This project was funded in part by the National Institutes of Health Project number 5P20GM103499-19 through the Student Initiated Research Project Program. This work was also partially supported by Clemson&#39;s Creative Inquiry Program. We also acknowledge Nicholas L&#39;Amoreaux and Aon Ali who initially worked on creating this protocol, publishing their first paper cited here14.

1. Spherical polymersome formation using a solvent injection method
<ol>
	<li>Dissolution of polyesters in organic solvent 
	NOTE: Only one polyester should be dissolved in its respective organic solvent at a time to form polymersomes.
	<ol>
		<li>Dissolve polyesters PEG-PLA or PEG-b-poly(lactic-co-glycolic acid) (PEG-PLGA) in dimethyl sulfoxide (DMSO) at a concentration of 1.5% weight. Specifically, dissolve 0.015 g of selected polyester in 1 mL of DMSO (15 mg/mL). Full dissolution of the polymer may require periods of up to 15 min of vortexing.</li>
	</ol>
	</li>
	<li>While the polyester is dissolving in organic solvent, set up the solvent injection apparatus according to Figure 1.
	<ol>
		<li>Place a stir plate directly below the vertical syringe pump. Place a 5 mL glass vial with 1 mL of Type II deionized water and a miniature stir bar on the stir plate.</li>
		<li>Adjust the syringe pump&#39;s height to allow for the tip of the needle to be fully immersed in Type II deionized water.</li>
		<li>Set the infusion rate of the syringe pump to 5 &#181;L/min. 
		NOTE: If a small volume syringe pump is used, the adapter with the syringe can be set up on a ring stand. If a large volume syringe pump is used, the pump can be placed vertically on a lab jack to adjust the height.</li>
	</ol>
	</li>
	<li>Perform the solvent injection by drawing the organic solvent and polyester solution (step 1.1.1) into a 27 G needle with a &#189;&#34; needle length.
	<ol>
		<li>Place the needle into the syringe pump and make sure it is entirely secure. Adjust the pusher block to touch the syringe plunger&#39;s end.</li>
		<li>Start the stir plate so that the water is spinning at 100 rpm, and then start the syringe pump.</li>
	</ol>
	</li>
	<li>Once the syringe pump has fully infused the organic solvent and polymer into the stirring water, remove from the stir bar and cap the glass vial.</li>
	<li>Characterize the polymersomes via dynamic light scattering (DLS).
	<ol>
		<li>Take 1 mL of water, now with a small percentage of organic solvent and polymer, and place in a 1 mL cuvette.</li>
		<li>Using the settings from Table 1, perform DLS by placing a 1 mL cuvette into the system and set up the run. Read and collect the polymersome intensity-weighted diameter and polydispersity index (PDI). 
		NOTE: A plastic cuvette works fine in this case, as the amount of organic solvent is very low. However, a glass cuvette will work as well if any concerns exist.</li>
	</ol>
	</li>
	<li>Confirm spherical polymersome formation using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).
	<ol>
		<li>Optimize TEM and SEM protocols based on the equipment available. Representative results were obtained at 120 kV in the TEM and 5.0 kV in the SEM.</li>
		<li>If polymersomes are not visible using EM, apply uranyl acetate as a background stain. 
		&#8203;NOTE: Details on TEM and SEM imaging for the shape modulation of polyester-based polymersomes can be found in reference14. Information on electron microscopy techniques for soft nanoparticles is detailed in reference16.</li>
	</ol>
	</li>
</ol>
2. Dialysis to remove organic solvent
<ol>
	<li>Wash a 300 kDa dialysis membrane according to protocols provided by the manufacturer.</li>
	<li>Add 1 mL of polymersome solution into the reservoir of the dialysis device.</li>
	<li>Place the dialysis device in a 250 mL beaker with 150 mL of Type II deionized water on a stir plate. Set the stir plate to a speed that allows for gentle movement of the dialysis device and leave to stir overnight. 
	NOTE: If a vortex is formed during dialysis, the speed needs to be decreased.</li>
	<li>After the dialysis is completed, extract the 1 mL polymersome solution from the dialysis device. Characterize the polymersome solution, following step 1.5. 
	NOTE: Collection of this information is relevant to determine the success of the shape modulation protocol, as one should be able to identify an increase in PDI if the polymersome has been elongated.</li>
</ol>
3. Dialysis against salt gradients
<ol>
	<li>Create 150 mL of desired salt buffer, with either 50 mM, 100 mM, or 200 mM concentration of sodium chloride based on the final desired polymersome properties. In general, increased salt concentration leads to increased polymersome elongation.</li>
	<li>Take the polymersome solution that was dialyzed and characterized and re-place into the dialysis device. Place the loaded dialysis device into 150 mL of the desired salt solution and allow to gently stir for 18 h. 
	&#8203;NOTE: Shape-modulated polymersomes can be stored and maintain their shape in an isotonic solution for periods of up to 7 days.</li>
</ol>
4. Shape modulated polymersome characterization
<ol>
	<li>After the shape modulation, perform polymersome characterization via DLS, TEM, and SEM. If polymersomes are not visible using EM, apply uranyl acetate as a background stain.</li>
	<li>Perform DLS measurements as described in step 1.5, paying particular attention to PDI measurements compared to spherical polymersomes, as a change in PDI suggests effective shape change in polymersomes.</li>
	<li>Ensure the use of appropriate controls for imaging, especially non-shape modulated polymersomes, to ensure the method&#39;s success.</li>
</ol>

<ol>
	<li>Varma, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+self-assembled+nanoparticles+for+drug+delivery.">Recent advances in self-assembled nanoparticles for drug delivery.</a> Current Drug Delivery. 17 (4), 279-291 (2020).</li><li>Salatin, S., Maleki Dizaj, S., Yari Khosroushahi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+surface+modification,+size,+and+shape+on+cellular+uptake+of+nanoparticles.">Effect of the surface modification, size, and shape on cellular uptake of nanoparticles.</a> Cell Biology International. 39 (8), 881-890 (2015).</li><li>Baio, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+activation+of+pH-sensitive+cell+penetrating+peptides+attached+to+gold+surfaces.">Reversible activation of pH-sensitive cell penetrating peptides attached to gold surfaces.</a> Chemical Communications. 51 (2), 273-275 (2015).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoporous+silica+nanoparticles+for+drug+and+gene+delivery.">Mesoporous silica nanoparticles for drug and gene delivery.</a> Acta Pharmaceutica Sinica B. 8 (2), 165-177 (2018).</li><li>Champion, J. A., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+target+geometry+in+phagocytosis.">Role of target geometry in phagocytosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (13), 4930-4934 (2006).</li><li>Banerjee, A., Qi, J., Gogoi, R., Wong, J., Mitragotri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nanoparticle+size,+shape+and+surface+chemistry+in+oral+drug+delivery.">Role of nanoparticle size, shape and surface chemistry in oral drug delivery.</a> Journal of Controlled Release. 238, 176-185 (2016).</li><li>Kolhar, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+shape+effects+to+target+antibody-coated+nanoparticles+to+lung+and+brain+endothelium.">Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium.</a> Proceedings of the National Academy of Sciences. 110 (26), 10753-10758 (2013).</li><li>Meng, F., Zhong, Z., Feijen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+polymersomes+for+programmed+drug+delivery.">Stimuli-responsive polymersomes for programmed drug delivery.</a> Biomacromolecules. 10 (2), 197-209 (2009).</li><li>Iqbal, S., Blenner, M., Alexander-Bryant, A., Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes+for+therapeutic+delivery+of+protein+and+nucleic+acid+macromolecules:+from+design+to+therapeutic+applications.">Polymersomes for therapeutic delivery of protein and nucleic acid macromolecules: from design to therapeutic applications.</a> Biomacromolecules. 21 (4), 1327-1350 (2020).</li><li>Discher, D. E., Ahmed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersomes.">Polymersomes.</a> Annual review of biomedical engineering. 8, 323-341 (2006).</li><li>Seifert, U., Berndl, K., Lipowsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+transformations+of+vesicles:+Phase+diagram+for+spontaneous-+Curvature+and+bilayer-coupling+models.">Shape transformations of vesicles: Phase diagram for spontaneous- Curvature and bilayer-coupling models.</a> Physical Review A. 44 (2), 1182-1202 (1991).</li><li>Rikken, R. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+polymersomes+into+predictable+morphologies+via+out-of-equilibrium+self-assembly.">Shaping polymersomes into predictable morphologies via out-of-equilibrium self-assembly.</a> Nature Communications. 7, 1-7 (2016).</li><li>Kim, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersome+stomatocytes:+Controlled+shape+transformation+in+polymer+vesicles.">Polymersome stomatocytes: Controlled shape transformation in polymer vesicles.</a> Journal of the American Chemical Society. 132 (36), 12522-12524 (2010).</li><li>L'Amoreaux, N., Ali, A., Iqbal, S., Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+prolate+polymersomes+for+enhanced+co-delivery+of+hydrophilic+and+hydrophobic+drugs.">Persistent prolate polymersomes for enhanced co-delivery of hydrophilic and hydrophobic drugs.</a> Nanotechnology. 31 (17), 175103 (2020).</li><li>Salva, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymersome+shape+transformation+at+the+nanoscale.">Polymersome shape transformation at the nanoscale.</a> ACS Nano. 7 (10), 9298-9311 (2013).</li><li>Skoczen, S. L., Stern, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Nanoparticles+Intended+for+Drug+Delivery.">Characterization of Nanoparticles Intended for Drug Delivery.</a> Methods in Molecular Biology. 1682, (2018).</li><li>Bhattacharjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLS+and+zeta+potential+-+What+they+are+and+what+they+are+not.">DLS and zeta potential - What they are and what they are not.</a> Journal of Controlled Release. 235, 337-351 (2016).</li><li>Men, Y., Li, W., Lebleu, C., Sun, J., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+polymersome+shape+using+the+Hofmeister+effect.">Tailoring polymersome shape using the Hofmeister effect.</a> Biomacromolecules. 21 (1), 89-94 (2020).</li><li>Decuzzi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape+effects+in+the+biodistribution+of+intravascularly+injected+particles.">Size and shape effects in the biodistribution of intravascularly injected particles.</a> Journal of Controlled Release. 141 (3), 320-327 (2010).</li><li>Liu, Y., Tan, J., Thomas, A., Ou-Yang, D., Muzykantov, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shape+of+things+to+come:+Importance+of+design+in+nanotechnology+for+drug+delivery.">The shape of things to come: Importance of design in nanotechnology for drug delivery.</a> Therapeutic Delivery. 3 (2), 181-194 (2012).</li><li>Tao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-specific+polymeric+nanomedicine:+Emerging+opportunities+and+challenges.">Shape-specific polymeric nanomedicine: Emerging opportunities and challenges.</a> Experimental Biology and Medicine. 236 (1), 20-29 (2011).</li><li>L'Amoreaux, N., Ali, A., Iqbal, S., Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+prolate+polymersomes+for+enhanced+co-delivery+of+hydrophilic+and+hydrophobic+drugs.">Persistent prolate polymersomes for enhanced co-delivery of hydrophilic and hydrophobic drugs.</a> BioRxiv. , 796201 (2019).</li><li>Chandler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfaces+and+the+driving+force+of+hydrophobic+assembly.">Interfaces and the driving force of hydrophobic assembly.</a> Nature. 437 (7059), 640-647 (2005).</li><li>Wong, C. K., Mason, A. F., Stenzel, M. H., Thordarson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+non-spherical+polymersomes+driven+by+hydrophobic+directional+aromatic+perylene+interactions.">Formation of non-spherical polymersomes driven by hydrophobic directional aromatic perylene interactions.</a> Nature Communications. 8 (1), (2017).</li><li>Abdelmohsen, L. K. E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+characterization+of+polymersome+morphologies+via+light+scattering+techniques.">Shape characterization of polymersome morphologies via light scattering techniques.</a> Polymer. 107, 445-449 (2016).</li><li>Men, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonequilibrium+Reshaping+of+polymersomes+via+polymer+addition.">Nonequilibrium Reshaping of polymersomes via polymer addition.</a> ACS Nano. 13 (11), 12767-12773 (2019).</li><li>Meeuwissen, S. A., Kim, K. T., Chen, Y., Pochan, D. J., van Hest, J. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+shape+transformation+of+polymersome+stomatocytes.">Controlled shape transformation of polymersome stomatocytes.</a> Angewandte Chemie. 123 (31), 7208-7211 (2011).</li><li>Wong, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Faceted+polymersomes:+A+sphere-to-polyhedron+shape+transformation.">Faceted polymersomes: A sphere-to-polyhedron shape transformation.</a> Chemical Science. 10 (9), 2725-2731 (2019).</li><li>Chidanguro, T., Ghimire, E., Simon, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-transformation+of+polymersomes+from+glassy+and+crosslinkable+ABA+triblock+copolymers.">Shape-transformation of polymersomes from glassy and crosslinkable ABA triblock copolymers.</a> Journal of Materials Chemistry B. 8 (38), 8914-8924 (2020).</li><li>Van Oers, M. C. M., Rutjes, F. P. J. T., Van Hest, J. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+polymersomes:+A+cross-linker-induced+shape+transformation.">Tubular polymersomes: A cross-linker-induced shape transformation.</a> Journal of the American Chemical Society. 135 (44), 16308-16311 (2013).</li><li>Che, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+dependent+shape-transformation+of+azide-decorated+polymersomes.">Pathway dependent shape-transformation of azide-decorated polymersomes.</a> Chemical Communications. 56 (14), 2127-2130 (2020).</li><li>Haryadi, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspherical+nanoparticle+shape+stability+is+affected+by+complex+manufacturing+aspects:+Its+implications+for+drug+delivery+and+targeting.">Nonspherical nanoparticle shape stability is affected by complex manufacturing aspects: Its implications for drug delivery and targeting.</a> Advanced Healthcare Materials. 8 (18), 11-13 (2019).</li><li>Katterman, C., Pierce, C., Larsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+nanoparticle+shape+modulation+and+polymersome+technology+in+drug+delivery.">Combining nanoparticle shape modulation and polymersome technology in drug delivery.</a> ACS Applied Bio Materials. , (2021).</li></ol>

The authors have nothing to disclose.

Self-assembled systems are notoriously uncontrollable. Their final properties, including size, shape, and structure, are driven by the chosen amphiphile&#39;s hydrophobic properties and the solvent environment selected. Amphiphilic block copolymers tend towards spherical shapes, which minimizes Gibb&#39;s free energy and leads to the thermodynamic equilibrium23, thus forming polymersomes. Because of their equilibrium nature, polymersomes are significantly more challenging to elongate or alter in shape and, therefore, less studied than solid nanoparticle counterparts. The solvent-driven method has been extensively studied in tandem with PEG-b-polystyrene (PS) based polymersomes12,13,24,25,26,27. However, due to the need to re-optimize protocols for a solvent-based approach for each block copolymer used, this method has not been widely adopted to change the shapes of polymersomes made from alternative materials. Other methods used to modulate polymersome shape are highly specific to the polymersome chemistry; some introduce perylene groups24,28 or other crosslinkers29,30,31 to initiate the desired collapse and elongation. The specificity of the approach to the polymeric material limits its widespread applicability. This method uses salt as the driver for creating an osmotic pressure gradient between the internal, vesicle-encapsulated water and the external saltwater. This can be more universally applied than the alternative solvent-driven method, as various polymers require various solvents to ensure success in the shape modulation. Salt-based shape modulation of polymersomes has worked with PEG-b-PS18, poly(dimethyl siloxane)-g-poly(ethylene oxide)15, and now, with a recent publication, polyester-based polymersomes14. The chemistry-independent success of this method is promising towards the development of more targeted drug delivery systems. Notably, the applicability of this approach to the use of polyesters could have widespread biologic benefits, as these materials are biodegradable.
The first dialysis step is crucial. The solvent injection method of polymersome formation utilizes water-miscible solvents that can become trapped in the hydrophobic membrane of polymersomes during formation, making their membranes more permeable to things like salt. The initial dialysis of formed polymersomes in water ensures that the solvent has been removed from the polymer backbone, demonstrated by a decrease in polymersome diameter (Figure 3). This makes the polymersome less permeable to salt molecules introduced in the second dialysis step, which forms the pressure gradient across the polymersome membrane.
When creating shape-modulated polymersomes, it is expected that each sample will possess polymersomes that are of various shapes and sizes. The benefits of membrane-bound drug delivery, including but not limited to payload protection, extended circulation time, and increased cellular uptake, outweigh the drawbacks of polydispersity. This fact necessitates that images are taken in tandem with DLS data each time polymersomes are prepared. It has been noted that an increase in PDI denotes a shift from spherical to non-spherical particles32, but a direct correlation between &#916;PDI and specific shape has not yet been identified. This is especially important with lower salt concentrations, where there is a large batch to batch variability in &#916;PDI (Figure 4).
This method has only been applied to modulating the shape of the two polyester block copolymers that form polymersomes presented in this paper (PEG-PLA and PEG-PLGA). Other types of PEG-based polymersome systems have been altered for the shape via other salt gradients or solvent gradients, which are reviewed elsewhere33, though there has not been much study into the shape modulation of non-PEG based polymersome systems. Polymersomes are the equilibrium state of PEG-PLA and PEG-PLGA in this paper due to their molecular weights, which provide hydrophilic fractions that lead to membrane bound vesicles10. Thus, following the method directly with an alternative polymer may yield different results. In general, this method can be used to control the shapes of notoriously uncontrollable, self-assembled polymersomes formed from polyester block copolymers.

Shape modulation is a relatively new and efficient way to improve nanoparticle-mediated drug delivery. Not only does the change in morphology increase the surface area of particles, which in turn allows for a greater carrying capacity, but it also has implications across the board to improve stability, circulation time, bioavailability, molecular targeting, and controlled release1. Polymersomes, the nanoparticle of focus in this method, tend to thermodynamically self-assemble into a spherical shape, which has proven to be impractical in cellular uptake and is more easily detected in the immune system as a foreign body. Being able to elongate the structure into a prolate or a rod will allow the drug carrier to evade macrophages by mimicking native cells and more successfully deliver to their desired target2,3,4,5,6,7. The significant benefits of polymersomes, including membrane-bound protection of payloads, stimuli-responsiveness of the membrane, and dual encapsulation of hydrophilic and hydrophobic drugs8,9,10, that make them strong candidates for drug delivery are maintained during shape modulation.
There are many different methods in modulating polymersomes&#39; shapes, and each comes with its respective advantages and disadvantages. However, most of these methods fall into two categories: solvent-driven and salt-driven osmotic pressure change11. Both approaches aim to overcome the bending energy present after polymersomes are formed in a spherical equilibrium shape. By introducing an osmotic pressure gradient, polymersomes can be forced to bend into elongated structures despite strong bending energies11,12.
The solvent-based method explores shape change inspired by the work of Kim and van Hest13. They plasticized polymersomes in an organic solvent and water mixture to trap the organic solvents in the vesicle membrane and drive water out of the vesicle core. Eventually, the particle&#39;s internal volume is so low that it elongates. While this method has shown promise, it lacks practicality. This method requires different solvents for each individual polymeric backbone involved in the modulation. Therefore, it is not widely applicable to promote shape change. Conversely, the salt-based method is uniform and utilizes one universal driver that can introduce osmotic pressure to many block copolymer-based polymersomes.
This project utilizes the salt-based method introduced by L&#39;Amoreaux et al14. This protocol involves two rounds of dialysis. One aims at purifying and solidifying poly(ethylene glycol)-b-poly(lactic acid) (PEG-PLA) polymersomes by removing organic solvent that may have gotten trapped in the bilayer during production, and one that promotes the shape change. The second dialysis step introduces a 50 mM NaCl solution that creates an osmotic pressure gradient to drive the shape change. This method is supported by Salva et al., who note that hypertonic stress in a solution will cause the vesicle to shrink15. This method builds on a previously published method14 looking at two different polyester-based polymersomes and various salt gradients from 50-200 mM NaCl. Polyesters are used due to their biocompatibility and biodegradation. The salt gradient has varying effects on the shape depending on the hydrophobicity of the block copolymer backbone. It can be used to create prolates, rods, and stomatocytes. This salt-driven method was chosen because of the ease of replication and experimental versatility.

<table><tbody><tr><td>15*45 vials screw thread w/cap attached</td><td>Fisherbrand</td><td>9609104000</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Fisher Chemical</td><td>D128-1</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>BDH</td><td>BDH1115-1LP</td><td></td></tr><tr><td>Isoremp stirrers, hotplates, and stirring hotplates</td><td>Fisher scientific</td><td>CIC00008110V19</td><td></td></tr><tr><td>LEGATO 130 SYRINGE PUMP</td><td>kd Scientific</td><td>788130</td><td></td></tr><tr><td>PEG(1000)-b-PLA(5000), Diblock Polymer</td><td>Polysciences Inc</td><td>24381-1</td><td>note the molecular weights when replicating</td></tr><tr><td>Poly(ethylene glycol) (2000) Methyl ether-block-poly(lactide-co-glycolide) (4500)</td><td>Sigma aldrich</td><td>764825-1G</td><td>note the molecular weights when replicating</td></tr><tr><td>Single-Use Syringe/BD PrecisionGlide Needle combination, sterile, BD medical</td><td>BD medical</td><td>BD305620</td><td>Tuberculin</td></tr><tr><td>Sodium Chloride</td><td>BDH</td><td>BDH9286</td><td></td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td></td><td></td></tr></tbody></table>

modulating shape of polyester based polymersomes using osmotic pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Table 2 presents expected results when following the protocol step 1. Note that DMSO is used as a solvent for both PEG-PLA and PEG-PLGA in polymersomes formation. Deviation from this solvent is possible, as other water-miscible solvents will dissolve the copolymers but is expected to change results. It is expected that PDI will be less than 0.2, indicating the formation of monodisperse polymersomes17. Note that increasing hydrophobicity leads to increased deviation in both polymersome diameter and PDI. If upon running the protocol, polymersome diameters vary dramatically from those reported in this table; the most typical culprit is a low concentration of nanoparticles, shown by a low count rate for that sample.
Figure 2&#160;demonstrates non-shape modulated polymersomes&#39; appearance before the addition of salt gradients. Presented here are representative results from PEG-PLA based polymersomes following protocol step 1. Regardless of the block copolymer used, the TEM should indicate an overall spherical structure, with a thicker exterior line, indicative of a membrane. Without NaCl, PEG-PLA polymersomes present as spherical structures in SEM with a brush-like exterior layer of PEG observed through the rough surface presentation.
Figure 3&#160;shows expected changes in polymersomes post-dialysis (step 2). Regardless of the concentration of organic solvent used in polymersome formation, one-hour dialysis in water to remove the solvent will lead to the same overall average diameter, with solvent removal decreasing the polymersome diameter. When larger initial concentrations of organic solvent are used, larger diameter decreases are expected.
Figure 4&#160;provides representative DLS results post-shape change (step 4). Figure 4A shows that when making PEG-PLA polymersomes, modest changes in PDI are expected, which could indicate a change in shape but require imaging to confirm what specific shapes are forming with the polymersomes. Dialyzing PEG-PLA polymersomes against 50 mM NaCl can lead to the formation of prolates with aspect ratios around 2, although this is not a consistent result, demonstrated by large deviation in PDI14. Larger concentrations of salt can lead to the formation of more stomatocyte-like shapes, which is consistent with the current literature18. When dialyzing PEG-PLGA polymersomes, which are slightly more hydrophobic than PEG-PLA polymersomes, against salt, the increase in PDI is more consistent with elongation, with all explored salt gradients leading to an increase in PDI. Having a change in PDI (&#916;PDI) above one is encouraging towards the formation of elongated polymersomes. Again, imaging should be used to confirm which shapes are being created. Figure 4B shows that similar results should be observed when using a 50 mM salt gradient to cause a shape change, regardless of polyester hydrophobicity, while 100 mM and 200 mM NaCl salt gradients display the direct trend that &#916;PDI increases with increasing polyester hydrophobicity (i.e., PEG-PLGA should have a higher &#916;PDI after salt dialysis than PEG-PLA).
Figure 5&#160;provides some examples of polymersome shapes expected when running the protocol. Presented are representative TEM images of shape-modulated PEG-PLA polymersomes after dialysis in 0, 50, 100, or 200 mM NaCl. Recall that self-assembled polymersomes are less controllable than solid particles. Therefore, it is expected to see deviations in sizes and shapes in each sample, which are not observed when modulating the shape of more solid nanoparticles5,7,19,20,21. Knowledge from both Figure 4 and Figure 5 demonstrates this for PEG-PLA polymersomes dialyzed with 50 mM of NaCl; this sample presents with stomatocytes and elongated rods. As salt is increased to 100 mM, an increased number of rods formation with a decreased number of stomatocytes is observed. Finally, with dialysis against 200 mM NaCl, PEG-PLA polymersomes more consistently form prolates with modest aspect ratios (between 2 and 3). Running this protocol will lead to a distribution of nanoparticle shapes, as is the nature of self-assembled nanomedicine.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Setting</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Material Refractive Index</td>
			<td>Set to your material; 1.450 for polymer</td>
		</tr>
		<tr>
			<td>Dispersant</td>
			<td>[NaCl] used; complex solvent set up in DLS software</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>25 &#176;C</td>
		</tr>
		<tr>
			<td>Equilibration Time</td>
			<td>120 s</td>
		</tr>
		<tr>
			<td>Measurement angle</td>
			<td>173 &#176; Back Scatter</td>
		</tr>
		<tr>
			<td>Measurement duration</td>
			<td>Automatic</td>
		</tr>
		<tr>
			<td>Data Processing</td>
			<td>General purpose</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters to use when measuring the size and polydispersity index for polymersomes via dynamic light scattering before and after shape modulation.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Polyester Block Co-Polymer</td>
			<td colspan="3">Diameter</td>
			<td colspan="3">PDI</td>
		</tr>
		<tr>
			<td colspan="3">d, nm</td>
			<td colspan="3">-</td>
		</tr>
		<tr>
			<td>PEG-b-PLA</td>
			<td>202.5</td>
			<td>&#177;</td>
			<td>12.0</td>
			<td>0.06</td>
			<td>&#177;</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>PEG-b-PLGA</td>
			<td>139.6</td>
			<td>&#177;</td>
			<td>25.9</td>
			<td>0.16</td>
			<td>&#177;</td>
			<td>0.06</td>
		</tr>
		<tr>
			<td>PEG-b-PCL</td>
			<td>320.9</td>
			<td>&#177;</td>
			<td>98.8</td>
			<td>0.14</td>
			<td>&#177;</td>
			<td>0.06</td>
		</tr>
	</tbody>
</table>
Table 2: Average polymersome diameter and polydispersity index after solvent injection. This data is typical for PEG-PLA and PEG-PLGA polymersomes after solvent injection, following step 1.5. in the protocol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62548/62548fig01.jpg" /> 
Figure 1: Solvent injection apparatus. Created with BioRender. <a href="https://www.jove.com/files/ftp_upload/62548/62548fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62548/62548fig02.jpg" /> 
Figure 2: TEM and SEM images of spherical PEG-PLA polymersomes pre-salt dialysis.&#160;For TEM, Spherical particles were dried from a 0 mM NaCl suspension and stained with uranyl acetate. TEM Images were taken at 120 kV/60,000x direct magnification. SEM images were taken at 5.0 kV.&#160;Images are adapted from previously published results14. <a href="https://www.jove.com/files/ftp_upload/62548/62548fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62548/62548fig03.jpg" /> 
Figure 3: Spherical PEG-PLA based polymersome Z-Average diameter during dialysis. Dialysis removes organic solvent, which solidifies polymersome membranes and decreases polymersome diameter, as demonstrated for PEG-PLA based polymersomes. This figure is adapted from a pre-print22 and published in Nanotechnology14. <a href="https://www.jove.com/files/ftp_upload/62548/62548fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62548/62548fig04.jpg" /> 
Figure 4: Expected PDI change (&#916;PDI) for each polyester-based polymersomes after the addition of salt gradients. (A) &#916;PDI versus Concentration of NaCl for each Polyester-Based Polymersome. (B) &#916;PDI versus Polyester for each Concentration of NaCl. <a href="https://www.jove.com/files/ftp_upload/62548/62548fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62548/62548fig05.jpg" /> 
Figure 5: Sample TEM images of shape modulated PEG-PLA polymersomes after being dialyzed against 0, 50, 100, and 200 mM NaCl. In order to aid in the visualization of shapes formed through the salt dialysis, a key is provided at the top of the figure, denoting potential shapes of spheres (purple), rods (green), prolates (orange), and stomatocytes (red). As is usual for self-assembled systems, a variety of shapes and sizes are formed. Before the salt dialysis, spheres are consistently observed. The use of 50 mM and 100 mM sodium chloride gradients leads to a wide variety of shapes, including stomatocytes (50 and 100 mM), rods (50 mM), spheres (100 mM) and prolates (100 mM) denoted by arrows colored according to the key provided. Finally, the use of a 200 mM sodium chloride gradient leads to the formation of mainly prolate shapes, with some stomatocytes. <a href="https://www.jove.com/files/ftp_upload/62548/62548fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure at JoVE.com

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...

modulating-shape-polyester-based-polymersomes-using-osmotic

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.1K Views.  Clemson University. Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Video: Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Chemistry

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 ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...