Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University
Xu, J., Amiji, M. Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles. J. Vis. Exp. (59), e3612, doi:10.3791/3612 (2012).
More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression.
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11.
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.
1. Preparation of Plasmid DNA Encapsulated EGFR-Targeted Gelatin Nanoparticles
2. Characterization of EGFR-Targeted Nanoparticles
3. In Vitro Transfection Studies in Panc-1 Pancreatic Cancer Cells
4. Representative Results
1. Synthesis and Chatacterization of EGFR Targeted Nanoparticles
EGFR targeting peptide-modified nanoparticles were synthesized as the scheme showed in figure 1. The nanoparticles prepared by desolvation were characterized for particle size and zeta potential. The average size and surface charge of the particles prepared from thiolated gelatins with different degrees of thiolation are listed in Table 1. The mean particle diameters of different nanoparticles were between 150-250 nm. Thiolated nanoparticles have smaller size compared to gelatin nanoparticles, might due to the disulfide bridge formation inside particles. With different surface modifications, sizes of nanoparticles have increased. The zeta potentials of different formulations were around -20 mV. With SEM analysis, the sizes, surface morphology and spherical shape of nanoparticles were observed and corresponding to Zetasizer result. DNA loading efficiencies in gelatin nanoparticles and thiolated gelatin nanoparticles were higher than 95% (Table 1).
Figure 1. Chemical Reaction Scheme, illustrating surface modification of thiolated gelatin nanoparticles with epidermal growth factor receptor (EGFR) binding peptide through a poly(ethylene glycol) (PEG) spacer. Please click here to see a larger version of this figure.
Characterization of Nanoparticles
|Formulation||Nanoparticle Diameter (nm)||Zeta Potential (mV)||Plasmid DNA Loading Efficiency (%)|
|Gel NP||151.4 ± 23.5||-17.1 ± 5.23||95.6 ± 2.2|
|SH-Gel NP||132.6 ± 17.9||-24.6 ± 5.16||97.0 ± 3.8|
|SH-Gel-PEG||179.0 ± 30.9||-22.3 ± 9.50||95.8 ± 6.5|
|SH-Gel PEG Peptide||230.8 ± 41.5||-18.1 ± 4.02||94.8 ± 5.1|
Table 1. Particle size, surface charge, and plasmid DNA encapsulation efficiencies of control and EGFR-targeted gelatin and thiolated gelatin nanoparticles.
High-resolution C1S scans of electron spectroscopy for chemical analysis (ESCA) was used to analyze surface component of thiolated gelatin (SH-Gel NP), PEG-modified thiolated gelatin (SH-Gel PEG) and EGFR targeting peptide-modified thiolated gelatin nanoparticles (SH-Gel PEG Peptide). The results in Table 2 showed peak intensities of the C-H (hydrocarbon), C-O (ether), and C=O (carbonyl) groups at 285.0, 286.3, and 288.1 eV, respectively. The ether C-O signal has increased after PEG modification and decreased after peptide conjugation. While nitrogen composition has decreased after PEG modification and increased after peptide modification, which confirmed the presence of EGFR-targeting peptide on the nanoparticles. ESCA analysis has further confirmed PEG and peptide surface modification.
Electron Spectroscopy for Chemical Analysis of Nanoparticles Surface composition
|Formulation||C 1s (%)||O 1s (%)||N 1s (%)|
|SH-Gel PEG Peptide||56.7±0.8||25.9±0.7||12.3±0.6|
|Formulation||C-C (%)||C-O, N (%)||C=O (%)|
|SH-Gel PEG Peptide||33.1||42.8||24.1|
Table 2. C1S high-resolution scans of electron spectroscopy for chemical analysis (ESCA)
In order to examine the stability of encapsulated plasmid, nanoparticles were treated with protease or DNAse separately, simuntaneously or sequentially. After electrophoresis, the results in Figure 2 have shown that plasmid DNA encapsulated in all the nanoparticles are protected by nanoparticles and stable, comparable to naked plasmid DNA. These studied have shown that all these nanoparticles could encapsulate and preserve the plasmid structure after encapsulation.
Figure 2. Stability of plasmid DNA encapsulated in thiolated gelatin, PEG-modified thiolated gelatin, and EGFR peptide-modified thiolated gelatin nanoparticles by agarose gel electrophoresis. The nanoparticles were treated with 0.2 mg/ml of protease to prove plasmid DNA encapsulation within the nanoparticle matrix
2. Baseline EGFR Expression in Pancreatic Cancer Cells
Two human pancreatic adenocarcinoma cell lines (Panc-1 and Capan-1) were analyzed by western blot for EGFR expression. Human ovarian adenocarcinoma (SKOV3) and murine fibroblast ((NIH-3T3) cells were chosen as positive and negative controls, respectively. Beta-actin was analyzed as protein loading control. Panc-1 cells have shown higher EGFR expression compared to Capan-1 and this cell line was then used for the following in vitro studies
3. Cytotoxicity of Control and Surface-Modified Thiolated Gelatin Nanoparticles
In order to evaluate the cellular interaction of nanoparticles, cytotoxicity assays were carried out after treatment with nanoparticles. Based on the results in Figure 3, both the control and the surface-modified nanoparticles were relatively safe and biocompatible in Panc-1 cells even at high concentrations, with comparison to PEI. The following studies were carried out with 1mg/ml nanoparticles.
Figure 3. Percent cell viability as a function of nanoparticle formulation concentrations in Panc-1 cells as evaluated by tetrazolium dye (MTS) assay
4. Receptor Mediated Cell Uptake in Panc-1 Cells
To confirm surface accessibility of EGFR-targeting peptide and receptor-mediated endocytotic uptake of nanoparticles, a system was designed by labeling each component with different fluorescence for visualization of nanoparticles uptake and trafficking in cells. With this labeling system, plasmid DNA, nanoparticles and cell nucleus could be identified. Laser scanning confocal fluorescence microscopy was used to take images at different time points, from 15 minutes to 6 hours. By comparing the images of different formulations, peptide conjugated gelatin nanoparticles showed the fast uptake and plasmid release within 30 minutes. This result further proved that EGFR peptide-conjugated nanoparticles underwent facilitated endocytosis with quick interaction between the EGFR specific peptide and EGFR receptors on cell surface, which was much faster, compared to other nanoparticles, which underwent non-specific endocytosis.
Cell Trafficking Study
Figure 4. Confocal fluorescence microscopy analysis of DNA-encapsulated nanoparticle uptake and trafficking in Panc-1 cells. (red=rhodamine-labeled nanoparticles, green=PicoGreen-labeled plasmid DNA, and blue=DAPI-labeled nucleus). The laser power was 7 times less in last four figures of lower panel.
5. Qualitative and Quantitative In Vitro Transfection with Enhanced Green Fluorescent Protein
ELISA in Figure 5 and fluorescence microscopic analysis in Figure 6 were used to measure qualitative and quantitative GFP tranfection efficiency in Panc-1 cells upon administration of unmodified, PEG-modified and EGFR peptide-modified thiolated gelatin nanoparticles. Plasmids delivered by EGFR-targeted nanoparticles resulted in the highest level of GFP expression after 48 hours relative to other controls, including Lipofectin-complexed DNA.
Figure 5. GFP expression analyzed by ELISA plotted as a function of time post-administration of plasmid DNA in control and EGFR-targeted nanoparticles.
Fluoresence Microscopic Analysis for GFP transfection
Figure 6. Qualitative analysis of green fluorescent protein expression in Panc-1 cells by epifluoresence microscopy after 24, 48, 72 and 96 hours post-transfection with EGFP-N1. Lipofectin-DNA complex was used as a positive control.
6. In Vitro Tranfection with Wild-Type p53 Plasmid in Panc-1 Cells
Wild-type p53 plasmids pORF-hp53, with EF-1α / HTLV hybrid promoter were extracted from E. coli and encapsulated into nanoparticles to study the apoptotic therapeutic effect. Panc-1 cells were treated with particles for 6 hours and post-transfected for additional 24, 48, 72, and 96 hours.
Since p53 could induce apoptosis in cells and in order to accomplish this function, many downstream transcription factors would be involved and directly regulated by expression of wt-p53. Among them, Bax, caspase-3, caspase-9, DR5, PUMA and Apaf-1 would be up-regulated by expression of p53 and while Bcl-2, survivin would be down-regulated. In order to examine the levels of these transcription factors, mRNA was extracted from Panc-1 cells after 48 hours post-transfection and used for RT-PCR. The products were evaluated with gel electrophoresis and bands were analyzed with ImageJ. Based on the results showed in Figure 7, survivin decreased significantly with the treatment of EGFR targeted thiolated gelatin nanoparticles compared to other treatments, no obvious change was seen in Bcl-2, Bax and expression of caspase-3, caspase-9, DR5, PUMA and Apaf-1increased with targeted nanoparticles treatment.
Figure 7. The mRNA levels of downstream factors of wt-p53 expression were compared by RT-PCR after 48 hours post-transfection.
After wt- p53 transfection, Chromatin Condensation/ Membrane Permeability/ Dead Cell Apoptosis kit was used to differentiate apoptotic cells, necrotic cells and live cells with different dyes. iCys Research Imaging Cytometer from CompuCyte (Westwood, MA) was used to analyze and compare apoptosis levels after treatment. Compared to the negative control, apoptotic cells fold changes were calculated out and listed in Figure 8. EGFR targeted thiolated gelatin nanopaticles have showed the highest apoptotic cell population after post-transfection. Analysis of caspase 3/7 activity also showed that EGFR-targeted nanoparticles had rapid internalization and highest level of apoptotic activity in Panc-1 cells.
Figure 8. Cytometric analysis of pro-apoptotic activity in control wt-p53 transfected Panc-1 cells using iCys° Imaging Cytometer
Control and EGFR targeted thiolated gelatin nanoparticles were prepared with efficient DNA encapsulation and stability. The particle sizes of all of these systems were in the range of 150-250 nm in diameter. Zeta potential has proved that this system is a slightly negative system. With SEM analysis, the sizes of nanoparticles were the same with Zetasizer result. ESCA analysis could confirm PEG and peptide surface modification.
Western blot analysis showed that Panc-1 cells had a high EGFR expression levels and this cell line was used for the in vitro studies. Both the control and the surface-modified nanoparticles were relatively less cytotoxic in Panc-1 cells as compared to PEI.
Cell trafficking studies showed rapid uptake and plasmid release of EGFR-targeted nanoparticles in Panc-1 cells. Delivery of reporter plasmids DNA expressing with EGFR-targeted nanoparticles resulted in highest levels of GFP expression relative to other controls, including Lipofectin-complexed DNA. With the same system, transfection with wt-p53 plasmid triggered the downstream apoptotic pathway and induced rapid apoptosis in Panc-1 cells.
These preliminary results suggest that the EGFR-targeted thiolated gelatin nanoparticles can serve as a safe and efficient DNA delivery system for gene therapy as a treatment for pancreatic cancer.
No conflicts of interest declared.
This study was supported by the National Cancer Institute's Alliance in Nanotechnology for Cancer's Center for Cancer Nanotechnology Excellence (CCNE) grant U54-CA151881.
|Type B gelatin, Bloom 225||Sigma-Aldrich||G9391|
|pEGFP-N1 plasmid||Elim Biopharm||N/A|
|pORF-hp53 E. coli||Invitrogen||porf-hp53|
|Glyoxal solution (40wt. % in H2O)||Sigma-Aldrich||128465|
|QIA filter Plasmid Mega kit||Qiagen||12281|
|Beckman LE 80K Ultracentrifuge||Beckman Coulter Inc.||N/A|
|FreeZone 6 Liter Console Freeze Dry Systems||Labconco Corp.||7753020|
|mPEG-SCM, MW 2,000 Da||Laysan Bio Inc.||mPEG-SCM-2K-1g|
|MAL-PEG-SCM, MW 2,000 Da||Jenkem Technology||A5001-1|
|Zetasizer Nano||Malvern Instruments||Zetasizer Nano ZS|
|Hitachi 4800 field emission scanning electron microscope||Hitachi||S-4800 UHR FE-SEM|
|Quant-iT PicoGreen dsDNA Reagent and Kits||Invitrogen||P7589|
|Lipofectin Transfection Reagent||Invitrogen||18292011|
|DMEM||Mediatech, Inc.||10 013 CM|
|RPMI||Mediatech, Inc.||50 020 PB|
|Pierce BCA Protein Assay Kit||Thermo Fisher Scientific, Inc.||23225|
|iBlot Dry Blotting System||Invitrogen||IB1001|
|XCellSureLock Mini-Cell and XCell II Blot Module Kit CE Mark||Invitrogen||EI0002|
|Pierce ECL Western Blotting Substrate||Thermo Fisher Scientific, Inc.||32109|
|Kodak Digital X-ray Specimen (DXS) System||Kodak||N/A|
|CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)||Promega Corp.||G3580|
|BioTek SynergyHT plate reader||BioTek||N/A|
|Nanodrop 2000||Thermo Fisher Scientific, Inc.||N/A|
|One-step RT-PCR kit||Qiagen||210212|
|Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit||Invitrogen||V23201|
|Apo-ONE Homogeneous Caspase-3/7 Assay kit||Promega Corp.||G7790|
|Hybaid PCR Sprint Thermal Cycler||Thermo Fisher Scientific, Inc.||N/A|
|EGF Receptor Antibody||Cell Signaling Technology||2232|
|β-Actin Antibody||Cell Signaling Technology||4967|
|Anti-rabbit IgG, HRP-linked Antibody||Cell Signaling Technology||7074|
|Mouse Monoclonal GFP Antibody||Novus Biologicals||NB600-597|
|Goat Polyclonal GFP antibody (Alkaline Phosphatase)||Novus Biologicals||NB600-1502|
|Phosphatase Substrate Kit||Thermo Fisher Scientific, Inc.||37620|