EGFR-targeted Lipid Nanoparticles for Selective mRNA Delivery to the Placenta

Introduction:: Obstetric complications, such as preeclampsia and fetal growth restriction, stem from dysfunctional placental development and are a major cause of maternal and fetal morbidity and mortality. Despite the prevalence of these disorders, treatment options are severely lacking given ethical concerns regarding potential toxicity of small molecule therapeutics to the unborn fetus. Lipid nanoparticle (LNP)-mediated delivery of messenger RNA (mRNA) is a promising approach for the treatment of pregnancy disorders, as LNP composition can be tuned for selective organ delivery. Specifically, the use of targeting moieties can shunt LNP accumulation to the placenta—reducing off-target effects and minimizing fetal exposure. The placenta has rich expression of the epidermal growth factor receptor (EGFR); further enhanced EGFR expression is characteristic of many placental disorders. In this work, we utilize click chemistry to formulate EGFR-targeted LNPs for selective mRNA delivery to the placenta during pregnancy.

Materials and Methods:: LNPs encapsulating luciferase mRNA were synthesized via microfluidic mixing with the inclusion of increasing amounts of polyethylene glycol (PEG)-azide compared to PEG. EGFR IgG1 antibodies were labeled with dibenzocyclooctyne (DBCO). Azide-containing LNPs were incubated with DBCO-labeled antibodies in a click chemistry reaction to create EGFR-targeted LNPs. LNPs were physiochemically characterized using size, surface charge, encapsulation efficiency and relative pKa measurements. Cytotoxicity and luciferase mRNA LNP delivery were assessed in vitro 24 hours after treatment in the JEG-3 placental trophoblast cell line. In vivo mRNA LNP delivery was evaluated 6 hours after intravenous injection in pregnant mice using an in vivo imaging system (IVIS).

Results, Conclusions, and Discussions:: Using click chemistry, we synthesized LNPs with increasing ratios of PEG-azide: PEG (1:7, 1:5, 1:3, 1:2) thereby increasing available sites for antibody conjugation. EGFR LNPs demonstrated an increase in size and a decrease in surface charge, suggesting an effective click reaction. Luciferase mRNA delivery was evaluated in vitro and in vivo and compared to a standard LNP formulation S1 (no azide) and a standard azide LNP formulation A1 (azide, no antibody). Three out of four EGFR-LNP groups outperformed the standard S1 and A1 formulations in vitro. Specifically, the 1:2 and 1:3 LNP formulations resulted in approximately a 9- and 8- fold increase in luciferase delivery, respectively. No cytotoxicity was observed in vitro across all LNP treatment groups. Interestingly, the 1:5 LNP formulation demonstrated significantly higher bioluminescent signal in mouse placentas in vivo compared to S1 and A1 formulations, with no bioluminescent signal observed in the fetuses.

This work demonstrates the use of click chemistry to create EGFR-targeted LNPs capable of selective mRNA delivery to the placenta when compared to standard, non-targeted formulations. This targeted LNP platform holds great potential to be used for organ-specific delivery of nucleic acid therapies for a broad range of diseases. Specifically, EGFR-targeted LNPs can be employed to treat a variety of obstetric complications during pregnancy, such as preeclampsia and fetal growth restriction, while minimizing off-target effects and avoiding fetal exposure. Future work will investigate delivery of therapeutically relevant mRNA to cellular subtypes of the placenta.

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