The authors have no acknowledgements.

1. Nanoparticle Preparation
All steps involving solvent or dissolved/emulsified polymer should be performed in a chemical fume hood.
<ol>
	<li>Weigh 100 mg (+/- 5 mg) of poly(lactic-co-glycolic acid) (PLGA) and place in a 13 mm x 100 mm test tube.</li>
	<li>Transfer 1 ml solvent (ethyl acetate (EtAc) or dichloromethane (DCM)) to the sample with a glass serological pipette.</li>
	<li>Cover the top of the tube with a small piece of aluminum foil, and Parafilm the foil securely, being sure to wrap the Parafilm tightly across the top edge of the vial as well as the bottom edge of the foil.</li>
	<li>Mark the level of the solvent on the outside of the test tube. Let the polymer dissolve overnight, adding additional solvent the next day if evaporation occurs. 
	Note: Polymer can also be dissolved the day of nanoparticle preparation, if needed. Follow steps 1.1-1.3, then vortex on high until all polymer is completely dissolved (~10 min).</li>
	<li>Prepare a work area in the fume hood with the following equipment/materials next to the ultrasonicator: a vortexer, two small glass Pasteur pipettes with rubber bulbs, and a magnetic stir plate. Place a large beaker full of ice water on a stand below the ultrasonicator.</li>
	<li>Add 45 ml&#160;of 0.3% w/v Vitamin E-TPGS to a 200 ml glass beaker with a magnetic stir bar and set stirring speed to 360 rpm.</li>
	<li>Add 2 ml&#160;of 0.3% w/v Vitamin E-TPGS to a 13 mm x 100 mm glass test tube.</li>
	<li>Add drug: 
	For hydrophobic agents: add encapsulant directly to the polymer solution (being careful to avoid the walls of the test tube) and vortex the tube until encapsulant is homogenously dispersed. 
	For hydrophilic agents: emulsify encapsulant with the polymer solution before proceeding. Add up to 50 &#956;l&#160;of drug in water or buffer to the surface of the polymer solution. Ultrasonicate briefly to emulsify the drug with polymer (typically 10 sec, or as necessary to achieve a homogenous, opaque solution). This sonication step should be performed on ice.</li>
	<li>Place the open tube polymer/encapsulant near the vortexer. While holding the test tube containing Vitamin E-TPGS vertically and on high vortex, use a glass Pasteur pipette to add the polymer/encapsulant solution dropwise. Be careful to avoid the sides of the tube and drop the polymer solution directly onto the surface of the vortexing emulsifier. After the entire 1 ml&#160;of polymer solution has been added to the Vitamin E-TPGS, continue vortexing the solution (now an emulsion) for an additional 15 sec.</li>
	<li>Immediately transfer the emulsified polymer to the ultrasonicator. Keep the test tube immersed in ice water and sonicate the emulsion in three 10&#160;sec bursts (40% amplitude for a 700 W sonicator, 1/8 in&#160;probe tip size). Move the emulsion up and down the probe to ensure even sonication, being careful to avoid touching the probe to the sides or bottom of the test tube. Pause between each ten second sonication to allow the solution to cool before proceeding.</li>
	<li>Add 1-2 ml&#160;of 0.3% Vitamin E-TPGS from the stirring solution to the emulsion with a glass Pasteur pipette, which thins the emulsified polymer so that it will pour more easily. Empty the emulsion into the stirring solution. Repeat this step to wash out any remaining solution from the test tube into the stirring solution.</li>
	<li>Allow nanoparticles to harden while stirring for three hours. If the encapsulant is light-sensitive, the beaker may be wrapped in aluminum foil, leaving the top open to facilitate solvent evaporation.</li>
</ol>
2. Nanoparticle Collection
<ol>
	<li>Split the hardened nanoparticles into two Oak Ridge centrifuge tubes (30 ml&#160;nominal volume) and balance to within 0.1 g.</li>
	<li>Centrifuge nanoparticles in a fixed-angle rotor for 15 min at 17,000 x&#160;g. Longer centrifugation times will result in the collection of a higher fraction of smaller nanoparticles.</li>
	<li>Discard the supernatant, being careful not to disturb the nanoparticle pellet (water may be poured or pipetted off). Add 15 ml&#160;diH2O and use a water bath sonicator and/or vortexer to completely resuspend the nanoparticles.</li>
	<li>Combine the contents of the two centrifuge tubes into one and repeat steps 2.1-2.3 2x more, for a total of three washes in 30 ml&#160;of diH2O each time. The fluid volume of the last pellet resuspension should be 4-5 ml. 
	A weight ratio of 1:2 trehalose:polymer may be added at this point as a cryoprotectant (freeze a small aliquot of nanoparticles without trehalose for SEM imaging). Ice crystals that form during the freezing process may damage the particle surface and induce aggregation, and inclusion of trehalose has been shown to improve the uniform and complete resuspension of PLGA nanoparticles13.</li>
	<li>Transfer the nanoparticles to a preweighed 5 ml&#160;centrifuge tube and freeze at -80 &#176;C for a minimum of 30 min.</li>
	<li>Moving quickly so as not to let the frozen contents melt, uncap the tube and cover the top by securing lab tissue across the top with a rubber band. **If any melting occurs, refreeze before placing in the lyophilizer.</li>
	<li>Lyophilize 72 hr for a 5 ml&#160;volume. Store lyophilized particles in Parafilm-wrapped tube at -80 &#176;C.</li>
</ol>
3. Sizing and Surface Morphology
Scanning Electron Microscopy
<ol>
	<li>Samples for SEM should be prepared the day of imaging. Place a strip of double-sided carbon tape on an SEM stub. Permanent marker may be used to label the metal portion of the stub for later reference.</li>
	<li>Use a metal spatula to collect a small quantity of lyophilized nanoparticles and gently spread them across the surface of the tape. Brush the surface of the stub with a lab tissue or use compressed air to remove loose nanoparticles.</li>
	<li>Sputter coat the sample with gold-paladium for 30-120 sec. A longer sputter time will produce a smoother surface for imaging, although this may result in the loss of fine surface details.</li>
	<li>Typical parameters for visualizing particles are a working distance of 5-15 mm, beam strength of 5-12 kV, and a spot size of 1-3. Higher beam strengths may result in regional heating of the sample, which will alter the surface morphology of the particles. Microparticles are observed at 100X magnification and nanoparticles will be distinguishable at 3,000X magnification.</li>
	<li>The ideal sample will consist of a uniform coating of particles across the flat surface of the stub. Occasionally, secondary structures (including flakes or spheres) may result from lyophilization. Focusing on the edge of a flake, where the flake meets tape, or the top of a sphere will help to locate particles that can be easily visualized with SEM. Collect at least three images per batch in order to obtain a representative sample of particle size and morphology.</li>
</ol>

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PLGA particles are commonly prepared by single- or double-emulsion, a method that provides the ability to customize particle characteristics such as size, encapsulant, and surface properties7. These properties and others depend on many variables, including solvent type, feed ratios, emulsification method, and emulsifier type. However, in our experience, it is possible for a single experimenter to produce particles with consistent properties. Using the same protocol, a well-trained experimenter produced seven separate batches of nanoparticles with the following mean diameters: 317 nm&#177;99, 342 nm&#177;112, 298 nm&#177;104, 361 nm&#177;110, 339 nm&#177;115, 360 nm&#177;123, and 364 nm&#177;110. Averaging the well-trained experimenter&#39;s batches yields a mean diameter of 340 nm&#177;25 and mean within-batch standard deviation of 110 nm&#177;8. A second experimenter, trained by the first, used the same protocol to produce nanoparticles with an average diameter of 328 nm&#177;138. However, a third experimenter, who had been making nanoparticles independently for several months, followed the same protocol to produce nanoparticles with an average diameter of 220 nm&#177;70. Thus, it is possible to make particles with an expected range of diameters, but this depends on exact replication of subtle experimental technique. Pooling batches may enable production of large quantities of nanoparticles with identical aggregate properties. For example, when we surface modify nanoparticles for in vitro or in vivo study, multiple (up to 6) 200 mg batches are prepared, washed, collected, mixed into a single tube, and then divided for surface modification. This ensures that nanoparticle size, drug loading, charge, and fraction of surface sites available for modification will be comparable between two treatment groups. In another example, if the variable of interest is drug efficacy with different agents being loaded into different batches of nanoparticles, it is important to measure and control for variability in drug loading. It is critical that particle properties be measured for each batch of particles produced, and experiments to be designed and replicated to control for batch-to-batch variability.
Complete emulsion of the organic and aqueous phases is critical for forming small particles. Poly(vinyl alcohol) (PVA) is perhaps the most commonly used emulsifier. We and others have used a variety of emulsifying and stabilizing agents, including vitamin E-TPGS9, PVA14, spans15, and poloxamers16 (for thorough discussion of the wide range of agents used for production of PLGA particles, see review by Wischke et al.17). Properly emulsified polymer will appear as a homogenous, milky-white/opaque solution. A poor (or &#34;broken&#34;) emulsion will show macroscopic heterogeneity or granularity, and it may even separate into two visually distinct layers in the tube. This generally indicates a failure to form nanoparticles. The emulsion may break following step 1.9 if it is not immediately transferred to the ultrasonicator. If this occurs, vortex again until homogenous and immediately proceed to the sonicator. Separation should not occur following the sonication step (1.10). Once the particles have been dispersed into a larger aqueous volume (step 1.11), the solution should be uniformly opaque to slightly translucent. The translucent quality will increase as particle size decreases, and a blue hue may be observed for very small nanoparticles. This hue might not be observed if particles encapsulate a drug. If the emulsion fails to disperse uniformly into the large aqueous volume or appears granular, the experiment should not proceed. It is normal to observe some variation in the appearance of the particle pellet, as well as its cohesion. Some pellets may resuspend upon mild vortexing, while others may take several minutes of water bath sonication. Resuspension should not take longer than 5 min. Lyophilized particles may take a variety of textures from cotton-like to free-flowing.
One of the many advantages of PLGA as a nanoparticle material is the ability to optimize the fabrication process to create the desired size of particle for the intended application. Larger nanoparticles will encapsulate a higher fraction of hydrophobic drug (relative to weight of polymer) than small nanoparticles, however, drug release kinetics may also be affected18-20. The size of nanoparticles has a direct effect on mechanisms of particle internalization by cells, as well as their distribution in tissue, which can result in dramatic differences in delivery effectiveness . For example, the mononuclear phagocyte system will tag and remove agents in systemic circulation that are larger than 1 &#956;m. Small nanoparticles may be of interest for direct infusion into the brain, where larger nanoparticles would be trapped in the tight extracellular matrix; delivery of small nanoparticles might also facilitate passive targeting of tumors via the enhanced permeation and retention effect. Drug encapsulation efficiency varies widely, depending on the properties of the specific drug, the size of the particle, and the emulsifier (i.e.&#160;its solubility in water versus solvent). Single versus double emulsion will also affect drug loading and nanoparticle size. Encapsulation of hydrophobic agents via single emulsion may aid in the production of ultra-small nanoparticles, compared to the double emulsion method. We direct the reader to other resources for extensive discussion of this topic23-28. Lastly, alternative emulsification methods are available, including high-speed homogenization, and these other approaches may provide advantages for specific types of drugs or drug delivery applications29.
The single emulsion method presents an opportunity for variation of a wide range of formulation variables, each of which are capable of altering nanoparticle properties. For example, using dichloromethane (DCM) as a solvent instead will generally produce larger nanoparticles with a broader size distribution. Since ethyl acetate (EtAc) is miscible in water, the surface tension of the polymer droplet in the primary emulsion is decreased, producing smaller nanoparticles. DCM may be substituted for the same volume of EtAc. Alternative solvents or blends of solvents may be used to optimize drug encapsulation and particle properties. Other formulation parameters are also capable of altering particle size: for example, increasing solvent volume, solvent:polymer ratio, sonication intensity, or speed/duration of centrifugation will all result in a decrease in the size of collected particles. It is possible to size fractionate particles by performing sequential centrifugation (e.g.&#160;a quick, low speed centrifugation to first remove larger particles, followed by a longer spin to collect ultra-small particles) or filtering .
Here, we describe formulation parameters that enable preparation of PLGA particles with average diameters ranging from 220 nm to 1.98 &#956;m. These methods for producing particles composed of PLGA may be used to optimize particle design (size, encapsulant, and surface properties) for future drug delivery application in vitro or in vivo.

Polymers have become increasingly popular drug delivery vehicles in the last several decades for applications ranging from targeted tumor therapy1 to modulation of the immune system2. Polymer-encapsulated or&#160;conjugated drugs are frequently more effective than their freely delivered counterparts, since polymer-associated drug is protected from degradation. This protection translates to a longer biological half-life and potentially improved efficacy with reduced systemic side effects3-5.
Poly(lactic-co-glycolic acid) (PLGA) exhibits many of the ideal properties of a nanoscale delivery system, providing long term release of the encapsulated agent and degrading into the biocompatible products of lactic and glycolic acid . Small molecules, proteins, and nucleic acids that are encapsulated in PLGA have demonstrated enhanced activity in a variety of disease applications8. Importantly, the material platform facilitates easy customization of features such as size, charge, and surface display of ligands for targeting particles to specific tissues or for imaging purposes. Because PLGA is already used in humans in the form of biodegradable sutures (e.g. Purasorb, Purac Biomaterials, and Vicryl, Ethicon Inc.), the potential for clinical translation is high.
Oil-water (single) or water-oil-water (double) emulsion is one method by which PLGA can be used to encapsulate hydrophobic and hydrophilic drugs in micro- or nano-scale form. Briefly, PLGA is dissolved into an organic phase (oil) that is emulsified with a surfactant or stabilizer (water). Hydrophobic drugs are added directly to the oil phase, whereas hydrophilic drugs (water) may be first emulsified with the polymer solution prior to formation of particles. High intensity sonication bursts facilitate the formation of small polymer droplets. The resulting emulsion is added to a larger aqueous phase and stirred for several hours, which allows the solvent to evaporate. Hardened nanoparticles are collected and washed by centrifugation (Figure 1).
Here, we describe a technique for making PLGA nanoparticles using single and double emulsion, with ethyl acetate (EtAc) as the solvent and vitamin E- D-&#945;-Tocopherol polyethylene glycol succinate (TPGS) as the emulsifying agent. The use of vitamin E-TPGS offers several possible advantages over other types of stabilizing agents (e.g.&#160;poly(vinyl alcohol), PVA), including improved emulsification and encapsulation efficiency. Other groups have reported advantages of Vitamin E-TPGS, including the inhibition of P-gp, a transmembrane efflux protein, which is commonly overexpressed on cancer cells and known to contribute to drug resistance by shuttling drugs out of target cells. Additionally, we demonstrate how to characterize and size the particles using scanning electron microscopy (SEM) and provide further guidance on size modification via alteration of the protocol.

<table border="0" cellpadding="0" cellspacing="0" width="714">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(lactic co-glycolic acid)&#160; (PLGA)</td>
			<td style="width:129px;">Lactel</td>
			<td style="width:119px;">B6010-2P</td>
			<td style="width:251px;">Inherent Viscosity: 0.55-0.75 dl/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethyl acetate (EtAc)</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">270989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride (DCM)</td>
			<td style="width:129px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vitamin E TPGS</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">57668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly(vinyl alcohol)</td>
			<td style="width:129px;"></td>
			<td style="width:119px;"></td>
			<td>87-89% hydrolized, 30,000-70,000 Da</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:129px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonic Dismembrator</td>
			<td style="width:129px;">Fisher Scientific</td>
			<td style="width:119px;">Model 705</td>
			<td>700 W (Ultrasonicator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator tip</td>
			<td style="width:129px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>1/8 in</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortexer</td>
			<td style="width:129px;">VWR</td>
			<td style="width:119px;">58816-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiposition stirrer</td>
			<td style="width:129px;">Corning</td>
			<td style="width:119px;">MP5I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultracentrifuge</td>
			<td style="width:129px;">Beckman-Coulter</td>
			<td style="width:119px;">L8-80M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fixed angle rotor</td>
			<td style="width:129px;">Beckman-Coulter</td>
			<td style="width:119px;">50.2TI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath sonicator</td>
			<td style="width:129px;">Fisher Scientific</td>
			<td style="width:119px;">FS30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lyophilizer</td>
			<td style="width:129px;">Millrock</td>
			<td style="width:119px;">BT85</td>
			<td></td>
		</tr>
	</tbody>
</table>

plga nanoparticles formed by single- or double-emulsion with vitamin e-tpgs

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

Representative SEM images of PLGA microparticles and nanoparticles prepared using this protocol are shown in Figures 2a and 2b. Particles will appear as individual, nonfused spheres, with a smooth surface morphology and broad range of sizes distributed throughout the sample.
To determine the average diameter of each batch, measurements are taken directly from the SEM images using the &#34;measure&#34; function in ImageJ. A minimum of 150 measurements of the diameter of particles selected randomly from the field of view should be taken to acquire a representative size distribution. The average size of a batch of particles depends on formulation parameters. For example, in Figure 2a, microparticles (average diameter of 730 nm&#177;370) were formed with 1 ml&#160;of solvent to 100 mg of PLGA, using 0.05% Vitamin E-TPGS in the emulsifying phase, whereas in Figure 2b, nanoparticles (average diameter of 340 nm&#177;70) were formed with 4 ml&#160;of solvent to 200 mg of PLGA, encapsulating the hydrophobic drug camptothecin and using 0.3% Vitamin E-TPGS in the emulsifying phase&#160;(full formulation parameters are described in the figure caption). Generally speaking, higher concentration of emulsifier will produce smaller particles, with average diameters ranging between 220-2,000 nm being achievable by varying emulsifier concentration from 0.3-0.01% (Figure 3).
Our yields range from 74-98% (22 batches of nanoparticles with varying solvents, emulsifiers, and emulsification conditions, prepared by a single experimenter). Better yields may be observed for larger (&#62;150 nm) batches, since small nanoparticles are lost easily during the centrifugation and wash step, particularly when the wash supernatant is poured off the pellet. Careful pipetting of the fluid can help to avoid this issue. Higher speeds and/or longer centrifugation can also improve yield both by improving the collection of the ultra-small fraction of nanoparticles and also by compacting to the pellet so that less is lost in the wash step. The protocol described here is focused on producing particles with TPGS as the emulsifying agent and EtAc as the solvent, however, alternative solvents and/or emulsifying agents are possible. In general, we find that EtAc produces smaller and more uniformly sized nanoparticles than DCM (e.g.&#160;diameters of 540 nm&#177;190 versus 2,160 nm&#177;1,530 for EtAc versus DCM, 100 mg/ml PLGA in solvent, emulsified into 2 ml&#160;0.1% TPGS), presumably because EtAc is water-miscible, whereas DCM is not. PVA is a popular emulsifying agent that has been used extensively by us and other groups. We have observed variability in nanoparticles prepared from different batches of commercially available PVA. It is advisable, therefore, to consider utilizing the same stock of PVA when possible to improve reproducibility. In our hands, we find that PVA is a more effective emulsifier at much higher concentrations than TPGS (e.g.&#160;diameter of 140 nm&#177;48, 100 mg/ml PLGA in DCM, emulsified into 2 ml&#160;5% PVA).
Characterizing nanoparticles with SEM can be challenging due to differences in equipment and imaging process. It is therefore important to distinguish morphological features that indicate problems with particle formation versus artifacts that are introduced inadvertently while imaging. Fusing (Figures 4a and 4b), dimpling (Figure 4c), or cracking (Figure 4f) of particles is sometimes observed in the SEM images. These artifacts sometimes indicate poorly formed particles (e.g.&#160;in Figure 4a, the emulsifier concentration was too high to form discrete particles) but also could result from artifacts introduced by the characterization method (e.g.&#160;in Figure 4b, the sample heated during sputter coating, and in Figure 4c, local heating from the SEM beam altered the surface morphology of the particles while imaging). To avoid imaging artifacts, sputter coating time should be minimized and beam power kept low. Smoother morphologies are observed for longer sputter times, however, this can induce fusing of particles. Sputter times that are too short may make imaging difficult, as particles will become charged and move during image capture. Capturing images quickly will help to avoid prolonged beam exposure and distortion of images (Figures 4d and 4e). High beam exposures can also result in expansion and cracking of the nanoparticle surface (Figure 4f). If morphology artifacts cannot be minimized by altering the characterization parameters, it is possible that they represent true issues with the nanoparticles themselves. During the fabrication process, fusing may result from overheating due to high ultrasonication power, incomplete evaporation of solvent, incomplete resuspension during the wash phase, or failed lyophilization.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51015/51015fig1highres.jpg" src="/files/ftp_upload/51015/51015fig1.jpg" /> 
Figure 1. Schematic of nanoparticle fabrication. <a href="https://www.jove.com/files/ftp_upload/51015/51015fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51015/51015fig2highres.jpg" src="/files/ftp_upload/51015/51015fig2.jpg" /> 
Figure 2. SEM images and corresponding size distributions of PLGA microparticles and nanoparticles produced by the single emulsion method. In (A), particles were formed by emulsifying 100 mg PLGA dissolved in 1ml&#160;ethyl acetate into 2 ml&#160;of 0.05% Vitamin E-TPGS, and hardened in 45 ml&#160;0.01% Vitamin E-TPGS. The corresponding size distribution is shown in (C). In (B), particles were formed by emulsifying 200 mg PLGA and 40mg of camptothecin dissolved in 4mL ethyl acetate into 4 ml&#160;0.3% Vitamin E-TPGS, and hardened in 90 ml&#160;0.3% Vitamin E-TPGS. The corresponding size distribution is shown in (D). <a href="https://www.jove.com/files/ftp_upload/51015/51015fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51015/51015fig3highres.jpg" src="/files/ftp_upload/51015/51015fig3.jpg" /> 
Figure 3. Particle size depends on emulsifier concentration. Increasing Vitamin E-TPGS concentration resulted in smaller nanoparticles. Average diameters and corresponding SEM images are shown of particles prepared by emulsifying 100 mg of PLGA dissolved in 1 ml&#160;of ethyl acetate into (A) 0.01%, (B) 0.05%, (C) 0.1%, or (D) 0.3% Vitamin E-TPGS and hardened in 45 ml&#160;0.3% Vitamin E-TPGS. Error bars represent standard deviation of 200 diameter measurements made from 2 images on a single batch. Scale bar represents 5 &#956;m (A-C) and 200 nm (D).<a href="https://www.jove.com/files/ftp_upload/51015/51015fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51015/51015fig4highres.jpg" src="/files/ftp_upload/51015/51015fig4.jpg" /> 
Figure 4. Surface morphology artifacts observed with SEM. In (A), heavy fusing and sheet-like structures were present throughout the entire sample. The concentration of Vitamin E-TPGS was too high (1%) to form discrete particles. In (B), some regions of the sample were fused, however, large pockets of discrete nanoparticles existed, particularly where there were gaps in the sputter coat. We believe these fusing artifacts are due to heating during the sputter coat process. In (C), particles initially appeared smooth, however, sustained exposure of the sample to the SEM beam resulted in dimpling and ruffling. (D) shows a poor resolution image that was captured with minimal exposure of the sample to the SEM beam; particles appear smooth with a round morphology. In (E), the same sample is exposed for a longer period of time, and obvious distortion of their original morphology is observed. In (F), a long beam exposure at a high voltage setting (12 kV) produced heating and cracking of the particles. <a href="https://www.jove.com/files/ftp_upload/51015/51015fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS at JoVE.com

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

plga-nanoparticles-formed-single-or-double-emulsion-with-vitamin-e

Research

JoVE Journal

Bioengineering

64.8K Views.  Barrow Neurological Institute. We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to 1.98 µm.

Video: PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...