(T1430-01-04) Ionizable Lipid Nanoparticles for Dual siRNA/Chemotherapy Delivery for B-cell ALL Treatment

Purpose: While B-cell Acute Lymphoblastic Leukemia (B-ALL) continues to have increasing rates of overall survival, the prognosis for cases of relapse and those refractory to treatment remains poor. Improved tyrosine kinase inhibitors and the advent of both monoclonal/bispecific antibodies have significantly improved the lowering of minimal residual disease in relapsed/refractory cases of B-ALL, yet the development of treatment resistance remains prominent [1]. With regards to chemotherapy resistance, upregulation of proteins with oncogenic potential commonly generates resistance. These oncogenic proteins can be downregulated through the use of small interfering ribonucleic acids (siRNAs) which inactivate and initiate degradation of the specific protein transcripts. Nucleic acid therapeutics unfortunately struggle with poor stability and undesirable off-target effects when administered in vivo. Ionizable lipid nanoparticles (LNPs) pose as a delivery platform for nucleic acids that can mitigate these biological hurdles [2]. Additionally, these LNPs can be engineered to incorporate cytotoxic agents that can be synergistically enhanced by siRNA mechanisms of action. Herein, we developed ionizable LNPs capable of synchronously delivering siRNA along with a hydrazone-modified variant of doxorubicin, aldoxorubicin that conjugates to free thiols. To the best of our knowledge, this is the first treatment strategy using LNPs to synchronously deliver siRNA and chemotherapy to B-ALL cells.

Methods: LNPs containing thiolated cholesterol, DLin-MC3-DMA, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxy poly(ethylene glycol) (DSPE-mPEG) were fabricated using a flash nanoprecipitation technique [3]. LNPs were encapsulated with negative control siRNA and then conjugated with aldoxorubicin via incubation for 2 h. Rigorous characterization of the nanoparticles was done using dynamic light scattering (DLS), electrophoretic light scattering (ELS), and transmission electron microscopy (TEM) imaging. Encapsulation efficiency and concentration of loaded siRNA were determined using the Quantifluor® RNA assay. Conjugation efficiency and concentration of aldoxorubicin were measured using the absorbance at 485 nm using spectrophotometry. 48-hour CellTiter-Glo® 2.0 Luminescent Cell Viability Assays were performed on REH-CRL cells to determine the cytotoxicity of all LNP formulations compared to free aldoxorubicin. Lastly, Cy5-conjugated siRNA was loaded into LNPs to assess cellular uptake and intracellular delivery of payloads in REH-CRL cells using flow cytometry and confocal microscopy, respectively.

Results: LNPs before and after conjugation with aldoxorubicin were in the size range of 85 – 96 nm and exhibited a polydispersity index of less than 0.3. The surface charge of all LNPs fabricated was neutral, as shown by ELS (Table 1). TEM imaging for aldoxorubicin conjugated as well as siRNA LNPs confirms spherical morphology (Figure 1). The encapsulation efficiency of siRNA was 85.7% while the conjugation efficiency of aldoxorubicin for LNPs with or without siRNA was 65.1% and 66.5%, respectively. LNPs were inherently non-cytotoxic to REH-CRL cells at total lipid concentrations ranging from 33 to 0.26 μg/mL, and showed a cell viability of >85%, following 48-hour incubation. Cytotoxicity of aldoxorubicin was enhanced when conjugated to LNPs with 95% confidence intervals for half-maximal inhibitory concentrations being 0.39 ± 0.05, 0.16 ± 0.02, and 0.14 ± 0.02 μg/mL for free aldoxorubicin, aldoxorubicin LNPs without siRNA, and aldoxorubicin LNPs with siRNA, respectively (Figure 2). Furthermore, flow cytometry analysis demonstrated a time-dependent cellular uptake of Cy5-siRNA LNPs REH-CRL cells whereas confocal microscopy confirmed cytosolic release of Cy5-siRNA from LNPs in REH-CRL cells.

Conclusion: Thiolated cholesterol containing ionizable LNPs allowed successful encapsulation of siRNA and aldoxorubicin and maintained greater morphological stability. These LNPs were efficiently taken up by B-ALL cells and intracellularly delivered siRNA. Of note, aldoxorubicin LNPs demonstrated superior toxicity in REH-CRL cells as compared to free aldoxorubicin. These preliminary findings suggest the potential of LNPs for combinatorial treatment strategies containing functional siRNAs and chemotherapeutic agents.

References: [1] Molina JC, Shah NN. Monoclonal Antibody-Based Treatment and Other New Agents for B-Lineage Acute Lymphoblastic Leukemia. In: Litzow MR, Raetz EA, editors. Clinical Management of Acute Lymphoblastic Leukemia, Cham: Springer International Publishing; 2022, p. 295–328.
[2] Tenchov R, Bird R, Curtze AE, Zhou Q. Lipid Nanoparticles─From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 2021;15:16982–7015.
[3] Hughes KA, Misra B, Maghareh M, Samart P, Nguyen E, Hussain S, et al. Flash nanoprecipitation allows easy fabrication of pH-responsive acetalated dextran nanoparticles for intracellular release of payloads. Discover Nano 2024;19:4.

Acknowledgements: This work was supported by National Institutes of Health—National Institute of General Medical Sciences (NIH-NIGMS) Tumor Microenvironment Centers of Biomedical Research Excellence (TME CoBRE) grant [2P20GM121322-06].

Table 1. Physicochemical characteristics of LNPs. Size and polydispersity were acquired via dynamic light scattering while electrophoretic light scattering was used to determine surface charges. Sizes of nanoparticles are represented by z-average (d.nm). Data represented as mean ± standard deviation (n=3).

Figure 1. Negative stain transmission electron microscopy images of lipid nanoparticles with aldoxorubicin conjugation (A.) and siRNA encapsulation (B.), respectively. The scale bar is 100 nm.

Figure 2. REH-CRL cytotoxicity after 48-incubation with free aldoxorubicin or aldoxorubicin conjugated to LNPs with or without negative control siRNA encapsulated. Viability was determined using CellTiter-Glo® 2.0 Luminescent Cell Viability Assay. Data represented as mean ± standard deviation (n≥4). Half-maximal inhibitory concentration (IC50) presented in μg/mL. Significance compared to free aldoxorubicin was determined using 2-way ANOVA analysis; * p < 0.0001.