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Logo of diaMary Ann Liebert, Inc.Mary Ann Liebert, Inc.JournalsSearchAlerts
Diabetes Technology & Therapeutics
 
Diabetes Technol Ther. 2009 July; 11(7): 443–449.
PMCID: PMC2902226

Transfection of Rat Pancreatic Islet Tissue by Polymeric Gene Vectors

Han Chang Kang, Ph.D. and You Han Bae, Ph.D.corresponding author

Abstract

Background

In vitro genetic modification has been regarded as one option to improve the viability and functionality of pancreatic islets when used for transplantation in patients with diabetes, either as naked islets or in a type of bioartificial pancreas. In this approach, vector safety and poor transfection efficiency are major concerns.

Methods

In this study, the influence of in vitro transfection conditions on polyplexes constructed of polyethyleneimine (PEI) and plasmid DNA (pDNA) on the transfection efficiency was investigated by varying the transfection medium, the pDNA dose, and the amines of polycation/phosphates of pDNA (N/P) ratio.

Results

Ca2+-containing Krebs-Ringer-HEPES medium was more effective than RPMI 1640 medium by increasing transfection efficiency (2.5-fold). An increase in pDNA dose slightly reduced the transfection efficiency but had minimal influence on islet loss. However, the N/P ratio had a large effect on islet viability and transfection efficiency. For example, the PEI/pDNA ratio at N/P = 10 caused greater islet loss (56% vs. 28%) and 30-fold less transfection efficiency than at N/P = 5. Even under a set of best conditions selected from this study, mostly a fraction of cells located in the peripheral regions of an islet were transfected, and the viability and insulin secretion from the treated islets were not altered. However, it was found that the extent of apoptosis was noticeably higher (~16%) than in untreated islets (~2%).

Conclusions

These results suggest that the gene delivery efficacy to isolated islets can be improved by manipulating the transfection conditions. Polymeric vectors will broaden the options for islet transfection, which is currently limited to viral vectors.

Introduction

Upon transplantation, pancreatic islets face a detrimental environment containing an undeveloped vascular network, hypoxia, and autoimmunity, which induce apoptosis and seriously hamper islet viability and insulin secretion ability. This prompted investigators to develop long-acting islets through various means. As one strategy, genetic modification of islet cells following isolation from a pancreas has been attempted with various genes for immunomodulation (e.g., CTLA-4Ig1), β-cell proliferation (e.g., hepatocyte growth factor2), anti-hypoxia (e.g., myoglobin3), neovascularization (e.g., vascular endothelial growth factor4,5), anti-apoptosis (e.g., bcl-26,7), and cytoprotection (e.g., interleukin-48). It was hypothesized that such genetic modification would improve the viability and functionality of transplanted islets.913

Islet transfection has mostly been conducted using viral or liposomal gene carriers. The use of viral vectors raises several safety concerns (e.g., toxicity, mutagenesis, carcinogenesis, and antiviral responses)1417 and presents serious technical challenges (e.g., limited loading capacity, limited gene size, and difficulties in production/purification).1719 Liposomal vectors have shown poor gene expression,5,2022 toxicity to islets at high liposome/plasmid DNA (pDNA) ratios,21,22 and impaired insulin secretion.22 The drawbacks of using viral or liposomal vectors have spurred interest in testing polymeric vectors.

Polymeric vectors have favorable safety profiles (e.g., low immunogenicity and no oncogenesis) and good technical features (e.g., mass productivity, easy quality control, and unlimited pDNA loading capacity).17,23 Polyethyleneimine (PEI)21 and Superfect™ (Qiagen, Chatsworth, CA) (polyamidoamine)5 have been tested using islets and had poor outcomes. One possible reason for such a negative outcome could have been that the experimental conditions used in the published reports5,21 had been adapted from the protocols for non-pancreatic cells (e.g., 3T3, COS7, HepG2, Jurkat, etc.).

This study attempted to investigate how the transfection conditions of polyplexes made from branched PEI (25 kDa)/pDNA influenced the viability of rat islets and insulin secretion function. Three parameters of transfection medium, pDNA dose, and amines of polycation/phosphates of pDNA (N/P) ratio were examined.

Materials and Methods

Islet isolation and culture

Islets of Langerhans were isolated from male Sprague-Dawley rats (weighing 200–300 g; Charles River Laboratories, Wilmington, MA) by using standard collagenase digestion and discontinuous Ficoll density gradient methods.24 Islet yield was about 350 islet equivalents (IEQ) per pancreas at 2 days post-isolation. The islets were cultured in RPMI 1640 medium supplemented with 11.1 mmol/L glucose and 10% heat-inactivated fetal bovine serum under humidified air containing 5% CO2 at 37°C.

Preparation of PEI/pDNA polyplex

Polyplexes were prepared containing pDNA encoding either firefly luciferase (gWiz-Luc; Aldevron, Fargo, ND) or green fluorescent protein (GFP) reporter gene (gWiz-GFP; Aldevron) and branched PEI (25 kDa; Aldrich Chemical Co., Milwaukee, WI) in HEPES buffer (20 mM, pH 7.3) supplemented with 5% glucose. After pDNA and PEI were mixed under predetermined complexation conditions, the polyplexes (0.02 mL/μg of pDNA) were incubated for 30 min at room temperature.

Particle size and surface charge of PEI/pDNA complexes

The polyplexes (0.2 mL; 10 μg of pDNA) were added to HEPES buffer (20 mM, pH 7.3) supplemented with 5% glucose or transfection medium (1.8 mL). The concentration of pDNA in the polyplex solution was 5 μg/mL. Surface charge and particle size of the polyplexes were measured using a Zetasizer 3000HS (Malvern Instrument, Inc., Malvern, Worcestershire, UK) at a wavelength of 677 nm with a constant angle of 90° at room temperature.

In vitro islet transfection

Polymeric transfection of pancreatic islets was optimized using PEI and a luciferase reporter gene. The parameters tested were transfection medium (RPMI 1640 medium at a fixed [Ca2+] of 0.424 mmol/L and Ca2+-containing Krebs-Ringer-HEPES medium [Ca2+ (+)KRH] with varying [Ca2+] in a range of 0.127–2.54 mmol/L),25 pDNA dose (3–10 μg of pDNA/100 IEQ), and PEI/pDNA N/P ratio (N/P = 3–10). At 2 days post-isolation, the isolated islets were transferred to a Petri dish (10 cm in diameter) and were cultured in serum-free transfection medium (2 mL) such as RPMI 1640 medium with glucose (5.6 mmol/L) or Ca2+(+)KRH (4.74 mmol/L KCl, 1.19 mmol/L KH2PO4, 1.19 mmol/L MgCl2 · 6H2O, 119 mmol/L NaCl, 0.127–2.54 mmol/L CaCl2, 25 mmol/L NaHCO3, 10 mmol/L HEPES, and 5.6 mmol/L glucose). After a 1-h incubation, the islets (200 IEQ) were transfected with PEI/pDNA complexes. After an additional 4-h incubation, culture medium (RPMI 1640 medium with 11.1 mmol/L glucose and 10% heat-inactivated fetal bovine serum; 20 mL) was added to the transfected islets. At 12 h post-transfection, the islets were transferred into fresh culture medium.

To evaluate the extent of luciferase expression, the transfected islets were examined at 1 day post-transfection. Islets were first washed with Ca2+/Mg2+-free Dulbecco's phosphate-buffered saline. After addition of lysis buffer (60 μL), the islets were gently vortex-mixed and centrifuged at 13,400 g for 2 min. Supernatants were collected to analyze luciferase expression (20 μL) and protein levels (20 μL). Luciferase activity in the transfected islets was quantified using a luciferase assay kit (Promega Co., Madison, WI). The protein amount was evaluated using a BCA™ protein assay kit (Pierce Biotechnology Inc., Rockford, IL) to normalize luciferase expression.

In vitro islet toxicity

PEI/pDNA-mediated islet toxicity was examined in terms of remaining islets (%), apoptosis (per IEQ), and cell viability (per IEQ). At 1 day post-transfection, remaining transfected islets were counted, and the change in islet number before and after transfection was evaluated as follows:

equation M1

For cell viability (per IEQ), the transfected islets were transferred to a 96-well plate at a density of 10 IEQ per well and were cultured at their final volume (0.1 mL per well). At 1 day post-transfection, CellTiter-Blue® reagent (Promega) (20 μL per well) was added to the islets. After a 4-h incubation at 37°C, fluorescence was measured at 560 nm (excitation) and 590 nm (emission). Cell viability of transfected islets was expressed in relative fluorescent units (RFU) per IEQ and was compared to the cell viability of untransfected islets. After the cell viability assay was performed, Apo-One® caspase-3/7 reagent (Promega) (0.1 mL per well) was added to the islets. After a 2-h incubation, fluorescence was measured at 499 nm (excitation) and 521 nm (emission) to assess apoptosis. Transfection-induced apoptosis was also expressed as percentage apoptosis per IEQ, which was converted from RFU/IEQ by comparing results to fluorescence levels obtained from dispersed, dexamethasone (100 nmol/L)-treated islet cells.26

Insulin secretion of transfection islets

Glucose-stimulated insulin secretion was evaluated following islet transfection. At 12 h post-transfection, the transfected islets were transferred to a 24-well plate at a density of 20 IEQ per well and were cultured at their final volume (1 mL). At 1 day post-transfection, the islets were rinsed twice with RPMI 1640 medium, which was supplemented with 2.8 mmol/L glucose, for 20 min to remove secreted insulin. Then, the islets were incubated in fresh RPMI 1640 medium supplemented with 2.8 mmol/L glucose, and after 30 min of starvation, the medium was collected. The glucose-starved islets were then exposed to 16.7 mmol/L glucose in RPMI 1640 medium to stimulate insulin secretion, and samples were collected after 30 min. The medium samples from these conditions were stored at −20°C until analysis. The medium used contained 10% fetal bovine serum regardless of glucose concentration. Insulin secreted from the transfected islets in RPMI 1640 medium supplemented with 2.8 mmol/L glucose or 16.7 mmol/L glucose was evaluated by 125I-insulin radioimmunoassay (MP Biomedicals, Solon, OH). Insulin stimulation index values were calculated as follows:

equation M2

GFP expression

To monitor the location of transfected cells within an islet, pDNA encoding a GFP reporter gene was introduced using optimal transfection conditions. Transfection of the GFP gene was monitored as described in the in vitro islet transfection study. Following transfection, GFP expression from the transfected islets was evaluated using a laser scanning confocal microscope (model FV1000, Olympus, Center Valley, PA) with Olympus Fluoview™ installed at an excitation wavelength of 488 nm. Confocal images were sectioned every 15 μm of thickness, and images were to reconstruct images of whole cells.

Statistical analysis

All tests were performed with at least n = 3, and these results are expressed as mean ± standard error of the mean (SEM) except for particle size and surface charge of polyplexes, which are expressed as mean ± standard deviation (SD). Statistical analysis by Student's t test or one-way analysis of variance (ANOVA) with Tukey's HSD (Honestly Significantly Different) test was evaluated with P < 0.05 significance.

Results and Discussion

Various chemicals, biological agents, and ions can directly or indirectly influence biological functions. For pancreatic islets, glucose, amino acids, vitamins, and Ca2+ are known to effect viability and insulin secretion. β-Cells in an islet are tuned to produce and secrete insulin in response to glucose concentration in the blood and culture environment. During culture, glucose levels are linked to islet apoptosis; islets exposed to a glucose concentration of ≤2 mmol/L or ≥24.4 mmol/L for 48 h presented more apoptotic events than islets exposed to a glucose concentration of 5.5–11.1 mmol/L.27

Normal cell growth medium (e.g., RPMI 1640 medium) supplemented with amino acids and vitamins has frequently been used for cell culture and gene transfection. However, amino acids promote insulin exocytosis via KATP channels or voltage-dependent Ca2+ channels,28 and vitamins can directly or indirectly influence insulin secretion.2931 We reported that RPMI 1640 medium stimulated higher insulin release from an insulinoma RINm5F cell line than Ca2+(+)KRH {[Ca2+] =2.54 mmol/L is Ca2+2.54 mmol/L(+)KRH}.32 The negatively charged insulin interacted with the cationic polyplexes, leading to reduced cellular uptake of the polyplexes and decreased polymeric transfection.25

Thus, RPMI 1640 medium and Ca2+2.54 mmol/L(+)KRH were selected for comparison because RPMI 1640 medium contains several amino acids and vitamins, whereas Ca2+2.54 mmol/L(+)KRH does not. A glucose concentration of 5.6 mmol/L was used to minimize glucose-induced insulin secretion and apoptosis. As a starting point, PEI/pDNA complexes containing 5 μg of pDNA were prepared at N/P = 5. Rat islets (100 IEQ) were treated with polyplexes in either Ca2+2.54 mmol/L(+)KRH or RPMI 1640 medium ([Ca2+] =0.424 mmol/L). PEI/pDNA complexes (N/P = 5) in Ca2+2.54 mmol/L(+)KRH had larger particle sizes than the complexes in RPMI 1640 medium, although both particles had similar surface charges (Table 1). Nevertheless, islet transfection performed in Ca2+2.54 mmol/L(+)KRH resulted in an approximately 2.5-fold (P = 0.02 by unpaired Student's t test) higher luciferase expression than in RPMI 1640 medium (Fig. 1, columns). This result was consistent with our previous report in which insulin-secreting RINm5F cells were treated with different polyplexes.25

Fig. 1.
Effects of transfection medium on transfection efficiency (columns) and remaining islet number (solid circles) in islets transfected with PEI/pDNA polyplex at 1 day post-transfection. PEI/pDNA polyplexes were prepared at a pDNA dose of 5 μg ...
Table 1.
Particle Size and Surface Charge of PEI/pDNA Complexes in Various Buffers

The transfection media we compared did not show any significant difference in the number of remaining islets after transfection (as a percentage) (P = 0.30 by unpaired Student's t test); the number of intact islets was approximately 76% and 78% of the initial number of islets in RPMI 1640 medium and Ca2+2.54 mmol/L(+)KRH, respectively (Fig. 1, solid circles). These results are concurrent with our previous cell viability data using RINm5F cells transfected in both transfection media.25

Ca2+ influx mediated by insulin stimulants (e.g., glucose and sulfonylurea) increases intracellular [Ca2+], which modulates insulin exocytosis as well as β-cell apoptosis.33 The effects of extracellular [Ca2+] on PEI/pDNA-mediated islet transfection in the presence of cationic PEI were investigated in Ca2+(+)KRH having different [Ca2+] (i.e., 0.127–2.540 mmol/L). Particle size and surface charge of PEI/pDNA complexes (N/P = 5) presented similar values in Ca2+(+)KRH regardless of [Ca2+] (Table 1). Also, exposing islets to different extracellular [Ca2+] for 4 h did not result in any statistically significant differences in transfection efficiency (P = 0.53 by one-way ANOVA) (Fig. 1, columns). As shown in Figure 1 (solid circles), the remaining islets (as a percentage) after transfection were approximately 71–80% with no statistical significance (P = 0.29 by one-way ANOVA), although the values were somewhat decreased in lower extracellular [Ca2+]: e.g., 71 ± 2% in Ca2+2.54 mmol/L(+)KRH, 76 ± 2% in Ca2+0.635 mmol/L(+)KRH, and 80 ± 3% in Ca2+1.27 mmol/L(+)KRH. Transfection medium containing a low glucose concentration (5.6 mmol/L) may not stimulate Ca2+-mediated islet death because PEI is probably not an insulin stimulant. Nevertheless, the depletion of [Ca2+] from the transfection medium caused poor transfection25 because [Ca2+] is essential for gene expression.34 Thus, for further optimization, a condition of [Ca2+] = 0.635 mmol/L in Ca2+(+)KRH (Ca2+0.635 mmol/L(+)KRH) was used to reduce the risk of high [Ca2+]-mediated damages to islet and low/depleted [Ca2+]-mediated loss on gene expression.

Islet transfection using PEI/pDNA polyplexes was reported to be ineffective when a dose of 1.5 μg of pDNA was given for every 100 IEQ.21 Thus, pDNA dose dependency for PEI/pDNA (N/P = 5)-transfected islets in Ca2+0.635 mmol/L(+)KRH was investigated in the range of 3–10 μg of pDNA per 100 IEQ. From the doses used, transfection levels were the highest at a dose of 5 μg of pDNA per 100 IEQ. This level was similar to that at 3 μg of pDNA per 100 IEQ but was approximately 2.9-fold (P = 0.07 by one-way ANOVA with Tukey's HSD test) and 2.3-fold higher than for 7 and 10 μg of pDNA doses per 100 IEQ, respectively (Fig. 2, columns). IEQ losses were approximately 20–30% at all doses tested (P = 0.73 by one-way ANOVA); the remaining islets (as a percentage) were 77 ± 4% at 3 μg of pDNA, 76 ± 2% at 5 μg of pDNA, 78 ± 4% at 7 μg of pDNA, and 73 ± 3% at 10 μg of pDNA in Ca2+0.635 mmol/L(+)KRH (Fig. 2, solid circles). The lower transfection levels at higher pDNA doses (7 and 10 μg of pDNA per 100 IEQ) is most likely related to the reduced viability of individual cell types, such as α-, β-, δ-, and PP-cells, in an islet rather than the loss of whole islets, which was visually observed before and after treatments.

Fig. 2.
Effects of pDNA doses on transfection efficiency (columns) and remaining islet number (solid circles) in islets transfected with PEI/pDNA polyplex at 1 day post-transfection. PEI/pDNA polyplexes were prepared in Ca2+0.635 mmol/L(+)KRH and an N/P ...

After selection of a pDNA dose (5 μg of pDNA per 100 IEQ) and a transfection medium [Ca2+0.635 mmol/L(+)KRH], the PEI/pDNA N/P ratio was changed from 3 to 10. N/P ratios higher than N/P = 10 were excluded because of the potential toxicity of PEI/pDNA polyplexes to cells and animals. The transfection efficiency of PEI/pDNA polyplexes was highest at N/P = 5; this was 15-, eight-, and 30-fold higher than transfection levels using N/P = 3, N/P = 7, and N/P = 10, respectively (P < 0.002 by one-way ANOVA with Tukey's HSD test) (Fig. 3, columns). This optimum complexation ratio is consistent with results from liposomal islet transfection.21,22 The poor transfection of PEI/pDNA complexes at N/P = 3 could be caused by the negative surface charge of these particles in Ca2+0.635 mmol/L(+)KRH (Table 1) because negative surface charges cause less cellular uptake of polyplexes than positive surface charges. In addition, the low transfection levels of PEI/pDNA complexes at higher N/P ratios (N/P = 7 and 10) could be due to uncomplexed PEI-mediated islet loss because uncomplexed PEI induces greater cytotoxicity than PEI complexed with genes.35 These transfection results may explain why islet loss was not significant at N/P = 3 and why islet loss increased with higher N/P ratios (P < 0.001 by one-way ANOVA). Approximately 79 ± 3% of islets maintained their integrity at N/P = 3, 76 ± 2% at N/P = 5, 60 ± 2% at N/P = 7, and 44 ± 1% at N/P = 10 (P < 0.01 compared with N/P = 3 and N/P = 5 by one-way ANOVA with Tukey's HSD test) (Fig. 3, solid circles).

Fig. 3.
Effects of N/P ratios on transfection efficiency (columns) and remaining islet number (solid circles) in islets transfected with PEI/pDNA polyplex at 1 day post-transfection. PEI/pDNA polyplexes were prepared in Ca2+0.635 mmol/L(+)KRH) at a pDNA ...

From the above screening tests, the best transfection efficiency of PEI/pDNA-transfected islets was attained when PEI/pDNA polyplexes were prepared at an N/P ratio of 5 and at a ratio of 5 μg of pDNA per 100 IEQ and transfected into rat islets in Ca2+0.635 mmol/L(+)KRH. Under these conditions, the location of PEI/pDNA-mediated transfected cells in a single islet was monitored using a GFP gene because a single islet is composed of approximately 1,000 cells (α-, β-, δ-, and PP-cells). Transfected islets showed higher fluorescence intensity compared to untransfected islets (Fig. 4a). Also, their sectioned images indicated that PEI-mediated islet transfection mostly occurred in the peripheral region of the islet (Fig. 4b). This result could be caused by the size of PEI/pDNA polyplexes (~400 nm in a diameter) in Ca2+0.635 mmol/L(+)KRH, which might be too big for homogeneous islet transfection. Also, this peripheral transfection of islets indicates that PEI-mediated transfection might occur in non–β-cells because these cells reside in the peripheral regions of an islet, unlike β-cells, which are located in the core of islets. This result was consistent with the results from other studies.21

Fig. 4.
GFP expression of PEI/pDNA-transfected islets under optimal transfection conditions: (a) whole islet images and (b) sectioned images. The polyplexes containing 5 μg of pDNA prepared at N/P = 5 per transfected 100 IEQ ...

The number of viable cells in a single islet is another critical factor for islet transfection. In particular, the viability of β-cells is directly linked to the success of islet grafts. Thus, under the optimum transfection conditions defined above, cell viability (per IEQ) and apoptosis (per IEQ) were evaluated. Transfected and untreated control islets had similar cell viabilities (per IEQ), whereas the extent of apoptosis (per IEQ) of transfected islets (~16%) was about eightfold higher than that of the control islets (~2%) (Fig. 5).

Fig. 5.
Cell viability (per IEQ), apoptosis (per IEQ), and insulin secretion of transfected islets under optimal transfection conditions: Ca2+0.635 mmol/L(+)KRH, 5 μg of pDNA per 100 IEQ, and N/P = 5. Transfected ...

In addition, the insulin-secreting ability of transfected islets was evaluated to determine whether transfection affects the cellular function of β-cells. In this study, the insulin stimulation index was considered instead of secreted insulin (e.g., pM or μIU) because insulin secretion of islets is strongly dependent on islet size36,37 and β-cell proportion (e.g., 600–800 cells in an islet).38 Glucose-stimulated insulin secretion index values of PEI-transfected islets were similar in value to that of untransfected islets. These insulin stimulation indices suggest that insulin secretion of transfected islets, especially β-cells, was not impaired. This result was supported by the distribution of GFP-transfected cells in an islet.

Peripheral transfection may be useful for delivering therapeutic genes of proteins to be secreted, such as vascular endothelial growth factor for angiogenesis and CTLA-4Ig for immunomodulation because these genes do not require homogeneous transfection of the whole islet. However, further development of custom-designed polymeric systems, which can control size, surface charge, and functional modification of polyplexes, may enable more homogeneous transfection of islets. For therapeutic genes that translate into non-secreted proteins, such as bcl-2 for anti-apoptosis, homogeneous transfection should result in improved viability and functionality of transplanted islets during islet grafts.

The use of polymeric vectors rather than viral vectors may allow therapeutic genes to be delivered in a safer manner. Additionally, unlike viral vectors, polymeric vectors are particularly well suited for the simultaneous delivery of multiple therapeutic genes, because of their unlimited gene loading capacity.

Conclusions

This study demonstrated that the optimization process is important in improving polymeric islet transfection. The optimum conditions of PEI-mediated islet transfection investigated in this study are an N/P ratio of 5, a ratio of 5 μg of pDNA per 100 IEQ, and Ca2+0.635 mmol/L(+)KRH as the transfection medium. The transfection conditions identified in this study may be useful for PEI-mediated transfection of islets with therapeutic genes such as vascular endothelial growth factor and CTLA-4Ig. Although this study used branched PEI, a similar optimization process can be applied to various functionalized polymeric vectors to achieve more efficient gene transfection in pancreatic islets.

Acknowledgments

This work was partially supported by grant DK 56884 from the National Institutes of Health. The authors acknowledge Deepa Mishra for her proofreading.

Author Disclosure Statement

No competing financial interests exist.

References

1. Gainer AL. Korbutt GS. Rajotte RV. Warnock GL. Elliott JF. Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival. Transplantation. 1997;63:1017–1021. [PubMed]
2. Garcia-Ocana A. Takane K. Reddy V. Lopez-Talavera J. Vasavada R. Stewart A. Adenovirus-mediated hepatocyte growth factor expression in mouse islets improves pancreatic islet transplant performance and reduces beta cell death. J Biol Chem. 2003;278:343–351. [PubMed]
3. Tilakaratne HK. Yang B. Hunter SK. Andracki ME. Rodgers VG. Can myoglobin expression in pancreatic beta cells improve insulin secretion under hypoxia? An exploratory study with transgenic porcine islets. Artif Organs. 2007;31:521–531. [PubMed]
4. Cheng K. Fraga D. Zhang C. Kotb M. Gaber AO. Guntaka RV. Mahato RI. Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets. Gene Ther. 2004;11:1105–1116. [PubMed]
5. Mahato RI. Henry J. Narang AS. Sabek O. Fraga D. Kotb M. Gaber AO. Cationic lipid and polymer-based gene delivery to human pancreatic islets. Mol Ther. 2003;7:89–100. [PubMed]
6. Rabinovitch A. Suarez-Pinzon W. Strynadka K. Ju Q. Edelstein D. Brownlee M. Korbutt G. Rajotte R. Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. Diabetes. 1999;48:1223–1229. [PubMed]
7. Contreras JL. Bilbao G. Smyth CA. Jiang XL. Eckhoff DE. Jenkins SM. Thomas FT. Curiel DT. Thomas JM. Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic bcl-2 gene. Transplantation. 2001;71:1015–1023. [PubMed]
8. Smith DK. Korbutt GS. Suarez-Pinzon WL. Kao D. Rajotte RV. Elliott JF. Interleukin-4 or interleukin-10 expressed from adenovirus-transduced syngeneic islet grafts fails to prevent beta cell destruction in diabetic nod mice. Transplantation. 1997;64:1040–1049. [PubMed]
9. Prud'homme GJ. Draghia-Akli R. Wang Q. Plasmid-based gene therapy of diabetes mellitus. Gene Ther. 2007;14:553–564. [PubMed]
10. Narang AS. Mahato RI. Biological and biomaterial approaches for improved islet transplantation. Pharmacol Rev. 2006;58:194–243. [PubMed]
11. Ren J. Jin P. Wang E. Liu E. Harlan DM. Li X. Stroncek DF. Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation. J Transl Med. 2007;5:1. [PMC free article] [PubMed]
12. Merani S. Shapiro AM. Current status of pancreatic islet transplantation. Clin Sci (Lond) 2006;110:611–625. [PubMed]
13. Balamurugan AN. Bottino R. Giannoukakis N. Smetanka C. Prospective and challenges of islet transplantation for the therapy of autoimmune diabetes. Pancreas. 2006;32:231–243. [PubMed]
14. Barbu AR. Akusjrvi G. Welsh N. Adenoviral-induced islet cell cytotoxicity is not counteracted by bcl-2 overexpression. Mol Med. 2002;8:733–741. [PMC free article] [PubMed]
15. Giannoukakis N. Trucco M. Gene therapy for type 1 diabetes. Am J Ther. 2005;12:512–528. [PubMed]
16. McCabe C. Samali A. O'Brien T. Cytoprotection of beta cells: rational gene transfer strategies. Diabetes Metab Res Rev. 2006;22:241–252. [PubMed]
17. Van Linthout S. Madeddu P. Ex vivo gene transfer for improvement of transplanted pancreatic islet viability and function. Curr Pharm Des. 2005;11:2927–2940. [PubMed]
18. Kapturczak MH. Flotte T. Atkinson MA. Adeno-associated virus (aav) as a vehicle for therapeutic gene delivery: improvements in vector design and viral production enhance potential to prolong graft survival in pancreatic islet cell transplantation for the reversal of type 1 diabetes. Curr Mol Med. 2001;1:245–258. [PubMed]
19. Phillips AJ. The challenge of gene therapy and DNA delivery. J Pharm Pharmacol. 2001;53:1169–1174. [PubMed]
20. Saldeen J. Curiel DT. Eizirik DL. Andersson A. Strandell E. Buschard K. Welsh N. Efficient gene transfer to dispersed human pancreatic islet cells in vitro using adenovirus-polylysine/DNA complexes or polycationic liposomes. Diabetes. 1996;45:1197–1203. [PubMed]
21. Benhamou PY. Moriscot C. Prevost P. Rolland E. Halimi S. Chroboczek J. Standarization of procedure for efficient ex vivo gene transfer into porcine pancreatic islets with cationic liposomes. Transplantation. 1997;63:1798–1803. [PubMed]
22. Lakey JRT. Young ATL. Pardue D. Calvin S. Albertson TE. Jacobson L. Cavanagh TJ. Nonviral transfection of intact pancreatic islets. Cell Transplant. 2001;10:697–708. [PubMed]
23. Kang HC. Lee M. Bae YH. Polymeric gene carriers. Crit Rev Eukaryot Gene Expr. 2005;15:317–342. [PubMed]
24. Chae SY. Kim SW. Bae YH. Effect of cross-linked hemoglobin on functionality and viability of microencapsulated pancreatic islets. Tissue Eng. 2002;8:379–394. [PubMed]
25. Kang HC. Bae YH. Polymeric gene transfection on insulin-secreting cells: sulfonylurea receptor-mediation and transfection medium effect. Pharm Res. 2006;23:1797–1808. [PubMed]
26. Ranta F. Avram D. Berchtold S. Dufer M. Drews G. Lang F. Ullrich S. Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes. 2006;55:1380–1390. [PubMed]
27. Mellado-Gil JM. Aguilar-Diosdado M. Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines. Biol Proced Online. 2005;7:162–171. [PMC free article] [PubMed]
28. McClenaghan NH. Barnett CR. O'Harte FP. Flatt PR. Mechanisms of amino acid-induced insulin secretion from the glucose-responsive BRIN-BD11 pancreatic B-cell line. J Endocrinol. 1996;151:349–357. [PubMed]
29. Sone H. Ito M. Sugiyama K. Ohneda M. Maebashi M. Furukawa Y. Biotin enhances glucose-stimulated insulin secretion in the isolated perfused pancreas of the rat. J Nutr Biochem. 1999;10:237–243. [PubMed]
30. Rathanaswami P. Pourany A. Sundaresan R. Effects of thiamine deficiency on the secretion of insulin and the metabolism of glucose in isolated rat pancreatic islets. Biochem Int. 1991;25:577–583. [PubMed]
31. Barker CJ. Leibiger IB. Leibiger B. Berggren PO. Phosphorylated inositol compounds in β-cell stimulus-response coupling. Am J Physiol Endocrinol Metab. 2002;283:E1113–E1122. [PubMed]
32. Kang HC. Functionalized polymeric gene delivery systems for pancreatic β-cell line, islets [Ph.D. thesis] Salt Lake City, UT: University of Utah, Department of Pharmaceutics and Pharmaceutical Chemistry; 2007.
33. Iwakura T. Fujimoto S. Kagimoto S. Inada A. Kubota A. Someya Y. Ihara Y. Yamada Y. Seino Y. Sustained enhancement of Ca2+ influx by glibenclamide induces apoptosis in RINm5F cells. Biochem Biophys Res Commun. 2000;271:422–428. [PubMed]
34. Quesada I. Rovira J. Martin F. Roche E. Nadal A. Soria B. Nuclear KATP channels trigger nuclear Ca2+ transients that modulate nuclear function. Proc Natl Acad Sci U S A. 2002;99:9544–9549. [PubMed]
35. Boeckle S. von Gersdorff K. van der Piepen S. Culmsee C. Wagner E. Ogris M. Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer. J Gene Med. 2004;6:1102–1111. [PubMed]
36. Lehmann R. Zuellig RA. Kugelmeier P. Baenninger PB. Moritz W. Perren A. Clavien PA. Weber M. Spinas GA. Superiority of small islets in human islet transplantation. Diabetes. 2007;56:594–603. [PubMed]
37. MacGregor RR. Williams SJ. Tong PY. Kover K. Moore WV. Stehno-Bittel L. Small rat islets are superior to large islets in in vitro function and in transplantation outcomes. Am J Physiol Endocrinol Metab. 2006;290:E771–E779. [PubMed]
38. Calne R. Cell transplantation for diabetes. Philos Trans R Soc Lond. 2005;360:1769–1774. [PMC free article] [PubMed]

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