Along with rigorous characterization, mRPZ was applied for in vitro cell transfection. Briefly, Cy5-tagged, eGFP-encoding mRNA-loaded mRPZ nanoparticles were suspended in Opti-MEM and administered to HEK293t in serum-containing media. After 48 hours, cells were fixed and stained with a nuclear dye (DAPI), and the resulting fluorescence was imaged with a microscope and quantified with a plate reader (Figure and S19). The presence of Cy5 located within the fixed cells, coupled with the expression of eGFP, indicates the successful delivery of mRNA by mRPZ and the subsequent expression of its corresponding protein. For comparison, cells treated with mRNA@ZIF-8 are absent of Cy5 and eGFP signals. Given that PEI is a known transfection reagent, mRNA-PEI transfection was also performed as a comparison, utilizing the same nitrogento-phosphate ratio of 9.3 as mRPZ. Interestingly, mRPZ performs five to ten times better than mRNA-PEI (PEI N/P=9.3) in HEK293t, HeLa, and CHO cells (Figure ). Specifically in HEK cells, mRPZ utilizing an N/P ratio of 9.3 outperforms mRNA-PEI complexes with an optimized N/P ratio of 28-30 (Figure ), which is consistent across PEI isoforms (Figure ). This performance increase may be explained by released ZIF-8 precursors shielding the polycationic charge of PEI and improving the mRNA release. Additionally, across all three cell lines, mRPZ outperforms Lipofectamine 2000 (Lipo 2k), and mRPZ performs better than or similar to Lipo MM, except in CHO cells. cell lines were above 92% (except Lipo MM) and 88%, respectively. This demonstrates that mRPZ is efficiently taken up by cells and causes minimal toxicity. Comparing the relative eGFP expression in all cell lines, mRPZ induces eGFP expression in 82.2%, 76.1%, and 49.5% of live cells for HEK293t, HeLa, and CHO cells, respectively (Figure ). In comparison to positive Lipofectamine controls, mRPZ performs equally well or slightly worse depending on the cell line and the specific Lipofectamine control. Interestingly, the Median Fluorescence Index (MFI), normalized with the percentage of GFP-positive cells, shows mRPZ performs similarly to Lipofectamine controls on a singlecell basis, except in comparison to Lipo MM in CHO cells (Figure ). mRPZ also outperforms mRNA-PEI at an N/P ratio of 9.3 and outperforms or equivalent performs to mRNA-PEI at an N/P ratio of 28 (PEI N/P=28) in terms of normalized MFI and percentage of GFP positive cells (Figure ). The normalized MFI measurement captures the intensity of eGFP expression on a single-cell basis. The increased MFI caused by mRPZ may allude to more mRNA strands being delivered into a single cell. Using nanoparticle tracking to obtain particle concentrations, we backcalculated the number of mRNA strands per nanoparticle to be about 33 (Figure ). Compared to known lipid nanoparticles, this is 11 to 16-fold more strands per particle, 10 providing some reasoning as to why the percentage of Cy5 and GFP positive cells are lower for mRPZ, but normalized MFI is higher or equivalent. Additional cell lines, RAW264.7, A549, and 231, were tested using mRPZ (Figure ). Generally, we find mRPZ underperforms compared to commercial lipid complexes, except in RAW 264.7 mouse macrophages where they perform equivalently to Lipo 2k. In comparison to mRNA-PEI, mRPZ outperforms or equivalently performs mRNA-PEI at both N/P ratios of 9.3 and 28.