Name: therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles
Uploaded: 2012-01-04T13:00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles at JoVE.com

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.

<ol>
	<li>Vimalachandran, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+prevention+of+pancreatic+cancer.">Genetics and prevention of pancreatic cancer.</a> Cancer. Control. 11, 6-14 (2004).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+evaluation+of+thiol-modified+gelatin+nanoparticles+for+intracellular+DNA+delivery+in+response+to+glutathione.">Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione.</a> Bioconjug. Chem. 16, 1423-1432 (2005).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+glycol)-modified+thiolated+gelatin+nanoparticles+for+glutathione-responsive+intracellular+DNA+delivery.">Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery.</a> Nanomedicine. 3, 32-42 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+gene+therapy+with+systemically+administered+sFlt-1+plasmid+DNA+in+engineered+gelatin-based+nanovectors.">Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors.</a> Cancer. Gene. Ther. 14, 488-498 (2007).</li><li>Kommareddy, S., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+and+pharmacokinetic+analysis+of+long-circulating+thiolated+gelatin+nanoparticles+following+systemic+administration+in+breast+cancer-bearing+mice.">Biodistribution and pharmacokinetic analysis of long-circulating thiolated gelatin nanoparticles following systemic administration in breast cancer-bearing mice.</a> J. Pharm. Sci. 96, 397-407 (2007).</li><li>Kaul, G., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-targeted+gene+delivery+using+poly(ethylene+glycol)-modified+gelatin+nanoparticles:+in+vitro+and+in+vivo+studies.">Tumor-targeted gene delivery using poly(ethylene glycol)-modified gelatin nanoparticles: in vitro and in vivo studies.</a> Pharm. Res. 22, 951-961 (2005).</li><li>Bardeesy, N., DePinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+biology+and+genetics.">Pancreatic cancer biology and genetics.</a> Nat. Rev. Cancer. 2, 897-909 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+novel+peptide+ligand+of+epidermal+growth+factor+receptor+for+targeted+delivery+of+therapeutics.">Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics.</a> FASEB. J. 19, 1978-1985 (2005).</li><li>McCormick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+gene+therapy:+fringe+or+cutting+edge.">Cancer gene therapy: fringe or cutting edge.</a> Nat. Rev. Cancer. 1, 130-141 (2001).</li><li>Green, D. R., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+functions+of+the+tumour+suppressor+p53.">Cytoplasmic functions of the tumour suppressor p53.</a> Nature. 458, 1127-1130 (2009).</li><li>Barton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+the+p53+tumour+suppressor+gene+in+human+pancreatic+cancer.">Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer.</a> Br. J. Cancer. 64, 1076-1082 (1991).</li></ol>

No conflicts of interest declared.

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.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Type B gelatin, Bloom 225</td>
 <td>Sigma-Aldrich</td>
 <td>G9391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-iminothiolane hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>I6256</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pEGFP-N1 plasmid</td>
 <td>Elim Biopharm</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pORF-hp53 E. coli</td>
 <td>Invivogen</td>
 <td>porf-hp53 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glyoxal solution (40wt. % in H2O) </td>
 <td>Sigma-Aldrich</td>
 <td>128465</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Sigma-Aldrich</td>
 <td>410225-250G </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>QIA filter Plasmid Mega kit</td>
 <td>Qiagen</td>
 <td>12281 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Beckman LE 80K Ultracentrifuge</td>
 <td>Beckman</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FreeZone 6 Liter Console Freeze Dry Systems</td>
 <td>Labconco</td>
 <td>7753020 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mPEG-SCM, MW 2,000 Da </td>
 <td>Laysan</td>
 <td>mPEG-SCM-2K-1g </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MAL-PEG-SCM, MW 2,000 Da </td>
 <td>Jenkem Technology</td>
 <td>A5001-1 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Zetasizer Nano</td>
 <td>Malvern</td>
 <td>Zetasizer Nano ZS </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hitachi 4800 field emission scanning electron microscope</td>
 <td>Hitachi</td>
 <td>S-4800 UHR FE-SEM </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Quant-iT PicoGreen 
 dsDNA Reagent and Kits </td>
 <td>Invitrogen</td>
 <td>P7589 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lipofectin Transfection Reagent</td>
 <td>Invitrogen</td>
 <td>18292011 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Mediatech cellgro</td>
 <td>10 013 CM </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Mediatech cellgro</td>
 <td>50 020 PB </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pierce BCA Protein Assay Kit</td>
 <td>Thermo Scientific</td>
 <td>23225</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>iBlot Dry Blotting System</td>
 <td>Invitrogen</td>
 <td>IB1001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>XCell&nbsp;SureLock Mini-Cell and XCell II Blot Module Kit CE Mark</td>
 <td>Invitrogen</td>
 <td>EI0002 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pierce ECL Western Blotting Substrate </td>
 <td>Thermo Scientific</td>
 <td>32109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Kodak Digital X-ray Specimen (DXS) System </td>
 <td>Kodak</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
 <td>Promega</td>
 <td>G3580</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BioTek SynergyHT plate reader</td>
 <td>BioTek</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nanodrop 2000</td>
 <td>Thermo Scientific</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>One-step RT-PCR kit</td>
 <td>Qiagen</td>
 <td>210212</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chromatin Condensation/Membrane Permeability/Dead Cell Apoptosis Kit </td>
 <td>Invitrogen</td>
 <td>V23201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Apo-ONE Homogeneous Caspase-3/7 Assay kit</td>
 <td>Promega</td>
 <td>G7790</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hybaid PCR Sprint Thermal Cycler</td>
 <td>Thermo Scientific</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGF Receptor Antibody </td>
 <td>Cell signaling</td>
 <td>2232</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&beta;-Actin Antibody </td>
 <td>Cell signaling</td>
 <td>4967</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG, HRP-linked Antibody </td>
 <td>Cell signaling</td>
 <td>7074</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse Monoclonal GFP Antibody</td>
 <td>Novus Biologicals</td>
 <td>NB600-597</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Goat Polyclonal GFP antibody (Alkaline Phosphatase)</td>
 <td>Novus Biologicals</td>
 <td>NB600-1502</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphatase Substrate Kit</td>
 <td>Thermo Scientific</td>
 <td>37620</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles

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.

Watch this Scientific Journal Video about Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles at JoVE.com

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

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. ...

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