Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning
April 16th, 2018
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.
The overall goal of this protocol is to fabricate degradable versions of thermoresponsive hydrogels with controlled length scales and geometries. This method can help address key challenges in the scenario of biomedical materials such as facilitating long term, minimally invasive local drug delivery, creating responsive scaffolds for tissue engineering, or designing targeted nanotherapeutics. The main advantage of our technique is a rapid reverse biohydrazone chemistry which allows us to control the size and shape of the degradable hydrogels by changing the polymer precursor mixing geometry.
This flexibility lets us fabricate folic gels, microgels, nanogels, and nanofibers all with the same starting material. First, prepare about two milliliters each of hydrazide and aldehyde-functionalized thermoresponsive precursor polymer solutions with concentrations between five and 40%by weight and 10 millimolar phosphate-buffered saline. Use single barrel syringes to load the precursor polymer solutions into separate barrels of a 2.5 milliliter, 1:1 double barrel syringe.
Next, connect a 1.5 inch static mixer to a 2.5 milliliter 1:1 double barrel syringe. If desired, attach an 18 gauge or 20 gauge 1.5 inch needle to the static mixer. Then, cut from a soft, silicone rubber sheet of the desired thickness.
A piece about the size of a standard microscope slide. Use a punch set to punch at least one hole of the desired shape into the silicone rubber sheet. Place the silicone rubber mold on a clean glass slide, ensuring that all holes are backed by the glass.
Gently press the mold against the slide to fix it in place. Silicone rubber sheeting may also be used as backing to ensure good substrate adhesion. Co extrude the hydrazide and aldehyde functionalized polymer solutions through the static mixer into the mold wells until the wells are filled completely.
Cover the mold with a second clean, glass slide and wait for gelation to finish. Then, remove the top slide and use a spatula to separate the hydrogels from the silicone rubber mold. Lift the mold from the slide to recover the hydrogels.
Prior to the procedure, fabricate a microfluidic chip for the microparticle generation. Synthesize hydrazide and aldehyde functionalized poly NIPAM. To begin, prepare two milliliters each of 6%by weight solutions of hydrazide and aldehyde functionalized poly NIPAM in deionized water.
Load the polymer precursor solutions into separate five milliliter syringes. Next, prepare 150 milliliters of a 1%by weight solution of sorbitan monooleate in heavy paraffin oil. Load this solution in two standard 60 milliliter syringes.
Mount the four syringes in separate infusion syringe pumps. Next, connect a 45 centimeter length of silicon tubing to the microfluidic chip outlet. Place the end of the outlet tubing in a waste container.
Connect the polymer solution syringes to the polymer inlet channels on the microfluidic chip with 30 centimeters lengths of silicon tubing. Connect one paraffin oil solution syringe to the upstream oil inlet channel with silicon tubing. Connect the other oil syringe to both downstream oil inlet channels using silicon tubing and a U-shaped syringe joint.
Set the flow rates of the pumps equipped with oil syringes to between 1.1 and 5.5 milliliters per hour. Flow oil through the microfluidic chip for at least 30 minutes to prime the chip and to confirm that the chip is defect free. Then, simultaneously deliver both polymer solutions to the primed chip at 03 mL per hour.
Allow the flow of polymer and oil to equilibrate for 30 minutes to an hour. To prevent premature gelation of the precursor polymers at the microfluidic nozzle, it is essential to carefully prime the tubing and start the flow of polymer exactly at the same time to prevent back flow into either polymer reservoir. Once uniform gel microparticles are forming at the nozzle of the microfluidic chip, collect the particles in a plastic centrifuge tube with an active magnetic stir bar.
Stop the syringe pumps and the stir motor when the oil has been consumed. Once the gel microparticles have settle in the tube, decant the paraffin oil with a pipette. Add to the flask 10 milliliters of pentane for every 5 mL of microparticles.
Vigorously mix the emolution for about a minute. Allow the particles to settle for one to two hours before decanting the pentane with a pipette. Repeat this pentane wash at least five times.
Transfer the washed gel microparticles to a small, glass scintillation vial. Resuspend the particles in one to two milliliters of deionized water. To begin this procedure, prepare five milliliters each of 1%solutions of hydrazide and aldehyde-functionalized poly NIPAM in deionized water.
Heat five milliliters of the hydrazide-functionalized poly NIPAM solution to 70 degrees celsius while stirring. Monitor the solution as it becomes opaque and confirm that no visible precipitate forms. Then, add 0.25 milliliters of the aldehyde-functionalized poly NIPAM solution drop-wise to the heated hydrazide-functionalized poly NIPAM solution over the course of five to 10 seconds.
Stir the mixture for 15 minutes at 70 degrees celsius. Then, remove the mixture from the oil bath and allow it to cool to room temperature over night to obtain the nanogels for purification. Place the nano gel mixture in a 3500 kilodalton molecular weight cut off dialysis membrane.
Dialyze the nanogels against dionized water six times over six hour cycles to remove non-cross-linked polymers. If desired, lyophilize the purified nanogels for storage. To begin the gel nanofiber fabrication procedure, prepare one milliliter each of 15%by weight solutions of hydrazide and aldehyde-functionalized POEGMA in deionized water.
Then, prepare two milliliters of a 5%by weight solution of high moleculor weight polyethylene oxide in deionized water. Combine each POEGMA precursor solution with one milliliter of the polyethylene oxide solution. Load the precursor solutions into separate barrels of the double barrel syringe.
Next, connect a 1.5 inch static mixer and a blunt tipped 18 gauge needle to a 2.5 milliliter, 1:1 double barrel syringe. Mount the double barrel syringe on an infusion syringe pump. Then, mount an aluminum disc electrospinning collector 10 centimeters from the end of the needle.
Ground a high-voltage power supply at the collector and connect the power supply to the needle. Set the syringe pump to 0.48 milliliters per hour and the power supply to 10 kilovolts. Simultaneously start the syringe pump and the power supply to initiate electrospinning.
Continue until the desired thickness is reached or the precursors are exhausted. After nanofiber production has finished, turn off and disconnect the electrospinning assembly. Soak the collected gel nanofibers in deionized water for 24 hours to remove the polyethylene oxide.
Double barrel syringe delivery produced hydrazone cross-linked POEGMA bulk hydra-gels with a range of mechanics, gelation times, and transparencies depending on the precursor polymer concentration, the reactive functional group density, and the number of ethylene oxide repeat units on the monomers. The flow rates of both the paraffin oil and the polymer precursor solutions affected the sizes of hydrozone cross-linked poly NIPAM gel microparticles fabricated with the microfluidic device. Reversible temperature dependent swelling was observed with hot stage optical microscopy.
The gel micro particles degraded to their oligomeric precursors over time. The sizes of self-assembled hydrazone cross-linked poly NIPAM nanogels varied with the aldehyde to hydrazide functionalized polymer precursor-mass ratio but remained highly mono dispersed. As the mass ratio increased, less thermal deswelling was observed.
Like the gel microparticles, the nanogels degraded to the oligomeric precursors over time. Hydrazone crosslinked POEGMA hydrogel nanofibers with diameters on the order of 300 nanometers were produced by reactive electrospinning. Nanofiber hydration was about two orders of magnitude faster than hydration of a bulk gel with the same polymer composition.
The nanofibers retained their nanofibrous morphology for over eight weeks before degrading under physiological conditions. Faster degradation occurred in acidic environments. The nanofibers were mechanically robust in both the dry and swollen states.
After watching this video, we hope you have a good understanding of how to create degradable, thermo-responsive hydrogels by controlling the geometry at which the gel precursor polymers are made. Remember to ensure the gelation time of the polymer precursor matches the needs of each particle. Gelation that is too fast will inhibit the microgel and the nanofiber fabrication.
Gelation that is too slow will result in the fully congeeled morphologies. These techniques pave the way for researchers in the areas of drug delivery and tissue engineering. and design well-defined hydrogel scaffolds for controlling the rate or location of drug release or stimulating tissue regeneration.
Overview
This article presents protocols for the fabrication of degradable thermoresponsive hydrogels using hydrazone cross-linking. The methods allow for the creation of hydrogels at bulk, microscale, and nanoscale levels, including gel nanoparticles and nanofibers.
Key Study Components
Area of Science
- Biomedical materials
- Hydrogel fabrication
- Tissue engineering
Background
- Thermoresponsive hydrogels have applications in drug delivery and tissue scaffolding.
- Controlling the size and shape of hydrogels is crucial for their functionality.
- Hydrazone cross-linking provides a versatile method for hydrogel synthesis.
- The study addresses challenges in creating responsive materials for biomedical use.
Purpose of Study
- To develop a method for fabricating degradable thermoresponsive hydrogels.
- To enable controlled geometries and length scales for various applications.
- To facilitate the creation of responsive scaffolds and targeted nanotherapeutics.
Methods Used
- Preparation of hydrazide and aldehyde-functionalized polymer solutions.
- Use of a double barrel syringe and static mixer for co-extrusion.
- Microfluidic chip fabrication for microparticle generation.
- Dialysis for purification of nanogels.
Main Results
- Successful fabrication of folic gels, microgels, nanogels, and nanofibers.
- Demonstrated control over hydrogel size and shape through polymer precursor mixing.
- Establishment of a reliable method for producing uniform gel microparticles.
- Effective removal of non-cross-linked polymers from nanogels.
Conclusions
- The presented protocols enable versatile hydrogel fabrication for biomedical applications.
- Hydrazone cross-linking is a promising approach for creating responsive materials.
- Future applications may include advanced drug delivery systems and tissue engineering scaffolds.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables the fabrication of degradable thermoresponsive hydrogels across multiple length scales, addressing a key translational barrier in smart material development: the lack of biologically relevant degradation in conventional thermoresponsive polymers. By enabling renal clearance through hydrolytically degradable hydrazone cross-links, the method supports the translation of thermoresponsive hydrogels for applications requiring controlled residence time, such as metabolism-regulated drug delivery and transient tissue scaffolds. The approach provides a versatile platform for generating hydrogels with tunable size, shape, and degradation kinetics, facilitating preclinical evaluation of responsive materials under physiologically relevant conditions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by providing degradable, stimuli-responsive scaffolds that mimic dynamic extracellular matrix conditions.
- Operational Value: Supports functional target validation through tunable hydrogel degradation that allows assessment of target engagement over defined time windows.
- Predictive Value: Enhances lead identification by allowing evaluation of drug release kinetics and cellular responses in degradation-competent environments.
Screening & Assay Development
- Assay Readiness: Produces uniform hydrogel microparticles and nanogels suitable for high-throughput screening of compound release and cellular uptake.
- Quantitative Output: Enables measurement of degradation-dependent changes in hydrogel swelling, mesh size, and drug diffusion as functional readouts.
- Reproducibility: Microfluidic and electrospinning methods provide scalable, standardized production of degradable hydrogel particles with controlled size distribution.
Translational & Preclinical Research
- Disease-Relevant System: Generates thermoresponsive hydrogels that degrade to oligomeric precursors below renal filtration threshold, supporting evaluation of clearance and biocompatibility.
- Translational Continuity: Bridges discovery and preclinical work by providing degradable scaffolds that maintain thermoresponsive function while enabling safety assessment via degradation product recovery.
- Mechanistic De-risking: Allows assessment of hydrogel-mediated drug delivery and tissue regeneration under conditions where material clearance mimics physiological elimination pathways.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from early target validation through lead optimization and preclinical development, providing degradable hydrogel platforms that can be adapted for screening, formulation, and mechanistic studies.
- Discovery Biology: Supports hypothesis testing by enabling spatiotemporal control of bioactive presentation via temperature- and degradation-responsive hydrogels.
- Screening: Delivers standardized hydrogel microparticles and nanofibers for consistent compound screening and cellular response analysis.
- Analytics: Provides quantitative readouts on degradation kinetics, swelling behavior, and release profiles that inform structure-activity and structure-property relationships.
- Translational Research: Enables preclinical evaluation of hydrogel-based systems where degradation to clearable oligomers reduces long-term retention risk.
- Enterprise Reuse: Establishes a modular platform applicable to various water-soluble polymers, allowing reuse across multiple therapeutic areas and modality programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by enabling evaluation of smart materials in degradation-competent, physiologically relevant contexts.
- Operational Value: Offers standardized, scalable fabrication across length scales (bulk, micro, nano) using shared precursor chemistry and adaptable equipment.
- Strategic Value: Reduces late-stage failure risk by addressing biocompatibility and clearance concerns early in development.
- Portfolio Impact: Supports risk-adjusted advancement by generating preclinical data on degradation, clearance, and transient functionality.
Implementation Considerations
- Requires expertise in polymer chemistry, hydrogel formation, and stimuli-responsive material characterization.
- Needs access to microfluidic systems, electrospinning equipment, and dialysis or purification infrastructure for nanogel isolation.
- Demands standardization of precursor synthesis and functionalization to ensure reproducible hydrazone cross-linking and degradation profiles.
- Involves adaptation considerations when applying the method to different polymers or adjusting degradation rates via cross-linker stoichiometry.
- Includes practical limitations such as gelation time sensitivity, which must be matched to fabrication method to avoid premature or incomplete network formation.
Biopharma FAQs
Chapters in this video
0:04
Title
0:49
Fabrication of Hydrazone-crosslinked Bulk Poly(Oligoethylene Glycol Methacrylate)(POEGMA) or Poly(N-Isopropylacrylamide) (PNIPAM) Hydrogels
2:41
Fabrication of Hydrazone-crosslinked PNIPAM Gel Microparticles via Reactive Microfluidics
6:18
Thermally-driven Reactive Self-assembly of Hydrazone-crosslinked PNIPAM Nanogels
7:34
Fabrication of Hydrazone- crosslinked POEGMA Nanofibers via Reactive Electrospinning
9:28
Results: Bulk, Microscale, and Nanoscale Thermoresponsive Hydrogels
11:17
Conclusion
