This work was funded by the Golfers Against Cancer, Carolina Center for Nanotechnology Excellence Pilot grant, University Cancer Research Fund and National Health Institute K-12 Career Development Award.

1. Synthesis of PLGA-b-PEG polymer
<ol>
 <li>Poly(D,L-lactide-co-glycolide) (PLGA) with terminal carboxylate groups (PLGA-carboxylate) is dissolved in any solvent for PLGA (as mentioned in materials section) at a concentration of 5mM. PLGA can be dissolved at this concentration with gentle stirring.</li>
 <li>Both NHS (molecular weight 115.09) and EDC (molecular weight 191.7) are dissolved in the PLGA solution at a concentration of 25mM. (Both EDC and NHS are added in a stoichiometric excess of 5 times compared to PLGA). PLGA-carboxylate is converted into PLGA-NHS by adding EDC and NHS to PLGA-carboxylate solution with gentle stirring for about 1 hour.</li>
 <li>The reaction product PLGA-NHS is precipitated out by adding the washing solution methanol. Approximately 10 times volume excess of methanol is added to the solution. The solution is centrifuged at 2000 x g to precipitate out the PLGA-NHS and discard the supernatant (removes the traces of EDC and NHS. This procedure of washing with methanol is repeated at least three times.</li>
 <li>The PLGA-NHS pellet is dried under a vacuum for 30 minutes to remove any traces of the washing solution.</li>
 <li>The PLGA-NHS pellet is now re-dissolved in the same solvent at the same concentration that was used initially to dissolve PLGA. The heterobifunctional PEG (amine-PEG-carboxylate) is then added to the PLGA solution at a concentration of 5mM (stoichiometric ratio of 1:1). The mixture solution is incubated for 24 hours with constant stirring.</li>
 <li>After 24 hours, the reaction product PLGA-b-PEG block copolymer is precipitated out by adding the washing solution methanol in excess. Repeat the washing and centrifugation process as mentioned above three times. This will remove all excess unreacted PEG.</li>
 <li>The PLGA-b-PEG block copolymer is dried under vacuum.</li>
</ol>
2. PLGA-b-PEG nanoparticle preparation
Nanoparticles with PLGA core covered with PEG at the surface can be prepared with these diblock copolymers. A variety of different hydrophobic drugs can be encapsulated in such nanoparticles. Fluorescent compounds can be encapsulated in the nanoparticles or can be conjugated to PLGA and thus these nanoparticles can be used for fluorescence imaging.
Nanoprecipitation method is used to make nanoparticles especially when the desired cargo to be encapsulated is highly hydrophobic in nature.
<ol>
 <li>The PLGA-b-PEG block copolymer and the drug/cargo (to be encapsulated) are dissolved in any solvent which dissolves PLGA. PLGA can be dissolved by many common solvents, including acetonitrile, DCM, tetrahydrofuran, acetone or ethyl acetate. The choice of solvent is critical, as it influences the properties of nanoparticle. Hence, appropriate solvent should be used in this step.</li>
 <li>The polymer/drug mixture is then added dropwise to 3-5 volumes of stirring water giving a final polymer concentration of around 3 mg/mL. (Fig. 2)</li>
 <li>The stirring is continued for 2 hours under reduced pressure to allow the nanoparticles to form by self-assembly and remove traces of the organic solvent.</li>
 <li>Harvesting and purification: The nanoparticles are then concentrated by centrifugation at 2,700xg for 10 min using an Amicon filter (MWCO 20KDa), washed, and reconstituted in PBS. This removes all the un-entrapped drug/cargo. Basic biophysical characterizations, such as size, surface charge, and drug loading efficiency can be performed to better understand the properties of nanoparticles.</li>
</ol>
3. Storage
Freeze-drying is a commonly used method to store nanoparticles11. Freeze-drying will preserve the physical and chemical characteristics of the nanoparticles for long term stability12. The freeze drying process can cause stress on the particles and destabilize the formulation, so cryo-protectants (protection from freezing stress) and lyo-protectants (protection from drying stress) are commonly used. The choice of these protectants is determined by the desired length of storage time13.
<ol>
 <li>In freeze-drying, there is total solidification of the sample by freezing below its Tg.</li>
 <li>In the drying step, the ice is removed by sublimation. Temperature and pressure should be optimized to achieve an efficient freeze-drying process.</li>
</ol>
4. Representative results:
Characterization of PLGA-b-PEG Di-block Copolymer
Different techniques can be used to confirm the successful conjugation of polymers. The composition of PLGA-b-PEG can be characterized using a 400 MHz 1H nuclear magnetic resonance (NMR). Molecular weight of the formed product (PLGA-b-PEG) can be verified by Gel permeation chromatography (GPC). The PLGA-b- PEG molecular weight distribution curve and elution time should be different from PLGA and PEG alone. In combination, these techniques should characterize the formed product and determine whether the conjugation reaction was successful.
Characterization of PLGA-b-PEG nanoparticles
Particle size and size distribution can be measured by dynamic light scattering. Different parameters in the nanoprecipitation process affect the size of particles. Molecular weights of the polymers used initially (both PLGA and PEG) also effect the particle size distribution. Transition electron microscopy (TEM) can also be used to confirm the size distribution and structure of the nanoparticles as seen in figure 3. The particle size range is generally in the nm range. Large particle sizes with uneven size distribution could indicate either an error in the conjugation reaction or the nanoprecipitation method needs optimization. In addition, surface zeta potential can be measured by ZetaPALS. 
The drug/cargo loading efficiency can be quantified with standard HPLC.
The particles are dissolved in organic solvent and HPLC can be performed to measure the absorbance of the drug/cargo (Fig. 4). The drug release kinetic study can be done where known fixed quantities of the nanoparticles are dialyzed in 30 Slide-A-Lyzer MINI Dialysis units. At fixed time intervals, the content in the dialysis unit is collected and equal volume of organic solvent is added to dissolve the nanoparticles. HPLC is done on these samples to quantify the drug/cargo content.
<img alt="Figure 1" src="/files/ftp_upload/3398/3398fig1.jpg" /> 
Figure 1. EDC/NHS chemistry
<img alt="Figure 2" src="/files/ftp_upload/3398/3398fig2.jpg" /> 
Figure 2. Nanoprecipitation method for preparing polymeric nanoparticles. The organic solution of a solvent (acetonitrile or DCM) containing the PEG-PLGA diblock and the drug or cargo to be loaded into the particle is added dropwise to 3-5 mL of stirring H2O.
<img alt="Figure 3" src="/files/ftp_upload/3398/3398fig3.jpg" /> 
Figure 3. Transmission Electron Microscopy of nanopartices. A TEM image of PEG-PLGA nanoparticles containing wortamin. Phosphotungstic acid was used as a contrast agent.
<img alt="Figure 4" src="/files/ftp_upload/3398/3398fig4.jpg" /> 
Figure 4. Controlled release of drug from nanoparticle. Release of Paclitaxel from nanoparticles after dialysis in PBS. At the noted time, particles were removed from dialysis cassettes and solublized in acetonitrile. The solution was measured by HPLC. Two separate lots of nanoparticles were compared.

<ol>
	<li>Drotleffa, S., Lungwitz, U., Breuniga, M., Dennis, A., Blunk, T., Tessmarc, J., Go&#235;pferich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+polymers+in+pharmaceutical+and+biomedical+sciences.">Biomimetic polymers in pharmaceutical and biomedical sciences.</a> European Journal of Pharmaceutics and Biopharmaceutics. 58, 385-407 (2004).</li><li>Bulte, J. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanoparticles in Biomedical Imaging. 3, (2008).</li><li>Omid, C., Farokhzad, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Nanotechnology+on+Drug+Delivery.">Impact of Nanotechnology on Drug Delivery.</a> ACS NANO. 3, 16-20 (2009).</li><li>Li, Y. -. P., Pei, Y. -. Y., Xian-Ying, Z., Zhou-Hui, G., Zhao-Hui, Z., Wei-Fang, Y., Jian-Jun, Z., Jian-Hua, Z., Xiu-Jian, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+PLGA+nanoparticles+as+protein+carriers:+synthesis,+preparation+and+biodistribution+in+rats.">PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats.</a> Journal of Controlled Release. 71, 203-211 (2011).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (2008).</li><li>Jeong, B., Bae, Y. H., Lee, D. S., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+block+copolymers+as+injectable+drug-delivery+systems.">Biodegradable block copolymers as injectable drug-delivery systems.</a> Nature. 388, 860-862 (1997).</li><li>Yoo, H. S., Park, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate+receptor+targeted+biodegradable+polymeric+doxorubicin+micelles.">Folate receptor targeted biodegradable polymeric doxorubicin micelles.</a> Journal of Controlled Release. 96, 273-283 (2004).</li><li>Cheng, J., Teply, B. A., Sherifi, I., Sung, J., Luther, G., Gu, F. X., Levy-Nissenbaum, E., Radovic-Moreno, A. F., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+Functionalized+PLGA-PEG+Nanoparticles+for+In+Vivo+Targeted+Drug+Delivery.">Formulation of Functionalized PLGA-PEG Nanoparticles for In Vivo Targeted Drug Delivery.</a> Biomaterials. 28, 869-876 (2007).</li><li>Gu, F., Zhang, L. F., Teply, B. A., Mann, N., Wang, A., Radovic-Moreno, A. F., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+engineering+of+targeted+nanoparticles+by+using+self-assembled+biointegrated+block+copolymers.">Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers.</a> Proceedings of the National Academy of Science. 105, 2586-2591 (2008).</li><li>Sanna, V., Pintus, G., Roggio, A. M., Punzoni, A., Posadino, A. M., Arca, A., Marceddu, S., Bandiera, P., Uzzau, S., Sechi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Biocompatible+Nanoparticles+for+the+Delivery+of+(-)-Epigallocatechin+3-Gallate+to+Prostate+Cancer+Cells.">Targeted Biocompatible Nanoparticles for the Delivery of (-)-Epigallocatechin 3-Gallate to Prostate Cancer Cells.</a> J. Med. Chem. 54, 1321-1332 (2011).</li><li>Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-drying+of+nanoparticles:+Formulation,+process+and+storage+considerations.">Freeze-drying of nanoparticles: Formulation, process and storage considerations.</a> Advanced Drug Delivery Reviews. 58, 1688-1713 (2006).</li><li>Holzer, M., Vogel, V., M&#228;ntele, W., Schwartz, D., Haase, W., Langer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physico-chemical+characterisation+of+PLGA+nanoparticles+after+freeze-drying+and+storage.">Physico-chemical characterisation of PLGA nanoparticles after freeze-drying and storage.</a> European Journal of Pharmaceutics and Biopharmaceutics. 72, 428-437 (2009).</li><li>Lee, M. K., Kim, M. Y., Kim, S., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoprotectants+for+Freeze+Drying+of+Drug+Nano-Suspensions:+Effect+of+Freezing+Rate.">Cryoprotectants for Freeze Drying of Drug Nano-Suspensions: Effect of Freezing Rate.</a> Journal of Pharmaceutical Sciences. 98, 4808-4817 (2009).</li><li>Wang, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalized+targeted+nanoparticles+for+therapeutic+applications.">Biofunctionalized targeted nanoparticles for therapeutic applications.</a> Expert opinion on biological therapy. 8, 1063-1070 (2008).</li><li>Jeong, B., Bae, Y. H., Kim, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+release+from+biodegradable+injectable+thermosensitive+hydrogel+of+PEG-PLGA-PEG+triblock+copolymers.">Drug release from biodegradable injectable thermosensitive hydrogel of PEG-PLGA-PEG triblock copolymers.</a> J. Control Release. 63, 155-163 (2000).</li><li>Gref, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+long-circulating+polymeric+nanospheres.">Biodegradable long-circulating polymeric nanospheres.</a> Science. 263, 1600-1603 (1994).</li></ol>

No conflicts of interest declared.

The nanoprecipitation method using diblock co-polymers represents a simple, fast method to engineer polymeric nanoparticles. The resulting nanoparticles are composed of a hydrophobic core which can be utilized for the delivery of poorly soluble compounds. The surface hydrophilic layer enables excellent aqueous solubility while providing a moiety for potential further conjugation to a targeting ligand.
There are many nanoparticle platforms, including liposomes, polymeric nanoparticles, dendrime...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>EDC</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
			<td>Conjugation Reagent</td>
		</tr>
		<tr>
			<td>NHS</td>
			<td>Thermo Scientific</td>
			<td>24500</td>
			<td>Conjugation Reagent</td>
		</tr>
		<tr>
			<td>amine-PEG-carboxylate</td>
			<td>Laysan Bio Inc.</td>
			<td>Nh2-PEG-CM-5000</td>
			<td>Polymer (Can use any PEG MW, 5000 is listed here)</td>
		</tr>
		<tr>
			<td>PLGA-carbxylate</td>
			<td>Lactel</td>
			<td>B6013-2</td>
			<td>Polymer</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>34856</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Acetonitrile &gt;99% purity</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Methanol &gt;99% purity</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Wash</td>
		</tr>
	</tbody>
</table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

Watch this Scientific Journal Video about Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique at JoVE.com

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

formulation-diblock-polymeric-nanoparticles-through-nanoprecipitation

Research

JoVE Journal

Bioengineering

37.2K ビュー.  University of North Carolina School of Medicine. この記事では、ジブロック共重合体を用いたポリマーをベースとしたナノ粒子を合成するナノ析出の方法を説明します。私たちは、ジブロックの合成共重合体、ナノ析出技術、及び潜在的なアプリケーションについて説明します。

ビデオ: Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

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 ...

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, ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

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 ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...