Name: intra-lymph node injection of biodegradable polymer particles
Uploaded: 2014-01-02T20:30:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Intra-lymph Node Injection of Biodegradable Polymer Particles at JoVE.com

This work was funded in part by the PhRMA foundation and a Research and Scholar Award from the University of Maryland, College Park. We thank Prof. Darrell Irvine for support of the initial work conducted in the completion of "In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles."5

All animal studies in this protocol were completed in compliance with Federal, State, and local guidelines, and using protocols reviewed and approved by the University of Maryland&#39;s Institutional Animal Care and Use Committee (IACUC).
1. Synthesis of Lipid-stabilized Micro- and Nanoparticles
<ol>
	<li>In a 7 ml&#160;glass vial(s), combine DOPC, DSPE-PEG, and DOTAP lipids at a 60:20:20 molar ratio to prepare a master lipid mix.
	<ol>
		<li>To synthesize a single sample: Transfer 242.9 &#956;l, 287.4 &#956;l, and 71.9 &#956;l, of DOPC, DSPE-PEG, and DOTAP respectively, into the vial using 2 ml&#160;glass serological pipettes.</li>
		<li>To synthesize multiple samples: Multiply each lipid volume above by the number of samples and combine into a single vial, then transfer equal aliquots of this lipid mixture into vials corresponding to each sample to be prepared.</li>
		<li>Dry lipids under a gentle stream of nitrogen gas for 10 min, or place in vacuum oven overnight.</li>
	</ol>
	</li>
	<li>In a single, empty 20 ml&#160;glass vial, dissolve 80 mg of PLGA in 5 ml&#160;of dichloromethane for each particle sample to generate a 16 mg/ml&#160;polymer stock solution.</li>
	<li>Add 5 ml&#160;of polymer solution to the vial(s) containing the dried lipids, cap, and vortex for 30 sec.</li>
	<li>To synthesize microparticles:
	<ol>
		<li>Begin sonicating the organic phase containing the polymer, lipid, and other water-insoluble cargo on ice at 12 W using a sonicator.</li>
		<li>Create the water-in-oil (w/o) emulsion by using a pipette to add 500 &#181;l&#160;of distilled H2O, or H2O containing 1 mg of peptide, protein, or other water soluble cargo.</li>
		<li>Continue sonicating for 30 sec&#160;at 12 W on ice, gently rocking the vial up and down and side to side around the sonicator tip to ensure complete emulsification.</li>
		<li>Create the water-in-oil-in-water (w/o/w) emulsion by pouring the w/o emulsion into 40 ml of H&#173;2O in a 150 ml&#160;beaker.</li>
		<li>Homogenize for 3 min&#160;at 16,000 rpm using a digital homogenizer.</li>
		<li>Add a magnetic stir bar, transfer the beaker to a stir plate, and allow the w/o/w emulsion to stir overnight to remove the excess solvent.</li>
	</ol>
	</li>
	<li>To synthesize nanoparticles:
	<ol>
		<li>Begin sonicating the organic phase containing the polymer, lipid, and other water insoluble cargo on ice at 14 W.</li>
		<li>Create the w/o emulsion by using a pipette to add 500 &#181;l&#160;of distilled H2O, or H2O containing 1 mg of peptide, protein, or other water soluble cargo.</li>
		<li>Continue sonicating for 30 sec&#160;at 14 W on ice.</li>
		<li>Create the w/o/w emulsion by pouring the w/o emulsion to 40 ml&#160;of H&#173;2O in a 150 ml&#160;beaker and sonicating for 5 min&#160;at 16 W on ice. Gently rock the vial up and down and side to side around the sonicator tip to ensure complete emulsification.</li>
		<li>Add a magnetic stir bar, transfer the flask to a stir plate and allow the w/o/w emulsion to stir overnight to remove the excess solvent.</li>
	</ol>
	</li>
	<li>The next morning, wash and collect particles:
	<ol>
		<li>Pour emulsion through 40 &#181;m Nylon mesh cell strainer into a 50 ml&#160;conical tube.</li>
		<li>Centrifuge particles for 5 min&#160;at 5,000 x g for microparticles or 5 min&#160;at 24,000 x g for nanoparticles.</li>
		<li>Decant supernatant and wash particles by resuspending in 1 ml&#160;of H&#173;2O.</li>
		<li>Transfer suspended particles to a 1.5 ml&#160;microcentrifuge tube.</li>
		<li>Centrifuge for 5 min&#160;at 5,000 x g for microparticles or 5 min&#160;at 24,500 x g for nanoparticles.</li>
		<li>Wash particles twice more by removing supernatant, resuspending in 1 ml&#160;H2O, and centrifuging as in step 1.6.5. After washing, suspend particles in 1 ml&#160;H2O for immediate use, or lyophilize for extended storage.</li>
	</ol>
	</li>
</ol>
2. Measurement of Synthesis Yield
<ol>
	<li>Preweigh an empty 20 ml&#160;glass vial. Add 100 &#181;l&#160;of particle suspension to the preweighed vial after pipetting up and down with a micropipette to mix.</li>
	<li>Lyophilize the particles or dry under a gentle stream of nitrogen.</li>
	<li>Weigh the vial containing the dried polymer. Determine the particle yield in the vial by subtracting the original weight of the vial from the mass of the vial containing the dried particles.</li>
	<li>Determine the overall particle yield by multiplying the particle mass in the vial by the dilution factor. To determine percent yield, divide the particle mass by the maximum theoretical input mass and multiply by 100%.</li>
</ol>
3. Determination of Particle Size
<ol>
	<li>Clean the supplied cuvette-style glass fraction cell by filling with deionized water and wiping with cotton-tipped swab. Transfer 10 ml&#160;of distilled H2O to the cleaned faction cell, add magnetic micro stir bar, and load the fraction cell into the cell mount of the particle size analyzer.</li>
	<li>Adjust the magnetic stirring speed in the particle analyzer to achieve complete mixing in the fraction cell and close the compartment door.</li>
	<li>Align the lasers to the fraction cell using the instrument software interface.</li>
	<li>Use the instrument software interface to record a baseline reading with the fraction cell containing only distilled H&#173;2O.</li>
	<li>Pipette the original particle suspension up and down with a micropipette to mix.</li>
	<li>Pipette 10 &#181;l&#160;of particle suspension (typically approximately 0.5 mg) into the fraction cell. Ensure the volume of particle sample added to the cell is sufficient to generate signal strength in the appropriate range as indicated on the instrument software interface. The actual mass of particles required is dependent on percent yield and the optical properties of the particle sample.</li>
	<li>Close the particle size analyzer compartment door and measure particle size using a refractive index of 1.60 for PLGA.</li>
	<li>Use the software interface to calculate particle diameter using a number basis.</li>
</ol>
4. Visualization of Particles
<ol>
	<li>Pipette particle suspension up and down with micropipette to mix. Dilute particle suspension to 1 mg/ml&#160;in deionized water.</li>
	<li>Prepare a microscope slide by adding 3 &#181;l&#160;of diluted particle suspension and mounting a coverslip at a 45&#176; angle to avoid bubble formation. Place the slide on microscope stage and image using the appropriate filter sets for each fluorescent cargo.</li>
</ol>
5. Preparation of Mice for i.LN. Injection
<ol>
	<li>Prepare tracer dye solution:
	<ol>
		<li>Prepare a 0.1% (w/v) solution of tracer dye by dissolving 10 mg of dye powder with 10 ml&#160;of distilled H2O.</li>
		<li>Sterilize the dye solution into a glass vial using a 0.2 &#181;m syringe filter.</li>
	</ol>
	</li>
	<li>One day prior to injection, anesthetize mouse using isoflurane according to an IACUC approved animal protocol. To evaluate depth of anesthesia, perform a toe pinch reflex test and monitor breathing rate to ensure a respiratory rate of approximately 100-140 breaths per minute.</li>
	<li>Shave hair at base of tail and hindquarter using clippers while the mouse is anesthetized. Remove&#160;hair from the ventral side of the animal and laterally around to the dorsal side just above the joint of the hind leg (hip).</li>
	<li>Inject tracer dye.
	<ol>
		<li>For each dye injection, use a micropipette to transfer 10 &#956;l&#160;of dye solution into a microcentrifuge tube, and aspirate the entire 10 &#956;l&#160;into a 31G&#160;needle attached to a 1 ml&#160;syringe.</li>
		<li>Inject 10 &#956;l&#160;of dye solution subcutaneously on each side of the tail base where the hair was clipped, reloading in between injections.</li>
	</ol>
	</li>
	<li>Remove remaining hair by applying a mild depilatory cream via cotton swabs. Be sure to coat the area in between the hind thigh and abdomen.</li>
	<li>Allow depilatory cream to incubate on skin for 3 min. After incubation, wet gloved hand with warm H2O and gently rub depilatory cream into skin.</li>
	<li>Immediately remove depilatory cream by wetting gloved hand with warm H2O and rubbing tail base and hindquarter. Repeat until excess depilatory is removed, making sure to keep hand wet to avoid irritation.</li>
	<li>Remove residual depilatory from mouse by wetting a soft cloth or paper towel with warm H2O and in a single motion, wiping lower portion of mouse. Avoid a rubbing motion to prevent abrasion or skin damage to the mouse.</li>
	<li>Allow mouse to recover under a heat lamp and return to holding.</li>
</ol>
6. i.LN. Injection of Particles
<ol>
	<li>On the following day, anesthetize mouse using isoflurane according to an IACUC approved animal protocol.</li>
	<li>Examine the mouse to confirm drainage of tracer dye into each inguinal lymph node. The lymph node should be visible as a dark spot near the hind thigh and abdomen.</li>
	<li>Prepare particle injection solution:
	<ol>
		<li>Resuspend particles in distilled H2O at desired injection concentration. For each injection, use a micropipette to transfer 10 &#181;l&#160;of particle solution into a microcentrifuge tube.</li>
		<li>Aspirate the entire 10 &#181;l&#160;into a 31G insulin needle attached to a 1 ml&#160;syringe.</li>
	</ol>
	</li>
	<li>Inject particle dose:
	<ol>
		<li>After visualizing LN, tighten skin around LN using thumb, index finger, and middle finger to pull skin taunt and allow for controlled placement of the injection volume.</li>
		<li>Approach the LN with the needle at a 90&#176; angle to the skin and penetrate the skin over the dyed LN to a depth of 1 mm.</li>
		<li>Slowly inject the entire volume. During injection, observe the LN volume through the skin to confirm injection by visible LN enlargement.</li>
	</ol>
	</li>
	<li>Allow the mouse to recover under a heat lamp and return to holding or conduct additional testing.</li>
</ol>
For relevant analysis techniques (e.g.&#160;histology, flow cytometry) see JoVE Articles 265, 1743, and 3054 and Current Protocols in Immunology, chapters 5 and 2115-22.

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Production costs and access fees for this articles were partially sponsored by HORIBA, Ltd.

The technique described in this protocol allows controlled delivery of vaccines to LNs and to LN-resident antigen presenting cells. Biomaterial encapsulated cargo can be localized within the LN, enabling manipulation of the doses of one or more types of cargo delivered to the LN microenvironment. The localization and controlled release from polymer particles has been shown to generate a potent cellular and humoral immune response at significantly lower doses than conventional approaches. Further, through the manipulation of biomaterial carrier size, the primary mode of cellular processing can be modulated between direct uptake of nanoparticles or extracellular cargo release from larger microparticles5.&#160;These results establish the feasibility of i.LN. biomaterial delivery as a platform for therapeutic vaccine delivery.
The synthesis of PLGA particles by emulsion/solvent evaporation has been widely employed in drug delivery applications23,24. Thus potential challenges associated with this technique relate mostly to successful identification and deposition of vaccines in the LN target site. Although the use of tracer dye facilitates the visualization of the targeted inguinal LNs, the target size and depth beneath the skin are small. Thus, the authors recommend allotting time and mice for practicing the preparation and injections of mice. During animal preparation (i.e.&#160;shaving and application of depilatory), care should be taken not to cut the mice on the ventral side of the animal where the angle of the leg with the abdomen makes the skin more prone to injury from clippers. Additionally, all depilatory should be removed with warm water to prevent animals from ingesting the cream during normal grooming behavior. To practice LN injections, a higher tracer dye concentration can be administered and practice animals can be euthanized, and then injected multiple times. Following injection mice can be necropsied and the size of LNs from injected animals can be compared with an uninjected control LN. One limitation of this technique is the physical limit of the injection volume that can be loaded into the LN structure. Our protocol suggests an injection volume of 10 &#181;l&#160;in mice, though other studies have reported larger injection volumes at least as high as 20 &#181;l.13 However, direct delivery of vaccines via i.LN. injection permits dramatic dose-sparing so the function of these vaccines should generally not be limited by volume constraints.
As noted, changing the physical property of the particles (i.e.&#160;size) is an effective mechanism to alter the pathway or outcomes induced by biomaterials and encapsulated cargos in LN tissue. The emulsion/solvent evaporation protocol can easily be modified to alter physical or chemical properties such as surface charge or functionality, and the rate of biodegradation/cargo release23,24. For example, the release kinetics can be tuned through alternative polymer compositions, and surface function can be altered using modified lipid compositions or poly(vinyl alcohol). The cargo loaded in particles can be easily manipulated to contain different antigens or adjuvants for target pathogens. The advantage of this approach is achieved through the combination of&#160;i.LN. delivery with local, controlled release of cargo from biomaterials. This synergy establishes a platform that can be exploited to efficiently generate adaptive immune responses using minute doses and with reduced nonspecific/systemic side effects.

The lymph nodes (LNs) are the command centers of the immune system. At this immunological site, antigen presenting cells prime na&#239;ve lymphocytes against specific foreign antigens to activate cellular and humoral immune responses. LNs have thus become an attractive target for delivery of vaccines and immunotherapies. Unfortunately, most vaccine strategies result in inefficient, transient delivery of antigen and adjuvants to the lymphoid tissue1.&#160;Approaches that improve the targeting and retention of vaccine components in LNs could therefore have a significant impact on the potency and efficiency of new vaccines.
One strategy for circumventing the challenge of LN targeting that has demonstrated great interest in new clinical trials is direct, intra-LN (i.LN.) injection2-4.&#160;These trials employed ultrasound guidance to deliver vaccines to LNs as a simple outpatient procedure. Compared to traditional peripheral injection routes, this approach resulted in significant dose-sparing and improved efficacy in therapeutic contexts including allergies and cancer2-4.&#160;These studies employed i.LN. injection of soluble vaccines (i.e.&#160;biomaterial-free) which were rapidly cleared by lymphatic drainage. Therefore, multiple injections- or cycles of multiple injections- were administered to achieve these impressive therapeutic effects. Improved retention in the LN could enhance the interaction between antigen and/or adjuvant and immune cells, further improving the potency of immune cell priming. This potential is supported by recent studies that show kinetics of antigen and adjuvant delivery play a critical role in determining the specific immune response generated5-7.&#160;Further, localizing and minimizing drug and vaccine doses could reduce or eliminate systemic effects, such as chronic inflammation.
Biomaterials have been studied extensively to enhance the potency and efficiency of vaccines1,8,9. Encapsulation in or adsorption on biomaterial carriers can physically shield cargo from degradation and overcome solubility limitations. Another notable feature of biomaterial carriers, such as polymeric micro- or nanoparticles, is the ability to coload several classes of cargo and, subsequently, release these cargos over controlled intervals. However, a significant limitation that continues to hinder biomaterial vaccines and immunotherapies in vivo&#160;is inefficient targeting of immune cells and limited trafficking to lymph nodes. For example, peripheral injection of biomaterial vaccines through conventional routes (e.g.&#160;intradermal, intramuscular) typically exhibit poor LN targeting, with up to 99% of the injected material remaining at the site of injection4,10. More recently, the size of biomaterial vaccine carriers has been tuned to improve preferential trafficking or drainage of these vaccines to LNs through interstitial flow8,10.&#160;These advances have led to enhanced cellular and humoral immune responses, underscoring the importance of targeting and engineering the LN environment for new vaccines.
This paper presents a vaccination protocol that combines lipid-stabilized polymer particles and i.LN. delivery to generate controlled release vaccine depots5,11.&#160;Building on recent studies employing surgical techniques for i.LN. in mice6,7,12,13,&#160;we developed a quick, nonsurgical strategy for injecting biomaterial vaccines in small animals5. Combining i.LN. delivery with biomaterial vaccine carriers potently enhanced CD8 T cell response within 7 days after a single injection of controlled release vaccine depots5.&#160;A strong humoral response (i.e.&#160;antibody titers) was also generated; both enhancements were linked to increased retention of vaccine components in lymph nodes that was mediated by controlled release from the biomaterial carriers. Interestingly, the size of vaccine particles altered the fate of these materials once in the LNs: nanoscale particles showed heightened direct uptake by cells, while larger microparticles remained in the extracellular LN environment and released cargo (e.g.&#160;adjuvant) that was taken up by LN-resident antigen presenting cells5.&#160;These data suggest two pathways that could be exploited for new vaccines by controlling the size of biomaterials injected i.LN.
In this article biodegradable lipid-stabilized polymer particles (micro- and nanoscale) are synthesized using a modified double emulsion strategy5,11.&#160;Particle properties are characterized by laser diffraction and microscopy. These particles are then injected directly into the inguinal LNs identified nonsurgically using a common, nontoxic tracer dye14. Post-injection analysis of LNs by histology or flow cytometry can be used to verify the distribution of particles within the LN environment, as well as to monitor cellular uptake and retention of particles over time. For protocols detailing histological processing and flow cytometry, readers are referred to recent JoVE articles and journal reports15-22. Typical results demonstrate local LN targeting of these depots that could be exploited to achieve potent, efficient immune responses or to tailor immunity for target pathogens.

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		<col />
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	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">1,2-dioleoyl-sn-glycero-3-phosphocholine1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850375</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] ammonium salt (DSPE-PEG)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">880128</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polylactic-co-glycolic acid (PLGA)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2191</td>
			<td style="width:167px;">Lactide:Glycolide (50:50). MW 30,000-60,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dichloromethane (DCM)</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH1113</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:71px;">Vetone</td>
			<td style="width:119px;">502017</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nair</td>
			<td style="width:71px;">Nair</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Evans blue tracer dye</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">AAA16774-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">U-100 BD Ultra-Fine Short Insulin Syringes, 31 G 5/16 in needle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD328418</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Single-Use Needles, BD Medical, 21 G, 1.5 in needle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD305167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringes with BD Luer-Lok Tip, BD Medical, 1 ml</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD309628</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon Cell Strainers, Sterile, Corning, 40 &#181;m</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-949</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vybrant DiD Cell-Labeling Solution</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">V-22887</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluoresbrite YG Microspheres 6.00 &#181;m</td>
			<td style="width:71px;">Polysciences</td>
			<td style="width:119px;">17149</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluoresbrite YG Microspheres 0.05 &#181;m</td>
			<td style="width:71px;">Polysciences</td>
			<td style="width:119px;">17156</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovalbumin, Purified</td>
			<td style="width:71px;">Worthington Biochemical</td>
			<td style="width:119px;">LS003056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qsonica Sonicator Ultrasonic Processor Q125</td>
			<td style="width:71px;">Qsonica</td>
			<td style="width:119px;">Q125</td>
			<td style="width:167px;">1/8 in diameter microtip probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Turrax T 25 digital homogenizer</td>
			<td style="width:71px;">IKA</td>
			<td style="width:119px;">YO-04739-22</td>
			<td style="width:167px;">10 G dispersing element</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent Microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-83</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laser Diffraction Particle Size Distribution Analyzer</td>
			<td style="width:71px;">Horiba</td>
			<td style="width:119px;">LA-950</td>
			<td style="width:167px;">Including provided cuvette-style glass fraction cell</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Professional 8685 Peanut Classic Clippers</td>
			<td style="width:71px;">Wahl</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

intra-lymph node injection of biodegradable polymer particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Expected results for the protocols presented in this manuscript can be divided into three categories: particle synthesis, animal preparation, and particle injection.
Figure&#160;1 depicts the synthesis and characterization of biodegradable polymer particles, stabilized by amphiphilic lipids. Results of the emulsion/solvent evaporation synthesis protocol (Figure&#160;1A) can be qualitatively assessed by visual inspection of the final emulsions generated; particle batches should be homogenous, stable emulsions with an opaque appearance. Complications include emulsions that cream or flocculate, often due to improper storage of lipid stabilizers. To avoid this instability, lipids should be stored at -80 &#176;C in a dehydrated state or in a sealed vial purged with nitrogen. Quantitative assessment of particle synthesis can be performed using laser diffraction or dynamic light scattering to analyze size distribution (Figure&#160;1B). Expected results include tightly-distributed, monomodal particle sizes, indicating a uniform population of particles. The synthesis parameters described in this manuscript generate number averaged distributions centered at approximately 100 nm or 3 &#181;m for nanoparticles and microparticles, respectively. Further qualitative assessment of particle synthesis can be achieved through modification of the above protocol to incorporate multiple classes of fluorescent cargo. In Figure&#160;1C, microscopy images of microparticles loaded with a fluorescent peptide (FITC, green), a lipophilic dye (DiD, red), and an overlay image (yellow) confirm creation of particles within the desired size range and encapsulation of peptide within the volume of the particle.
The first two panels of Figure&#160;2 summarize the expected results of animal preparation for the i.LN. injection strategy described in this paper. The methodology involves marking inguinal LNs by peripheral injection of a nontoxic tracer to identify the location for subsequent i.LN. injection of particles (Figure&#160;2A)5.&#160;As noted, drainage of the tracer dye following subcutaneous injection at the tail base will enable visualization of the inguinal LNs (Figure&#160;2B)5.&#160;Ingestion&#160;of approved depilatory creams can pose hazards to the mice. Thus, care should be taken to thoroughly remove all cream applied, paying particular attention to paws, and the ventral side of the mice. Depilatory should be removed using a wet, soft cloth or wet paper towel in a single, smooth motion. Avoid rubbing to remove cream, as this can lead to abrasions on the exposed skin of the mice.
Confirmation of local of delivery to the inguinal LN can be evaluated through observation or histology. The LN volume can be monitored visually during injection as an indicator of successful injection. Expected results include efficient cargo distribution throughout the LN structure, without significant leakage to adjacent tissues or cells. Further, as injected fluid displaces/dilutes the tracer in the LN, dye concentration/coloring should become less intense after injection. Observation of the tissue should reveal an intact, but enlarged LN due to fluid injection. Potential challenges include injecting too rapidly or missing the LN, both of which can cause elution of the volume into surrounding subcutaneous tissue. These undesirable outcomes can be confirmed by necropsy or histology, where the particle suspension will be observed spreading to cells and tissue remote from nodes targeted for injection. In contrast, an expected result would be the identification of an enlarged inguinal LN due to containment of particles within the LN structure. Histological processing of excised LNs can definitively confirm delivery of cargo to the lymphoid tissue, as shown in Figures&#160;2C and 2D. Note that the particles in Figure&#160;2 incorporate fluorescent cargo to allow for visualization of cargo during injection, as well as during histological processing and fluorescent microscopy.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50984/50984fig1.jpg" width="600px" /> 
Figure 1. Synthesis and Characterization of Lipid Stabilized Particles. A) Schematic diagram describing the synthesis of lipid-stabilized particles prepared by emulsion/solvent evaporation. B) Size distributions of microparticles (solid line, diameter = 2.8 &#956;m) and nanoparticles (dashed line, diameter = 113 nm). C) Fluorescent microscopy images of particles loaded with fluorescently-labeled peptide and a fluorescent particle dye. Labels: peptide (green) and particle (red). <a href="https://www.jove.com/files/ftp_upload/50984/50984fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50984/50984fig2.jpg" width="600px" /> 
Figure 2. i.LN. Injection and Distribution of Biodegradable Particles within LN. A) Methodology for i.LN. injection. B) Visualization of LNs in a mouse through skin (upper image) and following necropsy (lower image)5.&#160;C) Histological staining of a LN confirming deposition and distribution of fluorescently-labeled polymer microparticles (particles, green; T-cells, red; B-cells, blue). D) Fluorescently-labeled nanoparticles (50 nm, left image) and microparticles (6 &#181;m, right image) in LNs 24 hr&#160;after injection. <a href="https://www.jove.com/files/ftp_upload/50984/50984fig2highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Intra-lymph Node Injection of Biodegradable Polymer Particles at JoVE.com

Intra-lymph Node Injection of Biodegradable Polymer Particles

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

intra-lymph-node-injection-of-biodegradable-polymer-particles

Research

JoVE Journal

Bioengineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

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, ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule,&#160;Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Sustained Administration of &amp;#946;-cell Mitogens to Intact Mouse Islets &lt;em&gt;Ex Vivo&lt;/em&gt; Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.