Eye delivery of lipid nanoparticle mRNA for lanosterol synthase improves cataracts in rats

We subsequently examined the hemolytic activity of pB-UC18 LNPs in vitro. As illustrated in Fig. 2c, pB-UC18 LNPs induced no hemolytic activity at a final mRNA concentration of 2 μg mL^-1 under physiological pH, whereas obvious hemolytic activity occurred at acidic pH. These findings indicated that pB-UC18 LNPs were biocompatible at physiological pH. Furthermore, pH-sensitive membrane disruption of red blood cells mediated by pB-UC18 LNPs within the endosomal pH range suggested the endosomal escape potential of pB-UC18 LNPs.

To probe the uptake and endosomal escape of mRNA formulations, cells were treated with pB-UC18 LNPs encapsulating fluorescently labeled mRNA payloads (Cy5-labeled mRNA). After 4 h of treatment, we observed that partial Cy5 fluorescence signals colocalized with the endo-lysosome-specific tracers, while the residual signals were distributed throughout the cytoplasm. These observations suggested that pB-UC18 LNPs could facilitate the uptake of mRNA payloads and induce effective endosomal escape (Fig. 2d). To further elucidate the mechanism of cellular uptake, we examined the effects of various inhibitors of endocytosis on the uptake efficiency of pB-UC18 LNPs using flow cytometry. As shown in Fig. 2e, three inhibitors, chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis), methyl-β-cyclodextrin (MβCD, an inhibitor of caveolae-mediated endocytosis), and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, an inhibitor of macropinocytosis), showed significant inhibitory effects on particle uptake, indicating that pB-UC18 LNPs are internalized into cells via all three pathways.

We next designed hLSS mRNA according to its protein sequence (Supplementary Table 1). The coding sequence of hLSS mRNA was shown in Supplementary Table 2. To enhance the stability and translational capacity of mRNA, 5-methoxyuridine (5moU) was introduced into hLSS mRNA during in vitro transcription³². The single and clear band shown in the agarose gel validated the purity of 5moU-modified hLSS mRNA (Supplementary Fig. 2). Subsequently, we prepared hLSS mRNA-loaded pB-UC18 LNPs (hereinafter referred to as mLNPs) and tested whether exogenous hLSS mRNA delivered by pB-UC18 LNPs could be successfully translated into corresponding hLSS proteins. Immunofluorescence imaging confirmed elevated LSS levels in mLNPs-treated 293T, HeLa, and SRA01/04 cells (Fig. 2f). Moreover, western blot analysis further indicated that mLNPs induced upregulation of LSS expression (Fig. 2g and h).

Notably, despite being an avascular tissue, the mature lens possesses a unique microcirculatory system that facilitates the transport of mRNA formulations from the anterior chamber to the lens^33,34. In vivo bioluminescence imaging further demonstrated dose-dependent enhancement of mRNA delivery efficiency and protein expression after IC injection (Fig. 3e and f). At the same dose tested, pB-UC18 LNPs displayed more potent ocular delivery efficiency than clinically approved SM-102 LNPs (Fig. 3e and f).

Since the eye is one of the most elaborate organs, we conducted comprehensive safety assessments of mLNPs after IC injection. The cornea and lens were first assessed 24 h after IC injection using a slit-lamp microscope. In comparison with the uninjected group, the sham group and mLNPs group exhibited no significant ocular lesions (including eyelid irritation and redness, corneal opacification, and edema) except for corneal puncture marks (Fig. 4c). Further analysis of histological samples stained with hematoxylin and eosin (H&E) revealed that IC injection of mRNA formulations caused no apparent histological alterations in the eyes (Fig. 4d) and other major organs including the heart, liver, spleen, lungs, and kidneys (Supplementary Fig. 4), or structural changes in the ocular tissue (Fig. 4d). Besides, there were no statistically significant differences between the uninjected group and mLNPs group in routine blood parameters as well as hepatic and renal function (Supplementary Fig. 5).

H&E staining and TEM ultrastructural analysis reflected regular arranged lens fiber cells with no obvious vacuoles in the healthy group (Fig. 5e and f). However, irregular cellular arrangements and considerable vacuoles within the lens cortex were observed for the untreated and eLNPs cataract model groups (Fig. 5e and f). In contrast, these alterations were rare within the lens for the mLNPs cataract model group.

Taken together, the therapeutic effects observed in both cataract models demonstrated that ocular delivery of in vitro-transcribed hLSS mRNA via lipid nanoparticles was able to ameliorate both nuclear and galactose cataract in rats.

The purity of 5moU-modified hLSS mRNA (APExBio) was determined by 1% native agarose gel before encapsulation. LNP formulations were prepared through the previously reported method³¹. Briefly, pB-UC18 was synthesized via the condensation reaction followed by reduction of the Schiff base intermediate³¹. pB-UC18, along with helper lipids DOPE (Avanti) and PEGylated lipids DMG-PEG2k (TargetMol), were dissolved in ethanol at a molar ratio of 40:40:0 or 40:40:1 to form the ethanol phase. Meanwhile, mRNA was diluted with phosphate-buffered saline (PBS) to obtain the aqueous phase. The ethanol phase and mRNA aqueous phase were then rapidly mixed at an N:P ratio of 2:1 and a volume ratio of 1:9 and allowed to stand for 10 min prior to use.

The particle size and zeta potential of LNPs stored at ambient temperature for different time periods were measured using dynamic light scattering (Brookhaven, 90Plus PALS) and analyzed using the Particle Solution software. The particle size of freshly prepared pB-UC18 LNPs was also assessed by transmission electron microscopy (Thermo Fisher Scientific, Talos L120C).

Blood samples from Wistar rats were collected with EDTA-coated anticoagulant tubes. Red blood cells (RBCs) were isolated via centrifugation at 95 g for 10 min, washed, and suspended in PBS with pH 5.4 and 7.4. hLSS mRNA-loaded pB-UC18 LNPs at a mRNA concentration of 4 μg mL⁻¹ were then incubated with an equal volume of RBCs (4%, v/v) for 1 h at 37 °C and centrifuged (95 g) for 10 min at ambient temperature for imaging. RBCs lysed with 1% Triton X-100 were used as a positive control, while RBCs treated with PBS served as a negative control.

SRA01/04 cells obtained from Dr. Jun Zhao's lab were seeded at a density of 1.5 × 10⁴ cells per well in a 96-well plate. Following overnight culture, cells were pre-incubated with either PBS or inhibitors including CPZ (MedChemExpress, 10 μg mL⁻¹ final concentration), MβCD (MedChemExpress, 5 mM final concentration), and EIPA (MedChemExpress, 10 μg mL⁻¹ final concentration) for 30 min at 37 °C and then exposed to pB-UC18 LNPs containing Cy5-labeled mRNA (APExBio) at a final mRNA concentration of 2 μg mL⁻¹ for 4 h. After washing twice with PBS, cells were subjected to flow cytometric analysis (Agilent, NovoCyte 2000R) with the NovoExpress software. Gating strategy depicted in Supplementary Fig. 6. For confocal microscopy imaging, HeLa cells (C6330, Beyotime) were seeded at a density of 4 × 10⁴ cells per confocal dish (35 mm, Biosharp). Following overnight culture, cells were exposed to pB-UC18 LNPs containing Cy5-labeled mRNA at a final mRNA concentration of 2 μg mL⁻¹ for 4 h, stained with Lyso-Tracker Green (Beyotime), and imaged with confocal microscopy (Leica, TCS SP8).

293T (C6008, Beyotime), HeLa, and SRA01/04 cells were seeded in 96-well plates at densities of 2 × 10⁴, 1 × 10⁴, and 1.5 × 10⁴ cells per well, respectively, and allowed to culture overnight. Cells were then treated with pB-UC18 LNPs containing 200 ng of hLSS mRNA. Cells treated with PBS served as a control. Twenty-four hours post-treatment, cells were fixed by ethanol at −20 °C following supernatant removal, permeabilized with Triton X-100 (0.5%, v/v), and blocked with a bovine serum albumin solution (Servicebio, 5%, w/v) for 30 min. Cells were then incubated with LSS polyclonal antibody (Proteintech, Cat No. 18693-1-AP, 1:100) at room temperature for 90 min and FITC goat anti-rabbit IgG (Abclonal, Cat No. AS011, 1:100) at room temperature for 90 min and imaged using a fluorescence microscope (ZEISS, Vert.A1).

293T cells were seeded at a density of 1.2 × 10⁵ cells per well in a 24-well plate and allowed to culture overnight. Cells were then treated with pB-UC18 LNPs containing 1200 ng of hLSS mRNA for 24 h at 37 °C. Subsequently, cells were harvested and suspended in RIPA buffer supplemented with PMSF (Beyotime, 1 mM final concentration) for 60 min at 4 °C. The protein concentration in the lysate was quantified using a BCA protein assay kit (Beyotime). Samples were then diluted with loading buffer, heated for 10 min at 100 °C, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, proteins were then transferred onto a polyvinylidene difluoride membrane blocked with blocking buffer. Subsequently, the membranes were probed with antibodies against LSS (Proteintech, Cat No. 18693-1-AP, 1:500) or β-actin (Beyotime, Cat No. AA128, 1:7000) in blocking buffer for 1 h at 37 °C and washed three times with washing buffer. The blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Abclonal, Cat No. AS014, 1:7000) or horseradish peroxidase-conjugated goat anti-mouse IgG (Beyotime, Cat No. A0216, 1:7000) for 1 h at room temperature, washed three times with washing buffer, incubated with the western blotting substrate (Beyotime), and imaged with an imaging system (Thermo Fisher Scientific, iBright FL1000). Quantification of Western blot bands was done by densitometry using the ImageJ software.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Southern University of Science and Technology (protocol # 20200330-16) and conducted in accordance with the guidelines for the care and use of laboratory animals. Wistar rats were housed under standard conditions (12 h light and 12 h dark cycles, 20–22 °C, 40–60% humidity) with food and water provided ad libitum. Three-week-old Wistar rats were anesthetized using isoflurane before mydriasis with eyedrops containing 0.5% phenylephrine and 0.5% tropicamide and injected with 1.5 μL of FLuc mRNA-loaded pB-UC18 LNPs at a FLuc mRNA (APExBio) dose of 300 ng per injection through IVT, SCJ, IC, or SR injection. At 4 h, 26 h, or 48 h post-injection, D-luciferin potassium salt was administered intraperitoneally at a dose of 125 mg kg⁻¹. Ten minutes later, rats were anesthetized with isoflurane, imaged using a small animal imaging system (PerkinElmer, Lumina LT) and analyzed using the Living Image software. At 4 h and 48 h post-injection, the eyeballs were further harvested and dissected into the lenses and other tissues for ex vivo imaging. In a separate experiment, the eye, heart, liver, spleen, lungs, and kidneys were harvested 4 h post-injection for ex vivo imaging. For the dose response study, Wistar rats receiving 1.5 μL of FLuc mRNA-loaded pB-UC18 LNPs at different mRNA doses (40, 120, and 200 ng per injection) through IC injection were imaged at 4 h post-injection. FLuc mRNA-loaded SM-102 LNPs prepared at a SM-102:FLuc mRNA weight ratio of 10.75:1 and a SM-102 (Macklin):DSPC (Avanti):cholesterol (Sangon Biotech):DMG-PEG2k (TargetMol) molar ratio of 50:10:38.5:1.5 were used as a positive control⁴⁴.

Three-week-old Wistar rats were anesthetized using isoflurane before mydriasis with eyedrops containing 0.5% phenylephrine and 0.5% tropicamide and injected with 1.5 μL of mLNPs at a hLSS mRNA dose of 300 ng per injection through IC injection. Eyes from untreated rats served as a control. At 24 h post-injection, ocular tissues were harvested for preparation of paraffin-embedded eye tissue sections using the standard procedures. Slices were then blocked with 5% BSA at room temperature for 30 min after antigen retrieval with TE buffer (pH 9) and incubated overnight at 4 °C with LSS polyclonal antibody (Proteintech, Cat No. 18693-1-AP, 1:100) and Alexa Fluor 488-labeled goat anti-rabbit IgG (Servicebio, Cat No. GB25303, 1:400) at room temperature for 50 min. Slices were further stained with a DAPI staining solution (Servicebio) for 10 min at room temperature, rinsed three times with PBS, and incubated with Tissue Autofluorescence Bursting Agent (Servicebio) in droplet form for 5 min. After sealing, slices were imaged using a digital slide scanner (3DHISTECH, Pannoramic MIDI).

Wistar rats aged 3 or 8 weeks were anesthetized and intracamerally injected with 1.5 μL of mLNPs according to the above protocol. Rats were then euthanized 24 h post-injection, and the lenses were excised and weighed. Subsequently, 20 μL of lanosterol analog, cholesterol-d7 (1.0 mg mL⁻¹, served as an internal standard), was added to lenses, which was then homogenized in 600 μL of chloroform/methanol mixture (v:v, 1:2). Then, 300 μL of chloroform and 450 μL of saturated potassium chloride solution were added. The mixture was vortexed for 5 min and centrifuged at 3500 g for 5 min. The upper aqueous phase was then extracted twice with chloroform (300 μL each), combined with the previous lower organic phase, and evaporated under a nitrogen stream. Samples were resuspended in 100 µL of methanol for LC-MS quantitative analysis (SCIEX, 4000 QTRAP). The operating conditions for LC-MS were as follows: the ion source, atmospheric pressure chemical ionization, was set to run in the positive ion mode; the chromatographic column (1.8 μm, Agilent) with dimensions 100 × 4.6 mm was kept at 40 °C; the injection volume was 5 μL; chromatographic separation was done under isocratic conditions, with a mobile phase of isopropanol/acetonitrile/water/formic acid (48.75:48.75:2.4975:0.0025%, v/v/v/v) and flow rate of 0.8 mL min^-1.

Seven-week-old male Wistar rats receiving 1.5 μL of intracamerally injected mLNPs according to the above protocol were subjected to ocular slit-lamp (Bolan, BL-66B) examination 24 h post-injection. Uninjected rats and rats receiving a sham procedure (cornea puncture only) were included as two control groups. Blood samples from different groups were collected for measuring routine blood parameters and hepatic and renal function. Meanwhile, the major organs, including the eye, heart, liver, spleen, lungs, and kidneys, were harvested for H&E staining using the standard procedures.

Wistar rats with eyes open at approximately 14 days of age were randomly divided into four groups: the healthy group (without injection of sodium selenite), the untreated cataract model group (without injection of formulations), the eLNPs-treated cataract model group, and the mLNPs-treated cataract model group. At 14 days of age, one eye of each rat was intracamerally injected with eLNPs or equivalent mLNPs containing 200 ng of hLSS mRNA, while the contralateral eye from the same rat was left untreated for all treated groups. At 15 days of age, the selenium-induced cataract model was established by subcutaneous injection of sodium selenite solutions at a dose of 3.14 mg kg⁻¹. At 16 and 18 days of age, the same eyes of rats were further injected with the above dose of eLNPs or mLNPs through the anterior chamber. At 25 days of age, rats underwent slit-lamp examination to assess the stage of cataract development according to the cataract classification⁴⁵. At 27 days of age, rats were euthanized, and their lenses were photographed, fixed, and subjected to H&E staining and transmission electron microscopy (Hitachi, HT7800).

The galactose-induced cataract model was established by intraperitoneal injection of D-galactose solutions (50%, w/v, Aladdin) into Wistar rats once daily at a dose of 10 mL kg⁻¹ for three days (from 31 to 33 days of age) and twice daily at a dose of 15 mL kg⁻¹ for the following three weeks (from 34 to 54 days of age). At 54 days of age, rats were randomly divided into three groups: untreated cataract model group, eLNPs-treated cataract model group, and mLNPs-treated cataract model group. Age-matched rats without injection of both D-galactose and formulations served as the healthy control. For all treated groups, eLNPs or equivalent mLNPs containing 300 ng of hLSS mRNA were injected into the anterior chamber of two eyes every four days (from 54 to 62 days of age). Meanwhile, slit-lamp examination was conducted every four days (from 54 to 62 days of age). The area of each lens and the turbidity area within the lens were calculated using the ImageJ software. At 69 days of age, ocular tissues were harvested for H&E and TUNEL staining using the standard procedures.

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