Published: February 7th, 2021
This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.
These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.
Injectable hydrogels are an emerging tool to deliver therapeutic cells and drugs to the body in a controlled fashion1. These materials can be loaded with drugs or cells and can be administered in a minimally invasive manner through direct injection to superficial tissues or by catheter delivery to deep tissues. In general, injectable hydrogels are composed of water-swollen polymer networks that are crosslinked together by transient, physical interactions. At rest, these crosslinks provide a solid-like structure to the gels, but upon application of sufficient mechanical force these crosslinks are temporarily disrupted, transforming the material into a liquid-like state that can easily flow2. It is these rheological properties that allow physical hydrogels to shear-thin and flow through small needle diameters during injection3. After injection, the polymer network of the material reforms, allowing it to self-heal and rapidly form a solid-like gel in situ4,5. These structures can act as slow-release depots for drugs or scaffolds for tissue regeneration6,7. These materials have been used in diverse applications encompassing drug delivery technology, regenerative medicine, and immunoengineering1,8,9,10,11,12.
Both natural materials (e.g., alginate and collagen) and synthetic materials (e.g., poly(ethylene glycol) (PEG) or similar hydrophilic polymers) have been developed as biocompatible injectable hydrogel materials13,14,15. Many natural materials exhibit batch-to-batch variation affecting reproducibility4,16. These materials are often temperature-sensitive, curing upon reaching physiological temperatures; thus, handling these materials poses additional technical and logistical challenges17. Synthetic materials allow for more precise chemical control and excellent reproducibility, but these materials can sometimes be subject to adverse immune responses that limit their biocompatibility, a critical feature for in vivo therapeutic applications6,18,19. Recent efforts have shown there are many complex design criteria involved in engineering an injectable hydrogel material, including optimizing mechanical properties, polymer network mesh size, bioactive molecular cues, biodegradability, and immunogenicity of the material20,21,22,23,24,25,26. All of these factors must be considered depending on the application of interest, which means that a modular, chemically tunable platform is ideal for satisfying a wide breadth of applications.
The present methods describe the formulation and the use of an injectable polymer-nanoparticle (PNP) hydrogel platform that exhibits tunable mechanical properties, a high degree of biocompatibility and low immunogenicity, and presents sites for conjugating bioactive molecular cues27,28,29,30,31,32,33. These PNP hydrogels are composed of hydrophobically-modified cellulose polymers and self-assembled core-shell nanoparticles comprising poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA)27,34 that interact to produce a supramolecular network. More specifically, the dodecyl-modified hydroxypropylmethyl cellulose polymers (HPMC-C12) dynamically interact with the surface of PEG-PLA nanoparticles and bridge between these nanoparticles to form this polymer network27,34. These dynamic, multivalent interactions allow the materials to shear-thin during injection and rapidly self-heal after administration. The PNP hydrogel components are easily fabricated through simple one-pot reactions and the PNP hydrogel is formed under mild conditions by simple mixing of the two components35. Due to the ease of fabrication, this hydrogel platform is highly translatable at scale. The mechanical properties and mesh size of PNP hydrogels are controlled by altering the weight percent of the polymer and nanoparticle components in the formulation. Prior studies with this platform indicate that PNP hydrogels are highly biocompatible, biodegradable, and non-immunogenic28,30,31. Overall, these hydrogels present wide utility in biomedical applications encompassing post-operative adhesion prevention, tissue engineering and regeneration, sustained drug delivery and immunoengineering.
Prior to beginning this protocol, it is necessary to synthesize HPMC-C12 and PEG-PLA using previously published methods27,28,29,30,31,36,37,38.
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.
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.
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.
4. Characterizing in vitro drug release
5. Characterizing thermal stability of gel-encapsulated insulin
6. Assessing cell viability
7. Assessing cell settling
PNP hydrogel fabrication and characterization
PNP hydrogels are formed through the mixing of the two primary components - hydrophobically-modified HPMC polymers and PEG-PLA nanoparticles (Figure 1a). Therapeutic cargo is most easily incorporated into the additional buffer used to dilute the nanoparticle component prior to hydrogel preparation. For downstream biomedical characterization, it is convenient to use an elbow mixing method that enables simple and reproducible mixing of the two components (Figure 1b). After adequate mixing, the hydrogel should feel firm in the syringe, but yield under pressure and extrude from a standard needle (21G shown) (Figure 1c). After injection, the hydrogel should rapidly set into a solid-like material that resists flow from gravity. To fully characterize the hydrogel and ensure consistent batch-to-batch products, samples should be analyzed using several different experiments on a rheometer. The shear-thinning and self-healing capabilities of the gel will be easily observed using a flow sweep protocol and step-shear protocol, respectively (Figure 2a,b). For stiffer gels, such as the 2:10 formulation, the user should look for viscosity to decrease at least two orders of magnitude during the flow sweep as the shear rate is increased from 0.1 to 100 s-1, which simulates the mechanical conditions during injection. The step-shear protocol should reveal an orders-of-magnitude decrease in viscosity under the high-shear steps, and a rapid return (<5 s recovery time) to baseline viscosity during the low shear steps. Characterization of the storage and loss moduli using an oscillatory shear frequency sweep experiment in the linear viscoelastic regime should reveal solid-like properties at frequency ranges from 0.1-100 rad s-1 (Figure 2c). In particular, there should typically not be a crossover of the shear storage and loss moduli that is observable at low frequencies for stiffer formulations like the 2:10 hydrogels. Such a crossover event may indicate issues in the quality of the starting materials, either the modified HPMC or PEG-PLA polymer, or the size and dispersity of the PEG-PLA nanoparticles. It should be noted that a crossover event can be expected for weaker hydrogel formulations, such as the 1:5 hydrogel. Oscillatory shear amplitude sweeps on PNP hydrogels reveal that the materials do not yield until high stress values are applied, indicating these materials possess a yield stress, a threshold amount of stress required for the material to flow.
Characterizing release kinetics from PNP hydrogels
An essential step in designing PNP gels for drug delivery is the characterization of drug release kinetics from a chosen formulation. There are several techniques for this, but a simple in vitro methodology provides useful data during early formulation development (Figure 3a). Varying the polymer content of the PNP hydrogels through modulating the amount of HPMC-C12 or NPs is the most straightforward way to tune the mechanical properties and mesh size of these hydrogels, which can have a direct impact on the diffusion of cargo through the polymer network and rate of release from the materials (Figure 3b). For cargo that is larger than the dynamic mesh size (i.e., high molecular weight or large hydrodynamic radius), researchers should expect a slow, dissolution-mediated release of cargo from the hydrogel depot. Formulations with dynamic mesh sizes greater than or equal to the size of the cargo will allow for diffusion-mediated release that can be described using traditional models of cargo diffusion and release46,47,48,49. Based on the shape of the release curve, researchers can reformulate the hydrogel to tune it towards slower (e.g., increase the polymer content) or faster (e.g., decrease the polymer content) release.
Assessing stability of therapeutic cargo
Determining the stability of the therapeutic cargo in a hydrogel formulation is critical before commencing preclinical or cellular studies. Compared to other synthetic methods for encapsulating drugs, PNP hydrogels incorporate cargo in a gentle manner by mixing into the bulk material, and it is unlikely that encapsulation will damage the cargo. These studies indicate that PNP hydrogels can also stabilize cargo that is susceptible to thermal instability, such as insulin, considerably extending shelf life and reducing reliance on cold storage and distribution (Figure 4). It is important to evaluate the condition of the cargo immediately after encapsulation into the hydrogel as well as after extended periods of storage. These data show that insulin remains stable in hydrogels after 28 days of storage under continuous thermal and mechanical stress, using a simple fluorescence assay for measuring insulin aggregation. An alternative technique for cases where an appropriate plate assay is unavailable would be to perform circular dichroism measurements of the cargo, which is particularly useful for determining the secondary structure of protein drugs.
Determining cell viability and dispersion in PNP hydrogels
Many therapeutic cells require adhesion motifs to remain viable, and thus inclusion of integrin motifs like arginine-glycine-aspartic acid (RGD) peptides is an important step in adapting PNP hydrogels for cellular therapies50. The modular PEG-PLA polymer comprising the NPs enables chemical functionalization of the PEG corona through simple "click" chemistries28,51. In this example, cell-adhesive RGD peptides were attached to the PEG-PLA polymer to promote cell engagement with the PNP hydrogel structure. Formulations lacking adhesion sites will have low cell viability as encapsulated cells fail to proliferate compared to cells encapsulated in formulations with these adhesion motifs (Figure 5a,b). Encapsulated cells can be labeled with calcein AM or another appropriate fluorescent dye (e.g., CFSE) to facilitate cell counting with a fluorescence microscope. During optimization, viability should be compared to unmodified PNP hydrogels to assure integrin-functionalized formulations are providing enhanced viability and proliferation. If integrin-functionalized formulations are providing similar efficacy as unmodified hydrogels, this may indicate a failure in the conjugation chemistry used to incorporate the adhesion motifs.
Researchers should expect encapsulated cells to be evenly dispersed through the hydrogel medium when using an appropriate hydrogel formulation. This will allow for consistent and predictable dosing of cells during hydrogel administration and should translate to local retention of cells in the hydrogel after administration. The distribution of cells can be easily determined using fluorescence microscopy techniques. Cells can be labeled with an appropriate dye and then imaged using confocal microscopy. The images can be assessed visually (Figure 5c) and also quantitatively (Figure 5d) using ImageJ software to measure the average fluorescence intensity along the vertical axis of the image (or along whichever axis cell-settling due to gravity is expected to occur). If the hydrogel formulation is too weak to support the cells in suspension over prolonged timeframes, cell settling will occur, as observed in the 1:1 formulation in Figure 5. Increasing the polymer content can resolve issues with inhomogeneous cell dispersion due to settling.
Figure 1: Polymer-nanoparticle (PNP) hydrogels are easily formed by mixing two components. (a) The first component is a solution of dodecyl-modified hydroxypropylmethyl cellulose (HPMC-C12), and the second component is a solution of poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles along with any therapeutic cargo. Gentle mixing of these two components yields an injectable hydrogel, where the HPMC-C12 polymers are physically crosslinked by dynamic, multivalent interactions with the PEG-PLA nanoparticles. (b) Photograph demonstrating gel formulation by mixing with two syringes, each one containing one component of the PNP hydrogel. By connecting the two syringes with a Luer-lock elbow connector, the two components can be easily mixed under sterile conditions to yield a bubble-free hydrogel pre-loaded into a syringe for immediate use. The NP solution is dyed blue for the purpose of demonstration. (c) Demonstration of the injection of PNP hydrogels and their re-solidification. (i) PNP hydrogel in a syringe with an attached 21G needle. (ii) Injection places the hydrogel under shear which temporarily breaks the interactions between polymer and nanoparticles, creating a fluid-like consistency. (iii) Post-injection, the dynamic polymer-nanoparticle interactions rapidly reform, allowing the hydrogel to self-heal into a solid. (iv) The solid hydrogel does not flow under forces weaker than its yield stress, such as gravity. The PNP hydrogel is dyed blue for the purpose of demonstration. Please click here to view a larger version of this figure.
Figure 2: Rheological characterization of two PNP hydrogel formulations. Formulations are denoted as polymer wt.%: NP wt.%. (a) Steady shear flow sweeps from low to high shear rate of PNP hydrogels. Viscosity as a function of shear rate characterizes shear-thinning properties. (b) Viscosity as a function of oscillating shear rates between low shear rates (white background; 0.1 s−1) to high shear rates (red background; 10 s−1) demonstrating self-healing properties of PNP hydrogels. Shear rates are imposed for 30 s each. (c) Elastic storage modulus G′ and viscous loss modulus G″ as a function of frequency at a constant 1% strain for various PNP hydrogel formulations. (d) Amplitude sweeps at a constant frequency of 10 rad/s to characterize elastic storage modulus G′ and viscous loss modulus G″ of PNP hydrogels as a function of stress. This rheological characterization can be used as comparison for quality control. This figure has been adapted from Grosskopf et al.28 Please click here to view a larger version of this figure.
Figure 3: In vitro release of bovine serum albumin (BSA) from PNP hydrogels. Formulations are denoted as polymer wt.%: NP wt.%. (a) Schematic describing the experimental in vitro release protocol. Aliquots are removed from PNP hydrogel-loaded capillary tubes over time. (b) The in vitro release of BSA from 1:10 PNP, 2:5 PNP and 2:10 PNP reported as the mass collected by the specified time point divided by the total mass collected during the assay (data shown as mean ± SD; n = 3). BSA was detected through absorbance measurements. Please click here to view a larger version of this figure.
Figure 4: Thermal stability of insulin encapsulated in PNP hydrogels by ThT assay. Formulations are denoted as polymer wt.%: NP wt.%. Insulin encapsulated in both 1:5 and 2:10 PNP hydrogel remained unaggregated for over 28 days at stressed aging conditions of 37 °C and constant agitation. Time to aggregation for insulin formulated in PBS was 20 ± 4 h (mean ± SD, aggregation threshold 750,000 AFU). Data presented as an average of n = 4 experimental replicates (AFU, arbitrary fluorescence units). This figure has been adapted from Meis et al.38 Please click here to view a larger version of this figure.
Figure 5: Cell viability and cell settling in PNP hydrogels. (a,b) Cell viability studies in PNP hydrogels with human mesenchymal stem cells (hMSCs). (a) Representative images of viable hMSCs in 1:5 PNP hydrogels with and without the cell-adhesive arginine-glycine-aspartic acid (RGD) motif conjugated to the PEG-PLA NPs. hMSCs were calcein-stained for 30 min prior to confocal imaging. Scale bar represents 100 µm. (b) Cell viability on Day 6 defined as number of fluorescent cells in the image relative to number of fluorescent cells on Day 1 (data shown as mean ± SD; n = 3). (c,d) Cell encapsulation and settling experiments with hMSCs. (c) Maximum intensity images of calcein AM-stained hMSCs encapsulated in 1:1 PNP hydrogel (top row) and 1:5 PNP hydrogel (bottom row) across 4 hr to quantify cell settling. Scale bar represents 1 mm. (d) Average horizontal pixel intensity of hMSCs along the vertical profile of the hydrogel. This figure has been adapted from Grosskopf et al.28 Please click here to view a larger version of this figure.
Polymer-Nanoparticle (PNP) hydrogels are easily fabricated and enable the long-term local delivery of therapeutic cells and drugs through minimally invasive administration via direct injection or catheter delivery. These protocols describe the formulation of PNP hydrogels and the characterization methods for assuring quality of the resulting materials. Supramolecular PNP hydrogels are scalable to manufacture and are formed through the simple mixing of modified cellulose polymers and polymeric core-shell nanoparticles. The present methods describe facile procedures to form gels pre-loaded in syringes through simple elbow mixing protocols. Through quality control metrics of each of the component parts, such as DLS to monitor the NP size and distribution, one can reproducibly formulate PNP hydrogel materials with consistent rheological properties. Through varying the amount of HPMC-C12 or NPs, one can modulate the mesh size and stiffness of the resulting PNP hydrogel. These properties can be tuned to best suit a particular biomedical application, and with the rheological methods detailed here researchers can characterize the shear-thinning and self-healing properties of PNP hydrogels as they optimize the platform for their specific applications. Methods for in vitro release studies are also described; researchers can use these studies to characterize the relative timescale of release of drugs of interest, informing future in vivo studies. Using stability studies, researchers can also assess the ability of these materials to help preserve the biological structure and stability of sensitive biotherapeutics over time and extreme temperatures, with compelling potential applications to reduce the cold chain dependence of biotherapeutics. Finally, with simple cell viability assays, cell growth and migration within PNP materials can be evaluated, with potential applications in cell therapies and scaffolds.
Our group has found many compelling applications for the PNP hydrogel platform27. PNP hydrogels have been used for slow delivery of subunit vaccines, enabling matched kinetic release profiles of antigens and adjuvants to boost the magnitude, duration, and quality of the humoral immune response31. PNP hydrogels have been found to have a smaller mesh size than most commonly used hydrogels, so they are effective at slowing diffusion and slowly releasing molecular cargo. The unique tissue adherence properties and mechanical properties of PNP hydrogels have also been utilized to form physical barriers to prevent adhesions arising from surgery by spraying the hydrogels over large surface areas of organs following surgery30. PNP hydrogels have also been shown to be effective cell delivery vehicles, and the mechanical properties actually shield cells from the mechanical forces occurring in the syringe needle during injection, improving cell viability29. When the NPs are conjugated with a cell-adhesive peptide, cells can attach and engage with the PNP matrix to remain viable. Using this approach, PNP hydrogels have been shown to improve the local retention of injected stem cells compared to methods using liquid vehicles28. In addition, PNP hydrogels have been shown to prevent thermally-induced aggregation of encapsulated insulin, even under harsh stressed aging conditions, suggesting that these materials may be able to reduce the need to refrigerate temperature-sensitive drugs38.
Overall, the methodologies described here will allow research groups to fabricate and explore PNP hydrogels as a biomaterial. These protocols provide the lab-scale synthesis techniques to fabricate enough hydrogel material to pursue both in vitro and in vivo studies. The studies described above indicate that the dynamic crosslinks of these materials enable it to be suitable for a range of biomedical applications by allowing active motility of entrapped cells while restricting passive diffusion of molecular cargo. It is anticipated that researchers will find the PNP platform an accessible and powerful tool to improve clinical outcomes through controlled drug delivery and to study basic biological mechanisms such as cell recruitment and mechanobiology.
These authors have nothing to disclose.
This research was financially supported by the Center for Human Systems Immunology with Bill & Melinda Gates Foundation (OPP1113682) and the Bill & Melinda Gates Foundation (OPP1211043). C.M.M. was supported by a Stanford Graduate Fellowship and the Stanford Bio-X William and Lynda Steere Fellowship. A.K.G. is thankful for a National Science Foundation Graduate Research Fellowship and the Gabilan Fellowship of the Stanford Graduate Fellowship in Science and Engineering. S.C. was supported by the National Cancer Institute of the National Institutes of Health under Award Number F32CA247352. The authors would also like to warmly acknowledge Appel Lab members including Dr. Gillie Roth, Dr. Anthony Yu, Dr. Lyndsay Stapleton, Dr. Hector Lopez Hernandez, Dr. Andrea d'Aquino, Dr. Julie Baillet, Celine Liong, Ben Ou, Emily Meany, Emily Gale and Dr. Anton Smith for their effort and time in helping the Appel Lab to develop these protocols over the years.
|PNP hydrogel injection
|22G, 4 in hypodermic needles
|In vitro release studies
|384-well plates, black, clear bottom
|Dynamic light scattering (DLS)
|96-well plates, black
|96-well plates, clear
|Cell viability and settling studies
|Thermo Fisher Scientific
|Cell viability and settling studies
|In vitro release studies
|Centrifugal filter units
|Cell viability and settling studies
|DLS Plate Reader
|DynaPro II Plate Reader
|Dynamic light scattering (DLS)
|In vitro release studies
|In vitro release studies
|Luer elbow connector
|PNP hydrogel formulation
|Luer lock syringe
|PNP hydrogel formulation
|Phosphate Buffered Saline (1x)
|PNP hydrogel formulation
|Synergy H1 Hybrid Multi-Mode Plate Reader
|Recombinant human insulin
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