine(Fig. 7a).
We used the p53 plasmid cloned with a mCherry reporter geneto visualize and analyze the morphology of PSMT-p53 NP treatedcells. The p53-mCherry plasmid was delivered by the PSMT car-rier and the expression of p53 protein was confirmed by the red
K.C.T. Nguyen et al. / Colloids and Surfaces B: Biointerfaces 119 (2014) 126–136 135
F r cells1 ee p53
flptaeptca
ig. 8. Detection of apoptosis in PSMT/p53 plasmid NP15 treated cells. HeLa cance5 min with 500 mM PI to detect apoptosis. PSMT/Topo vector plasmid NP15 and fr
uorescence of the reporter gene. The expression of p53 mCherrylasmid was seen only in PSMT/p53-mCherry treated cells. Cellsreated with free p53-mCherry plasmid DNA did not show notice-ble expression of p53 protein (Fig. 7b). Interestingly, the p53xpressing cells not only showed red fluorescence, but also mor-
hological changes, which indicated possible therapeutic effects ofhe p53-mCherry plasmid/PSMT treatment. Therefore, the PSMTarrier could possibly be used for the successful delivery of a ther-peutic gene.
were treated with PSMT/p53 plasmid NP15. After 24 h, the cells were stained for plasmid treated HeLa cells were used as controls.
PSMT-p53 NP treated cells showed a rounded morphology,and their stage of apoptosis was examined. PI accumulates inthe nucleus of apoptotic cells because the membranes of apop-totic cells are weaker and are penetrable even by a hydrophobicdye. Therefore, PI can traverse the outer membrane and nuclear
membrane of damaged cells, and indicate whether they are under-going apoptosis. Cells treated with free p53 plasmid DNA did notshow PI accumulation in the nucleus, while PSMT-p53 NP-treatedcells displayed both morphological changes and PI accumulation
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36 K.C.T. Nguyen et al. / Colloids and Sur
t 24 h post-transfection. However, control cells maintained a nor-al morphology and did not show PI accumulation (Fig. 8). These
esults indicate that p53 plasmid delivery resulted in cellular dam-ge which ultimately led to apoptosis. Functional anti-cancer geneelivery with a PSMT carrier appears to be a strategy to pursue inhe fight against cancer. Additionally, a PSMT carrier could be use-ul in therapeutic gene delivery studies conducted in vitro. Furthern vivo experiments with this carrier should to be performed, andhe in vitro transfection results need to be validated.
cknowledgements
This research was supported by the Leading Foreign Researchnstitute Recruitment Program through the National Researchoundation of Korea (NRF) funded by the Ministry of Education,cience and Technology (MEST) (2011-0030034).
ppendix A. Supplementary data
Supplementary material related to this article can be found,n the online version, at http://dx.doi.org/10.1016/j.colsurfb.014.05.003.
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