Our protocol facilitates the formulation of polymer-nanoparticle hydrogels for use as biomaterials. We hope researchers will develop this material for translational applications and to explore basic biological questions. PNP hydrogels are easily injected through small diameter needles and catheters, but quickly self-heal after injection.
This allows for the non-invasive controlled delivery of drugs and cells over long timescales. This technology pushes the boundaries for localized therapy and extended drug release, with implications for wide raging conditions from cancer to tissue regeneration to passive immunization. To synthesize nano-particles by nano precipitation, add 50 milligrams of PEG-PLA polymer to an eight milliliter glass scintillation vial and add one milliliter of acetonitrile to the vial.
Vortex to fully dissolve. Next add 10 milliliters of ultra pure water to a 20 milliliter glass scintillation vial with a small stir bar and place the vial on a stir plate set to 600 revolutions per minute. Use a 200 microliter pipette to add one milliliter of the polymer solvent solution dropwise to the vial of water.
Core-shell nanoparticles will form as the polymer solvent solution is rapidly dispersed throughout the water. Verify the particle size by dynamic light scattering according to standard protocols. Then transfer the nano-particle solution into a centrifugal filter unit to concentrate the solution to less than 250 microliters and resuspend the nanoparticles in an appropriate buffer.
To prepare the hydrogel, add 333 milligrams of 6%HPMC-C12 stock solution to a one milliliter Luer lock syringe and add 500 microliters of the 20%nanoparticle stock solution and 167 microliters of PBS to an eight milliliter vial. After mixing, use a needle to fill another one milliliter Luer lock syringe with the diluted nano-particle solution and attach the two syringes to an elbow mixer. Mix the two solutions for approximately 60 cycles until a homogeneous, opaque white hydrogel material has formed.
To measure the rheological properties of the formulated hydrogel, inject the appropriate volume of hydrogel according to the selected geometry gap into the center of a serrated rheometer plate and use oscillatory and flow tests to measure the mechanical properties of the sample. To characterize drug release from the hydrogel, first, prepare a glass capillaries by using epoxy to seal one end of each tube. When the epoxy has set, use a four-inch 22 gauge hypodermic needle to inject 100 to 200 microliters of hydrogel into a minimum of three tubes per sample, and carefully add 200 to 300 microliters of PBS onto each volume of hydrogel.
At the appropriate time points, according to the anticipated timescale of drug release, use a needle to carefully remove the PBS from each capillary without disturbing the hydrogel surface and add a fresh volume of PBS. At the completion of the study, analyze the collected PBS aliquots with an appropriate method to quantify the amount of drug released at each time point. To characterize the thermal stability of gel encapsulated insulin, load both insulin and thioflavin T into the hydrogel as demonstrated and use a 21 gauge needle to inject 200 microliters of the cargo and probe loaded hydrogel into at least three wells of a black 96-well plate per sample.
Then seal the plate with an optically clear adhesive plate seal to prevent evaporation and insert the plate into a plate reader equipped with temperature control, shaking and a kinetic read programming. To assess hydrogel encapsulated cell viability, use a 21 gauge needle to inject 150 microliters of hydrogel containing the appropriate concentration of cells into each of three wells per sample in a clear bottom 96-well plate and add 100 microliters of the appropriate cell medium into each volume of hydrogel. On day one of culture, replace the supernatant on each hydrogel at the appropriate time point for each sample group with 50 microliters of two millimolar Calcein AM solution.
After a 30 minute incubation, image the center of each well by confocal microscopy. To evaluate the ability of the encapsulated cells to settle in a syringe before injection, dilute the cells of interest to a one times 10 to the six cells per milliliter in PBS concentration and stain the cells with 50 microliters of two millimolar Calcein AM for 10 minutes at room temperature. At the end of the incubation, mix the cells with 500 to 700 microliters of hydrogel as demonstrated and use a 21 gauge needle to inject 100 to 200 microliters of cell containing hydrogel into the bottom of at least one cuvette per sample.
Then image the cuvettes lying flat on their sides on the stage of a confocal microscope immediately after injection and at one and four hours after seeding to observe whether the cells have settled in the hydrogel or whether they have remained suspended. The shear thinning and self-healing capabilities of the gel can be observed using flow sweep and step-shear protocols, respectively. Characterization of the storage and loss moduli using an oscillatory shear frequency sweep experiment in the linear viscoelastic regime at frequency ranges from 0.1 to 100 radians per second reveals the solid-like properties.
There should typically not be a crossover of the shear storage and loss moduli observed at low frequencies for stiffer formulations, while crossover events can be expected for a weaker hydrogel formulations. Varying the polymer content of the PNP hydrogels can have a direct impact on the diffusion of cargo through the polymer network and the rate of release from the materials. PNP hydrogels can also stabilize cargo that is susceptible to thermal instability, considerably extending the cargo shelf life and reducing the reliance on cold chain storage and distribution.
The inclusion of integrin motifs can be useful for adapting PNP hydrogels for cellular therapies. Encapsulated cells can be fluorescently labeled to facilitate their visualization and quantification. For example, formulations lacking adhesion sites will have a low cell viability as encapsulated cells fail to proliferate compared to cells encapsulated and formulations with adhesion motifs, such as RGD.
We are still exploring how changes in the formulation affect the rheological characteristics and dynamic mesh of the polymer matrix. We also use FRAP to study the diffusion of molecules within the hydrogel. These materials can be used to ask new biological questions about how sustained delivery might affect drug delivery, vaccine development or cancer immunotherapy.
