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. ...

therapeutic-gene-delivery-transfection-human-pancreatic-cancer-cells

Research

JoVE Journal

Bioengineering

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...