Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Summary

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.

Abstract

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.

Introduction

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 target^2,3,4,5,6,7. The significant benefits of polymersomes, including membrane-bound protection of payloads, stimuli-responsiveness of the membrane, and dual encapsulation of hydrophilic and hydrophobic drugs^8,9,10, that make them strong candidates for drug delivery are maintained during shape modulation.

There are many different methods in modulating polymersomes' 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 energies^11,12.

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'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'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.

Protocol

1. Spherical polymersome formation using a solvent injection method

1. Dissolution of polyesters in organic solvent
   NOTE: Only one polyester should be dissolved in its respective organic solvent at a time to form polymersomes.
   1. 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.
2. While the polyester is dissolving in organic solvent, set up the solvent injection apparatus according to Figure 1.
   1. 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.
   2. Adjust the syringe pump's height to allow for the tip of the needle to be fully immersed in Type II deionized water.
   3. Set the infusion rate of the syringe pump to 5 µL/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.
3. Perform the solvent injection by drawing the organic solvent and polyester solution (step 1.1.1) into a 27 G needle with a ½" needle length.
   1. Place the needle into the syringe pump and make sure it is entirely secure. Adjust the pusher block to touch the syringe plunger's end.
   2. Start the stir plate so that the water is spinning at 100 rpm, and then start the syringe pump.
4. 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.
5. Characterize the polymersomes via dynamic light scattering (DLS).
   1. Take 1 mL of water, now with a small percentage of organic solvent and polymer, and place in a 1 mL cuvette.
   2. 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.
6. Confirm spherical polymersome formation using Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM).
   1. 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.
   2. If polymersomes are not visible using EM, apply uranyl acetate as a background stain.
      ​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.

2. Dialysis to remove organic solvent

1. Wash a 300 kDa dialysis membrane according to protocols provided by the manufacturer.
2. Add 1 mL of polymersome solution into the reservoir of the dialysis device.
3. 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.
4. 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.

3. Dialysis against salt gradients

1. 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.
2. 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.
   ​NOTE: Shape-modulated polymersomes can be stored and maintain their shape in an isotonic solution for periods of up to 7 days.

4. Shape modulated polymersome characterization

1. 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.
2. 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.
3. Ensure the use of appropriate controls for imaging, especially non-shape modulated polymersomes, to ensure the method's success.

Results

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...

Discussion

Self-assembled systems are notoriously uncontrollable. Their final properties, including size, shape, and structure, are driven by the chosen amphiphile's hydrophobic properties and the solvent environment selected. Amphiphilic block copolymers tend towards spherical shapes, which minimizes Gibb'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...

Materials

List of materials used in this article

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