W5 - Optimizing Hemocompatibility Assays Using Benchmark Nanoparticles for Translational Gene Delivery Platforms

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

CK

Catie King

Biomedical Engineering Research Intern
Oregon Health and Science University, University of Wisconsin-Madison, United States

Co-Author(s)

PJ

Paula Josic Dominovic

Post Doctoral Scholar
Oregon Health & Science University
Portland, Oregon, United States
Araylia-Marie L. Martinez

Biomedical Engineering Research Intern
Oregon Health and Science University, Clark College
Battleground, Washington, United States

Last Author(s)

Deirdre Anderson

Scientist
Oregon Health & Science University
Portland, Oregon, United States

Primary Investigator(s)

MH

Monica Hinds

Professor
Oregon Health & Science University, United States

Introduction: : The use of nanoparticles (NPs) for therapeutic drug or gene delivery has long been a major aim of tissue engineering efforts to treat chronic diseases, such as cardiovascular disease and cancer [1]. NP administration techniques, such as intravenous or intra-arterial injection, allow immediate contact with blood cells and plasma proteins [2]. The interaction between the NP material and the blood components, including platelets, fibrin, and factor XII, will dynamically alter the coagulation response, which has the potential to lead to thrombosis [2]. Polymeric NPs (PNPs) have shown promise for targeted gene delivery. However, before assessing novel polymer formulations for PNPs, we must optimize assays to understand the material activation of the contact pathway of clotting, specifically factor XIIa (FXIIa) activation and fibrin. Here, we present methoxy-PEG gold nanoparticles (AuNPs) and kaolin powder as negative and positive controls, respectively, in a human platelet-poor plasma experimental environment. We hypothesize that AuNPs will have no significant effects on fibrin or FXIIa compared to plasma, and kaolin will significantly increase FXIIa and decrease the time for fibrin generation compared to both plasma and AuNPs. This work will lay the groundwork in analyzing the effect of future PNPs on the coagulation cascade by providing the baseline metrics for NP induced coagulation.

Materials and

Methods: : Human pooled platelet-poor plasma was prepared from six healthy adult volunteers and stored in -80°C until use (3 male, 3 female). AuNPs and kaolin powder suspensions were prepared in a range of concentrations (0, 2.5, 5, 10, 20 and 40 μg/mL) and combined with thawed, diluted pooled plasma at 37°C to assess FXIIa concentration and the time to initiate fibrin generation. The FXIIa assay utilizes S-2302, a chromogenic substrate, as FXIIa cleaves S-2302 at a known proportion. As FXII is activated, the S-2302 elicits a color change, thus a faster rate of color change signifies more FXIIa in solution. In the fibrin generation assay, calcium chloride was used to initiate the formation of a fibrin clot. The time to initiate fibrin generation was measured by a change in the turbidity of the NP-plasma samples. Both assay outputs were evaluated using the TECAN Infinite® MPlex plate reader with a kinetic cycle set at 60 seconds for an hour with absorbance 405 nm at 37°C. MATLAB® was used to compare fibrin and FXIIa generation between AuNPs, kaolin, and diluted plasma without NPs.

Results, Conclusions, and Discussions:: The FXIIa assay showed that kaolin powder had more FXIIa generation than AuNPs at all tested concentrations (Figure 1A). As the NP concentrations increased, the gap between FXIIa generation in kaolin and gold only increased. Notably, at 2.5 μg/mL, kaolin powder still had considerably more FXIIa in solution (0.316±0.033 optical density % per hour) than diluted plasma (0.097±0.036) and AuNPs (0.098±0.008). Kaolin also showed faster fibrin clot initiation times than AuNPs at all concentrations (Figure 1B). Kaolin’s take off time (422.2±107.9 seconds) was considerably smaller than diluted plasma (1265±71.8) and AuNPs (1528±276.3) at 2.5 μg/mL. However, the initiation times of kaolin and AuNPs drew closer as concentrations of NPs increased.

The results of these studies conclude that Kaolin and AuNPs are valid positive and negative controls, respectively, for fibrin initiation time and FXIIa generation assays. The highest concentrations of AuNPs (40 and 20 μg/mL) decreased time for fibrin clotting, making these concentrations unreliable for a negative control. This is likely due to optical interference by the NPs. However, AuNPs at lower concentrations, such as 2.5 μg/mL, did not show major differences compared to diluted plasma, while kaolin did, making them valid negative and positive controls respectively.

We are currently working on complexating a novel PNP with eGFP-mRNA and running the same assays with the polyplex at differing N/P ratios (molar ratio of nitrogen containing groups in the PNP to phosphate containing groups in the mRNA) and concentrations. Since positive and negative controls for hemocompatibility have been successfully established, we have expected values to draw comparisons for future testing of PNPs. If the PNP exhibits hemocompatibility, further testing will be done in vitro, and subsequently in vivo, to determine its potential as a carrier of genetic material for gene therapy.

While hemocompatibility is crucial in the understanding of a NP’s biocompatibility, immunogenicity also plays a role in the safety and efficacy of a NP in targeted gene/drug delivery. Additional future work will include complement assay optimization with these established controls and the novel eGFP-mRNA NPs.

Acknowledgements and/or References (Optional): : [1] A. M. Flores, J. Ye, K.-U. Jarr, N. Hosseini-Nassab, B. R. Smith, and N. J. Leeper, “Nanoparticle Therapy for Vascular Diseases,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 39, no. 4, pp. 635–646, Apr. 2019, https://doi.org/10.1161/atvbaha.118.311569.
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[2] K. de la Harpe, P. Kondiah, Y. Choonara, T. Marimuthu, L. du Toit, and V. Pillay, “The Hemocompatibility of Nanoparticles: A Review of Cell–Nanoparticle Interactions and Hemostasis,” Cells, vol. 8, no. 10, p. 1209, Oct. 2019, https://doi.org/10.3390/cells8101209.
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