Name: Forma modulada de poliéster baseado em polimeroses usando pressão osmótica (Vídeo) | JoVE Journal
Uploaded: 2021-04-21T15:00:00.000+00:00
Duration: 6 min 1 s
Description: Aprenda a criar polymersomas alongados usando pressão osmótica para melhorar a liberação de fármacos. Este método simples permite encapsular simultaneamente drogas hidrofóbicas e hidrofílicas, com potencial para atravessar a barreira hematoencefálica.

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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>

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<li>Rikken, R. S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shaping+polymersomes+into+predictable+morphologies+via+out-of-equilibrium+self-assembly%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Polymersome+stomatocytes%3A+Controlled+shape+transformation+in+polymer+vesicles%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">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/pubmed?term=((Persistent+prolate+polymersomes+for+enhanced+co-delivery+of+hydrophilic+and+hydrophobic+drugs%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">Persistent prolate polymersomes for enhanced co-delivery of hydrophilic and hydrophobic drugs.</a> Nanotechnology. 31 (17), 175103(2020).</li>
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<li>Men, Y., Li, W., Lebleu, C., Sun, J., Wilson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tailoring+polymersome+shape+using+the+Hofmeister+effect%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((Size+and+shape+effects+in+the+biodistribution+of+intravascularly+injected+particles%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">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/pubmed?term=((The+shape+of+things+to+come%3A+Importance+of+design+in+nanotechnology+for+drug+delivery%5BTitle%5D)+AND+%22Therapeutic+Delivery%22%5BJournal%5D)">The shape of things to come: Importance of design in nanotechnology for drug delivery.</a> Therapeutic Delivery. 3 (2), 181-194 (2012).</li>
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<li>Men, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonequilibrium+Reshaping+of+polymersomes+via+polymer+addition%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Nonequilibrium Reshaping of polymersomes via polymer addition.</a> ACS Nano. 13 (11), 12767-12773 (2019).</li>
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<li>Chidanguro, T., Ghimire, E., Simon, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shape-transformation+of+polymersomes+from+glassy+and+crosslinkable+ABA+triblock+copolymers%5BTitle%5D)+AND+%22Journal+of+Materials+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((Tubular+polymersomes%3A+A+cross-linker-induced+shape+transformation%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">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/pubmed?term=((Pathway+dependent+shape-transformation+of+azide-decorated+polymersomes%5BTitle%5D)+AND+%22Chemical+Communications%22%5BJournal%5D)">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/pubmed?term=((Nonspherical+nanoparticle+shape+stability+is+affected+by+complex+manufacturing+aspects%3A+Its+implications+for+drug+delivery+and+targeting%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">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>
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</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 s...

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

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 polymer...

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 ...

Nanoparticle shape influences cellular uptake, but limited work has been done to change the shape of polymersomes. This protocol demonstrates how to elongate polymersomes for enhanced drug delivery applications. This technique provides a simple approach that can create many different polymersome shapes with potential applications in drug delivery, pseudo cells, or for other unexplored fields.Elongated polymersomes can simultaneously deliver hydrophobic and hydrophilic drugs and more easily in to the cells. This technique could help shuttle drugs across the blood-brain barrier, addressing a major medical challenge. To begin, dissolve 0.015 grams of selected polyester in one milliliter of DMSO by vortexing for 15 minutes, and set up the solvent injection apparatus.Place the start plate directly below the vertical syringe pump, then place a five milliliter glass bile with one milliliter of type two deionized water in a miniature star bar on the stir plate. Adjust the syringe pumps height to allow for the tip of the needle to be fully immersed in type two deionized water and set the infusion rate of the syringe pump to five microliters per minute. Perform the solvent injection by drawi

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

Research

JoVE Journal

Bioengineering

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

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.

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

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.

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

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.

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

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.

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

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.

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

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

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.

Assembly and Characterization of Polyelectrolyte Complex Micelles

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Aprenda a criar polymersomas alongados usando pressão osmótica para melhorar a liberação de fármacos. Este método simples permite encapsular simultaneamente drogas hidrofóbicas e hidrofílicas, com potencial para atravessar a barreira hematoencefálica.

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

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