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High School Student with a Traditionally Underrepresented Background

HS Poster

Poster 61 - Electrostatic MicroGels for Biologic Delivery

Saturday, October 26, 2024
10:00 AM - 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: While growth factors are key in regulating cell behavior and tissue repair, their clinical use in regenerative medicine encounters numerous obstacles. The short half-life of growth factors, resulting from poor stability or rapid clearance, necessitates frequent doses, increasing both costs and compliance challenges.
MicroGels (MGs) have the potential to transport various therapeutics due to their electrostatic nature. Individual MGs can be paired with biomolecules and serve as a site delivery vehicle. For example, a positively charged MG can be paired with a negatively charged biomolecule, like glial cell line-derived neurotrophic factor (GDNF). Together the GDNF and MG will generate an electrostatic attraction and can be transported to the desired location. Additionally, stem cells (SC) can be fostered on these oppositely charged bonded MGs, giving cells room to proliferate and allowing them to extend through small gaps found throughout the MG. From there, MGs coupled with SCs can be implanted into an injury site, leading the SCs to differentiate, promoting rapid recovery.
This study investigates the effect of the MicroGel’s pH on the resulting particle charges.

Materials and

Methods: Gelatin:
Gelatin was chosen for its biocompatible, biodegradable, and nonimmunogenic properties, with type A porcine gelatin having a positive charge and type B bovine gelatin a negative charge due to different treatment processes.

Crosslinking:
Cross-linking stabilized the MicroGels (MGs) at room temperature by promoting bond formation between polymer chains. Ethylene Dichloride (EDC) and N-hydroxysuccinimide (NHS) were used for cross-linking, improving the MGs' stability.

MG Synthesis:
For MG synthesis, type A gelatin dissolved in acetic acid produced positively charged MGs (AMGs), while type B gelatin in sodium hydroxide produced negatively charged MGs (BMGs). These solutions were dispelled into vegetable oil, washed, and cross-linked with EDC and NHS.

Hydrogel:
Due to weak electrostatic interactions, MGs were encapsulated in a gelatin gel to promote bonding, with equal masses of AMGs and BMGs added.

Measuring Charges:
Zeta potential (ZP) was measured by breaking down MGs into nanoparticles, flash freezing, and testing the solution with a Zetasizer.

Differentiating MGs:
AMGs and BMGs were initially dyed green and red, respectively, for differentiation, but this proved ineffective. Instead, differentiation relied on MG size and location, considering their theorized bonds.

Results, Conclusions, and Discussions: Results
MGs exhibited a strict spherical morphology throughout the entire formation process. These observations were later confirmed through microscopic imaging, which showed matching diameters across an individual MG. According to size results, AMGs displayed an average size of 229.09µm, while the BMGs displayed a size of 278.702µm. This variation in size between the MGs allowed for easy differentiation in microscopic imaging. Additionally, zeta potential (ZP) results validated the hypothesis that AMGs and BMGs will develop positive and negative charges respectively. AMGs displayed a mean ZP of 22.7 mV, indicating a strong positive charge, and BMGs displayed a mean ZP of -8.29 mV, indicating a weak negative charge. AMGs and BMGs were then combined in a 1:1 ratio and when suspended in PBS, MGs displayed slight clumping, indicating evidence of weak interactions. As shown by Fig.1, when the AMGs and BMGs were suspended in a gelatin gel, there was further evidence of interactions. The BMGs were dispersed throughout the well plate. and created electrostatic attractions with AMGs in between.

Discussion
While the MGs demonstrated electrostatic interactions, due to the BMGs having only a slight negative charge, the interactions were very weak. Further investigations should focus on manipulating the BMGs’ charge to a more extreme negative. This would cause much stronger bonds to be formed between the AMGs and BMGs, hopefully leading to a solid structure following an incubation and/or oven period. This solid structure could be used to foster SC proliferation and differentiation. Furthermore, achieving extreme charges would facilitate biologic delivery, as stronger bonds between the BMGs and positive biomolecules would lead to efficient and accurate conveyance.

Acknowledgements (Optional):

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TOBO, C.P.E., 2024. Optimizing localized delivery of biological cargo via gelatin. Thesis. (M.A). Saint Louis University
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