Potent Gene Silencing In Vitro at Physiological pH Using Chitosan Polymers

Abstract

Among non-viral cationic polymers, biodegradable chitosan has during the last decade become an attractive carrier for small interference RNA (siRNA) delivery. Currently, degradation of macromolecules in the lysosomes is assumed to be a major barrier for effective siRNA transfection. Hence, transfection protocols are focused toward endosomal release mechanisms. In this work, we have tested 3 novel chitosan polymers and their siRNA delivery properties in vitro. To obtain efficient gene silencing of our model gene, S100A4, various transfection parameters were investigated, such as pH, nitrogen/phosphate ratio, photochemical internalization (PCI), media for complex formation, and cell lines. Our results showed that 2 linear chitosan polymers demonstrated excellent siRNA gene silencing, better than Lipofectamine 2000. The silencing effect was achieved without PCI treatment, under physiological pH, and with no observable reduction in cell viability.

Introduction

Presently, several biodegradable non-viral carriers in combination with small interfering RNA (siRNA) molecules are under investigation for therapeutic applications. These include, for example, atelocollagen (Azuma et al., 2010), chitosan (Kim et al., 2011), and a biocompatible linear cyclodextrin-based polymer (Davis et al., 2010).

The natural polysaccharide chitosan is obtained from deacetylation of chitin, a component from the exoskeleton of crustaceans. Chitosan consists of D-glucosamine and N-acetyl-D-glucosamine linked by glycosidic bonds. Advantageous properties like low toxicity, low immunogenicity, and high biocompatibility bears promise of chitosan as an effective carrier for gene therapy applications. Moreover, when the primary amines are protonated at acidic pH, many cationic sites are available for binding to the anionic siRNA molecule (Mao et al., 2010). However, chitosan is reported to have a poor transfection efficacy at physiological pH (Strand et al., 2008; Opanasopit et al., 2010).

Chitosan has a weak pH buffering capacity compared to polyethylenimine (PEI), which is assumed to be a crucial step for escaping the endosomal compartments (Boe et al., 2008; Mao et al., 2001). However, modification of chitosan with imidazole offers an improved endosomal pH buffering mechanism, leading to enhanced transfection efficiency (Ghosn et al., 2008). An alternative strategy to overcome the endosomal barrier is a method called photochemical internalization technology (PCI) (Berg et al., 1999). PCI makes it possible to both target the desired site and significantly enhance the delivery of molecules and particles entrapped in endosomal and lysosomal vesicles. The PCI method relies on illumination by activating a pre-endocytosed photosensitizer (PS) localized in the endosomes and lysosomes. Upon light activation, the PS generates reactive oxygen species, eventually leading to rupture of the endocytic membrane, and thereby releasing the entrapped material to cytosol. In the field of gene silencing, siRNA molecules have previously been transfected successfully in combination with PCI in vitro with various carriers including jetSI and jetSI-ENDO reagents, Lipofectamine, various branched polyethylenimine (B-PEI) formulations, dextran nanogels, unmodified β-cyclodextrin, and trimethylated chitosan (Boe et al., 2007, 2008; Oliveira et al., 2007; Boe et al., 2010; Raemdonck et al., 2010; Varkouhi et al., 2011). Here, we have evaluated 3 novel chitosan polymers and present an optimized in vitro transfection protocol for effective siRNA gene silencing using the linear chitosan-75.7 polymer.

Materials and Methods

Cell culture

The OHS (osteosarcoma) (Fodstad et al., 1986), the RMS (melanoma) (established at The Norwegian Radium hospital), and the SKBR3 (breast cancer) (American Type Culture Collection) cells were grown in RPMI 1640 medium (Bio Whittaker) supplemented with 10% fetal bovine serum (PAA Laboratories) and 2 mM l-glutamine (Gibco-BRL). Maintenance of cells took place in a moist atmosphere at 37°C, containing 5% CO₂. Cells were regularly tested for mycoplasma infections.

siRNA

The rational siRNA design algorithm (Reynolds et al., 2004) was used to design a specific siRNA molecule (sequence 5′-UGAGCAAGUUCAAUAAAGA-3′, 3′-ACUCGUUCAAGUUAUUUCU-5′, 481–499) targeting the S100A4 mRNA sequence (Genbank accession no. NM_002961). To ensure a good stability of the siRNA duplexes, the GC content was limited to be within a 30%–70% range, and synthesized with dTdT overhangs at their 3′ ends. Prior to ordering the S100A4 siRNA molecules (Eurogentec), a basic local alignment search tool (BLAST) search was aligned to the human genome database to eliminate siRNAs with considerable similarity to other genes. In addition, the Silencer^® Select Negative Control No. 2 siRNA molecule (Ambion) with a composition of its bases not hybridizing to human DNA was employed. Dried oligonucleotides were dissolved in nuclease free water and stored at −20°C. Diluted aliquots of 20 μM were used for transfection.

Transfection

Two linear chitosan polymers, of molecular weight (mw) of 75.7 and 150 kDa, and one self-branched chitosan polymer, mw 40 kDa, were evaluated as siRNA-carriers for gene silencing. These chitosan polymers are completely deacetylated [fraction of acetylated units (FA)=0) and were gifts from Dr. Sabina Strand at the Dept. of Biotechnology, the Norwegian University of Science and Technology, Trondheim, Norway. B-PEI (25 kDa) and Lipofectamine 2000 were purchased from Sigma Aldrich and Invitrogen, respectively. Prior to transfection, the chitosan polymers were diluted to 0.1 mg/mL in sterile water. Complex formation between chitosan and siRNA was prepared in either 100 μL sterile water (default) (B-BRAUN), RPMI 1640 medium (BioWhittaker), or Dulbecco′s phosphate buffered saline (PBS) (1×) containing Ca/Mg (PAA), while when inducing complex formation between siRNA and B-PEI, the mixture was incubated in 200 μL RPMI serum-free medium. siRNA transfection (50 nM) for Lipofectamine 2000 was performed according to producer guidelines. After 30 minutes incubation at 25°C, the complexation solutions with chitosan was added to 1900 μL RPMI serum-containing medium, in 12-well plates with (only for chitosan-75.7) or without PS [disulfonated tetraphenylporphine (TPPS_2a)=0.5 μg]. Wells with B-PEI-containing complexes had a total volume of 1 ml, with or without PS (TPPS_2a=0.5 μg). Subsequently, the plates were incubated for 18 hours, and cells were washed 3 times with serum-free medium to remove the transfection solution. Cells with PS were exposed to light 4 hours after the washing steps, and harvested 24 hours following illumination, and cells without PS were reincubated for 28 hours before harvesting. In either case, cells were reincubated with 500 μL RPMI serum-containing medium after the washing steps. A blue light with an irradiance of 5.1 mW/cm² was used as a light source. The cells were harvested 24 hours after light treatment and protected from light by aluminum foil throughout the procedure from transfection start to cell harvesting. Transfections were conducted at pH 7.4, with a siRNA concentration of 50 nM, and under a cell confluency of ∼50%. Validation of transfection parameters are described in the results section.

Photochemical internalization technology

The photosensitizer, TPPS_2a, was obtained from Porphyrin Products. Cells were illuminated with light from a Lumisource prototype (PCI Biotech AS) containing 4 fluorescent tubes (Osram 18W/67) with fluorescence peaking around 420 nm.

Quantitative reverse transcriptase–polymerase chain reaction of S100A4 levels

Total RNA was isolated with the GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich). Approximately 1 μg of total RNA from sample was reverse transcribed with the qScript cDNA synthesis kit (Quanta Biosciences). The primers were designed with the Primer Express software (Applied Biosystems). The S100A4 primer pair (forward sequence 5′-AAGTTCAAGCTCAACAAGTCAGAAC-3′; reverse sequence 5′-CATCTGTCCTTTTCCCCAAGA-3′) amplifies a 79-bp-long segment from exon 2 and 3, while the TATA box-binding protein (TBP) (forward sequence 5′-GCCCGAAACGCCGAATAT-3′; reverse sequence 5′-CGTGGCTCTCTTATCCTCATGA-3′) and the human acidic ribosomal phosphoprotein P0 (RPLP0) (forward sequence 5′-CGCTGCTGAACATGCTCAAC-3′; reverse sequence 5′-TCGAACACCTGCTGGATGAC-3′) were housekeeping genes used as controls. The 3 primer sets were ordered from Eurogentec. Real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) detection was performed with the Perfecta SYBR Green Supermix for iQ (Quanta Biosciences).

The qRT-PCR was prepared by making a mix of 60 μL, consisting of 10 μL cDNA template, 30 μL Perfecta SYBR Green Supermix for iQ (Quanta Biosciences), 300 nM per primer, and 16.4 μL sterile water. To ensure reliable duplicates, 2×25 μL of the prepared mix was pipetted onto the PCR plate (Bio-Rad). The following qRT-PCR protocol was used on the iCycler machine (Bio-Rad): initial denaturation/Accustart Taq DNA polymerase activation for 3 minutes at 95°C, 42 cycles with 10 seconds denaturation at 95°C and 35 seconds annealing/extension at 60°C, and with 2 holds, the first for 20 seconds at 95°C and the second hold for 1 minute at 55°C. A melting curve analysis consisting of 80 steps lasting for 10 seconds was included in the end. To calculate the gene expression from the qRT-PCR, the Gene Expression Macro software, version 1.1 (Bio-Rad) was utilized according to the ΔΔC_T method (Vandesompele et al., 2002). The S100A4 mRNA expression was normalized to the mean mRNA level of the duplicates from two housekeeping genes, namely TBP and RPLP0, and relative to an untreated control.

Cell viability

Cells were treated as described under transfection. The medium (500 μL) was changed 26 hours after the washing steps, and 100 μL MTS [3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] solution (Promega) was applied to every well, making a final volume of 600 μL (1:6 dilution). After reincubation for 2 hours, absorbance was measured at 490 nm. Cells were protected from light by aluminum foil throughout the experiments.

Zeta potential and complex sizes

The complexes consisting of chitosan-75.7 and siRNA molecules were prepared the same way as those used for transfection and were measured for complex size (Z-average radius) at 25°C on a Zetasizer Nano ZS (Malvern Instruments Ltd.). The same instrument and temperature was used to determine the Zeta potential by diluting the complexes in double distilled water with a zeta potential of −20 mV.

Results

Evaluation of 3 different chitosan polymers for siRNA delivery

Three chitosan polymers were evaluated for siRNA delivery in the OHS cell line (Fig. 1). We used 50 nM S100A4 siRNA combined with various chitosan polymers. Efficient gene silencing was observed with the linear chitosan polymers, chitosan-75.7 and chitosan-150, where the S100A4 mRNA levels decreased with 95% and 90%, respectively. In contrast, with the self-branched chitosan-40 polymer, the S100A4 mRNA level was only reduced with 25%. To ensure that the silencing effect was caused by siRNA molecules against S100A4 mRNA transcripts and not an effect of the chitosan polymers itself, we also employed a negative siRNA control for each carrier. A minor decrease in S100A4 mRNA levels (10%–15%) was detected in samples treated with negative siRNA control.

FIG. 1.

Evaluation of small interference RNA (siRNA) transfection capacity using 3 chitosan polymers in the osteosarcoma (OHS) cell line. The concentration of siRNA under transfection was 50 nM, while the weight of chitosan was 1.64 μg for each polymer (linear chitosan-75.7, linear chitosan-150, and self-branched chitosan-40). White and gray bars represent negative siRNA control and S100A4 siRNA, respectively. Each bar represents the mean value of 3 individual experiments, with standard error of the mean (±SEM).

Optimization of chitosan-75.7 concentrations for siRNA gene silencing

The linear chitosan-75.7 polymer was optimized for siRNA delivery in the OHS cell line (Fig. 2A). A critical factor for efficient siRNA gene silencing is the nitrogen/phosphate (N/P) ratio. The N/P ratio was optimized in our system by keeping a constant siRNA concentration of 50 nM, while variable amounts of chitosan-75.7 was used [(0.65 μg (N/P 0.8), 0.98 μg (N/P 1.2), 1.31 μg (N/P 1.6), and 1.64 μg (N/P 2)]. Our results showed that S100A4 mRNA levels decreased by increasing N/P ratio, with 2 as the most effective (95% silencing). Lipofectamine 2000 was used as positive control and resulted in 85% silencing.

FIG. 2.

Evaluation of various nitrogen/phosphate (N/P) ratios of chitosan-75.7/S100A4 siRNA upon gene silencing (A), cell viability (B), and complex sizes (C) in the OHS cell line. The concentration of siRNA (50 nM) was kept constant for all samples, while concentrations of chitosan-75.7 ranged from N/P ratio 0.8 to 2 (N/P 0.8, 1.2, 1.6, and 2) corresponding to chitosan weighting 0.65–1.64 μg (0.65 μg, 0.98 μg, 1.31 μg, and 1.64 μg). Transfection efficacies and cell viability were compared to Lipofectamine 2000. White and gray bars represent negative siRNA control and S100A4 siRNA, respectively (A and B). Each bar represents the mean value of 3 individual experiments±SEM.

Evaluation of cell viability for chitosan-75.7

Low toxicity is an essential feature for a nucleic acid carrier, not only for a possible future clinical gene therapy protocol, but also to prove that the achieved gene silencing effect is caused by the specific siRNA given. We utilized MTS assays to monitor cell toxicity for chitosan-75.7 and Lipofectamine 2000 (Fig. 2B). No toxicity was observed for the chitosan-75.7-transfected samples, while a minor decrease in cell viability was observed with Lipofectamine 2000 (∼15%).

Evaluation of complex sizes for chitosan-75.7

The particle size distribution between chitosan-75.7 and siRNA was determined for N/P 0.8, 1.2, 1.6, and 2 (Fig. 2C). Complexes were incubated in sterile water for 30 minutes (default complexation time). The smallest complexes were obtained with N/P 0.8 and N/P 1.2 with similar complex sizes (radius ∼50 nm), while complex sizes increased heavily at N/P 1.6 (radius ∼850 nm) and N/P 2 (radius ∼1000 nm).

Evaluation of transfection parameters for chitosan-75.7

To investigate the influence of other transfection parameters on silencing efficiency, we varied media for complex formation and pH. Our results showed highest siRNA silencing activity when using sterile water (95%), while RPMI 1640 serum-free medium and PBS were less effective with 40% and 55% silencing, respectively (Fig. 3A). Of note, silencing efficacy did not change significantly in samples treated with negative siRNA control. As the sterile water resulted in the highest transfection efficiency, sterile water was used as the default complexation solution throughout the work. Next, we investigated S100A4 siRNA silencing efficacy under 3 different pH settings in RPMI 1640 medium (Fig. 3B). Results showed a clear increase of S100A4 mRNA levels when pH was raised (7.4<7.8<8.2) in samples treated with S100A4 siRNA. In contrast, samples treated with negative siRNA control were unaffected by the pH changes.

FIG. 3.

Evaluation of chitosan-75.7/S100A4 siRNA gene silencing under various transfection media and pH values in the OHS cell line. Complexes containing 50 nM S100A4 siRNA at an N/P ratio of 2 were prepared in RPMI 1640 medium without serum, phosphate buffered saline (PBS), and sterile water, respectively (A). The same chitosan-75.7/S100A4 siRNA concentration (N/P 2) was transfected in RPMI 1640 medium with pH values ranging from 7.4 to 8.2 (pH 7.4, 7.8, and 8.2) (B). White and gray bars represent negative siRNA control and S100A4 siRNA, respectively. Each bar represents the mean value of 3 individual experiments±SEM.

Light-directed siRNA delivery with chitosan-75.7

To examine whether chitosan-75.7/S100A4 siRNA complexes were restricted by endosomal and lysosomal entrapment and to investigate the possibility for targeted delivery by the use of light, we combined chitosan-75.7/S100A4 siRNA complexes with PCI treatment. Our results showed that PCI did not improve the silencing effect of chitosan-75.7/S100A4 siRNA complexes considerably at N/P ratios of 0.8, 1.2, and 1.6 compared to samples without PCI treatment. With an N/P of 0.8, no significant difference in the S100A4 mRNA expression level was achieved with PCI. A slightly improved gene silencing effect was obtained with an N/P of 1.2, reducing the mRNA expression level from 76% to 56% with PCI treatment. At an N/P of 1.6, the highest chitosan concentration tested with PCI, the mRNA level decreased with about 10% in the PCI treated samples, compared to non-treated PCI samples (Fig. 4). A previously optimized ratio of siRNA/B-PEI (N/P 3) was used as a positive control for light-directed siRNA delivery (Boe et al., 2008). Light-directed targeting above 80% was detected in the siRNA/B-PEI samples (difference in S100A4 mRNA levels with and without PCI).

FIG. 4.

Evaluation of PCI-mediated chitosan-75.7/S100A4 siRNA delivery in the OHS cell line. The 25-kDa branched polyethylenimine (B-PEI) carrier was used as a positive control for photochemical internalization (PCI)-mediated siRNA delivery. White and gray bars represent samples without or with photosensitizer, respectively. Each bar represents the mean value of 3 individual experiments±SEM.

Zeta potential and time-lapse study of complex sizes

As the N/P ratio of 2 demonstrated the highest S100A4 gene silencing effect with chitosan-75.7/S100A4 siRNA, we examined the particle size distribution and zeta potential. We found that complexes had a zeta potential close to 0 mV in sterile water. In addition, the complex size of N/P 2 was determined at 3 time-points in sterile water (Fig. 5). Complexes increased significantly in size with increasing complexation times. After 30 minutes (default complexation time), complexes had an average radius of ∼1000 nm.

FIG. 5.

Evaluation of complex sizes measured at chitosan-75.7/S100A4 siRNA complexes after 3 time-points: 15 minutes, 30 minutes, and 45 minutes. Chitosan-75.7 (1.64 μg) was complexed to S100A4 siRNA (1.4 μg) at an N/P ratio of 2. Each bar represents the mean value of 3 individual experiments±SEM.

siRNA delivery with chitosan-75.7 in the RMS and SKBR3 cell lines

In order to examine the siRNA delivery potential of chitosan-75.7 in other cell types, the melanoma cell line RMS and the breast cancer cell line SKBR3 were also tested (Fig. 6). In these cell lines the N/P ratio was increased to 5, as this ratio resulted in the highest silencing effect (around 60% silencing was measured in both cell lines).

FIG. 6.

Evaluation of chitosan-75.7/S100A4 siRNA gene silencing in the RMS and SKBR3 cell lines. The concentration of siRNA under transfection was 50 nM and the N/P ratio was 5. White and gray bars represent negative siRNA control and S100A4 siRNA, respectively. Each bar represents the mean value of 3 individual experiments±SEM.

Discussion

A safe, efficient, and non-viral nucleic acid carrier with in vivo potential still largely remains elusive for gene therapy purposes. In this work, 3 novel biodegradable chitosan polymers were evaluated for siRNA delivery in vitro. Recently, siRNA delivery strategies in vitro with chitosan polymers have been focused towards improved cellular uptake of complexes with for instance hyaluronic acid and thiolated chitosan (Ravina et al., 2010; Varkouhi et al., 2010), or toward endosomal release mechanisms with either imidazole or PCI (Ghosn et al., 2008; Varkouhi et al., 2011). The illumination based method PCI offers the possibility to both target the area of interest and release entrapped material from the endosomes and lysosomes, as exploitation of the TPPS_2a photosensitizer depends on complexes localized in either of these organelles. In contrast to Varkouhi and colleagues, PCI treatment combined with the linear 75.7 kDa chitosan did not improve siRNA delivery considerably in this study. An explanation for this may be that Varkouhi and colleagues used a different type of chitosan polymer in their study, a trimethylated chitosan polymer. Surprisingly, neither a modification of the chitosan-75.7 kDa polymer nor endosomal release mechanisms like PCI was required to induce effective siRNA gene silencing in our study. This indicates that our chitosan-75.7/S100A4 siRNA complexes were able to overcome a possible endosomal barrier, possibly by the previously suggested proton sponge mechanism in late endosomes (pH 5.0–6.0) (Huang et al., 2005). To elucidate this further we investigated the importance of pH in the medium. Our results showed that by changing the pH in the transfection medium, the siRNA silencing efficiency was impaired by increasing pH (7.4>7.8>8.2). An increase in pH may reasonably limit/abolish proton sponge activity and thereby reduce delivery of siRNA into cytosol. The pH sensitivity upon transfection when using chitosan polymers is in agreement with earlier reports (Strand et al., 2008; Opanasopit et al., 2010). Importantly, our results showed the possibility for 95% siRNA silencing of our model gene S100A4, under physiological pH, without any observable reduction in cell viability. The transfection efficiency of chitosan polymers is dependent on the molecular weight and degree of deacetylation, as decreasing either of these parameters affected the transfection of plasmid DNA negatively (Huang et al., 2005). Our linear chitosan polymers have molecular weights of 75.7 kDa and 150 kDa, as well as being completely deacetylated (FA=0), which most likely explain the high level of siRNA silencing obtained in this study. Our study also indicates that gene silencing with chitosan/siRNA is dependent on complex sizes, as the highest achieved gene silencing was obtained with the largest complex measured. The lower silencing efficacy measured with chitosan-40 is possibly due to the self-branched structure or the lower molecular weight compared to the 2 linear chitosan polymers tested. Comparable, linear based PEI formulations have been found more versatile and efficient for transfection compared to branched PEI formulations (Poulain et al., 2000). Furthermore, the transfection efficacies of PEI have been shown to rise upon increasing molecular weight of the carrier (25 kDa>1.8>0.8) (Boe et al., 2008). In our study we obtained only a moderate siRNA silencing effect (around 60%) with chitosan-75.7 in the RMS melanoma and in the SKBR3 breast cancer cell line, while a superior effect was measured in the OHS cell line (95%). Despite the lower transfection efficiency obtained in the SKBR3 cell line with chitosan-75.7, this was still higher than for Lipofectamine 2000, where a transfection efficiency of 49% is previously documented (Invitrogen). Recently, unmodified β-cyclodextrin and siRNA was transfected with PCI in the OHS cell line and later in the RMS cell line. Here, the same concentrations of carrier/siRNA was used in the 2 cell lines, but reoptimization of the illumination dose was necessary to obtain a significant gene silencing in the RMS cell line compared with the OHS cell line (Boe et al., 2010). This may explain why we had to increase the N/P ratio using chitosan 75.7 in our present study to achieve a considerable gene silencing.

In conclusion, we have here evaluated 3 novel biodegradable chitosan polymers for in vitro siRNA delivery. By optimizing various transfection parameters we show potent siRNA silencing at physiological pH, with no observable reduction in cell viability, using a completely deacetylated 75.7-kDa linear chitosan polymer.

Footnotes

Acknowledgments

The authors are grateful for the Ph.D. grant allocated by the Norwegian Gene Therapy Programme, the Norwegian Radium Hospital. We would also like to thank Dr. Sabina Strand at the Department of Biotechnology, the Norwegian University of Science and Technology, for providing the chitosan polymers.

Author Disclosure Statement

No competing financial interests exist.

References

AZUMA

, NAKASHIRO

, SASAKI

, GODA

, ONODERA

, TANJI

, YOKOYAMA

, HAMAKAWA

2010. Anti-tumor effect of small interfering RNA targeting the androgen receptor in human androgen-independent prostate cancer cells. Biochem. Biophys. Res. Commun., 391:1075–1079.

BERG

, SELBO

P.K.

, PRASMICKAITE

, TJELLE

T.E.

, SANDVIG

, MOAN

, GAUDERNACK

, FODSTAD

, KJOLSRUD

, ANHOLT

et al. 1999. Photochemical internalization: a novel technology for delivery of macromolecules into cytosol. Cancer Res., 59:1180–1183.

BOE

, LONGVA

A.S.

, HOVIG

2007. Photochemically induced gene silencing using small interfering RNA molecules in combination with lipid carriers. Oligonucleotides, 17:166–173.

BOE

, LONGVA

A.S.

, HOVIG

2008. Evaluation of various polyethylenimine formulations for light-controlled gene silencing using small interfering RNA molecules. Oligonucleotides, 18:123–132.

BOE

S.L.

, LONGVA

A.S.

, HOVIG

2010. Cyclodextrin-containing polymer delivery system for light-directed siRNA gene silencing. Oligonucleotides, 20:175–182.

DAVIS

M.E.

, ZUCKERMAN

J.E.

, CHOI C

, SELIGSON

, TOLCHER

, ALABI

C.A.

, YEN

, HEIDEL

J.D.

, RIBAS

2010. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature, 464:1067–1070.

FODSTAD

, BROGGER

, BRULAND

, SOLHEIM

O.P.

, NESLAND

J.M.

, PIHL

1986. Characteristics of a cell line established from a patient with multiple osteosarcoma, appearing 13 years after treatment for bilateral retinoblastoma. Int. J. Cancer, 38:33–40.

GHOSN

, KASTURI

S.P.

, ROY

2008. Enhancing polysaccharide-mediated delivery of nucleic acids through functionalization with secondary and tertiary amines. Curr. Top. Med. Chem., 8:331–340.

HUANG

, FONG

C.W.

, KHOR

, LIM

L.Y.

2005. Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation. J. Control. Release, 106:391–406.

10.

KIM

H.S.

, HAN

H.D.

, ARMAIZ-PENA

G.N.

, STONE

R.L.

, NAM

E.J.

, LEE

J.W.

, SHAHZAD

M.M.

, NICK

A.M.

, LEE

S.J.

, ROH

J.W.

et al. 2011. Functional roles of Src and Fgr in ovarian carcinoma. Clin. Cancer Res., 17:1713–1721.

11.

MAO

H.Q.

, ROY

, TROUNG-LE

V.L.

, JANES

K.A.

, LIN

K.Y.

, WANG

, AUGUST

J.T.

, LEONG

K.W.

2001. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J. Control. Release, 70:399–421.

12.

MAO

, SUN

, KISSEL

2010. Chitosan-based formulations for delivery of DNA and siRNA. Adv. Drug Deliv. Rev., 62:12–27.

13.

OLIVEIRA

, FRETZ

M.M.

, HOGSET

, STORM

, SCHIFFELERS

R.M.

2007. Photochemical internalization enhances silencing of epidermal growth factor receptor through improved endosomal escape of siRNA. Biochim. Biophys. Acta, 1768:1211–1217.

14.

OPANASOPIT

, TECHAARPORNKUL

, ROJANARATA

, NGAWHIRUNPAT

, RUKTANONCHAI

2010. Nucleic acid delivery with chitosan hydroxybenzotriazole. Oligonucleotides, 20:127–136.

15.

POULAIN

, ZILLER

, MULLER

C.D.

, ERBACHER

, BETTINGER

, RODIER

J.F.

, BEHR

J.P.

2000. Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives. Cancer Gene Ther., 7:644–652.

16.

RAEMDONCK

, NAEYE

, HOGSET

, DEMEESTER

, DE SMEDT

S.C.

2010. Prolonged gene silencing by combining siRNA nanogels and photochemical internalization. J. Control. Release, 145:281–288.

17.

RAVINA

, CUBILLO

, OLMEDA

, NOVOA-CARBALLAL

, FERNANDEZ-MEGIA

, RIGUERA

, SANCHEZ

, CANO

, ALONSO

M.J.

2010. Hyaluronic acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an application for pDNA and siRNA delivery. Pharm. Res., 27:2544–2555.

18.

REYNOLDS

, LEAKE

, BOESE

, SCARINGE

, MARSHALL

W.S.

, KHVOROV

A.A.

2004. Rational siRNA design for RNA interference. Nat. Biotechnol., 22:326–30.

19.

STRAND

S.P.

, ISSA

M.M.

, CHRISTENSEN

B.E.

, VARUM

K.M.

, ARTURSSON

2008. Tailoring of chitosans for gene delivery: novel self-branched glycosylated chitosan oligomers with improved functional properties. Biomacromolecules, 9:3268–3276.

20.

VANDESOMPELE

, DE PRETER

, PATTYN

, POPPE

, VAN ROY

, DE PAEPE

, SPELEMAN

2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol., 3research0034–research0034.11.

21.

VARKOUHI

A.K.

, LAMMERS

, SCHIFFELERS

R.M.

, VAN STEENBERGEN

M.J.

, HENNINK

W.E.

, STORM

2011. Gene silencing activity of siRNA polyplexes based on biodegradable polymers. Eur. J. Pharm. Biopharm., 77:450–457.

22.

VARKOUHI

A.K.

, VERHEUL

R.J.

, SCHIFFELERS

R.M.

, LAMMERS

, STORM

, HENNINK

W.E.

2010. Gene silencing activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan. Bioconjug. Chem., 21:2339–2346.