After evaluating mRNA length, we conducted in vitro transfections to assess mRNA expressibility. Briefly, Cy5-eGFP-mRNA samples encapsulated within ZIF-8 were prepared with nuclease-free water, methanol, and ethanol and isolated with centrifugation. The particles were then introduced to HEK293t cells. As controls, mRNA samples exfoliated from mRNA@ZIF-8 (Exf. mRNA@ZIF-8), prepared with all mentioned solvents, were applied to transfect the same cells using Lipo MM. Following a 24-hour incubation, cells were fixed, and fluorescence was observed under a microscope, with quantification performed using a plate reader (Figure ). The presence of Cy5 (red) indicated mRNA uptake, while eGFP (green) indicated expressed mRNA. Interestingly, mRNA@ZIF-8 synthesized with any of the mentioned solvents did not induce eGFP expression, and no Cy5 signal was visible. The lack of Cy5 signal suggests a delivery issue with mRNA@ZIF-8 as exfoliated mRNA delivered with Lipo MM exhibited eGFP expression. Additionally, eGFP expression levels were compared among exfoliated mRNA samples. While particles synthesized with water and ethanol showed statistically similar eGFP expression, samples synthesized with methanol exhibited significantly lower eGFP expression, consistent with our earlier findings that mRNA is incompatible with the synthetic conditions in methanol. mRNA@ZIF-8 synthesized with any solvent maintained mRNA length and expressibility, but significant mRNA degradation occurred with methanol-based synthesis conditions and when mRNA was incubated with ZIF-8 in water or methanol. Interestingly, ZIF-8-mRNA co-incubation causes degradation in all solvents tested except PBS and EtOH for up to 4 hours (Figure ). Moreover, the deviation of mRNA@ZIF-8 particles synthesized in water from simulated ZIF-8 PXRD patterns and morphology indicated that these biomimetic mineralization concentrations were insufficient for uniform ZIF-8 nanoparticle formation. Based on these findings, ethanol-based coprecipitation was identified as the optimal solvent for mRNA encapsulation in ZIF-8. Ethanol-based coprecipitation maintained mRNA length better than tested solvents when dried and stored for up to 7 days (Figure ) and was stable in PBS and EtOH post-synthesis (Figure ). Having identified a compatible synthetic strategy for one-pot coprecipitation of mRNA@ZIF-8, we spearheaded the delivery issue of mRNA@ZIF-8.