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

Summary

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.

Abstract

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. 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.

Introduction

By definition, microgels are hydrogels of any shape with an equivalent diameter of approximately 0.1-100 μm¹. Because of their size and characteristics, polymeric microgels present a versatile tool for advancing drug delivery and tissue engineering systems. While bulk hydrogels are extensively utilized as tissue engineering scaffolds and drug delivery vehicles with great success^2-4, a recent shift to microscale control of scaffolds provides a unique opportunity for microgels to be used as base materials for building scaffolds. In addition, microgels have a high surface area to volume ratio for cellular interactions and in solution have a low viscosity that makes them ideal for injections. Finally, microgels can be formed using numerous polymers by a variety of methods dependent on the desired microgel characteristics, making them highly customizable for a variety of applications.

Techniques to produce microgels include suspension, emulsion, dispersion, or precipitation polymerizations. Emulsion and suspension polymerizations typically require organic solvents and surfactants or stabilizers to form the microgels. The nature of these methods yield a highly disperse particle size distribution⁵. Dispersion and precipitation reactions render particles with a lower polydispersity⁶; however particles formed by dispersion polymerization still require the use of stabilizing agents⁶. Microgels formed by precipitation reactions are unique in that they form particles of uniform size and shape without the use of stabilizers or surfactants. Microgel formation is achieved when growing polymer chains phase separate from the continuous phase by enthalpic or entropic precipitation⁷. Precipitation polymerization is often at high temperatures that can be lowered by the use of kosmotrophic salts, which decrease the solubility of the polymer in the solvent⁸. This work focuses on microgels formed from poly(ethylene glycol) (PEG) by a photopolymerized precipitation reaction under biologically-compatible conditions, with variations to alter microgel properties and encapsulate molecules for drug delivery applications.

Previous studies with PEG hydrogels show that the polymerization conditions greatly influence the physical and mechanical properties of hydrogels, namely the hydrogel water content and compressive modulus^3,9. These crosslinked materials are of interest because the relationship between structural and physical properties described by Flory¹⁰ can be utilized to tailor the crosslinked hydrogel for specific applications. These principles are similar for microgels. Precipitation-formed PEG microgels have been found to have potential for regenerative medicine¹¹, however further investigation into the microgel properties was necessary to enhance their repertoire for future biomedical applications. This report describes the procedure to fabricate microgels by precipitation reaction and alter characteristics, such as microparticle diameter, polydispersity index (PDI), density, and swelling, that would be important to further develop these materials for drug delivery or regenerative medicine.

Protocol

1. Preparing Solutions for Use in Microgel Fabrication

1. Before beginning the precipitation reaction, make the necessary solutions and warm them to 37 °C. The required solutions include 0.5% photoinitiator, 1.5 M Na₂SO₄, buffer solution, 200 mg/ml PEG-diacrylate (PEG-DA) solution, and 1x phosphate buffered saline (PBS). Note: Acrylate all PEG precursors according to published methods and store at -20 °C under argon until use¹².
2. Weigh out photoinitiator and dissolve in deionized (DI) H₂O for a 0.5 (w/v)% solution. It is important to protect the solution from exposure to UV light.
   1. After full dissolution, filter through a 0.22 μm filter. This solution can be made in advance and stored at 4 °C in the dark. Note: Decrease the stirring time to approximately 1 hr by heating the solution to 40 °C while stirring.
3. For 1x PBS, add 1 tablet PBS to 1 L DI H₂O and filter through a 0.22 μm filter. PBS can be made in advance and stored at 4 °C.
4. Measure 39.4 ml of phosphate buffered saline (PBS, pH=7.4) and add 612 μl triethanolamine (TEOA), and 260 μl of 6 M hydrochloric acid (HCl). Note: TEOA is very viscous; preheat TEOA to 37 °C prior to pipetting.
   1. Adjust the pH of the buffer so that it is approximately 7.7 and filter through a 0.22 μm filter (final pH ~7.8). The buffer solution can be scaled up, made in advance, and stored at 37 °C.
5. Make the PEG-DA solution prior to beginning the reaction each time; decreased reactivity has been observed if the solution is stored. Weigh out PEG-DA and add PBS/TEOA/HCl buffer to final concentration of 200 mg/ml. Note: Add buffer slowly to carefully control PEG concentration because PEG swells in solution.
   Note: This solution is highly customizable. Options for customizing microgels include: changing the starting molecular weight of PEG to alter microgel properties, using a degradable PEG precursor to fabricate degradable microgels, sterile filtering all of the solutions for biologically sterile microgels, completing the process of formation in a laminar flow hood, and finally adding functional groups to the surface by incorporating them in the PEG solution.
6. Weigh out Na₂SO₄ and dissolve in DI H₂O for a 1.5 M solution. For best results, the sodium sulfate solution must also be made just prior to beginning the reaction.
   1. Heat and vortex the solution until Na₂SO₄ is completely dissolved. Filter through 0.22 μm filter as necessary.

2. Microgel Fabrication

1. Place 127.5 μl of PBS/TEOA/HCl buffer, 10 μl of 0.5% photoinitiator, and 25 μl of 200 mg/ml PEG solution into the tubes where the microgels will be formed; typically 2 ml microcentrifuge tubes.
   1. Heat these tubes along with the 1.5 M Na₂SO₄ solution to 37 °C.
   2. Mix the tubes while heating. Note: This microgel protocol can be scaled up.
2. To encapsulate molecules into the microgels, add the desired molecule (e.g. ovalbumin) to the PBS/TEOA/HCl buffer, photoinitiator, and PEG solution. After crosslinking the molecule will be trapped in the microgel network.
3. Complete each microgel reaction one at a time. Take the first tube and add 87.5 μl of 1.5 M Na₂SO₄.
4. Mix tube by pipetting 3-5x; alternatively wait 30 sec. After mixing, the salt concentration will be evenly distributed and the solution should be clear.
5. Place the tube under the UV light and crosslink for 30 sec. Following crosslinking there should be a cloudy layer on top of the solution, this layer contains the microgels. See Figure 1 for reaction scheme.
6. Add 750 μl PBS to form a pellet upon centrifugation and mix well.
7. Wash the microgels 5 x by centrifuging for 2 min at 4,000 x g to form a pellet and exchanging the supernatant with PBS. Note: Do not disturb the microgel pellet while buffer exchanging, it may not be possible to remove all the supernatant.

3. Microgel Size and Polydispersity

1. Pipette 30 μl of microgels from the pellet formed from centrifuging in 1 ml of DI H₂O. For best results, sonicate the microgel solution in a water bath sonicator for 1 hr (50-60 Hz and 1.4 A) or mix vigorously to suspend the microgels.
2. Pipette 15 μl of the microgel solution onto a clean glass slide and put #1.5 cover slip on top. Flip the slide and cover slip over onto a laboratory tissue and push down on the slide to remove excess solution and ensure that the microgels are in a single layer.
3. Capture several images of the microgels using a DIC 100X oil objective. Typically 5 images are taken per sample and 3 samples per batch are recommended for a total of 15 images. Note: no batch to batch variation has been observed to date.
4. Measure the microgels to obtain a representative diameter.
5. Calculate PDI using the following volume based equation in which N is the total number of microgels and V_i is the volume of microgel i.

4. Microgel Density

1. Prepare dextran 7, 6, 5, 4, 3, and 1 (w/v)% solutions in DI H₂O to determine microgel density. These densities were calculated to be 1.022, 1.020, 1.018, 1.016, 1.014, and 1.010 g/cm³, respectively.
2. Add 1 ml of 7% dextran solution to a 15 ml centrifuge tube.
   1. Slowly pipette 1 ml of 6% dextran solution to form a distinct layer atop the 7% dextran.
   2. Wait 5 min then add 1 ml of the 5% solution.
   3. Continue until all dextran solutions are layered in order of decreasing dextran concentration.
3. Carefully layer the microgels above the 1% dextran solution.
4. Centrifuge the gradient for 10 min at 4,000 x g and 5 °C. Microgel position after centrifugation allowed for simple calculation of density¹³.

5. Microgel Equilibrium Swelling

1. To measure the swollen mass (M_s) of the microgels, defined as the mass after equilibrium swelling (48 hr), first wash the microgels 5x in DI H₂O and swell for a minimum of 48 hr in preweighed microcentrifuge tubes.
2. Centrifuge the microgels for 10 min at 4,000 x g.
3. Remove all supernatant and record the wet mass. Use filtering methods to ensure all excess water is removed to obtain more accurate results.
4. Freeze the microgels at -80 °C then lyophilize to constant mass to obtain the dry mass (M_d).
5. Calculate the water content in the microgels by Water% = (M_s - M_d) / M_d x 100.
6. Determine polymer content by Polymer% = 100 - Water% .
7. Perform statistical analysis on all data using SAS with n-way ANOVA and Tukey post-hoc test. Differences are noted when p<0.05.

Results

Microgel size is dependent on polymerization conditions. Figure 2A illustrates how microgel diameter increases with increasing PEG starting molecular weight and crosslinking time. Representative images of microgels used for sizing for various molecular weights and crosslinked for 30 sec are shown in Figures 2C-G. For PEG with a molecular weight of 3,000 Da, the microgel average diameter increased from 1.65±0.26 to 2.20±0.54 μm as UV exposure increased from 30-600 sec (Figure 2B

Discussion

Physical properties of PEG microgels were examined for changes in polymerization conditions. For this precipitation reaction, a buffer solution, 20% (w/v) PEG-diacrylate (MW 3,000, 4,600, or 6,000 Da) solution, and photoinitiator were mixed and warmed to 37 °C. Addition of 1.5 M Na₂SO₄, a kosmotrophic salt that increases the interactions between water molecules, caused PEG to momentarily precipitate upon its addition. This effect is more prominent with higher molecular weight PEG⁸

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline (PBS)	MP Biomedical	2810305
Triethanolamine (TEOA)	J.T. Baker	9468-01	Preheat to 37 °C prior to pipetting
Hydrochloric acid (HCl)	BDH Aristar	BDH3028
Sodium Sulfate	J.T. Baker	3891-01
Irgacure 2959	Ciba	029891301PS04
Ovalbumin (OVA)	Invitrogen	34782
PEG 1,500	Alfa Aesar	A16241
PEG 3,000	Fluka	03997-1KG
PEG 4,000	Alfa Aesar	A16151
PEG 4,600	Sigma	373001-250G
PEG 6,000	Fluka	03394-1KG
PEG 10,000	Alfa Aesar	B21955
Dextran 70	TCI	D1449