Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

Prior to beginning this protocol, it is necessary to synthesize HPMC-C₁₂ and PEG-PLA using previously published methods^{27,28,29,30,31,36,37,38}.

1. Nanoparticle (NP) synthesis by nanoprecipitation

NOTE: This section describes synthesis of a single batch of NPs, producing 250 µL of 20 wt% NPs in buffer solution (50 mg of dry PEG-PLA polymer per batch). Notes for scaling up the number of batches are provided at relevant steps.

1. Measure 50 mg of PEG-PLA polymer into an 8 mL glass scintillation vial and add 1 mL of acetonitrile. Vortex to fully dissolve.
   NOTE: To scale up the number of batches, linearly scale this step and add the total amount of polymer and solvent needed in a single vial.
2. Add 10 mL of ultrapure water into a 20 mL glass scintillation vial with a small stir bar. Put on a stir plate set to 600 rpm.
   NOTE: If step 1.1 has been scaled, it is still necessary to have an individual scintillation vial to precipitate into for each equivalent batch. For example, for 200 mg of polymer dissolved in 4 mL of acetonitrile, prepare 4 x 20 mL scintillation vials.
3. To form NPs by nanoprecipitation, add 1 mL of polymer solvent solution dropwise into the water using a 200 µL pipette. Stir for 2 min. The PLA block of the PEG-PLA is not soluble in water, and as a result, core-shell NPs will self-assemble with the hydrophobic PLA blocks as the core and the hydrophilic PEG blocks as the shell.
4. Verify the particle size by dynamic light scattering (DLS).
   NOTE: This procedure is specifically written for a commercially available plate reader with the associated software package (see Table of Materials). For using alternative instruments, refer to the sample preparation methods described by the instrument's manufacturer.
   1. Dilute 20 µL of NP solution with 80 µL of ultrapure water (analysis concentration: 1 mg/mL PEG-PLA NPs). Add 30 µL per well to a clear bottom black 384-well plate (analyze in triplicate).
   2. Measure the hydrodynamic radius and polydispersity of each sample with a DLS plate reader using preset protocol options in the software package. As an example of a typical protocol, set the data collection parameters to acquire 5-10 DLS measurements of 2-5 s duration per acquisition and then report a mean particle size and distribution per well, calculated from the globular proteins model. To form hydrogels with consistent rheological properties, the resulting particles should be 30-50 nm in hydrodynamic diameter with a polydispersity (PD) < 0.2.
      NOTE: If the NPs are smaller than desired, use a solution of 75% acetonitrile / 25% dimethyl sulfoxide (DMSO) in step 1.1. Increasing the percentage of DMSO in the solvent solution will generally increase particle size.
5. Transfer the NP solution from the 20 mL scintillation vial into a centrifugal filter unit. Centrifuge at 4500 x g for 1 h to concentrate the NP solution to <250 µL.
6. Resuspend in desired buffer, such as phosphate-buffered saline (PBS), to 20 wt% NPs. Pipette the contents of the centrifugal filter unit into a tared microcentrifuge tube or glass scintillation vial on a mass balance. Use a small amount (50-100 µL) of buffer to rinse the filter and ensure collection of all NPs. Add buffer to reach a total mass of 250 mg.
   NOTE: Batches can be pooled during resuspension. NP stock solutions can be stored at 4 °C for approximately 1 month. Do not freeze. For longer storage, verify size and polydispersity by DLS before use.

2. Hydrogel formulation and encapsulation of drugs or cells

NOTE: This section describes preparation of 1 mL of 2:10 PNP hydrogel formulation, with 2:10 denoting 2 wt% HPMC-C₁₂ and 10 wt% NPs (12 wt% total solid polymer) and 88 wt% buffer solution, drug cargo solution, or cell suspension. The formulation percentages can be varied to yield hydrogels with a range of mechanical properties. For example, 1:5 PNP hydrogels were used for the cell settling and viability experimental results shown.

1. Prepare stock solution of 6 wt% HPMC-C₁₂ in PBS (or other buffer of choice). Dissolve for 48 h to ensure the polymer is fully dispersed.
   NOTE: The HPMC-C₁₂ stock solution is stable for months at room temperature. However, storage at 4 °C is recommended to inhibit microbial growth.
2. Add 333 mg of 6 wt% HPMC-C₁₂ stock solution into a 1 mL Luer lock syringe.
3. Add 500 µL of 20 wt% NP stock solution to a microcentrifuge tube. Add 167 µL of PBS and pipette to mix. Using a needle, fill another 1 mL Luer lock syringe with the diluted NP solution.
   NOTE: To load drug cargo, calculate the desired final concentration of the drug in the hydrogel and load the appropriate amount into the 167 µL of PBS that is mixed with the NPs. If a molecular probe is necessary for an in situ assay, such as for monitoring drug stability, load the probe in a similar manner as described above for loading drug cargo. To load cells, calculate the desired final cell concentration in the hydrogel and load the appropriate number of cells into the 167 µL of PBS that is mixed with the NPs.
4. Mix the two hydrogel components (HPMC-C₁₂ and NPs) using an elbow mixing method³⁵.
   1. Attach the Luer elbow connector to the syringe containing NP solution (optionally also containing drug cargo or cells). Push the NP solution through the elbow until the meniscus is visible at the open end. Pull back slightly and connect the syringe containing HPMC-C₁₂ solution.
      ​NOTE: It is important to minimize air in the elbow connection to prevent the formation and dispersal of bubbles throughout the hydrogel during the mixing process. When mixing cells with the elbow mixer, take care to mix more gently as mixing too rapidly may subject the cells to high shear forces, leading to cell death.
   2. Pump the two solutions back and forth through the elbow mixer for approximately 60 cycles until a homogeneous, opaque white hydrogel material has formed.
   3. Push the entire volume of hydrogel into one syringe. Remove the empty syringe and draw the plunger back on the gel-loaded syringe to recover material from the elbow connector. Cap with a needle or plug.
      ​NOTE: It is necessary to account for ~300 µL of lost hydrogel volume due to the dead space in the mixing process. For example, if 700 µL of final hydrogel volume is desired, 1 mL of hydrogel should be prepared. The hydrogel formulation process can be scaled up by using larger syringes. However, for stiff hydrogel formulations, such as 2:10, it can become difficult to mix and inject from syringes larger than 3 mL in volume due to the increasing ratio of syringe barrel to elbow or needle diameter.
   4. Store the hydrogel in the syringe at room temperature. However, if drugs are encapsulated, storage at 4 ˚C is recommended unless the drug manufacturer specifies otherwise. Do not freeze the material.

3. Measuring rheological properties of hydrogel formulations

NOTE: This protocol is specifically used with the commercial rheometer mentioned in the Table of Materials with a 20 mm serrated plate geometry. For using other instruments, refer to the manufacturer's instructions for sample preparation.

1. Formulate at least 700 µL of PNP hydrogel for rheological characterization.
2. Inject material in the center of the serrated rheometer plate. The amount will vary depending on the chosen geometry gap. For reference, a 700 µm gap requires ~400-500 µL of material.
3. Lower the rheometer to the trim gap (500-1000 µm) and slowly turn the top rheometer plate as it makes contact with the PNP hydrogel to ensure the gap is filled evenly and completely.
4. Inspect the loading of the PNP hydrogel such that it covers the entire rheometer plate surface. Use a spatula or plastic trimmer to gently trim and remove any excess material, such that it has a very slight bulge out of the plate.
5. Lower the rheometer to the final geometry gap and verify the sample is cleanly loaded.
6. Measure the mechanical properties of the sample using oscillatory tests, such as amplitude or frequency sweeps, or flow tests, such as flow sweeps or step tests.
   ​NOTE: In the representative data shown, oscillatory amplitude tests are run at a constant frequency of 10 rad/s. Oscillatory frequency sweeps are run at a constant 1% strain, within the linear viscoelastic regime of the amplitude sweep. Flow sweeps were run from high shear rates to low shear rates³⁹. All tests are completed with 10 points per decade of data collected and at room temperature. The test parameters may need to be adjusted depending the properties of the formulation. Subjecting stiffer PNP materials such as 2:10 formulations to high shear rates can cause the material to be ejected from the rheometer plates, resulting in inaccurate mechanical characterization, and requiring reloading of the sample between subsequent tests. Representative data shown below can be used for comparison during quality control testing.

4. Characterizing in vitro drug release

1. Prepare capillary tubes by cutting glass capillary tubes to desired length. Seal one end of each tube by using a disposable spatula or pipette tip to push a small amount of epoxy into the end of the tube to form a plug. Allow epoxy to set per manufacturer's recommended time.
   NOTE: The tube must be shorter than the length of the injection needle. Tubing with 2-3 µm inner diameter is recommended such that a length of 2.5 in will contain at least 300 µL of total volume.
2. Formulate at least 500 µL of a PNP hydrogel material in a syringe containing the drug of interest. Prepare each sample group in a separate syringe.
3. Inject 100-200 µL of the PNP hydrogel at the bottom of each capillary tube using a long hypodermic needle (22G, 4 inch). Prepare at least three tubes (triplicate) per sample group.
4. (Optional) Place filled capillary tubes in a conical centrifuge tube and centrifuge for 1 min at 1000 x g to ensure the surface of the hydrogel is uniform. This step may need to be repeated, altering time and speed as necessary to smooth the surface of the material.
   CAUTION: Ensure centrifuge is well balanced.
5. Carefully fill 200-300 µL of PBS on top of the hydrogel in the capillary tube using a syringe and needle or pipette. Do not contact or disturb the surface of the hydrogel. Seal the tube with a cap or plug or cover with at least two layers of paraffin film.
6. (Optional) Incubate samples at 37 °C to simulate in vivo conditions.
7. Carefully completely remove the PBS from each capillary, without disturbing the hydrogel surface, using a syringe and needle at chosen time points depending on the anticipated time scale of drug release. Replace the volume removed with fresh PBS. Store aliquots under appropriate conditions.
   NOTE: The recommended volumes and time points in steps 4.3, 4.5 and 4.7 can be optimized to capture in vitro drug release over a range of timescales, depending on how much drug is loaded in the material and how quickly it releases into the supernatant. A sample of selected time points could be 6 h, 1 day, 3 days, 1 week and 2 weeks for a slow-releasing drug. Aliquots can also be analyzed as they are acquired rather than stored.
8. At the completion of the study, analyze aliquots with an appropriate method such as ELISA, HPLC or fluorescence assay to quantify the amount of drug released at each time point^40,41,42. The appropriate detection method will vary depending on the drug of interest.
   ​NOTE: In vitro release studies are useful for comparing release between different hydrogel formulations or drug cargo. The in vitro release timescale does not often directly indicate an expected time scale of release in vivo.

5. Characterizing thermal stability of gel-encapsulated insulin

1. Formulate at least 1.2 mL of PNP hydrogel per sample group. Following the procedure described in section 2.3, load both insulin (drug cargo) and thioflavin T (ThT) (molecular probe) into the PNP hydrogel.
   NOTE: The primary mechanism of aggregation and, therefore, inactivation of insulin is through the formation of amyloid fibrils. ThT is a suitable molecular probe because it produces a strong fluorescence signal in the presence of amyloid fibrils, allowing for in situ monitoring of insulin aggregation. Depending on the drug cargo of interest, aggregation may be monitored through different methods. For the representative data shown, insulin was loaded to a final concentration of 6.7 or 10 mg/mL and ThT to a final concentration of 25 µM.
2. Using a 21 G needle, inject 200 µL of PNP hydrogel per well into a black 96-well plate. Each sample group should be measured in at least triplicate. Seal plate with an optically clear adhesive plate seal to prevent evaporation.
3. Insert plate into a plate reader equipped with temperature control, shaking, and kinetic read programming and begin read protocol. Representative data was acquired with a commercially available plate reader (see Table of Materials) using the following conditions:
   1. Stressed aging conditions: continuous linear shaking (410 cpm, 5 mm) at 37 ˚C.
   2. Data acquisition: excitation/emission 450 nm/482 nm at 20 min intervals
      ​NOTE: If a plate reader with temperature control, shaker, and kinetic read capabilities is not available, the plate can be placed on a shaker plate in an incubator and read manually at above wavelengths at selected time points.
4. Plot data as mean fluorescence signal over time for each group. Time to aggregation can be quantified by defining an arbitrary signal threshold⁴³.
   NOTE: For the representative data shown below, the threshold was defined as 750,000 arbitrary fluorescence units (AFU). This value was chosen to be above the measured baseline while still sufficiently capturing the onset of aggregation indicated by a sharp fluorescent signal increase.
5. Terminate assay when samples aggregate or visually begin to dehydrate.

6. Assessing cell viability

1. Formulate at least 2 mL of PNP hydrogel containing the desired cell concentration following above protocols (normally 1 – 5 x 10⁶ cells/mL). Prepare each sample group in a separate syringe.
2. Using a 21G needle, inject 150 µL PNP hydrogel into each well in a clear bottom 96-well plate; each well is one replicate. Each sample group should have 3-5 replicates per time point. Centrifuge the plate at 50 x g for 2 min to spread the hydrogel evenly in the well.
3. Add 100 µL of the appropriate cell media on top of the hydrogel. Remove this media each day and add 100 µL new media.
4. On Day 1, remove the media on top of the hydrogel for the designated replicates for that time point for each sample group. Add 50 µL of 2 mM calcein AM solution on top of the hydrogels. Incubate for 30 min.
   NOTE: Calcein AM can be used to identify and label live cells. In live cells, the non-fluorescent calcein AM is converted to a green-fluorescent calcein, by intracellular esterases after acetoxymethyl ester hydrolysis.
5. Image the center of each well in a 96-well plate using a confocal microscope. Survey an area of least 300 µm² with a z-stack spanning at least 150 µm. Use confocal wavelength settings to capture the fluorescence of calcein (excitation/emission: 495 nm/515 nm).
6. Repeat step 6.4 and 6.5 for each subsequent time point as desired.
7. To analyze each image, collapse all z-stack images into a single plane maximum intensity image using FIJI or similar software. Quantify the number of fluorescent cells in each image. The ratio of the number of fluorescent cells at each time point compared to the number of fluorescent cells at Day 1 is the relative cell viability in the PNP hydrogels.

7. Assessing cell settling

1. Calculate the number of cells required to formulate 500-700 µL of PNP hydrogel at a final concentration of 5 x 10⁶ cells/mL. Suspend cells in 1 mL of PBS at a concentration of 1 x 10⁶ cells/mL. Stain cells by adding 50 µL of 2 mM calcein AM. Incubate the cells with the dye for 10 min.
2. Centrifuge cells at appropriate conditions, remove the PBS and resuspend the cells in the volume of PBS needed to form 500-700 µL of the desired PNP hydrogel.
   NOTE: The recommended speed and duration to centrifuge each specific cell type is typically provided in the product documentation.
3. Formulate 500-700 µL of PNP hydrogel with the stained cells (5 x 10⁶ cells/mL) following protocol section 2.
4. Using a 21G needle, inject 100-200 µL of PNP hydrogel containing the stained cells in the bottom of a cuvette. Three replicates should be performed for each sample. Move the needle back and forth within the cuvette while injecting to prevent bubble formation.
5. Immediately (time t=0), image the cuvettes lying on their side over the entire flat cuvette rectangle area at the base of the cuvette. Use the confocal tile scanning capabilities to image the entire well area and image a z-stack in 3D across a 100 µm depth. For later visualization, either use the confocal microscope software to stitch together all of the individual tiles and perform a maximum intensity projection to form a single image of the large area or use FIJI software on a personal computer^44,45.
6. Following imaging, stand the cuvettes upright.
7. Image at 1 h and 4 h to observe if cells have settled in the hydrogel or whether they remain suspended.
   NOTE: These time points are suggestions and can be modified as desired.
8. To analyze each image, collapse all z-stack images into a single plane maximum intensity image. Using FIJI or similar software, quantify the cell distribution by measuring the fluorescence intensity down the center vertical profile of the cuvette to determine the degree of settling.