Quantitative Pharmacology to Interrogate Mechanisms of Lipoplex Destabilization by Serum Proteins
Authors: Abbiati Roberto Andrea , Yeung Bertrand Z. , Jaiprasart Pharavee , Lu Ze , Wientjes Guill M. , Au Jessie L‐S 
Published: November 2023

Short interfering RNA (siRNA) represents an option to correct genetic lesions. Due to its instability and unfavorable transport properties (high MW, large size, negative charges), siRNA is commonly delivered as a complex with cationic liposomes or Lipoplex. Lipoplex, ~100–200 nm diameter, is multilamellar comprising alternating layers of lipids and siRNA (located on both the surface (siRNAsurface) and inner layers (siRNAinner)). This study investigated the kinetics of Lipoplex destabilization by serum proteins and the changes in destabilization products, as functions of siRNA and protein concentrations, and used quantitative pharmacology (QP) modeling to infer likely mechanisms.Experiments used five methods to separate, identify and quantify the products of Lipoplex destabilization by fetal bovine serum (FBS), i.e., ultrafiltration, centrifugation, confocal microscopy with quantitative image analysis, gel electrophoresis, and RT‐qPCR (quantitative reverse transcriptase polymerase chain reaction). The results show the products of Lipoplex destabilization depended on concentrations of both proteins and siRNA, with free or degraded siRNA as a major product (maximal release of 65%). Computational models were developed in MATLAB, are based on a system of ordinary differential equations solved with a variable‐step and variable‐order solver. Model parameters were determined by assigning initial estimates, followed by optimization using MATLAB fminsearch algorithm to obtain the best‐fit parameter values (i.e., lowest sum of squared errors (SSE) between model‐simulated and experimental results).We evaluated four models based in part on literature data on protein binding, Lipoplex structure and changes in protein‐Lipoplex interactions, and on our experimental results. Each model described a mechanism or a combination of mechanisms resulting in concentration‐dependent siRNA release. Two models, one depicting cooperative protein binding to Lipoplex and one depicting replacement of low affinity proteins from their Lipoplex binding sites by high affinity proteins, yielded unsatisfactory fit to the experimental results as indicated by the high SSE (5327–6835). In contrast, extending these models to include the additional feature of saturable or limited cationic lipid binding sites for which protein and siRNA compete yielded better fitting results (SSE of 4010 and 376).In summary, this study demonstrates the release of siRNA from Lipoplex due to interactions with serum proteins was dependent on the protein:siRNA molar concentration ratio (Ratio), and that the likely mechanism involves proteins competing and thereby releasing siRNAsurface from the cationic lipid binding sites, with accelerated siRNA release when high abundance low affinity proteins are replaced by low abundance high affinity proteins at higher Ratio, until all siRNAsurface is released.Support or Funding InformationSupported in part by research grants RO1 CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS.