Effective mRNA Inhibition in PANC-1 Cells in Vitro Mediated via an mPEG–SeSe–PEI Delivery System

Yuefeng Zhang; Bin Yang; Yajie Liu; Wenjie Qin; Chao Li; Lantian Wang; Wen Zheng; Yulian Wu

doi:10.1248/bpb.b15-00645

Abstract

RNA interference (RNAi)-mediated gene therapy is a promising approach to cure various diseases. However, developing an effective, safe, specific RNAi delivery system remains a major challenge. In this study, a novel redox-responsive polyetherimide (PEI)-based nanovector, mPEG–SeSe–PEI, was developed and its efficacy evaluated. We prepared three mPEG–SeSe–PEI vector candidates for small interfering glyceraldehyde-3-phosphate dehydrogenase (siGADPH) and determined their physiochemical properties and transfection efficiency using flow cytometry and PEG_11.6–SeSe–PEI polymer. We investigated the silencing efficacy of GADPH mRNA expression in PANC-1 cells and observed that PEG_11.6–SeSe–PEI/siGADPH (N/P ratio=10) polyplexes possessed the appropriate size and zeta-potential and exhibited excellent in vitro gene silencing effects with the least cytotoxicity in PANC-1 cells. In conclusion, we present PEG_11.6–SeSe–PEI as a potential therapeutic gene delivery system for small interfering RNA (siRNA).

RNA interference (RNAi) technology, which utilizes small interfering double-stranded RNAs (siRNAs) to induce the silencing of sequence-specific mRNAs, has attracted extensive research attention for its potential medical applications since its initial introduction in 1998.¹⁾ RNAi provides a new approach to treat diseases by effectively silencing pathogenic genes.^2,3) Delivery of siRNAs using nucleic acid vectors is necessary to avoid rapid degradation by RNases in the cell and tissue microenvironment. However, finding an efficacious delivery system remains a major obstacle for use in gene therapy.⁴⁾ An ideal carrier should be safe, highly efficient, and targeted. Potential vectors can be classified into two groups: viral delivery systems; and nonviral delivery systems.⁵⁾ The former include lentivirus⁶⁾ and adenovirus⁷⁾ vectors, while the latter includes liposomes,⁸⁾ cationic polymers,⁹⁾ cationic peptides,¹⁰⁾ and nonionic surfactant vesicles (Noisome).¹¹⁾ Although the viral vectors possess higher efficiency, their side effects including immunogenicity and increased risk of neoplasias are a source of concern.¹²⁾ Compared with viral carriers, a nonviral delivery system may be considered more attractive due to increased regulatory control and better safety profile.^13,14)

Numerous nonviral vectors have been extensively studied. Polyetherimide (PEI) vectors are considered the gold standard with a relatively high transfection efficiency.^15–17) However, rapid aggregation and cytotoxicity due to the highly positive charge have limited their application.¹⁸⁾ One strategy to overcome these limitations is to modify PEI with polyethylene glycol (PEG) chains,^19–22) although PEGylation can result in decreased cellular internalization of DNA and affect the gene release from lysosomes.^20,23,24) Therefore, we developed a novel PEG-detachable PEI-based polymer, mPEG–SeSe–PEI, that addresses this issue. By taking advantage of higher intracellular glutathione (GSH) levels, the diselenide bond between PEG and PEI can be easily cleaved in the cytoplasm to facilitate the release of DNA. The novel redox-responsive polycation with minimized cytotoxicity allows easier endosomal escape and excellent transfection efficacy for delivering plasmid DNA (pDNA) in HepG2 cells.^20,25) We preliminarily evaluated the efficiency of mPEG–SeSe–PEI to deliver siRNA versus pDNA and found that it was more effective in carrying siRNA.

In this study, the optimal conditions for mPEG–SeSe–PEI to condense and deliver siRNA were investigated. The autoassembling complexes of mPEG–SeSe–PEI/siRNA, PEG–PEI/siRNA, and PEI/siRNA were prepared and their physicochemical properties were determined. The ability to condense siRNA was detected in the gel retardation assay, and zeta-potentials and particle sizes of those complexes were characterized using dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM). The internalization efficiency was determined by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM), and cytotoxicity was assessed in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reverse transcription-polymerase chain reaction (RT-PCR) assay was performed to investigate the efficacy of RNA interference.

MATERIALS AND METHODS

Chemicals and Reagents

siRNA targeting GADPH (siGADPH) (sense: 5′-UGA CCU CAA CUA CAU GGU UTT-3′ and antisense: 5′-AAC CAU GUA GUU GAG GUC ATT-3′), negative control siRNA (siNC) (sense: 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and antisense: 5′-ACG UGA CAC GUU CGG AGA ATT-3′), Cy3-labeled siGADPH, and fluorescein amidite (FAM)-labeled siGADPH were designed and synthesized by GenePharma (Shanghai, China). The commercial transfection reagent Lipofectamine2000 (Lipo2000) was purchased from Life Technologies (Carlsbad, CA, U.S.A.).

Cell Culture

The human pancreatic cancer cell line PANC-1 was purchased from the Shanghai Institute for Biological Sciences (Shanghai, China). The cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) to which 10% fetal bovine serum and 1% penicillin–streptomycin were added at 37°C in a fully humidified atmosphere of 5% CO₂.

Synthesis of mPEG–SeSe–PEI and PEG–PEI

Three types of mPEG–SeSe–PEI polymers with different grafting degrees and PEG–PEI were synthesized following the procedures and using the materials described in our previous study.²⁰⁾ The mPEG–SeSe–PEI and PEG–PEI polymers were dissolved in double-distilled water (ddH₂O), diluted to a concentration of 2 mg/mL, and then stored at −20 and 4°C, respectively.

Preparation of siGADPH Complexes

The N/P ratio was used to measure the molar ratio between the positive amino groups of PEI and the negative phosphate groups of siRNA. The siGADPH complexes at different N/P ratios were formed as follows: the appropriate amount of siRNA and a corresponding amount of mPEG–SeSe–PEI, PEG–PEI, or PEI were dissolved separately in Opti-MEM (Life Technologies). The two solutions were mixed thoroughly and maintained at room temperature for 30 min.

Agarose Gel Retardation Assay

The agarose gel retardation assay was conducted to determine the ability of mPEG–SeSe–PEI, PEG–PEI, and PEI to condense siGADPH molecules. Briefly, siGADPH complexes containing different polymers at various N/P ratios (N/P=0, 5, 8, 10, 15, 20) and siGADPH (40 pmol) were prepared, mixed with loading buffer (TaKaRa, Dalian, China), loaded onto 1% agarose gel stained with 1 µg/mL ethidium bromide (Sigma-Aldrich, St. Louis, MO, U.S.A.), and electrophoresed at 100 V for 30 min in Tris–acetate–ethylenediaminetetraacetic acid (EDTA) buffer at room temperature. The bands of siRNA were visualized and recorded with the personal gel imaging system (Cell Biosciences, Santa Clara, CA, U.S.A.).

Transfection Efficiency Assay

PANC-1 cells were seeded in six-well plates and maintained with complete medium containing no antibiotics. Transfection was performed when cell confluence reached approximately 80%. First, a series of complexes was prepared by thoroughly mixing 100 pmol of FAM-labeled siRNA with the corresponding amounts of mPEG–SeSe–PEI, PEG–PEI, PEI, and Lipo2000 and then diluted with Opti-MEM to a specific volume before being added to the wells. After being exposed to these different complexes for 5 h, the PANC-1 cells were harvested. FCM was performed with BD FACS Canto-II (Becton, Dickinson and Company, Franklin Lakes, NJ, U.S.A.) to determine the transfection efficiency, and the data were processed with FlowJo software, version 7.6 (FlowJo, Ashland, OR, U.S.A.). Phosphate buffered saline (PBS)/siRNA served as the negative control, and Lipo2000/siRNA (a mixture of 7.5 µL Lipo2000 and 100 pmol FAM-labeled siRNA) was used as the positive control.

Physical Characteristics of siGADPH Complexes

The particle sizes and zeta-potentials of complexes dissolved in ddH₂O at different N/P ratios were determined by DLS analysis with a Zetasizer NanoS90 nanoparticle analyzer (Malvern Instruments Ltd., Malvern, U.K.). The transmission electron microscope (Tecnai 12 Bio-Twin, FEI/Philips, Hillsboro, OR, U.S.A.) was used to observe the morphology of the nanoparticles and images were captured with an Erlangshen ES500W camera (Gatan, Pleasanton, CA, U.S.A.).

Cell Viability Assay

The cytotoxicity of PEG_11.6–SeSe–PEI, PEG–PEI, and PEI in PANC-1 cells was assessed in the MTT assay. PANC-1 cells were seeded in 96-well plates at a density of 8000 cells/well with 200 µL of complete medium and incubated overnight. The culture medium was replaced with 200 µL of Opti-MEM containing different siRNA complexes at a series of N/P ratios. After being incubated for 5 h, the cells were cultured with normal complete DMEM again for another 19 h. Twenty microliters of MTT (Sigma-Aldrich) stock solution was added to each well and incubated for 4 h, and then the discarded medium and formazan crystals were dissolved with 150 µL of dimethyl sulfoxide (DMSO) (Sigma-Aldrich). Absorbance was detected with a Microplate Reader (Bio-Rad, Hercules, CA, U.S.A.) at a wavelength of 570 nm. Blank wells with 150 µL of DMSO were used as the internal control.

CLSM

PANC-1 cells were seeded into a 24-well plate for 24 h and then exposed to Cy3-labeled siRNA complexes containing different transfection reagents. After 5 h, the cells were fixed with 4% paraformaldehyde for 10 min and subsequently permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 5 min and stained with the DNA-staining reagent 0.5% 4′-6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for another 10 min. The cells were washed three times with PBS before every reagent change. CLSM (Carl Zeiss Co., Ltd., Gottingen, Germany) was then performed for fluorescent imaging.

RNA Isolation and Quantitative RT-PCR

PANC-1 cells were seeded into six-well plates and transfected with different complexes containing 100 pmol of siGADPH per well, and the transfection assay was performed with the method mentioned above. After transfection, PANC-1 cells were cultured with fresh complete medium for 48 h, the cells were harvested, and total RNA was extracted using Trizol Reagent H (Life Technologies) according to the manufacturer’s protocol. The concentration and quality of RNA were estimated with a NanoDrop1000 (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Total RNA (1000 ng) was reverse-transcribed with the Prime Script RT reagent kit (TaKaRa). Quantitative RT-PCR was performed in the 7500Fast System (Applied Biosystems, Foster City, CA, U.S.A.) using the Mix SYBR Kit (TaKaRa). The 2^−ΔΔCT method was used to analyze the relative GADPH mRNA expression level, and β-actin was used to normalize the results. The following primers were used: GADPH-forward, 5′-CTC ACT CAA GAT TGT CAG CAA TG-3′; GADPH-reverse, 5′-GGC AGT GAT GGC ATG GAC TGT-3′; β-actin-forward, 5′-ATG ATA TCG CCG CGC TCG TCG TC-3′; and β-actin-reverse, 5′-CGC TCG GCC GTG GTG GTG AA-3′.

Statistical Analysis

The Statistical Package for the Social Sciences version 13.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used for statistical analyses. Differences between two groups were compared using Student’s t-test. A p value of less than 0.05 was defined as significant. Data were acquired from at least three independent experiments and presented the mean±standard deviation (S.D.). All graphs and curves were drawn using GraphPad Software, Inc. in U.S.A. (GraphPad Software, Inc., CA, U.S.A.).

RESULTS

Preparation of mPEG–SeSe–PEI and siGADPH Complexes

The mPEG–SeSe–PEI and PEG–PEI copolymers were formulated, and the molecular weight of PEG and branched PEI used in this research was 5 and 25 kDa, respectively. We prepared mPEG–SeSe–PEI and PEG–PEI with different grafting levels by changing the feed ratio of PEI and PEG. The m value indicated the average number of PEG segments conjugated per PEI backbone, which was calculated according to the results of the ¹H-NMR spectrum. The calculated m values were 5, 6.9, and 11.6 for the corresponding polymers, denoted as PEG₅–SeSe–PEI, PEG_6.9–SeSe–PEI, and PEG_11.6–SeSe–PEI, respectively. PEG–PEI was synthesized with a PEG grafting level of 11.8. The different siGADPH complexes at various N/P ratios were then synthesized following the procedures described above.

Gel Electrophoresis Retardation Assay

The agarose gel electrophoresis retardation assay was performed with PEI as a control. As shown in Fig. 1, the bands of different siGADPH complexes disappeared at N/P ratios greater than 5, 10, 8, 8, and 10 for PEI/siGADPH (Fig. 1A), PEG_11.8–PEI/siGADPH (Fig. 1B), PEG₅–SeSe–PEI/siGADPH (Fig. 1C), PEG_6.9–SeSe–PEI/siGADPH (Fig. 1D), and PEG_11.6–SeSe–PEI/siGADPH (Fig. 1E), respectively. Only the naked anionic siRNA could be detected as a band in the gel. Therefore the results revealed that siGADPH could be completely condensed by PEI at the N/P ratio of 5, while PEG–PEI, PEG₅–SeSe–PEI, PEG_6.9–SeSe–PEI, and PEG_11.6–SeSe–PEI condensed all the siGADPH at the N/P ratio of 10, 8, 8, and 10, respectively.

Fig. 1. Images of Agarose Gel Electrophoresis Retardation Assay

The six lanes from left to right were N/P ratio of 0, 5, 8, 10, 15, and 20, respectively. Complexes of (A) PEI/siGADPH, (B) PEG_11.8–PEI/siGADPH, (C) PEG₅–SeSe–PEI/siGADPH, (D) PEG_6.9–SeSe–PEI/siGADPH, and (E) PEG_11.6–SeSe–PEI /siGADPH at various N/P ratios (0, 5, 8, 10, 15, 20) mixed with loading buffer were loaded on 1% agarose gel, electrophoresed at 100 V for 30 min in TAE buffer at room temperature. The assay was repeated three times.

Gene Transfection Efficiency

To determine the best vector from among these candidates, the transfection efficiency of various complexes at N/P ratios greater than 5 was evaluated using FCM. As shown in Fig. 2A, the maximum transfection efficiencies of PEI/siRNA (N/P=20), PEG₅–SeSe–PEI/siRNA (N/P=5), PEG_6.9–SeSe–PEI/siRNA (N/P=15), PEG_11.6–SeSe–PEI/siRNA (N/P=10), and PEG_11.8–PEI (N/P=10) were 54.1, 43.6, 59.8, 73.8, and 75.7%, respectively. The maximum transfection efficiency of the six different polyplexes was then compared with that of Lipo2000/siRNA, which was 73.2% (Fig. 2B). Compared with Lipo2000/siRNA, increased transfection efficacy was achieved with PEG_11.6–SeSe–PEI/siRNA at the N/P ratio of 10 (Fig. 2B).

Fig. 2. Gene Transfection Efficiency Were Determined with Flow Cytometry

(A) Transfection efficiency of different complexes at various N/P ratios. (B) The comparison of maximum transfection efficiency of different ployplexes. The data were presented as the mean±S.D. (n=3). Differences compared to the Lipo2000/siRNA group were defined as significant when p<0.05 (p<0.05).

Sizes and Zeta-Potentials of siGADPH Complexes

The particle sizes and zeta-potentials of different siGADPH complexes at the N/P ratio of 10 and PEG_11.6–SeSe–PEI/siGADPH at various N/P ratios were determined using DLS analysis. As shown in Table 1, at the N/P ratio of 10, the PEI/siGADPH (N/P=20) complexes had larger sizes and higher zeta-potentials than those of mPEG–SeSe–PEI/siGADPH and PEG–PEI/siGADPH. At the same N/P ratio, increasing the PEGylation level of mPEG–SeSe–PEI/siGADPH resulted in increased particle sizes and decreased zeta-potentials. The size distribution graphs of different polyplexes are presented in Figs. 3A–E. In addition, the proportion of nanoparticles with diameters of 50–100 nm for PEI/siGADPH, PEG_11.8–PEI/siGADPH, PEG_11.6–SeSe–PEI/siGADPH, PEG_6.9–SeSe–PEI/siGADPH, and PEG₅–SeSe–PEI/siGADPH were 0, 83.1, 76.9, 52.4, and 22.2%, respectively (Figs. 3A–E). Table 2 shows the particle sizes and zeta-potentials of PEG_11.6–SeSe–PEI/siGADPH at various N/P ratios. With increasing N/P ratio, the sizes of PEG_11.6–SeSe–PEI/siGADPH decreased and the zeta-potentials increased. The TEM images of PEG_11.6–SeSe–PEI/siRNA (N/P=10) particles showed that those nanoparticles were relatively regular spheres with good distribution (Figs. 3F, G).

Table 1. Particle Sizes and Zeta-Potentials of Different Nanoparticles at the N/P Ratio of 10

	Effective diameter (nm)	zeta-Potential (mV)
PEI/siGADPH	237.56±25.28	39.28±2.36
PEG11.8–PEI/siGADPH	78.82±13.44	32.89±2.27
PEG11.6–SeSe–PEI/siGADPH	68.75±11.26	33.30±2.38
PEG6.9–SeSe–PEI/siGADPH	56.09±10.24	35.33±2.62
PEG5–SeSe–PEI/siGADPH	42.55±5.25	37.70±2.58

Fig. 3. Sizes of Different Nanoparticles

(A)–(E) were the size distribution graphs of nanoparticles PEI/siGADPH, PEG_11.8–PEI/siGADPH, PEG_11.6–SeSe–PEI/siGADPH, PEG_6.9–SeSe–PEI/siGADPH, and PEG₅–SeSe–PEI/siGADPH, respectively. The heights of black bars denoted the proportions of nanoparticles with the diameter in range of 50–100 nm. (F) and (G) were the typical TEM images of PEG_11.6–SeSe–PEI/siGADPH(N/P=10) complexes, the little dark dots represented the nanoparticles. The data were presented as the mean±S.D. (n=3).

Table 2. Particle Sizes and Zeta-Potentials of PEG_11.6–SeSe–PEI/siGADPH at Various N/P Ratios

N/P	Effective diameter (nm)	zeta-Potential (mV)
5	64.41±13.89	30.35±2.61
10	60.75±15.00	33.30±2.38
15	55.15±10.06	35.21±2.35
20	48.02±10.25	36.33±2.28
30	41.89±8.13	39.70±1.62
40	39.96±7.35	41.73±4.41

MTT Cytotoxicity Analysis

The MTT assay was performed to estimate the cytotoxicity of PEG_11.6–SeSe–PEI, PEG–PEI, PEI, and Lipo2000 in PANC-1 cells. Lipo2000/siRNA was used as the positive control. As shown in Fig. 4, cell viability decreased dramatically when the N/P ratio increased, and the toxicity of the complexes increased in an N/P ratio-dependent manner. In addition, the toxicity of PEG_11.6–SeSe–PEI and PEG–PEI complexes were similar but weaker than that of the PEI complex. This discrepancy was especially apparent when the N/P ratio was high. The survival rate of cells treated with Lipo2000/siRNA was 86.41% and that of cells exposed to PEG_11.6–SeSe–PEI/siRNA (N/P=10) was 91.27%.

Fig. 4. Cytotoxicity of Various Complexes at Different N/P Ratios after Incubation for 5 h within PANC-1 Cells

The last bar denoted liposome (Lipo2000/siRNA complexes). The data were presented as the mean±S.D. (n=5). Differences compared to the control group were defined as significant when p<0.05.

CLSM

The internalization efficiency of different polyplexes in PANC-1 cells was confirmed with CLSM. As shown in Fig. 5, gray fluorescence denoting CY3-labeled siRNA overlapped with or was in close proximity to DAPI-labeled nuclei (dark gray fluorescence), indicating the intracytoplasmic localization of siRNA. Notably, siRNA accumulation (gray fluorescence) within the cytoplasm of cells incubated with Lipo2000/siRNA, PEG_11.6–SeSe–PEI/siRNA, and PEG–PEI/siRNA complexes was markedly greater than that in cells transfected with PEI/siRNA (Figs. 5A–D).

Fig. 5. The CLSM Images of PANC-1 Cells after Exposure to Different Ployplexes for 5 h

The gray fluorescence denoted the Cy3-labeled siRNA and the dark gray fluorescence denoted the DAPI-stained cell nucleus. Group pictures (A)–(D) were the cellular uptake of Lipo2000/siRNA, PEI/siRNA and PEG_11.8–PEI/siRNA, PEG_11.6–SeSe–PEI/siRNA polyplexes, respectively.

Efficacy of Gene Suppression

The RT-PCR assay was conducted to investigate the effects of different siGADPH complexes on GADPH mRNA levels in PANC-1 cells. As shown in Fig. 6, compared with the control, no significant decrease in GAPDH RNA was observed in the PEG_11.6–SeSe–PEI/siNC group. The degree of inhibition induced by Lipo2000/siGADPH, PEI/siGADPH, PEG–PEI/siGADPH, and PEG_11.6–SeSe–PEI/siGADPH was 76.58, 52.65, 61.28, and 75.64%, respectively. The inhibitory effect on GAPDH RNA levels by Lipo2000 and PEG_11.6–SeSe–PEI in medium containing serum was also investigated and found to be 51.95% and 71.30%, respectively.

Fig. 6. The Gene Silencing Efficiency Were Assessed with the RT-PCR Assay

The cells treated with Opti-MEM containing no complexes was the control.The data were presented as the mean±S.D. (n=6). Differences compared to the control group were defined as significant when p<0.05 (*** p<0.001).

DISCUSSION

PEI is a standard nonviral vector used for siRNA.^15,16) The presence of rich secondary amino groups in PEI leads to a high positive charge density.¹⁶⁾ The efficient nucleic acid condensation by PEI and PEI/siRNA complexes enabling effective cellular uptake can be attributed to the strong binding anions of nucleic acids and membrane proteins by their high-density surface cations. Moreover, the proton sponge effect resulting from the strong surface charge of PEI facilitates the escape of PEI/siRNA complexes from endosomes/lysosomes.¹⁷⁾ Conjugating the water-soluble PEG chains to PEI is widely applied to decrease PEI toxicity and enhance its biocompatibility.²⁴⁾ However, studies suggested that PEGylation of PEI attenuates the efficacy of pDNA transfection and improves the efficacy of siRNA-induced RNAi.¹⁹⁾ We previously developed mPEG–SeSe–PEI as a novel redox-responsive PEG-modified PEI-based polymer. The diselenide bonds between PEG and PEI segments can easily cleave in the setting of higher intracellular GSH levels, resulting in easier endosomal/lysosomal escape and improved pDNA transfection efficiency in HepG2 cells.^20,25)

In the present study, we evaluated the ability of mPEG–SeSe–PEI to deliver siRNA and screened for optimal conditions. The ability to condense siRNA was first estimated. Results from the gel retardation assay suggested that mPEG–SeSe–PEI and PEG–PEI were less efficient than PEI in condensing siRNA. Furthermore, the siRNA condensation by mPEG–SeSe–PEI decreased with increased PEGylation, which can be attributed to PEG-induced steric repulsion.^19,21) The gel retardation assay results also showed that PEG_11.6–SeSe–PEI had the same ability to condense siRNA as PEG_11.8–PEI, indicating that the diselenide bonds have no impact on the ability of mPEG–SeSe–PEI to condense siRNA.

The transfection efficiency of various complexes was then determined. The FCM results revealed that PEG_11.6–SeSe–PEI/siRNA at the N/P ratio of 10 had excellent delivery efficiency comparable to that of Lipo2000/siRNA and PEG–PEI/siRNA. Their transfection efficiency was significantly higher than that of PEI/siRNA, PEG₅–SeSe–PEI/siRNA, and PEG_6.9–SeSe–PEI/siRNA complexes. We also observed that there was no significant difference in transfection efficiencies among PEG_11.6–SeSe–PEI/siRNA (N/P=10), Lipo2000/siRNA, and PEG–PEI/siRNA (N/P=10) (p>0.05).

Particle size and zeta-potential are two vital physicochemical characters that are closely related to the cellular uptake efficacy of nanoparticles.^26–29) To elucidate the results shown in Fig. 2, the particle sizes and zeta-potentials of different siGADPH complexes were determined. At the same N/P ratio, increasing the PEGylation level of mPEG–SeSe–PEI/siGADPH led to increased particle size and decreased zeta-potential. The trends were consistent with those in other studies and could be explained by the assembly mechanisms.^26,30–32) The PEG layer conjugated to PEI can dramatically improve the water solubility of copolymers. As a result, mPEG–SeSe–PEI/siRNA or PEG–PEI/siRNA nanoparticles conform in a corona-like structure with the PEG shells outside, making their size smaller than that of PEI/siGADPH particles.^26,30–32) The appropriate size is essential for effective cellular internalization, and nanoparticles with a diameter of 50–100 nm were found to be favorable for endocytosis.^27–29) Nanoparticles either too large or too small impede endocytosis. We hypothesized that the appropriate size of PEG_11.6–SeSe–PEI/siRNA and PEG–PEI/siRNA may be responsible for their greater internalization efficacy. To investigate this, the proportions of nanoparticles with the diameter ranging from 50 to 100 nm were determined. The proportions for PEI/siRNA (N/P=20), PEG₅–SeSe–PEI/siRNA (N/P=5), PEG_6.9–SeSe–PEI/siRNA (N/P=15), PEG_11.6–SeSe–PEI/siRNA (N/P=10), and PEG–PEI (N/P=10) were 0, 76.9, 83.1, 52.4, and 22.2%, respectively, and the corresponding transfection efficiency of those complexes was 54.1, 73.8, 75.7, 59.8, and 43.6%, respectively. These results showed that a higher percentage of nanoparticles with diameters in the range of 50–100 nm corresponded with higher transfection efficiency, confirming previous results.^27–29) It was also noted that PEI/siGADPH had relatively high transfection efficiency although most polyplexes of PEI/siGADPH had diameters greater than 200 nm. It is assumed that the high positive surface charge may account for this. In addition, the typical CLSM images indicated that complexes of Lipo2000/siRNA, PEG–PEI/siRNA, and PEG_11.6–SeSe–PEI/siRNA have similar internalization efficiencies, while PEI/siRNA has relatively lower transfection efficiency. These findings confirmed the results of FCM.

The cytotoxicity assay findings indicated that transfected PEG_11.6–SeSe–PEI/siRNA (N/P=10) did not notably affect the viability of PANC-1 cells. Furthermore, the PEG layer may be responsible for the reduced toxicity of PEG_11.6–SeSe–PEI and PEG–PEI complexes.²⁴⁾

High transfection efficiency does not always lead to high inhibition efficiency, as only escaped siRNA can silence the target gene. Therefore, evaluating gene inhibition efficiency in the RT-PCR assay provides a more direct method to evaluate siRNA vectors. Markedly downregulated expression of GADPH was observed in cells treated with Lipo2000/siRNA and PEG_11.6–SeSe–PEI/siRNA. This suggests that PEG_11.6–SeSe–PEI is an excellent siRNA vector and is as effective as Lipo2000 (p>0.05). The finding that gene inhibition mediated by PEG–PEI/siGADPH was more efficient than that by PEI/siGADPH is in agreement with previously reported results.¹⁹⁾ Although PEG grafting levels, ability to condense siRNA, physicochemical characteristics, cytotoxicity, and internalization efficiency appear to be similar between PEG_11.6–SeSe–PEI/siRNA, PEG–PEI/siRNA, and PEG_11.6–SeSe–PEI/siRNA is more efficient than PEG–PEI/siRNA in gene silencing (p<0.01). This is believed to be due to the previously documented enhanced cargo release to the cytoplasm via the cleavage of diselenide bonds.²⁰⁾ In addition, the presence of serum significantly decreased the internalization efficiency of Lipo2000/siRNA (p<0.01) but not that of PEG_11.6–SeSe–PEI/siRNA (p>0.05). The outer PEG layer of PEG_11.6–SeSe–PEI can inhibit the nonspecific interaction between PEG_11.6–SeSe–PEI particles and protein in medium, which may account for this.^18,24) Overall, PEG_11.6–SeSe–PEI/siRNA is more stable and less vulnerable to serum than Lipo2000/siRNA.

While this study confirmed that PEG_11.6–SeSe–PEI is an efficient siRNA delivery system in PANC-1 cells in vitro, its stability and metabolism under physiological conditions are more complicated. In addition, several studies on PEGylated PEI-based nanovectors found varying results, possibly due to the differing molecular weights of PEI or PEG,^21,33) different ratios of PEG and PEI, and different methods of conjugation.²¹⁾ A further factor may be the addition of other compounds, such as bioinspired phosphorylcholine,³⁴⁾ hyaluronic acid,³⁵⁾ cyclic RGD,³⁶⁾ and γ-polyglutamic acid.³⁷⁾ Consequently, further studies on these modifications could achieve even more efficient, specific PEI-based nanovectors.

CONCLUSION

In summary, this study presents a new type of PEG-detachable PEI-based polymer, PEG_11.6–SeSe–PEI, that can completely encapsulate the siRNA at N/P ratios greater than 10 and condense siRNA into small spherical nanoparticles with good distribution. The PEG_11.6–SeSe–PEI/siGADPH (N/P=10) nanoparticles, 60.75±11.26 nm in diameter and with zeta-potentials of 33.30±2.38 mV, showed reduced cytotoxicity, excellent cell internalization efficiency, and ability to mediate significant gene suppression in PANC-1 cells even in the presence of serum protein. Therefore, PEG_11.6–SeSe–PEI is a superior siRNA carrier compared with PEI, PEG–PEI, or Lipo2000 and holds tremendous potential for clinical therapy applications.

Acknowledgment

This study was financially supported by The Natural Science Foundation of Zhejiang Province of China (Grant number: Q16H180002).

Conflict of Interest

The authors declare no conflict of interest.

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