Effects of local structural transformation of lipid-like compounds on delivery of messenger RNA

Lipid-like nanoparticles (LLNs) have shown great potential for RNA delivery. Lipid-like compounds are key components in LLNs. In this study, we investigated the effects of local structural transformation of lipid-like compounds on delivery of messenger RNA. Our results showed that position change of functional groups on lipid-like compounds can dramatically improve delivery efficiency. We then optimized formulation ratios of TNT-b10 LLNs, a lead material, increasing delivery efficiency over 2-fold. More importantly, pegylated TNT-b10 LLNs is stable for over four weeks and is over 10-fold more efficient than that of its counterpart TNT-a10 LLNs. Additionally, the optimal formulation O-TNT-b10 LLNs is capable of delivering mRNA encoding luciferase in vivo. These results provide useful insights into the design of next generation LLNs for mRNA delivery.


H NMR and MS (Supporting information).
In order to investigate the delivery efficiency of TNT-a 10 and TNT-b 8 to TNT-b 14 , we utilized a luciferase expression assay in Hep 3B cells, a human hepatoma cell line 5 . TNTs were formulated with DOPE, cholesterol, DMG-PEG 2000 and Fluc mRNA using the previously optimized formulation ratio (TNTs/DOPE/Cholesterol/ DMG-PEG 2000 = 20/30/40/0, Fig. 2). Cells were treated with freshly formulated TNT LLNs for 6 h and luciferase activity was then quantified. As shown in Fig. 2A, TNT-b 10 LLNs showed higher transfection efficiency than TNT-b 8 , TNT-b 12 , and TNT-b 14 LLNs. Moreover, TNT-b 10 LLNs was over 2-fold more efficient compared to TNT-a 10 , structure of which is similar to that of TNT-b 10 except the position exchange of hydroxyl and amino groups. These results indicate that not only the length of carbon chains but also the position of functional groups of lipid-like compounds have dramatic effects on delivery efficiency.
In order to evaluate the effects of phospholipids, we next formulated TNT-b 10 with DSPC or POPE, another two widely used phospholipids in nanoparticle formulations. Consistent with previous studies 5, 6 , DOPE-formulated LLNs were more efficient for mRNA delivery compared to DSPC and POPE-formulated LLNs (Fig. 2B). Consequently, DOPE was selected for further in vitro optimization of TNT-b 10 LLNs. Previously, we utilized an orthogonal experimental design to optimize the formulation ratio, which has been demonstrated as a useful tool 5 . In this study, we assigned three levels for each component, which provide 27 formulations (Supporting information, Table S1 and S2). Formulation 25 (F25) with a combination of TNT-b10/ DOPE/Cholesterol = 30/40/35 was 2-fold more efficient compared to the initial formulation (TNT-b10/DOPE/ Cholesterol = 20/30/40) (Fig. 3A). We then performed a correlation analysis of particle properties and delivery efficiency. We observed positive correlation between luciferase intensity and entrapment efficiency, while no significant correlation was found between luciferase intensity and particle size, zeta potential, or cell viability ( Fig. 3B-E).
Yet, we observed that the particle size of F25 increased significantly several hours after formulation ( In order to visualize cellular uptake of O-TNT-b 10 LLNs, we treated cells with O-TNT-b 10 LLNs encapsulated Alexa-Fluor 647 labeled RNA (red). 3 h after treatment, Hep 3B cells were fixed with formaldehyde. Nuclei and membrane were then stained with DAPI (blue) and Alexa-Fluor 488 conjugate of wheat germ agglutinin (green), respectively. A dramatic cellular uptake of O-TNT-b 10 LLNs was detected using fluorescence microscopy compared to a control group treated with free labeled RNA (Figs 5).
Lastly, in order to study delivery efficiency of O-TNT-b 10 LLNs in vivo, O-TNT-b 10 LLNs were administered in mice via three injection routes: intravenous (i.v.), intraperitoneal (i.p.), and subcutaneous (s.c.). Intravenous and intraperitoneal injections of O-TNT-b 10 LLNs showed high expression of luciferase in the liver and spleen. No detectable signal was observed in kidney, heart, and lung. No signal was detected in mice treated with subcutaneous injection of O-TNT-b 10 LLNs and intravenous injection of free mRNA. Interestingly, a significantly higher signal was detected in the spleen compared to the liver (over 10-fold) in mice with intravenous injections of O-TNT-b 10 LLNs (Fig. 6A). These results demonstrated that O-TNT-b 10 LLNs is capable of delivering mRNA in vivo and shows a unique expression in the spleen may have potential therapeutic applications for spleen disorders. A preliminary histology analysis indicated no significant pathological changes in all treated groups compared with the control group (Fig. 6B).

Discussion
In conclusion, we designed and synthesized TNT-b 8 to TNT-b 14 in order to investigate the effects of local structural transformation on mRNA delivery. TNT-b 8 to TNT-b 14 were composed of a phenyl ring, three amide linkers, and three amino lipid tails. Compared to the structure of TNT-a 10 , a previously reported lipid-like compound, TNT-b 8 to TNT-b 14 exchanged the positions of hydroxyl and amino groups. According to an in vitro luciferase assay, TNT-b 10 LLNs were 2-fold more efficient than TNT-a 10 LLNs, demonstrating the importance of local structural transformations. The correlation analysis of delivery efficiency and particle properties showed a positive correlation between delivery efficiency and mRNA entrapment percentage, consistent with our results reported previously 5 . Optimization of formulation ratios improved delivery efficiency over 2-fold. Because pegylation stabilized the LLNs, we identified an optimal formulation O-TNT-b 10 LLNs, 10-fold more efficient compared to TNT-a 10 LLNs formulated with the same formulation ratios. Cellular uptake of O-TNT-b 10 LLNs was visualized by fluorescence imaging analysis. More importantly, O-TNT-b 10 LLNs is capable of delivering mRNA  analysis between transfection efficiency and cell viability, particle size, zeta potential, and entrapment efficiency. Positive correlation between luciferase intensity and entrapment efficiency was observed, while no significant correlation was found between luciferase intensity and particle size, zeta potential, or cell viability.  To a solution of compound 1 (5 g) and triethylamine (Et 3 N, 10 mL) in DMF (20 mL) was added methanesulfonyl chloride (MsCl, 5 mL) dropwise. After reaction, the mixture was poured into ice-water (100 mL), filtered and dried overnight. Then the intermediate was reacted with excessive N-methyl-1-phenylmethanamine (20 g) overnight at 90 °C. The reaction mixture was purified using silica gel chromatography to obtain compound 2. The resulting compound 2 was hydrogenated at 130 psi in the presence of Pd/C at RT to give compound 3. Compound 3 (0.2 mmol), epoxide (0.9 mmol), and TEA (0.8 mmol) were mixed and stirred at RT for 30 min, then reacted at 150 °C under microwave for 5 h. The mixture was purified by silica gel chromatography to afford TNTs. Structures  Preparation and characterization of LLNs encapsulated FLuc mRNA. TNT LLNs were formulated with TNTs, phospholipid (DOPE, DSPC or POPE), cholesterol and FLuc mRNA as reported previously 5 . Briefly, TNT, phospholipid, and cholesterol were dissolved in ethanol at a molar ratio of 20:30:40, and mRNA was dissolved in 10 mM sodium acetate buffer (pH = 3).The ethanol phase was then mixed well with equal volume of mRNA solution (the weight ratio of TNT: mRNA = 10: 1). Finally, the formulated LLNs were diluted by equal volume of PBS. For in vivo experiments, LLNs were formulated using a microfluidic-based mixing device (Precision Nanosystems). The particle size of formulations was determined by dynamic light scattering using a Zetasizer NanoZS (Malvern, Worcestershire, UK) at a scattering angle of 173°. The encapsulation efficiency of mRNA in LLNs was quantified by a RiboGreen assay.

Cell transfection and formulation Optimization. Hep 3B cells purchased from American Type Culture
Collection (Manassas, VA) were seeded into 96-well opaque white plates in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% heat inactivated FBS at a density of 20,000 cells per well. After overnight incubation, cells were transfected by LLNs containing 200 ng of FLuc mRNA for 6 h. The culture medium containing formulations was then discarded, and luciferase activity was quantified using a SpectraMax M5 microplate reader after adding 50 μL of serum-free EMEM and 50 μL of Bright-Glo luciferase reagent (Promega). In order to optimize formulation ratio of TNT-b 10 LLNs, 27 formulations prepared using the ratio listed in Supplementary  Table S1 were evaluated with the above-mentioned method. Cells treated with free FLuc mRNA were used as a control group. Transfections were performed in triplicate.
Cytotoxicity assay. The cytotoxicity of formulations against Hep3B cells was determined using an MTT assay 5 . Hep 3B cells were seeded in 96-well clear plates (20,000 cells/well) overnight and were then treated with LLNs (equivalent to 200 ng of FLuc mRNA). After 6 h treatment, MTT was added and incubated for another 4 h. The medium was then removed, and 150 μL of dimethylsulfoxide was added. Ten minutes later, the absorbance at a wavelength of 570 nm was measured on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Cell viability was normalized by untreated cells.
Cryo-transmission electron microscopy. A small volume (3-5 μL) of samples was added to a lacey carbon coated 300 mesh copper grid. The grid was blotted and immediately plunged into liquid ethane to rapidly form a thin film of amorphous ice and then transferred to a Gatan 626 cryo-transfer holder (Gatan, Pleasanton, CA). Cryo-transmission electron microscopy (Cryo-TEM) images were taken at low dose conditions in a Tecnai F20 S/TEM (FEI, Hillsboro, OR). The frozen grids were kept under liquid nitrogen temperature at all times. Cellular uptake. Hep3B cells were seeded in a sterile 6-well plate and grown to about 70% confluent prior to treatment. Cells were then treated with free labeled RNA or O-TNT-b 10 LLNs containing labeled RNA (the final concentration of labeled RNA is 0.25 μg/mL). After 3 h treatment, cells were rinsed and fixed by 4% formaldehyde for 10 min. Cells were then washed three times and stained with DAPI and Alexa-Fluor 488 conjugate of wheat germ agglutinin (final concentration of 1 μg/mL). Cells were washed three times again and mounted onto slides using a ProLong diamond antifade mountant reagent. Images were taken using an ECLIPSE Ti inverted fluorescence microscopy (Nikon, Japan). mRNA delivery in vivo and histopathological analysis. All procedures in animal studies conducted at The Ohio State University were approved by the Institutional Animal Care and Use Committee (IACUC) and were consistent with local, state and federal regulations as applicable. C57BL/6 mice (3 mice/per group) were injected free FLuc mRNA or FLuc mRNA encapsulated O-TNT-b 10 LLNs at a dose of 0.5 mg/kg via three administration routes (i.v., i.p. and s.c.) 34 . Six hours after administration, D-luciferin substrate (150 μL, 30 mg/mL) were then i.p. injected into the mice. Eight minutes later, mice were sacrificed and organs including heart, liver, spleen, lung and kidney were harvested. The luminescence was immediately measured by a Xenogen IVIS imaging system (Caliper, Alameda, CA) and normalized against organ weight. For histopathological analysis, organs were fixed overnight in 10% formaldehyde and transferred to 70% ethanol. After paraffin embedding, sectioning and hematoxylin and eosin staining, histopathological examination was conducted using an ECLIPSE Ti inverted fluorescence microscopy (Nikon, Japan).