Name: preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation
Uploaded: 2017-05-22T15:00:00
Description: Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

This work was supported by NIH R03 NS087322-01 to Dong, X.

NOTE: The animal studies included in all procedures have been approved by the Institutional Animal Care and Use Committee at the University of North Texas Health Science Center.
1. Preparation of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Dissolve the excipients, phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), cholesteryl oleate (CO), and D-&#945;-tocopheryl polyethylene glycol succinate (TPGS), in ethanol to prepare stock solutions at 1 mg/mL. 
	NOTE: The stock solutions were aliquote...

<ol>
	<li>Bruno, B. J., Miller, G. D., Lim, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+and+recent+advances+in+peptide+and+protein+drug+delivery.">Basics and recent advances in peptide and protein drug delivery.</a> Ther Deliv. 4 (11), 1443-1467 (2013).</li><li>Mo, Z. C., Ren, K., Liu, X., Tang, Z. L., Yi, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-density+lipoprotein-mediated+drug+delivery+system.">A high-density lipoprotein-mediated drug delivery system.</a> Adv Drug Deliv Rev. 106 (Pt A), 132-147 (2016).</li><li>Lacko, A. G., Sabnis, N. A., Nagarajan, B., McConathy, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+as+a+drug+and+nucleic+acid+delivery+vehicle.">HDL as a drug and nucleic acid delivery vehicle.</a> Front Pharmacol. 6, 247-252 (2015).</li><li>Vaishya, R., Khurana, V., Patel, S., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+delivery+of+protein+therapeutics.">Long-term delivery of protein therapeutics.</a> Expert Opin Drug Deliv. 12 (3), 415-440 (2015).</li><li>Lasic, D. 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Modulation of release rate.</a> J Control Release. 67 (2-3), 249-260 (2000).</li><li>Patel, A., Patel, M., Yang, X., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+protein+and+peptide+drug+delivery:+a+special+emphasis+on+polymeric+nanoparticles.">Recent advances in protein and peptide drug delivery: a special emphasis on polymeric nanoparticles.</a> Protein Pept lett. 21 (11), 1102-1120 (2014).</li><li>Kuai, R., Li, D., Chen, Y. E., Moon, J. J., Schwendeman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+lipoproteins:+nature's+multifunctional+nanoparticles.">High-density lipoproteins: nature's multifunctional nanoparticles.</a> ACS Nano. 10 (3), 3015-3041 (2016).</li><li>Gursky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+stability+and+functional+remodeling+of+high-density+lipoproteins.">Structural stability and functional remodeling of high-density lipoproteins.</a> FEBS Lett. 589 (19 Pt A), 2627-2639 (2015).</li><li>McMahon, K. M., Thaxton, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+lipoproteins+for+the+systemic+delivery+of+short+interfering+RNA.">High-density lipoproteins for the systemic delivery of short interfering RNA.</a> Expert Opin Drug Deliv. 11 (2), 231-247 (2014).</li><li>McMahon, K. M., Foit, L., Angeloni, N. L., Giles, F. J., Gordon, L. I., Thaxton, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+high-density+lipoprotein-like+nanoparticles+as+cancer+therapy.">Synthetic high-density lipoprotein-like nanoparticles as cancer therapy.</a> Cancer Treat Res. 166, 129-150 (2015).</li><li>Lerch, P. G., F&#246;rtsch, V., Hodler, G., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+a+reconstituted+high+density+lipoprotein+for+therapeutic+applications.">Production and characterization of a reconstituted high density lipoprotein for therapeutic applications.</a> Vox Sang. 71 (3), 155-164 (1996).</li><li>Zhang, Z., Chen, J., Ding, L., Jin, H., Lovell, J. F., Corbin, I. R., Cao, W., Lo, P. C., Yang, M., Tsao, M. S., Luo, Q., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL-mimicking+peptide-lipid+nanoparticles+with+improved+tumor+targeting.">HDL-mimicking peptide-lipid nanoparticles with improved tumor targeting.</a> Small. 6 (3), 430-437 (2010).</li><li>Kim, Y., Fay, F., Cormode, D. P., Sanchez-Gaytan, B. L., Tang, J., Hennessy, E. J., Ma, M., Moore, K., Farokhzad, O. C., Fisher, E. A., Mulder, W. J., Langer, R., Fayad, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+step+reconstitution+of+multifunctional+high-density+lipoprotein-derived+nanomaterials+using+microfluidics.">Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics.</a> ACS Nano. 7 (11), 9975-9983 (2013).</li><li>Prathipati, P., Zhu, J., Dong, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+HDL-mimicking+&#945;-tocopherol-coated+nanoparticles+to+encapsulate+nerve+growth+factor+and+evaluation+of+biodistribution.">Development of novel HDL-mimicking &#945;-tocopherol-coated nanoparticles to encapsulate nerve growth factor and evaluation of biodistribution.</a> Eur J Pharm and Biopharm. 108, 126-135 (2016).</li></ol>

In this study, we demonstrate a simple method to prepare HDL-mimicking NPs for NGF encapsulation. Various NP delivery systems have been studied to deliver proteins. Currently, many NP preparations involve dialysis, solvent precipitation, and film hydration. These processes are generally complicated and challenging upon scale-up. During this NP development, it was determined that the lipids had strong adhesion to the glass wall of the container, which led to the difficulties in hydrating the thin film and efficiently mixi...

Macromolecules, such as proteins, peptides, and nucleic acids, have been emerging as promising medications and have gained considerable attention in past decades1,2. Due to their high efficacy and specific action modes, they exhibit great therapeutic potential for the treatments of cancer, immune disease, HIV, and related conditions3,4. However, physiochemical properties, such as their large molecular size, three-dimensional structure, surface charges, and hydrophilic nature, make the in vivo delivery of these macromolecules very challenging. This considerably impedes their clinical use4. Recent advancements in drug delivery systems, such as microparticles, polymer nanoparticles (NPs), liposomes, and lipid NPs, overcame these challenges and significantly improved the in vivo delivery of macromolecules. However, some drawbacks regarding these delivery cargoes have been revealed, including low drug loading capacity, low entrapment efficiency, short half-life, loss of bioactivity, and undesirable side effects5,6,7,8. Effective carrier systems remain an area of research interest. Moreover, the development of analytical methods to characterize drug-loaded NPs is more challenging for macromolecules than for small molecules.
High-density lipoprotein (HDL) is a natural NP composed of a lipid core that is coated by apolipoproteins and a phospholipid monolayer. Endogenous HDL plays a critical role in the transport of lipids, proteins, and nucleic acids through its interaction with target receptors, such as SR-BI, ABCAI, and ABCG1. It has been explored as a vehicle for the delivery of different therapeutic agents9,10,11,12. Various methods have been developed to prepare HDL-mimicking NPs. Dialysis is a popular approach. In this method, NPs are formed by hydrating a lipid film using sodium cholate solution. The salt is then removed through a 2-day dialysis with three buffers13. Sonication methods fabricate NPs by sonicating a lipid mixture for 60 min under a heating condition; the NPs are further purified through gel chromatography14. Microfluidics generates NPs via a microfluidic device, which mixes phospholipids and apolipoprotein A-I (Apo A-I) solutions by creating microvortices in a focusing pattern15. Clearly, these methods can be time consuming, harsh, and difficult for industrial scale-up.
In this article, we introduce the preparation and characterization of novel HDL-mimicking NPs for nerve growth factor (NGF) encapsulation. NGF is a disulfide-linked polypeptide homodimer containing two 13.6-kDa polypeptide monomers. A novel procedure to prepare the NPs by homogenization, followed by the encapsulation of NGF into the NPs, was developed. The NGF HDL-mimicking NPs were characterized for particle size, size distribution, zeta potential, and in vitro release. Their bioactivity was evaluated for neurite outgrowth in PC12 cells. The biodistribution of NGF HDL-mimicking NPs was compared with that of free NGF after intravenous injection in mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant Human Beta-NGF</td>
			<td>Creative Biomart</td>
			<td>NGF-05H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-a-Phosphatidylcholine (PC)</td>
			<td>Avanti</td>
			<td>131601P</td>
			<td>95%, Egg, Chicken</td>
		</tr>
		<tr>
			<td>Sphingomyelin (SM)</td>
			<td>Avanti</td>
			<td>860062P</td>
			<td>Brain, Porcine</td>
		</tr>
		<tr>
			<td>Phosphatidylserine (PS)</td>
			<td>Avanti</td>
			<td>840032P</td>
			<td>Brain, Porcine</td>
		</tr>
		<tr>
			<td>Cholesteryl oleate (CO)</td>
			<td>Sigma</td>
			<td>C9253</td>
			<td></td>
		</tr>
		<tr>
			<td>D-&#945;-Tocopheryl polyethylene glycol succinate (TPGS)</td>
			<td>BASF</td>
			<td>9002-96-4</td>
			<td>Vitamin E Polyethylene Glycol Succinate</td>
		</tr>
		<tr>
			<td>Protamine sulfate</td>
			<td>Sigma</td>
			<td>P3369</td>
			<td>meets USP testing specifications</td>
		</tr>
		<tr>
			<td>Apolipoprotein A1, Human plasma</td>
			<td>Athens Research &#38; Technology</td>
			<td>16-16-120101</td>
			<td>1mg in 671 &#181;l 10 mM NH4HCO3, pH 7.4</td>
		</tr>
		<tr>
			<td>Sepharose 4B-CL</td>
			<td>Sigma</td>
			<td>CL4B200</td>
			<td>Cross-linked agarose,&#160; gel filtration chromatography column filling material</td>
		</tr>
		<tr>
			<td>Sandwich ELISA Kit for NGF</td>
			<td>R&#38;D system</td>
			<td>DY008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>GE Healthcare Life Science</td>
			<td>SH30096.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>GE Healthcare Life Science</td>
			<td>SH30074.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>PC12 cells</td>
			<td>ATCC</td>
			<td>CRL-1721</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail collagen type I</td>
			<td>Sigma</td>
			<td>C3867</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>31414</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzethonium chloride</td>
			<td>Sigma</td>
			<td>B8879</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>Tekmar</td>
			<td>T 25-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Delsa Nano HC particle analyzer</td>
			<td>Beckman-Coulter</td>
			<td>Delsa Nano HC</td>
			<td></td>
		</tr>
		<tr>
			<td>Float-A-Lyzer G2 Dialysis Device</td>
			<td>Spectrum Laboratories</td>
			<td>G235036</td>
			<td>Molecule Cutoff 300 kDa</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendoff</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytron homogenizer</td>
			<td>Kinematica</td>
			<td>PT 1200C</td>
			<td></td>
		</tr>
		<tr>
			<td>DecapiCone</td>
			<td>&#160;Braintree Scientific Inc.</td>
			<td>DC-M200</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The engineering scheme of HDL-mimicking, &#945;-tocopherol-coated NGF NPs prepared by an ion-pair strategy is shown in Figure 1. To neutralize the surface charges of NGF, protamine USP was used as an ion-pair agent to form a complex with NGF. To protect the bioactivity, prototype HDL-mimicking NPs were engineered, first using homogenization; then, the NGF/protamine complex was encapsulated into the prototype NPs. Homogenization provided sufficient energy and successfully ...

Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

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

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 ...

The overall goal of this article is to introduce preparation and characterization methods for nerve growth factor-loaded, high-density lipoprotein-mimicking nanoparticles. Simple homogenization was used to prepare normal, high-density lipoprotein-mimicking nanoparticles. The procedure is simple and scalable.Moreover, the nanoparticle greatly improved entrapment efficiency, also NGF, and APOA1 in the nanoparticles. Various challenges to characterize a nanoparticle were overcome, including separate to the unloaded NGF from the NGF nanoparticles, conduct reliable in vitro radius studies, and the bioavailability of NGF nanoparticles. To begin, mix 10 microliters of NGF with 10 microliters of protamine USP in a 1.5 milliliter microcentrifuge tube and let it stand for 10 minutes at room temperature to form the complex.Next, add 59 microliters of phosphatidylcholine and 11 microliters of sphingomyelin to a glass vial. Also add four microliters of phosphatidylserine, 15 microliters of cholesteryl oleate, and 45 microliters of tocopheryl polyethylene glycol succinate. Mix and evaporate the ethanol under a gentle nitrogen stream for about five minutes.All excipients should form an oily thin film at the bottom of the glass vial. Add one milliliter of ultrapure water to the vial and homogenize at 9, 500 rpm for five minutes at room temperature to form the prototype nanoparticles, or NPs. Then, add the prepared complex to the prototype NPs.Incubate the suspension at 37 degrees Celsius for 30 minutes with stirring, by using a small stirring bar in the glass vial. Cool the NPs down by stirring at room temperature for another 30 minutes. After cooling, add 106 microliters or apolipoprotein A-1 and stir at room temperature overnight to form the final NGF HDL-mimicking NPs.Measure the particle size and zeta potential using a particle analyzer as per the manufacturer's instructions. A cross-linked gel filtration chromatographic column is used to separate the unloaded NGF from the NGF HDL-mimicking nanoparticles and determines entrapment efficiency of the NGF. For the column preparation, transfer 15 milliliters of sepharose 4B CL suspension to a 50 milliliter beaker.Stir the bead suspension and pour some into a column. Gently tap the column to get rid of bubbles. Allow the solvent to drain and the beads to settle for a few minutes.Continue to add the remaining suspension. Rinse the inside wall of the column to clean the beads. Drain the solvent until the solvent level is slightly above the top of the stationary phase.Then, wash and condition the column with 20 milliliters of 1X phosphate-buffered saline. To measure the entrapment efficiency of the NGF, load 200 microliters of NGF HDL-mimicking NPs onto the column and elute with 1X PBS. As before, collect a total of 12 one-milliliter fractions.Pretreat eight dialysis tubes by following the manufacturer's instructions. Add 30 milliliters of release medium to a 50 milliliter centrifuge tube. Then, warm it up to 37 degrees Celsius in a shaker with a 135 rpm shaking speed.Close the dialysis tube and place it into the release medium. Quickly withdraw 100 microliters of the release medium from outside the dialysis tube as the sample for time zero. Immediately put the sample into minus 20 degrees Celsius for later analysis.Add 100 microliters of fresh release medium into the centrifuge tube to replace the withdrawn sample. Then, start the timer. At 1, 2, 4, 6, 8, 24, 48, and 72 hours, withdraw 100 microliters of the release medium and replace it with 100 microliters of fresh medium.Immediately put the withdrawn samples into minus 20 degrees Celsius for later analysis. After a three-minute homogenization, consistent particle sizes were obtained for the prototype nanoparticles. The final NGF nanoparticles had particle sizes of 170 nanometers with a narrow and monodispersed size distribution.Unloaded NGF and nanoparticles were completely separated on the gel filtration column. Nanoparticles were eluded in fractions two through four. ELISA measurement indicated that free NGF was eluded in fraction 7 to fraction 10.The calculated entrapment efficiency of NGF was approximately 66%In vitro release of NGF nanoparticles is shown here. By adding PBS and BSA to the release medium, free NGF passed through the dialysis membrane with a recovery of over 85%Conversely, NGF nanoparticles showed a slow release profile with about 10%of NGF being released from the nanoparticles over 72 hours. NGF nanoparticles stimulated neurite outgrowth in PC12 cells to a similar extent as for free NGF.Neurite outgrowth was clearly observed under the microscope for both groups when dosed at 50 nanograms per milliliter. NGF nanoparticles considerably increased the plasma concentration and decreased the uptake of NGF in the mice liver, kidney, and spleen. HDL-mimicking nanoparticles assisted NGF, allowing for a long half-life in in vivo stability.Once mastered, monodispersed nanoparticles can be prepared in three minutes using a homogenization method if it is performed properly. While attempting this procedure, it is important to remember to use a gentle nitrogen stream to evaporate to the solvent and make sure no lipid residue sticking on the wall of the container after homogenization. After watching this video, you should have a good understanding of how to make HDL-mimicking nanoparticles using a homogenization method and how to characterize NGF loaded nanoparticles.

preparation-characterization-novel-hdl-mimicking-nanoparticles-for

Research

JoVE Journal

Bioengineering

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.

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. ...

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, ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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 ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

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 ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...