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 aliquoted and stored at -20 &#176;C. The cholesteryl oleate was stored in dark bottles. The PC, SM, PS, and CO stock solutions were stable for up to 6, 3, 12, and 12 months, respectively, at -20 &#176;C. The TPGS solution was stable for at least 12 months at -20 &#176;C.</li>
	<li>Mix 10 &#181;L of NGF (1 mg/mL in water) with 10 &#181;L of protamine USP (1 mg/mL in water) in a 1.5 mL microcentrifuge tube and let it stand for 10 min at room temperature to form the complex. 
	NOTE: The NGF and protamine stock solutions were aliquoted and stored at -20 &#176;C. Repeated freezing and thawing is not recommended for the NGF stock.</li>
	<li>Add 59 &#181;L of PC, 11 &#181;L of SM, 4 &#181;L of PS, 15 &#181;L of CO, and 45 &#181;L of TPGS to a glass vial. Mix and evaporate the ethanol under a gentle nitrogen stream for about 5 min; all excipients should form an oily, thin film at the bottom of the glass vial.</li>
	<li>Add 1 mL of ultrapure (type 1) water to the vial and homogenize at 9,500 rpm (8,600 x g) for 5 min at room temperature to form the prototype NPs.</li>
	<li>Add the complex prepared in step 1.2 to the prototype NPs and incubate at 37 &#176;C for 30 min with stirring by using a small stirring bar in the glass vial.
	<ol>
		<li>Cool the NPs down by stirring at room temperature for another 30 min. After this cools, add 106 &#181;L of Apo A-I (1.49 mg/mL) and stir at room temperature overnight to form the final NGF HDL-mimicking NPs.</li>
	</ol>
	</li>
</ol>
2. Characterization of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Measure the particle size and zeta potential using a particle analyzer (see the Materials Table) as per the manufacturer&#39;s instructions.</li>
	<li>Use a cross-linked agarose gel filtration chromatography column to separate the unloaded NGF from the NGF HDL-mimicking NPs and determine the entrapment efficiency of the NGF.
	<ol>
		<li>For the column preparation, transfer 15 mL of Sepharose 4B-CL suspension to a 50-mL beaker. Stir the bead suspension using a glass rod and pour some into a column (30 cm length &#215; 1 cm diameter, with a glass frit at the bottom). Gently tap the column to get rid of bubbles. Allow the solvent to drain and the beads to settle for a few min.</li>
		<li>Continue to add the remaining suspension. Rinse the inside wall of column to clean the beads. Drain the solvent until the solvent level is slightly above the top of the stationary phase. Wash and condition the column with 20 mL of 1x phosphate-buffered saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4).</li>
		<li>To determine the fractions containing unloaded NGF, load 200 &#181;L of NGF solution (10 &#181;g/mL) onto the gel filtration column (25 cm length x 1 cm diameter) and elute with 1x PBS.</li>
		<li>Collect a total of 12 fractions (1 mL for each fraction) and measure the concentration of NGF in each fraction using a Sandwich ELISA kit for NGF.</li>
		<li>Detect NGF from fractions 6 to 10 using the Sandwich ELISA kit. Obtain a chromatogram of unloaded NGF on the column. Wash the column with 20 mL of PBS.</li>
		<li>To determine the fractions containing NGF HDL-mimicking NPs, load 200 &#181;L of the NPs onto the column and elute with 1x PBS. Collect a total of 12 fractions (1 mL for each fraction) and measure the intensity of particles in each fraction using the particle size analyzer. Detect the NGF NPs from fractions 2 to 4. 
		NOTE: The intensity is a parameter measured by the particle analyzer, which tells how many nanoparticles may exist in the test solution. The more NPs exist in solution, the higher the intensity is. By this approach, we can confirm that the column can separate NGF NPs from unloaded NGF.</li>
		<li>To measure the entrapment efficiency of NGF, load 200 &#181;L of NGF HDL-mimicking NPs onto the column and elute with 1x PBS. Collect a total of 12 fractions (1 mL for each fraction).</li>
		<li>Measure the unloaded NGF concentration from fractions 6 to 10 using the Sandwich ELISA kit.</li>
		<li>Add the NGF amounts measured from fractions 6 to 10 together as the unloaded NGF and calculate the entrapment efficiency of NGF using the following equation. 
		%EE = (1 - unloaded NGF/total NGF added into NP) x 100% Equation (1)</li>
	</ol>
	</li>
</ol>
3. In Vitro Release of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Study free NGF (10 &#181;g/mL in water; n = 4) and NGF HDL-mimicking NPs (10 &#181;g/mL; n = 4) in parallel.</li>
	<li>Pre-treat 8 dialysis tubes (molecular weight cutoff: 300 kDa) by following the manufacturer&#39;s instructions.</li>
	<li>Prepare 5% bovine serum albumin (BSA) in PBS as the release medium. Add 30 mL of release medium to a 50-mL centrifuge tube and warm it up to 37 &#176;C in a shaker with a 135 rpm shaking speed. Place another 50 mL of release medium in the shaker for replacement.</li>
	<li>Add 400 &#181;L of release medium into the dialysis tube and quickly add 200 &#181;L of the tested sample to make enough volume for dialysis.</li>
	<li>Close the tube and quickly put the dialysis tube into the release medium (the centrifuge tube). Quickly withdraw 100 &#181;L of the release medium from the outside dialysis tube as the sample for time 0. Immediately put the sample into -20 &#176;C for later analysis. Add 100 &#181;L of fresh release medium into the centrifuge tube to replace the withdrawn sample. Start the timer.</li>
	<li>At 1, 2, 4, 6, 8, 24, 48, and 72 h, withdraw 100 &#181;L of the release medium and replace it with 100 &#181;L of fresh medium. Immediately put the withdrawn samples into -20 &#176;C for later analysis.</li>
	<li>After 72 h, take out all samples from -20 &#176;C and thaw them at room temperature. Measure the NGF concentrations of the release samples using the Sandwich ELISA kit.</li>
</ol>
4. Bioactivity of NGF HDL-mimicking Nanoparticles (Neurite Outgrowth Study)
<ol>
	<li>Culture PC12 cells in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 &#181;g/mL streptomycin, and 100 units/mL penicillin. Maintain the cells in a humidified incubator at 37 &#176;C and with 5% CO2.</li>
	<li>When the cells reach ~70-80% confluence, remove the culture medium and wash the cells with 1x PBS (approximately 2 mL per 10 cm2 culture surface area). Remove the wash solution. Trypsinize the cells by adding 0.25% Trypsin-EDTA solution (0.25% trypsin and 1 mM EDTA; 0.5 mL per 10 cm2 culture surface area) to the flask and incubate at 37 &#176;C until most cells are detached.</li>
	<li>Add two volumes of culture medium and spin down at 180 x g for 5 min. Remove the supernatant and resuspend the cell pellet in 5 mL of culture medium. Filter the cells through a 22 G, &#189; inch needle to break cell clusters.</li>
	<li>Split the cells at a ratio of 1:3 into a new cell culture flask. After incubating overnight, remove unattached PC12 cells. Allow the attached cells to remain for further growth.</li>
	<li>Repeat this procedure for 3 passages to select the subculture of PC12 that has a strong adhesion property. Pre-coat a 6-well plate with 800 &#181;L/well of rat tail collagen type I (100 &#181;g/mL).</li>
	<li>Trypsinize the selected PC12 cells as described in step 4.2 and filter them through a 22 G, &#189; inch needle to break the cell clusters. Count the cells with a hemocytometer. Seed the cells overnight at a density of 10,000 cells/well in the pre-coated 6-well plate in step 4.5 to allow the cells to attach to the plate.</li>
	<li>Dilute free NGF (10 &#181;g/mL) and NGF HDL-mimicking NPs (10 &#181;g/mL) with the culture medium (described in step 4.1) to prepare 0.5, 1, 5, 10, 50, and 100 ng/mL concentrations. Remove 2 mL of medium from each well and replace with 2 mL of the diluted free NGF or NGF HDL-mimicking NPs. Culture for 4 days.</li>
	<li>On day 4, change the medium to fresh medium (described in step 4.1) containing the corresponding treatment and continue the treatment for another 3 days.</li>
	<li>On day 7, visualize the cells with an inverted light microscope and image each well at random spots under 10X magnification.</li>
</ol>
5. Biodistribution of NGF HDL-mimicking Nanoparticles
<ol>
	<li>Use adult BALB/c mice (male, 25-30 g) to test the tissue distribution of NGF NPs. Randomly divide the mice into three groups of 3 mice per group. Use a cone-shaped plastic restraint (e.g., decapicone) to restrain a mouse and wipe the tail with ethanol to promote vasodilation and the visibility of the vein.</li>
	<li>Inject 100 &#181;L of either saline, free NGF, or NGF HDL-mimicking NPs to each group of mice (3 groups) through the tail veins at a dose of 40 &#181;g/kg of NGF. Use a 30&#189; G needle attached to a 1 mL syringe.</li>
	<li>At 30 min after the injection, anesthetize the mice using 3% inhaled isoflurane (in oxygen at flow rate of 2 L/min). Perform a tail and toe pinch to determine the depth of anesthesia. Collect blood by cardiac puncture.
	<ol>
		<li>Withdraw approximately 1 mL of blood from the heart. Euthanize each mouse by cervical dislocation.</li>
	</ol>
	</li>
	<li>Place the mouse carcass in dorsal recumbency. Open the abdomen using surgical scissors and move fat and intestine aside using a cotton swab to expose the liver, spleen, and kidney. Harvest these tissues and rinse them in 1x PBS to clean the blood.</li>
	<li>Immediately centrifuge the blood samples at 3,400 x g and 4 &#176;C for 5 min to obtain the plasma. Store the plasma and tissues at -80 &#176;C until the analyses.</li>
	<li>To analyze the tissue samples, move the samples from -80 &#176;C to 4 &#176;C and suspend 100 mg of tissue sample in a 10x volume of extraction buffer (0.05 M sodium acetate, 1.0 M sodium chloride, 1% triton X-100, 1% BSA, 0.2 mM phenylmethanesulfonyl fluoride, and 0.2 mM benzethonium chloride). Homogenize at 10,000 rpm and 4 &#176;C for 5 min.</li>
	<li>Sacrifice two untreated mice to collect blank plasma and tissues, as described in steps 5.3 and 5.4. Prepare NGF standard solutions using blank plasma or blank tissue homogenates.</li>
	<li>Determine the concentrations of NGF in the plasma and tissue homogenates using the Sandwich ELISA kit, as described above.</li>
</ol>

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The authors have nothing to disclose.

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 ...

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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 ...

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Research

JoVE Journal

Bioengineering

8.3K Views.  University of North Texas Health Science Center. 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. 

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

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 ...

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 ...

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

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 ...