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Biomaterials

Biomaterials - Poster Session B

Poster B9 - Development of Injectable, Supramolecular PLGA Nanoparticle-Polymer Hydrogels for 3D Stem Cell Culture

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Induced pluripotent stem cells (iPSCs) have tremendous potential to treat a wide range of degenerative, autoimmune, and genetic disorders, especially those with unmet clinical needs such as Alzheimer’s Disease (AD) and solid tumor cancers. However, effective targeted delivery of stem cells remains a significant hurdle in regenerative medicine. Many traditional methods, including bone marrow transplant and direct injection of liquid stem cell suspensions, are associated with high invasiveness, low retention, and low overall efficacy. The leading method for targeted delivery is hydrogel carriers, which are biomimetic polymer scaffolds capable of liquid retention and 3D stem cell suspension. However, traditional hydrogels often require invasive surgical implantation or harsh, non-biocompatible chemicals that solidify the gel in in-vivo temperatures post-liquid injection.

To address this need, we have designed an injectable, supramolecular nanoparticle-polymer (PNP) hydrogel platform utilizing noncovalent cross-linkages between octadecyl-modified hydroxypropyl methylcellulose (HPMC-C18) and poly(lactic-co-glycolic) acid (PLGA) nanoparticles for viable 3D culture and facile clinical delivery of iPSCs. Both cellulosic polymer and nanoparticle materials are highly biocompatible, FDA-approved, and widely used pharmaceutically. Our PNP hydrogel platform exhibits fine-tunable mechanical and chemical properties that can be tailored to preserve iPSC viability and motility in culture. Further applications include directing in-situ differentiation of iPSCs to specific mature cell types, such as neurons, for therapeutic delivery.

Materials and

Methods:  PLGA nanoparticles were synthesized microfluidically by flow rate-controlled flash nanoprecipitation. 5mg/ml PLGA in acetonitrile and 0.1% PVA were injected by syringe pump at a 11mL/min flow rate through a confined impinging jet (CIJ) mixer into a reservoir of 0.1% PVA. The precipitated nanoparticles were dialyzed to remove excess organic solvent. Tangential flow filtration and centrifugal filtration were then performed to concentrate the nanoparticle solution. Particles were characterized for size and zeta potential through dynamic light scattering at an absorbance of 0 and a refractive index of 1.46. Nanoparticle tracking analysis (NTA) was performed to determine the concentration (particles/mL) and to validate particle size. A 20µL aliquot was subjected to lyophilization for 24 hours to determine concentration by weight (mg/mL).

HPMC polymer was modified with a hydrophobic octadecyl (C18) chain through isocyanate coupling chemistry. Unmodified HPMC was dissolved in N-Methyl-2-pyrrolidone in an argon-purged flask. The catalyst N,N-Diisopropylethylamine, commonly known as Hünig’s base, was added with octadecyl isocyanate to initiate modification. The resulting polymer aggregates were dialyzed, lyophilized, and characterized by nuclear magnetic resonance spectroscopy to validate proper modification.

Physical mixing of PLGA nanoparticles and HPMC-C18 at a 4.5:2 weight percent ratio formed a supramolecular hydrogel. Rheology was performed to characterize the gel’s properties and potential as a clinically relevant platform for injectable stem cell delivery.

Results, Conclusions, and Discussions: Our synthesis yielded ~120nm PLGA nanoparticles with a polydispersity index (PDI) measurement of 0.115, indicating a sufficiently uniform and monodisperse solution of particles. NTA data further validated that particles were 144.8+-53.8nm in size. Smaller nanoparticles lead to stiffer hydrogels due to decreased interparticle spacing and increased surface area for hydrophobic interaction. DLS measurements showed that zeta potential was -27.01mV, indicating an electrostatically stabilized colloid solution. Nanoparticles were suspended at a concentration of 3.3*10^12 particles/mL or 68mg/ml, shown by NTA and lyophilization data respectively.

A supramolecular hydrogel formed when PLGA NPs and HPMC-C18 were mixed at 4.5:2 weight percent ratio. Dynamic oscillatory shear rheology of the gel revealed that the storage modulus (G’) was greater than the loss modulus (G’’) over the entire range of frequencies evaluated. The lack of crossover in low-frequency regions indicates solid-like characteristics. These data validate the targetability and retention of stem cell-laden PLGA:HPMC-C18 PNP hydrogels, as solid-like gels can suspend cells in one area and maintain firmness under innate bodily mechanical pressures.

To determine if the hydrogel exhibited injectable mechanical properties, we performed steady shear rheology to evaluate shear-thinning and self-healing ability. We observed that viscosity decreased steadily for approximately three orders of magnitude under high shear conditions that mimicked the shear force of a syringe needle, suggesting that the multivalent interactions of the structure were disrupted. When high-shear conditions were stopped, the gel displayed self-healing abilities. These results indicate that PLGA hydrogels can dynamically re-assemble the noncovalent network following injection.

The hydrogel’s solid-like property, coupled with its injectability, offers promise for better protection against necrosis or premature differentiation during delivery than traditional liquid stem cell injections. Overall, our development of a biocompatible, supramolecular PLGA PNP hydrogel platform offers a fine-tunable, minimally invasive administration route for iPSC delivery.

Acknowledgements (Optional):