Injectable supramolecular polymer–nanoparticle hydrogels enhance human mesenchymal stem cell delivery

Abstract Stem cell therapies have emerged as promising treatments for injuries and diseases in regenerative medicine. Yet, delivering stem cells therapeutically can be complicated by invasive administration techniques, heterogeneity in the injection media, and/or poor cell retention at the injection site. Despite these issues, traditional administration protocols using bolus injections in a saline solution or surgical implants of cell‐laden hydrogels have highlighted the promise of cell administration as a treatment strategy. To address these limitations, we have designed an injectable polymer–nanoparticle (PNP) hydrogel platform exploiting multivalent, noncovalent interactions between modified biopolymers and biodegradable nanoparticles for encapsulation and delivery of human mesenchymal stem cells (hMSCs). hMSC‐based therapies have shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery.


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shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery. and adipogenic lineages. 1,2 Compared to embryonic stem cells and induced pluripotent stem cells, hMSCs are broadly accessible from bone marrow, do not pose ethical concerns, and exhibit low teratoma formation and immunogenicity. 3 hMSCs, sometimes even referred to as human medicinal signaling cells, 4 also produce high levels of complex therapeutic paracrine molecules to aid in wound healing and recruit other cells throughout the body. 5 Due to these widespread capabilities, hMSCs are the most common cell type used in clinical cell therapy applications to treat myocardial infarctions, osteochondral defects, spinal cord injuries and graft versus host disease. 3,6,7 For hMSC therapies to be most effective they require high hMSC engraftment and localization after injection.
Additionally, the complex interactions between hMSCs and the inflammatory immune response limit the efficacy of hMSCs' therapeutic capabilities. 3 There are many challenges in the local delivery of stem cell therapeutics. 8 Cells encounter gravitational forces and strong bodily pressures diminishing cell retention at the injection site. 9,10 Injected cells must also interact with the immune system. The leading methods for local delivery involve hydrogels, which are biomimetic water swollen polymer networks that are capable of suspending cells in 3D. 8,11,12 Hydrogels can increase local cell retention and engraftment at the injection site compared to traditional liquid injections or infusion by acting as local niches to physically hold cells in place. 13 Additionally hydrogels can act as immunomodulatory protective barriers against infiltrating immune cells. 14 The benefits of encapsulating hMSCs in hydrogels have been shown in multiple clinical studies targeting an array of indications. 10,15,16 Yet, crosslinked hydrogels containing cells often require invasive implantation procedures. 17 The development of shear-thinning hydrogels has enabled quicker, less invasive administration procedures by injection through a needle. 16,[18][19][20][21][22][23] Injectable hydrogels enhance cell viability during the injection process compared to liquid vehicles by alleviating the mechanical forces cells experience when traveling through small diameter needles. 19,23 However, injectable shearthinning hydrogels are often formed through weak physical interactions and cannot persist long enough in the body to enhance cell retention compared to traditional liquid injections. 21 To circumvent this challenge, researchers have developed stimuli responsive and dynamic hydrogel materials that change properties in situ after injection. 12,13,20,[24][25][26] Hydrogels based on polymers exhibiting lower critical solution temperature (LCST) behavior that are injectable liquids at room temperature but gel in situ at physiological temperature after injection have been developed 13,20,24 ; however, these "thermogelling" hydrogels can be difficult to handle due to their inherent temperature sensitivity, often involve harsh complex chemistries, and often suffer from cells settling in the syringe prior to injection. 8 Moreover, many LCST-based hydrogels rely on high molecular weight polymers, including poly(N-isopropylacrylamide) (PNIPAM), which are not biodegradable. 27 Further, many other injectable hydrogel platforms are composed of natural materials such as collagen and alginate, and as such are subject to batch-to-batch variability and lack mechanical tunability, or are composed of expensive and poorly scalable proteinengineered materials. 8 It is important to note that while numerous reported studies have focused on developing hydrogels for enhanced delivery and retention of various cell types, engineering the material properties of hydrogels for robust hMSC delivery and retention has only rarely been investigated.
Here we present the use of a novel injectable shear-thinning supramolecular polymer-nanoparticle (PNP) hydrogel made from a mixture of dodecyl-modified hydroxypropylmethylcellulose (HPMC 12 ) and biodegradable nanoparticles comprising poly(ethylene glycol)block-poly(lactic acid) (PEG-PLA NPs) to enhance the local retention of hMSCs after injection. Dynamic PNP interactions have previously been shown to create injectable hydrogels exhibiting long-term delivery of therapeutics (small molecules and biologics) and enhance cell viability during injection. [28][29][30] These materials are injectable and biodegradable. The strong supramolecular interactions between the functional biopolymers and the NPs that give rise to the hydrogel structure yield in vivo depots capable of persisting for long timescales in the body. 31 In this article, we first identify the fundamental mechanical properties of hydrogels needed for the entire cell delivery process with in vitro experiments. We then demonstrate enhanced hMSC retention with PNP hydrogels in vivo compared to traditional liquid injections.
2 | RESULTS AND DISCUSSION 2.1 | Design of PNP hydrogels for cell delivery Cellulose derivatives are widely available, inexpensive, and biocompatible high molecular weight polymers commonly used as viscosifiers in pharmaceutical formulations, 32 cosmetics, 33 and a broad array of industrial applications. 34 In our hydrogel system, hydroxypropylmethylcellulose (HPMC) was modified with hydrophobic lipid dodecyl chains (C 12 ) using isocyanate coupling chemistry. 29 PEG-PLA NPs with a diameter of~30 nm were prepared using nanoprecipitation techniques, 29,35 yielding core-shell NPs with a hydrophilic PEG-based corona and a hydrophobic PLA-based core. To promote cellular adhesion and viability, the cell adhesion motif arginine-glycine-aspartic acid (RGD) was attached to the hydrophilic end of the PEG-PLA copolymer through a coppercatalyzed "click" reaction prior to nanoprecipitation. A 50:50 physical mixture of RGD-functionalized PEG-PLA polymer (RGD-PEG-PLA) and unmodified PEG-PLA polymer was used to create RGD-functionalized PEG-PLA NPs. F I G U R E 1 Polymer-nanoparticle (PNP) hydrogels for cell encapsulation and delivery. Aqueous solutions of RGD-functionalized PEG-PLA NPs, HPMC-C 12 , and hMSCs are mixed to form PNP hydrogels. These hMSC-loaded hydrogels are easily injected through high-gauge needles and can be used to enhance cell viability during injection and local retention at the injection site in immunocompetent mice  Another important material property of these hydrogels is the yield stress, which describes the stress required to initiate flow in a material.

| PNP hydrogel formation and mechanical properties
A higher yield stress corresponds to greater resilience and may lead to greater persistence in the body and greater cell retention. To determine the yield stress of our formulations, stress amplitude sweeps were performed at a constant angular frequency (ω = 10 rad/s; Figure 2d).
The stress at which G 0 and G 00 cross over one another, indicating a switch from "solid-like" to "liquid-like" behavior, is often characterized as the yield stress. 36 The yield stress may also be characterized through plotting stress as a function of shear rate ( Figure S1). As the polymer and NP concentrations are increased in the hydrogel formulations, the yield stress increases accordingly. While it is hypothesized that both longer relaxation time and higher yield stress can lead to increased material residence in the body, many other complex factors affect hydrogel persistence in vivo.  Alternatively, small and defined cell adhesive motifs can be synthetically introduced into materials to allow for cell adhesion. 38,39 Accordingly, we hypothesized that incorporation of RGD-functionalized PEG-PLA NPs into PNP hydrogels would promote cell viability. To understand the effects of RGD incorporation into the PNP hydrogel structure on cell growth, hMSCs were cultured over the course of 6 days in 1:5 PNP hydrogels prepared with and without the RGD-functionalized PEG-PLA NPs (Figure 4). These PNP hydrogels were prepared with a mixture of plain PEG-PLA NPs and RGD-functionalized PEG-PLA NPs with a concentration of RGD in the hydrogel of 500 μM, which has been shown previously to be sufficient to support hMSC viability. 39 In these experiments, hMSC viability was assessed through calcein staining on Days 1 and 6. The RGD motif enhanced cell viability over time and promoted hMSCs viability and expansion in the PNP hydrogel as an increase in viable cells was observed on Day 6 in the RGD-functionalized PNP hydrogel. hMSCs were viable in PNP with no RGD sequence after Day 1, but viable hMSCs drastically decreased by Day 6.

| PNP hydrogel mechanical properties control tissue integration
Upon administration, therapeutic cells must integrate with tissue at the injection location, by attaching and migrating into the native tissue. The cell delivery community lacks biomimetic techniques for understanding the entire cell delivery process in vitro. We developed a novel in vitro experimental procedure that closely follows the in vivo cell delivery process. A small injection (around 15 μl) of dye-labeled hMSC-loaded PNP hydrogel was injected into tissue mimetic soft collagen hydrogels (2.5 mg/ml) using a 3D bioprinter (Figure 5a). The PNP hydrogel was labeled with a dye by modifying the HPMC-C 12 polymer with FITC prior to hydrogel fabrication.
Following administration into the tissue mimic, both the PNP hydrogel and the encapsulated hMSCs were monitored over the course of 5 days (Figure 5b) with fluorescence confocal imaging. On Day 1, the 1:5 PNP hydrogel remained intact with cells completely suspended in the administered gel. In contrast, the 1:1 PNP hydrogel had already dissolved (i.e., dye-labeled hydrogel was no longer present) and hMSCs had completed settled into a dense layer at the bottom of the injection site. On Day 5, the 1:5 PNP hydrogel had dissolved, but hMSCs had uniformly migrated out of the injection site into the surrounding collagen hydrogel tissue mimic. These results indicate that a uniform cell suspension and more robust hydrogel structure with slower dissolution is required to improve the uniformity of cell delivery and enhance tissue integration. See Figure S2 for more replicates of representative images.

| PNP hydrogels enhance cell retention and Immunoprotection in vivo
To understand the utility of the PNP hydrogel system for cell delivery   were imaged across a z-stack of 500 μm with 10 μm increments. Maximum intensity projections were generated in FIJI image software. See Figure S2 for more replicates of representative images.

| Statistical analyses
All data are reported as the mean with error bars representing SD.
Data are classified as significant if p < .05 as determined by a Student's t test.