Accepted Manuscript
Title: Selective transfection with osmotically active sorbitol modified PEI nanoparticles for enhanced anti-cancer gene therapy
Author: Kim Cuc Thi Nguyen Muthunarayanan Muthiah Mohammad Ariful Islam R. Santhosh Kalash Chong-Su Cho Hansoo Park Il-Kwon Lee Hyeoung-Joon Kim In-Kyu Park Kyung A. Cho
PII: S0927-7765(14)00229-X
DOI: http://dx.doi.org/doi:10.1016/j.colsurfb.2014.05.003
Reference: COLSUB 6408
To appear in: Colloids and Surfaces B: Biointerfaces Received date: 3-2-2014
Revised date: 1-5-2014 Accepted date: 2-5-2014
Please cite this article as: K.C.T. Nguyen, M. Muthiah, M.A. Islam, R.S.
Kalash, C.-S. Cho, H. Park, I.-K. Lee, H.-J. Kim, I.-K. Park, K.A. Cho, Selective transfection with osmotically active sorbitol modified PEI nanoparticles for enhanced anti-cancer gene therapy, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.05.003
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Accepted Manuscript
Selective transfection with osmotically active sorbitol modified PEI
241
nanoparticles for enhanced anti-cancer gene therapy
242
Kim Cuc Thi Nguyena,f †, Muthunarayanan Muthiahb,f †, Mohammad Ariful Islamc, R 243
Santhosh Kalashb, Chong-Su Choc, Hansoo Parke, Il-Kwon Leed, Hyeoung-Joon Kimd, In- 244
Kyu Park b,f*, Kyung A Choa,f* 245
aDepartment of Biochemistry, Chonnam National University Medical School, Gwangju 501- 246
746, South Korea 247
bDepartment of Biomedical Science and Chonnam National University Medical School, 248
Gwangju, 501-746, South Korea 249
cDepartment of Agricultural Biotechnology and Research Institute for Agriculture and Life 250
Sciences, Seoul National University, Seoul 151-921, South Korea 251
dDepartment of Hematology-Oncology, Chonnam National University Hwasun Hospital, 252
Hwasun, Jeollanamdo 519-763, South Korea 253
e School of Integrative Engineering, Chung-Ang University, Dongjak-gu, Seoul 156-756, 254
South Korea 255
f BK21 PLUS Center for Creative Biomedical Scientists at Chonnam National University, 256
Chonnam National University Medical School, Gwangju 501-746, South Korea 257
Correspondence to: I.K. Park, Department of Biomedical Sciences, Chonnam National 258
University Medical School, Gwangju 501-746, South Korea. Tel.:+82 61 379 8481; fax: +82 259
61 379 8455.
260
Correspondence to: K.A Cho, Department of Biochemistry, Chonnam National University 261
Medical School, Gwangju 501-746, South Korea 262
E-mail addresses: [email protected] (I.-K. Park), kacho@ chonnam.ac.kr (K.-A. Cho) 263
264
† These authors contributed to the work equally.
265
Summary 266
Total Number of Words: 5,841 267
Total Figures: 9 268
269
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Abstract 269
270
Polysorbitol-mediated transporter (PSMT) has been previously shown to achieve high 271
transfection efficiency with minimal cytotoxicity. Polysorbitol backbone possesses osmotic 272
properties and leads to enhanced cellular uptake. The PSMT/pDNA nanoparticles were 273
prepared and the particle size, surface charge of the nanoparticles was determined for the 274
study. PSMT delivers genes into cells by the caveolae mediated endocytic pathway. Caveolae 275
expression is usually altered in transformed cancer cells. Transfection through the caveolae 276
may help PSMT to selectively transfect cancer cells rather than normal cells. Transfection of 277
the luciferase gene by PSMT was tested in various cell types including cancer cell lines, 278
primary cells, and immortalized cells. Luciferase transgene expression mediated by PSMT 279
was remarkably increased in HeLa cells compared to expression using the control carrier 280
Lipofectamine. Moreover, the toxicity of PSMT was comparable to the control carrier 281
(Lipofectamine) in the same cells. Selective transfection of cancer cells using PSMT was 282
further confirmed by co-culture of cancer and normal cells, which showed that transgene 283
expression was pre-dominantly achieved in cancer cells. A functional p53 gene was also 284
delivered into HeLa cells using PSMT and the selective transgene expression of p53 protein 285
in cancer cells was analyzed through western blotting and confocal microscopy. HeLa cells 286
transfected with PSMT/p53 plasmid nanoparticles showed cellular damage and apoptosis, 287
which was confirmed through propidium iodide staining.
288 289
Keywords: Transfection efficiency; Polyethylenimine; sorbitol; Caveolae-dependent 290
endocytosis.
291
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Introduction 292
Successful gene therapy depends on the efficient and safe delivery of a therapeutic gene. The 293
main obstacles to achieving efficient gene delivery lie in selection of the gene carrier and its 294
transfection efficiency [1, 2]. Viral gene carriers have enhanced transfection efficiencies, but 295
their associated toxicities limit their use as clinical vectors [3, 4]. Recently, the focus has 296
turned towards non-viral carriers, but such carriers still have to compete with viral carriers in 297
terms of transfection efficiency. The initial search for non-viral carriers focused on the use of 298
polylysine, while later studies examined transfection efficiencies achieved using 299
polyethylenimine (PEI) [5]. While PEI was efficient as a non-viral carrier, its non-degradable 300
nature produced toxicity in cells [6]. PEI was thus chemically modified to reduce its toxicity 301
and increase biocompatibility. These modifications included its conjugation with 302
biocompatible polymers such as chitosan, PEG, and hyaluronic acid. Degradable units were 303
also introduced into PEI for facilitating its degradation into smaller units after entering cells 304
[7, 8]. To some extent, di-sulphide modified PEI (ssPEI) also reduced toxicity when it was 305
formulated as a non-viral carrier for gene delivery. The ssPEI was stable in the extracellular 306
environment and reductive in the intracellular environment; these characteristics helped to 307
deliver the gene and facilitated degradation of the carrier over time [9]. However, issues 308
concerning accelerated cellular uptake and intracellular localization remained areas of 309
concern. Recently, polysorbitol-mediated transporter (PSMT), whose structure is based on 310
sorbitol diacrylate (SDA) and low molecular weight PEI (LMW PEI), was synthesized in our 311
group to facilitate cellular uptake of genetic material and enhance transfection efficiency in 312
cells [10]. The PSMT transporter system has a polysorbitol backbone possessing osmotic 313
properties. When the carrier binds to the cells, the osmotic condition of the cells are altered 314
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and the cellular uptake of the particle is enhanced. PSMT interacts with the cell through the 315
caveolae mediated pathway. It was hypothesized that osmotically active PSMT gene carrier 316
might be able to selectively transfect and treat caveolae over-expressing cancer cells with 317
tumor suppressor gene, as described in scheme 1. Compared to the currently available 318
transfection agents and polymeric carriers, cytotoxicity was greatly reduced with PSMT.
319
PSMT polymer was synthesized using Michael addition reaction, so the low molecular PEI 320
was conjugated alternatively to SDA. This was due to the fact that only non-toxic lower 321
molecular PEI was conjugated alternatively to hydrophilic SDA, and degradability was 322
assured with the ester linkage formed by SDA modification. Transfection efficiency is an 323
important and interesting phenomenon because it varies among different types of cells 324
according to their origin, proliferation status, cellular constituents, membrane architecture, 325
and receptors present on the membrane [11, 12]. Normal cells differ from cancer cells in their 326
membrane architecture, cellular uptakes, and various other characteristics which may 327
influence transfection efficiency [13]. Our previous study demonstrated that the selective 328
caveolae-dependent endocytic pathway played a significant role for increasing efficiency of 329
transfection by osmotic PSMT-mediated gene delivery [14]. The caveolae protein is over- 330
expressed in most of cancer cells, while it is less pronounced in normal cells [15-18]. Based 331
on such findings, we hypothesized that PSMT-based transfection of therapeutic genes would 332
be selectively enhanced in cancer cells compared to normal cells. A comparison of PSMT 333
mediated transfection in cancer cells and normal cells of various origins would provide 334
additional information regarding the use of PSMT as a carrier for selective gene delivery. We 335
were therefore interested to examine whether the transfection efficiency of PSMT is 336
consistent across different cell lines, or selectively depending on cellular caveolae activation.
337
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In this study, we have physico chemically characterized the PSMT/pDNA nanoparticles and 338
tested both the capability of PSMT to transfect various cell types and its cytotoxicity to cells.
339
Selective transfection using the carrier was confirmed by co-culture experiments performed 340
after differential labeling of normal cells and cancer cells. Finally, PSMT was utilized for 341
cancer cell-specific therapeutic gene delivery. Following intracellular delivery of p53 tumor 342
suppressor gene/PSMT nanoparticles, morphological changes and the apoptotic status of cells 343
were assessed with respective experiments.
344
345
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Materials and methods 345
2.1. Reagents 346
Branched PEI (bPEI) 25 kDa and Poly-L lysine were purchased from Sigma-Aldrich (St.
347
Louis, MO, USA). Plasmid gWiz-luc (Aldevron, Madison, Wisconsin, USA) was 348
transformed into competent Escherichia coli DH5α cells by a heat shock method. The gWiz- 349
luc was then propagated in bacterial cultures grown in Luria-Bertani (LB) media (BD, 350
Franklin Lakes, New Jersey, USA) containing 100 mg/mL kanamycin (Biosesang Inc., 351
Korea) and extracted and purified using a mini DNA-spin kit (Intron Biotechnol Co., Korea).
352
Lipofectamine 2000, Cell Trace™ Oregon Green, propidium iodide, and TOPO T-A cloning 353
vector were purchased from Invitrogen (Carlsbad, CA, USA). The CellTiter 96® AQueous 354
Non-Radioactive Cell Proliferation Assay (MTS assay) was purchased from Promega 355
(Madison, WI, USA) and used as per the manufacturer’s protocol. Glass coverslips were 356
purchased from Marienfeld (Lauda-Königshofen, Germany). Plasmid pLentiH1.4- 357
monomerRFP was purchased from Macrogen, (Seoul, Korea) and used for the over- 358
expression of Red fluorescent protein (RFP) reporter in cells. Primary antibody against p53 359
protein (Santa Cruz, Dallas, Texas, USA) was used to confirm expression of p53 protein by 360
western blotting.
361
2.1.1 Cell lines 362
Various cancer cell lines, including human cervical cancer (HeLa), BALB/c colon carcinoma 363
(CT-26), C57BL/6 colon carcinoma (MC38), and human breast adenocarcinoma cell line 364
(MCF7) were purchased from American Type Culture Collection (ATCC; Manassas, VA, 365
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USA) and used in this study. Both primary and immortalized cells were used for comparisons 366
of PSMT-mediated transfection. Human diploid fibroblasts (HDF) were isolated from human 367
foreskins as previously described [19] . Stable clonal lines of human brain microvessel 368
endothelia cells (HEN7) were generated from human fetal telencephalon by using retroviral 369
vector encoding human telomerase reverse transcriptase and beta-galactosidase. Primary 370
mouse peritoneal macrophages (M) were isolated from C57BL/6 mice as previously 371
described [20].
372
2.1.2 Synthesis of PSMT 373
PSMT composed of Low Molecular Weight (LMW) branched PEI and Sorbitol Di-Acrylate 374
(SDA) was synthesized via the Michael addition reaction as previously reported 10. In brief, 375
LMW branched PEI and SDA were separately dissolved in a vial with anhydrous DMSO.
376
Then, SDA was slowly dropped to PEI solution in a three-neck reaction flask under dry 377
nitrogen purge at a stoichiometric ratio of 1:1 of PEI to SDA. The reaction was performed at 378
80 °C for 24 h with magnetic stirring. Then, the reaction mixture was dialyzed against 379
distilled water (MWCO: 3500 Da) at 4 °C for 48 h and lyophilized. The final products were 380
stored at −70 °C for the steps.
381
2.2 Preparation and physico chemical characterization of PSMT/plasmid DNA 382
nanoparticles 383
The plasmid DNA binding ability of the PSMT was evaluated by electrophoresis through a 384
1.2% agarose gel containing ethidium bromide intercalating dye. The nitrogen-to-phosphate 385
(N/P) molar ratio was varied by adding predetermined PSMT concentrations to a fixed 386
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amount of the plasmid DNA. The PSMT and plasmid DNA solutions were mixed at N/P 387
ratios from 5 to 20 and vortexed briefly. bPEI 25kDa was taken as control. The complexes 388
were kept at room temperature for 30 min for complete complexation before being loaded 389
into an agarose gel. Electrophoresis was carried out at 100 V for 50 min, and DNA retention 390
was visualized under ultraviolet (UV) illumination. Particle sizes and zeta potentials were 391
measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK).
392
2.3 Cell culture and analysis of PSMT transfection efficiency 393
HDF, HeLa, CT-26, and MC-38 cells were cultured in Dulbecco’s Modified Eagle’s Medium 394
(DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA).
395
M and MCF7 cells were cultured in RPMI-1640 supplemented with 10% FBS. HEN7 cells 396
were cultured in endothelia basal medium (EGM-2 Bullet Kit, Lonza, Switzerland).
397
Cells were seeded in 24-well culture plates in a 5% CO2 atmosphere at 37°C. After reaching 398
confluence, the cells were washed 3 times with DPBS (Takara, Japan) and transfected with 1 399
μg of plasmid DNA complexed with either Lipofectamine 2000 prepared according to the 400
manufacturer’s instructions or PSMT at different charge ratios in Opti-MEM solution for 4 h, 401
followed by a 48 h post-incubation in culture medium containing 10% FBS at 37°C in a 5%
402
CO2 incubator. At 48 h post-transfection, the cells were lysed in 1x lysis buffer (Promega, 403
Madison, WI, USA). Transfection results were obtained by measuring the extent of transgene 404
expression in the lysate, as quantified by luciferase activity determined using the Luciferase 405
Assay System (Promega, Madison, WI, USA). A microplate luminometer (Microlumat Plus 406
LB96V, Berthold Technologies, Germany) was set for a 3-sec delay with signal integration 407
for 10 sec. Luciferase activity was normalized to the amount of total protein in a sample. A 408
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calibration curve created using a bovine serum albumin standard was used to measure protein 409
concentration. Transfection activity was expressed as relative light units (RLU) per milligram 410
of cellular protein.
411
2.4 Cytotoxicity of PSMT/plasmid nanoparticles 412
HDF and HeLa were seeded in 96-well tissue culture plates with DMEM medium containing 413
10% FBS. The cytotoxicity of PSMT/pLuc NP was evaluated by determining cell viability 414
after a 24 h incubation in DMEM media containing 10% FBS with different concentrations of 415
PSMT. Numbers of viable cells were determined by measuring mitochondrial reductase 416
activity using the tetrazolium-based colorimetric method (MTS assay, Promega) according to 417
the manufacturer’s instructions.
418
2.5 Comparison of transgene expression by PSMT/plasmid NP in transformed cancer 419
cells and normal cells.
420
HDF and HeLa cells were cultivated in DMEM at 37°C under humidified 5% CO2 for 3 days, 421
and were then sub-cultured after reaching confluence. Cells were seeded on Poly L-lysine 422
coated glass coverslips at a density of 1 x 104cells per well in DMEM medium containing 423
10% FBS for 24 h. The cells were then treated with PSMT/RFP reporter plasmid NP or 424
PSMT-p53 NP for 4 h in opti-MEM (Gibco, NY, USA) and then transferred to DMEM 425
medium containing 10% FBS for further incubation. After 24 h, the coverslips were washed 426
with DPBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature (RT).
427
The treated cells were washed 3 times with DPBS and permeabilized with 0.05% Triton X- 428
100 in DPBS for 10 min at RT. After washing, the samples were counter-stained with DAPI, 429
mounted on glass slides, and observed by confocal microscopy (Carl Zeiss Microimaging Inc.
430
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Oberkochen, Germany).
431
For the co-culture model, HeLa cells were first seeded on glass coverslips overnight and then 432
co-seeded with Oregon Green labeled-HDF on the same coverslips. After confirming the full 433
attachment of HDF, transfection experiments were performed using either 434
Lipofectamine/plasmid NP or PSMT/plasmid NP. The samples were then fixed, 435
permeabilized, and counter-stained with DAPI at 16 h post-transfection as described above.
436
2.6 Confirmation of transgene expression by western blotting after treatment with 437
PSMT/p53 tumor suppressor gene NP.
438
HeLa cells were seeded on 6-well plates one day before transfection with p53 plasmid. NP 439
formed with either Lipofectamine/p53 NP or PSMT-p53 NP was delivered to HeLa cells and 440
the intracellular proteins were collected at 24 h post-transfection by scrapping the treated 441
cells in lysis buffer [50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA, 60 mM Octyl-β-D- 442
glucopyranoside, 1 mM phenylmethylsulfonyl fluoride protease inhibitor cocktail (1:500)], 443
followed by brief sonication for 10 sec. The protein concentration of each sample was 444
measured using the Bradford assay. Protein samples were separated by SDS-PAGE and then 445
transferred to a PVDF membrane. The membrane was blocked in 5% skim milk for 1 h at RT 446
and incubated over-night with anti-p53 primary antibody at 4oC, followed by incubation with 447
peroxidase-conjugated anti-mouse secondary antibody for an additional 1 h. After washing 3 448
times, the membranes were developed using an enhanced chemiluminescence detection kit.
449
2.7 Detection of apoptosis induced by treatment with PSMT/p53 plasmid NP 450
HeLa cells were seeded on Poly-L lysine coated coverslips at a density of 1 x 104 cells per 451
well in DMEM medium containing 10% FBS for 24 h. The cells were then transfected with 452
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either p53 plasmid alone or PSMT/p53 plasmid NP (PSMT-p53 NP). At 24 h post- 453
transfection, the cells were stained with 500 mM PI in DMEM media supplemented with 454
10% FBS for 15 min in a 5% CO2 atmosphere at 37oC for detection of apoptosis.. After 455
staining, the cells were washed 3 times in DPBS, fixed with 4% PFA, permeabilized with 456
0.05% Triton X-100, and counter-stained with DAPI as described above.
457
2.8 Statistical analysis 458
Quantitative data are expressed as means ± SD. The means were compared using an 459
independent samples t-test. P values less than 0.05 were considered statistically significant.
460
461
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3. Results and Discussion 461
Hyperosmotic activity improves transfection capability by increasing the cellular uptake of 462
osmotically active gene carriers [21]. The PSMT transporter system has a polysorbitol 463
backbone possessing osmotic properties. As a result, the osmotically active component in 464
PSMT might also be responsible for the synergistically enhanced cellular uptake and 465
transfection efficiency of PSMT. In our previous study, the transfection efficiency with PSMT 466
was analyzed in human lung adenocarcinoma epithelial cells (A549), human lung bronchio- 467
alveolar carcinoma cells (H322), and human cervix epithelial carcinoma cells. All 3 of these 468
cell lines had originated from the cancers and showed enhanced transfection efficiency with a 469
PSMT carrier. We therefore wanted to confirm whether the use of PSMT would be beneficial 470
for enhancing transfection with all types of cells of different lineages.
471
3.1 Physico chemical characterization of PSMT/pDNA nanoparticles 472
PSMT/pDNA nanoparticles (NP) were completely retarded in a polyacrylamide gel at an N/P 473
ratio of 15, whereas bPEI/pDNA NP was retarded in the gel at an N/P ratio of 5 (Fig. 1a). The 474
particle sizes of the PSMT/pDNA NP ranged from 100 to 200 nm. (Figure 1b, Left). The 475
surface charge of the PSMT/pDNA was measured by zeta potential analysis and the charges 476
were negative at the lower N/P ratio 1, but the transition to the positive charge was observed 477
at the N/P ratio 3. Surface charge of the PSMT/pDNA NP at the N/P ratio 20 to 40 was within 478
the range between +20mV and +30mV (Figure 1b, Right).
479
3.2 Efficiency of PSMT transfection in the co-culture of cancer cells and normal cells 480
Two cell types representing cancer cells and normal cells were selected for this study to test 481
the effect of PSMT on transfection efficiency in various cell types. HeLa cells were chosen as 482
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a representative cell line for transformed cells, and HDF (human diploid fibroblasts) cells 483
were selected as normal cells. To determine transfection efficiency, luciferase plasmid was 484
mixed with varying concentrations of PSMT, and the complexes were incubated with HeLa 485
and HDF cells to examine luciferase expression (Figure 2). PSMT mediated luciferase 486
expression was significantly increased in HeLa cells when compared to luciferase expression 487
mediated by Lipofectamine. Lipofectamine and PSMT utilize different pathways for 488
transfection. Lipofectamine is a cationic carrier which was designed and made commercially 489
available for efficient transfection. It is reported that the liposomes generally utilize the 490
clathrin mediated uptake pathway [22], Lipofectamine's cationic lipid molecules are 491
formulated with a neutral co-lipid (helper lipid). The DNA-containing liposomes (with 492
positive charge on their surfaces) can fuse with the negatively charged endosomal membrane 493
of living cells, due to the neutral co-lipid mediating fusion of the liposome with the cell 494
membrane, allowing nucleic acid to cross into the cytoplasm and DNA contents to be 495
available to the cell for replication or expression. However, the results showed that transgene 496
expression in HeLa cells with the PSMT carrier (3*106) was approximately 30% higher than 497
that with Lipofectamine (2*106). PSMT/luciferase plasmid nanoparticles (NP) (PSMT/pLuc 498
NP) complexed at different charge ratios (N/P) of 10, 15, 20, and 30 were incubated with 499
HeLa cancer cells, and transfection properties were compared. The highest transfection 500
efficiency was achieved at N/P 15, where a lower amount of PSMT was used compared to the 501
complexes formed at N/P 20 and N/P 30. It was therefore evident that increasing the carrier 502
concentration did not increase transfection efficiency. The transfection efficiency of 503
PSMT/pLuc NP was somewhat reduced at higher charge ratios of 20 and 30 when compared 504
to N/P 10. N/P 15 was found to be the optimal charge ratio for transfection with PSMT, and 505
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higher concentrations produced decreased transfection accompanied by severe cytotoxicity.
506
These results suggest it may be feasible to use lower amounts of PSMT to enhance its 507
biocompatibility. Finally, the optimal charge ratio for transfection varied depending on cell 508
type. Interestingly, when using PSMT as a carrier, levels of transfection in HDF cells 509
remained at low or negligible levels irrespective of the N/P charge, which ranged from 10 to 510
30. However, the commercial lipid based carrier Lipofectamine produced considerable 511
transfection in both HDF and HeLa cells. This result suggests that the transfection property of 512
PSMT is selective in cancer cells, but not in normal cells. Transfection level varies between 513
the cancer cell and normal cell. Primary cells and immune cells are hard to transfect cells, but 514
the transfection of lipofectamine in HDF cells was comparatively enhanced than PSMT 515
transfection in those cells. Lipofectamine and PSMT follow a different pathway for cellular 516
uptake which influences the transfection capabilities. Lipofectamine utilizes the clathrin 517
mediated pathway and PSMT utilizes the caveolae mediated pathway. Transfection of the 518
PSMT was based on the caveolae expression in the cells. Some of the cancer cells have 519
reduced caveolae protein expression which in turn affected the transfection in those cells.
520
(Supplementary Fig 2 and Fig 4b). If PSMT were injected in an in vivo tumor model, the 521
enhanced expression should be predominantly found in the tumor region when compared to 522
normal regions, thus reducing unwanted off-target side effects by expressing the therapeutic 523
gene in an enhanced manner only in the tumor region.
524
To further confirm the selective transfection property of PSMT, we conducted experiments 525
using cancer cells and normal cells of different origins. Cancer cells including HeLa, BALB/c 526
colon carcinoma (CT-26), C57BL/6 colon carcinoma (MC38), and human breast 527
adenocarcinoma cell line (MCF7) were chosen to test the consistency of the PSMT carrier’s 528
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transporter properties. HDFs, immortalized cerebral endothelial cells (HEN7), and primary 529
mouse peritoneal macrophage (M) cells were selected to represent normal cells. From the 530
previous experiment using HeLa and HDF cells, we deduced that PSMT nanoparticles 531
formed at the charge ratio (N/P) of 15 should exhibit enhanced transfection properties. In this 532
new experiment, all cancer and normal cells were transfected with PSMT at N/P 15, and their 533
luciferase expression was compared. In comparison with previous studies [14], the 534
transfection was found to be high in cancer cells, but the transfection was not compared to 535
normal cells. We have compared with normal cells, primary cells and cells of different origin 536
in this study. In agreement with the former experiment, an enhanced transfection efficiency 537
with PSMT was found with cancer cells, which possess a highly active caveolae-mediated 538
pathway [19, 23-25], but not in normal cells (Scheme 1) (Figure 2). Among the cancer cell 539
lines tested, luciferase expression was more pronounced only in HeLa and MCF-7 cells 540
(Figure 4), both of which originated from a human source. We checked the caveolae 541
expression among the cancer cells, the caveolae protein was found to be abundant in HeLa 542
and MCF-7 cells. The caveolae expression is different within cancer cells, which is shown by 543
us and also by other studies [26]. But more detailed study is needed to confirm these 544
assessments, which is beyond the scope of this study. We have also blocked the endocytosis 545
pathway and confirmed the reduction in both caveolae mediated uptake and transgene 546
expression. (Fig.4b and supplementary Fig.2) 547
3.3 Cytotoxicity of PSMT in cells from different origins 548
The cytotoxicity of a carrier substance must be analyzed because it may drastically reduce the 549
viability of treated cells. If the carrier itself is toxic, the effect of the therapeutic gene cannot 550
be distinguished from effects of the carrier. Carrier toxicity may also produce unwanted side 551
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effects if it reaches non-targeted organs in the body. PSMT carrier consists of PEI carrying 552
numerous primary amine groups with a highly positive charge. This positive charge may 553
rupture cellular membranes due to strong electrostatic interaction with the plasma membrane 554
[27, 28]. We selected HeLa cancer cells and HDF normal cells to study the toxic effects of 555
PSMT and Lipofectamine carriers. Different amounts of PSMT carrier, equivalent to the 556
polymer concentrations used for nanoparticles formed at N/P ratios of 10 to 20, were 557
incubated with HeLa and HDF cells. One day after polymer treatment, toxicity was analyzed 558
using the MTS assay, which determines the number of viable cells among an entire 559
population of cells. The viability of HeLa and primary cells treated with Lipofectamine was 560
reduced in comparison to PSMT treated cells (Figure 3). A minimal toxicity was observed 561
with the carrier and the cell viability of > 80% was observed in cancer cells treated with 562
PSMT. HeLa cells treated with PSMT with an N/P of 10-20 showed > 60% viability.
563
However, in primary cells, toxicity increased with increasing concentrations of PSMT. While 564
the carrier was more toxic to primary cells than to cancer cells, PSMT was non-toxic at lower 565
concentrations. Based on the results of transfection efficiency and cell viability studies, the 566
PSMT with N/P 15 demonstrated the most enhanced transfection properties accompanied by 567
reduced toxicity among the N/P ratios tested. The PSMT/plasmid NP formed at N/P 15 was 568
used in further studies for comparisons with the Lipofectamine/plasmid complex.
569
Toxicity of the carriers against normal cells can be reduced when the carrier is targeted to 570
cancer cells. For targeting, the carrier has to be modified with targeting moieties like peptides, 571
aptides, carbohydrates etc. In the context of this study, it helps to understand that the carrier 572
does not mediate the cancer cell suppression and the suppression was due to the tumor 573
suppressor gene P 53 delivered with PSMT. In a previous study [14], the toxicity was tested 574
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against 293T normal cells and the carrier was not toxic to those cells. So the toxicity to 575
normal cell line is varying between cell to cell and it may be partially due to their different 576
origin and the expression level of survival related genes. HDF is a primary cell, which might 577
be more sensitive to the gene carrier. We are currently improving the carrier for achieving 578
cyto-compatibility to all kinds of normal cells.
579
580
3.4 Transgene expression after PSMT mediated reporter gene delivery 581
Following quantitative confirmation of transfection efficiency, gene expression and 582
visualization capabilities were analyzed to provide qualitative visualization of the effect of 583
transgene expression mediated by PSMT. We selected the RFP expressing plasmid for this 584
study. RFP plasmid was complexed with PSMT at N/P 15 and incubated with HeLa cancer 585
cells and HDF primary cells (Figure 5). Transgene expression of the red fluorescent protein 586
was observed only in the transfected cancer cells. The primary cells were not transfected and 587
were only visible due to DAPI staining; however, transfection and expression of RFP were 588
not detected in these cells. These results demonstrate that transgene expression mediated by 589
PSMT is more specific in cancer cells than in normal cells. Enhanced gene transfer by 590
osmotic PSMT is mediated through the selective caveolae endocytic pathway, which was 591
previously demonstrated by an author of this manuscript [14]. The fact that cancer cells have 592
more caveolae than normal cells might be the primary reason for this observation.
593
3.5 PSMT carriers selectively enhanced transfection in a co-culture study 594
In the previous experiment, the cells were treated with the PSMT/reporter plasmid complex 595
while in an environment specific for the cell type. We next investigated whether cancer cell- 596
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specific delivery of PSMT would be maintained when both cell types (cancer and normal 597
types) were present in the same milieu. Therefore, we co-cultured HeLa cancer cells and 598
HDF primary cells in the same environment (Figure 6). Primary cells were labeled with 599
Oregon green and the cancer cells were unlabeled. After labeling, the cells were co-cultured 600
and treated with PSMT/RFP plasmid NP. The expression of Red fluorescent protein was 601
observed only in the cancer cells and not in primary cells (Figure 6). This result indicates that 602
cancer cells were transfected with RFP and that primary cells labeled with Oregon green had 603
not been transfected. Therefore, we believe that intracellular uptake and transgene expression 604
with PSMT appears to be selective for transformed cells and does not occur in primary cells.
605
If a therapeutic gene is delivered using PSMT, expression of the therapeutic gene should be 606
more pronounced in the cancer cells than in normal cells, due to the caveolae mediated 607
transfection of PSMT in caveolae over-expressed cancer cells.
608
3.6 PSMT mediated delivery of functional p53 tumor suppressor gene for cancer 609
therapy 610
Successful reporter gene expression mediated by the PSMT suggests that a functional 611
therapeutic gene could also be delivered. The p53 tumor suppressor gene was selected as the 612
functional plasmid to be delivered using PSMT as the carrier, and the resulting anti-cancer 613
effects were analyzed. A delivered therapeutic plasmid generally has to cross numerous 614
internal and external barriers before reaching the target region. However, if the therapeutic 615
gene is delivered with a carrier molecule, the gene will be assisted in overcoming most of the 616
barriers and protected until it reaches the cancer region. Functional plasmid p53 can induce 617
apoptosis in the cancer cells, and proliferation of cancer cells can be reduced if the p53 gene 618
is over-expressed. Thus, PSMT carrier was employed in this study to achieve effective 619
Accepted Manuscript
delivery and expression of the p53 gene. The functional plasmid p53 was delivered with 620
PSMT carrier and its expression in HeLa cells was confirmed by western blotting (Figure 7a).
621
The expression of p53 protein was enhanced when delivered with PSMT compared to 622
delivery using the free p53 plasmid. Free p53 plasmid DNA has a negative charge and its 623
interaction with the negatively charged cell membrane will be greatly hindered. As a result, 624
free p53 plasmid should not be easily incorporated by cells. The PSMT mediated expression 625
of p53 was comparable to p53 expression mediated by Lipofectamine (Figure 7a).
626
We used the p53 plasmid cloned with a mCherry reporter gene to visualize and analyze the 627
morphology of PSMT-p53 NP treated cells. The p53-mCherry plasmid was delivered by the 628
PSMT carrier and the expression of p53 protein was confirmed by the red fluorescence of the 629
reporter gene. The expression of p53 mCherry plasmid was seen only in PSMT/p53-mCherry 630
treated cells. Cells treated with free p53-mCherry plasmid DNA did not show noticeable 631
expression of p53 protein (Figure 7b). Interestingly, the p53 expressing cells not only showed 632
red fluorescence, but also morphological changes, which indicated possible therapeutic 633
effects of the p53-mCherry plasmid/PSMT treatment. Therefore, the PSMT carrier could 634
possibly be used for the successful delivery of a therapeutic gene.
635
PSMT-p53 NP treated cells showed a rounded morphology, and their stage of apoptosis was 636
examined. PI accumulates in the nucleus of apoptotic cells because the membranes of 637
apoptotic cells are weaker and are penetrable even by a hydrophobic dye. Therefore, PI can 638
traverse the outer membrane and nuclear membrane of damaged cells, and indicate whether 639
they are undergoing apoptosis. Cells treated with free p53 plasmid DNA did not show PI 640
accumulation in the nucleus, while PSMT-p53 NP -treated cells displayed both morphological 641
changes and PI accumulation at 24 h post-transfection. These results were similar to those 642
Accepted Manuscript
observed in Lipofectamine /p53 treated cells. However, control cells maintained a normal 643
morphology and did not show PI accumulation (Figure 8). These results indicate that p53 644
plasmid delivery resulted in cellular damage which ultimately led to apoptosis. Functional 645
anti-cancer gene delivery with a PSMT carrier appears to be a strategy to pursue in the fight 646
against cancer. Additionally, a PSMT carrier could be useful in therapeutic gene delivery 647
studies conducted in vitro. Further in vivo experiments with this carrier should to be 648
performed, and the in vitro transfection results need to be validated.
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Acknowledgements:
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This research was supported by the Leading Foreign Research Institute Recruitment Program 653
through the National Research Foundation of Korea (NRF) funded by the Ministry of 654
Education, Science and Technology (MEST) (2011-0030034).
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Figure legends 657
Figure 1. Physico-chemical characterization of PSMT/pDNA nanoparticles. The 658
complexation between the PSMT carrier and the pDNA was analyzed by agarose gel 659
retardation assay. The particle size and surface charge of the PSMT/pDNA nanoparticles were 660
measured by dynamic light scattering and Zeta potential analysis, respectively.
661
Figure 2. Transfection efficiency of PSMT for cancer and primary cells. Transfection 662
efficiency of the PSMT carrier in different cell types was analyzed by the luciferase assay.
663
HeLa cancer cells and HDF primary cells were transfected with PSMT/pLuc NP at charge 664
ratios (N/P) of 10, 15, 20, and 30, and luciferase activity was measured after 48 h.
665
Lipofectamine/pLuc NP was used as a positive control to compare the transfection properties 666
of PSMT. Control cells alone were used as negative controls. Results represent the mean ± 667
SD (error bars) of 3 independent experiments. (*P < 0.05) 668
Figure 3. Cytotoxicity of PSMT on HeLa and HDF cells. Different concentrations of PSMT 669
were added to cells after 24 h of seeding. The MTS assay was performed 24 h after treatment 670
to determine cell viability. Non-treated cells (control) or cells treated with DNA (DNA 671
control) were used as negative controls, and Lipofectamine was used as a positive control.
672
Results represent the mean ± SD (error bars) of 3 independent experiments.
673
Figure 4. Transfection efficiency of PSMT in various cell lines a). PSMT/pLuc NP was 674
prepared at N/P 15 and incubated with cells at 24 h after seeding. Transfection efficiency of 675
PSMT/pLuc NP was analyzed in colon cancer cells (CT-26, MC38), breast cancer cells 676
(MCF7), primary cells (HDF and M), immortalized cells (HEN7), and cervical cancer cells 677
(HeLa) after 48 h. Non-treated cells were used as a negative control, and Lipofectamine/pLuc 678
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NP (Lipo/pLuc) was used as a positive control. Results represent the mean ± SD (error bars) 679
of 3 independent experiments. Effect of endocytosis inhibitor (β-methyl cyclodextrin) on 680
transfection efficiency of PSMT/DNA complexes in HeLa,CT-26 and MC 38 cells b).
681
Figure 5. RFP reporter gene transfection into HeLa and HDF using PSMT. HeLa and HDF 682
cells were seeded on coverslips for 1 day and then transfected with PSMT/RFP plasmid NP.
683
Confocal image was taken 48 h post-transfection. Cells were stained with DAPI to visualize 684
the nucleus.
685
Figure 6. Determination of selective transfection with PSMT NP in co-culture experiment.
686
HeLa cells were seeded on coverslip overnight and Oregon Green labeled HDF cells were co- 687
seeded. After the cells were fully attached to coverslips, RFP reporter plasmid was delivered 688
into the cells using PSMT. Confocal images were taken at 16 h post-transfection.
689
Figure 7. Therapeutic transgene expression mediated by PSMT in cancer cells. HeLa cancer 690
cells were treated with PSMT/p53 plasmid NP15 (PSMT/p53) and the expression of p53 691
protein was confirmed by western blotting. Lipofectamine/p53 (Lipo/p53) plasmid and 692
PSMT/Topo vector (PSMT/T-A vector) plasmid NP15 treated cells were used as controls. a) 693
p53-mCherry plasmid was delivered with PSMT to cancer cells for visualizing therapeutic 694
transgene expression. b) Cells were treated with PSMT/p53-mcherry plasmid NP, and the 695
confocal images were taken at 24 h post transfection.
696
Figure 8. Detection of apoptosis in PSMT /p53 plasmid NP15 treated cells. HeLa cancer cells 697
were treated with PSMT /p53 plasmid NP15. After 24 h, the cells were stained for 15 min 698
with 500 mM PI to detect apoptosis. PSMT /Topo vector plasmid NP15 and free p53 plasmid 699
treated HeLa cells were used as controls 700
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Scheme 1 703
704
Scheme 1. The therapeutic gene expression in the cancer cells is enhanced due to the specific 705
interaction of sorbitol component in the PSMT/pDNA to the caveolae protein which is found 706
to have high expression in cancer cells compared to normal cells. The normal cells have basal 707
level of caveolae protein leading to reduced uptake and expression of the PSMT/pDNA 708
nanoparticle.
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Figures 710
Figure 1 711
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Fig 2 714
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Figure 3 719
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Figure 4 a) 721
Control Lipo/pLuc PSMT/Pluc
0 1.0106 2.0106 3.0106
4.0106 HDF TG-M HEN-7 MC38 CT26 MCF7 HeLa
RLU/mg protein
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b) 723
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Figure 5 727
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Figure 6 732
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Figure 7a 743
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Figure 7b 745
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Figure 8 748
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Polysorbitol-mediated transporter (PSMT) nano carrier showed enhanced transfection 833
efficiency in caveolae over expressed cancer cells, which was confirmed by co-culture study 834
and p53/PSMT mediated apoptosis was detected through the apoptosis assays.
835 836 837
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837
Highlights 838
839
Transfection efficiency of PSMT was tested against different cell lines 840
Luciferase expression mediated by PSMT increased in HeLa cells than normal cells 841
Selective transfection using PSMT was confirmed by co-culture of both the cells 842
PSMT/p53 nanoparticles treated HeLa cells showed cellular damage and apoptosis 843
844 845 846
