Poster J10 - Reprogramming Inflamed Maternal Macrophages Toward Wound Healing Phenotypes in Murine Obese Pregnancy with Hydrogel Nanoparticles

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Grace K. Hayes (she/her/hers)

Undergraduate Research Assistant
University of Oklahoma
Oklahoma City, Oklahoma, United States

Co-Author(s)

Chloe Catelain (she/her/hers)

Graduate Research Assistant
University of Oklahoma
Norman, Oklahoma, United States
Harsh A. Joshi, PhD (he/him/his)

Postdoctoral Research Associate
University of Oklahoma, Oklahoma, United States
WZ

Wanke Zhao

Research Assistant
University of Oklahoma Health Sciences Center-Harold Hamm Diabetes Center, United States
RJ

Rachel Janssen

Senior Research Assistant
University of Oklahoma Health Sciences Center-Harold Hamm Diabetes Center, United States
Karen Jonscher, PhD

Associate Professor
University of Oklahoma Health Sciences Center-Harold Hamm Diabetes Center
Oklahoma City, Oklahoma, United States

Primary Investigator(s)

John R. Clegg, PhD (he/him/his)

Assistant Professor
University of Oklahoma, Norman
Norman, Oklahoma, United States

Introduction: Obese pregnancies are becoming increasingly common in Western countries, due to the high fat, high sugar “NASH” diet (ND). Studies demonstrate that ND can cause long-term negative side effects on offspring, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is caused by gestational reprogramming of fetal stem cells, which then differentiate into macrophages that exhibit altered inflammatory behavior. The delivery of anti-inflammatory cytokines to maternal tissue and placental targets is a possible therapeutic strategy to prevent developmental programming of MASLD. Cytokine biodistribution and pharmacokinetics can be improved with nanoparticles that extend cytokine half-life and decrease off-target accumulation. Thus, we evaluated the delivery of anti-inflammatory cytokines conjugated to poly(acrylamide-co-methacrylic acid) (P(AAm-co-MAA)) nanogels (NG), for their ability to target macrophages in vitro, redirect macrophage phenotype in vitro, and distribute to target maternal organs in vivo. We hypothesized that anti-inflammatory cytokine conjugated nanogels delivery would allow increased wound healing capabilities of macrophages. We also hypothesized that delivery efficiency of the cytokine conjugated nanogels to relevant organs in pregnant mice would be influenced by particle composition and maternal diet.

Materials and

Methods: P(AAm-co-MAA) nanogels were conjugated to recombinant murine IL-10 and dosed to RAW264.7 macrophage and L929 fibroblast co-cultures (1:16 macrophage: fibroblast ratio) at 1.5 ug/mL IL-10 for 24 hours. Scratch wounds were generated in the co-culture, and the kinetics of wound closure (24 hours) were compared for treatments of unconjugated nanogels (30 ug/mL), recombinant IL-10 (40 ng/mL), nanogel-cytokine conjugates, and an untreated control. Wound area was monitored for 24 hours using a bright-field automated EVOS M7000 microscope, taking images at 2-hour intervals. This data was then analyzed using Celleste software. To evaluate the biodistribution of IL10-conjugated P(AAm-co-MAA) nanogels, mice were fed two separate diets: chow diet (CD) and high fat, high sugar NASH diet (ND). Then, the mice were impregnated. On embryonic day 14.5, pregnant mice were injected with fluorescent IL-10-conjugated nanogel (NG-IL10), control nanogels (NG) or saline. Mice were sacrificed after 24 hours. Organs (liver, kidney, spleen, heart, lungs, pancreas, brain) were isolated, weighed homogenized, and diluted into 96-well plates. Then, Alexa Fluor 647 fluorescence intensity was measured for each organ using a plate reader. Data were processed using Microsoft Excel, with statistical analysis conducted in GraphPad Prism.

Results, Conclusions, and Discussions: None of the treated macrophages, including those treated with NG-IL-10, unconjugated NG, or free soluble IL-10 exhibited a significantly different rate of wound healing closure as compared to the untreated macrophages in vitro. This indicates that transient exposure of unstimulated RAW264.7 macrophages with IL-10 (and conjugates thereof) does not impact their wound healing phenotype. Biodistribution analysis revealed nanogel accumulation in the liver, kidney, spleen, and lungs of the pregnant mice, irrespective of maternal diet and nanogel conjugation. Nanogels also accumulated in the iWAT (inguinal white adipose tissue) and BAT (brown adipose tissue). No statistical significance was observed between accumulation levels in any organ due to maternal diets or the nanogel conjugation to IL-10. However, the result should be seen as preliminary given the limited sample size (n = 2-4 mice/group). Nanogels successfully accumulated in the placentas of both CD and ND mice. These results demonstrated that P(AAm-co-MAA) nanogels were delivered to major organs, adipose tissue, and the placenta for both unconjugated and IL-10 conjugated treatments. Further evaluation is necessary to determine the extent to which nanogel delivery to the placenta and major organs attenuates macrophage inflammation or resolves developmental consequences of obese pregnancy in mice.

Acknowledgements (Optional): Gracious acknowledgement for funding is extended to our sponsors at the Harold Hamm Foundation, and we thank our collaborators at the Harold Hamm Diabetes Center for their hard work and support on this project.