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. Author manuscript; available in PMC: 2022 Dec 19.
Published in final edited form as: ACS Appl Mater Interfaces. 2020 Jul 28;12(32):35835–35844. doi: 10.1021/acsami.0c08268

Surface-Functionalized PEGylated Nanoparticles Deliver Messenger RNA to Pulmonary Immune Cells

Xiyu Ke a,b, Lillie Shelton a,b, Yizong Hu a,c, Yining Zhu a,b, Emily Chow d, Haoyu Tang a, José Luis Santos e,*, Hai-Quan Mao a,b,c,f,*
PMCID: PMC9762545  NIHMSID: NIHMS1852234  PMID: 32659078

Abstract

Nanoparticles designed as messenger RNA (mRNA) carriers to deliver gene medicine have shown great potential to change the way lung disease states are managed. Controlling their delivery to the lung and the transgene expression in a specific population of cells remains a challenge. Here, we developed a series of nanoparticles with polyethylene glycol (PEG) corona prepared by condensing mRNA with PEG-grafted polyethyleneimine (PEI-g-PEG) with different PEG terminal functional groups and grafting ratios. PEGylated nanoparticles (PEG grafting ratio was 0.5%) with amino or amino acid terminal groups showed the highest transgene expression levels in the lung following systemic administration, and cell profiling analysis indicated that pulmonary immune cells contributed to the majority of expression. We also showed that these nanoparticles can be prepared by the flash nanocomplexation method, which is a scalable and reproducible process, yielding lyophilizable nanoparticles that were stable for at least 4 months at −20 °C. These results suggest that these surface-functionalized PEGylated nanoparticles may serve as desirable carriers to deliver mRNA to the lung for pulmonary immunomodulation.

Keywords: mRNA, PEGylated nanoparticles, terminal groups, lung delivery, pulmonary immune cells

Graphical Abstract

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1. INTRODUCTION

Messenger RNA (mRNA) based gene therapy has attracted broad attention for cancer immunotherapy, protein replacement therapy, intracellular reprogramming, and gene editing over the past two decades.1–3 Compared to plasmid DNA, mRNA therapeutics hold several advantages: (1) higher transfection efficiency in non-dividing cells; (2) bypassing nuclear translocation barrier; (3) transient and controllable gene expression; and (4) avoiding the risk of host genome integration.4 The naked mRNA is unable to permeate tissues and cell membrane barriers and mediate protein expression inside the target cells. Several carriers have been developed over the past few years to overcome these challenges for mRNA delivery. Cationic lipids and ionizable lipids have been explored as carriers to form lipid-based formulations (i.e. liposomes, lipoplexes and lipid nanoparticles (NPs)) with mRNA. By manipulating the chemical structure of the lipids as well as the chemical composition of lipid excipients, high level and tissue-specific gene expression could be achieved.5–9 As an alternative with a higher degree of structure flexibility, cationic polymers including polyethyleneimine (PEI), poly(amino acid)s and poly(β-amino ester)s and their derivates have been characterized for mRNA delivery.10–12 For in vivo applications, delivery of mRNA to tissues beyond the liver and targeting specific population of cells within an organ remain particularly challenging. A recent report on a polymer-lipid hybrid mRNA delivery revealed lung-targeted transfection primarily in endothelial cells.9, 13 In another study, a high-throughput method using DNA barcodes and the Cre-Lox system identified two lipid NPs that efficiently deliver mRNA to the splenic endothelial cells.14

Linear PEI is considered one of the most potent non-viral vectors for DNA delivery with several formulations currently in clinical trials.15–16 However, PEI has several limitations including high toxicity and low colloidal stability at physiological conditions as a result of its non-biodegradable nature and high overall positive charge.17–18 In order to address these limitations, our group and others has designed several carriers based on polyethylene glycol (PEG)-grafted PEI (PEI-g-PEG).19–22 These systems form micelle NP structures with PEI/DNA in the core and a PEG corona. Due to the anti-fouling effect of PEG, these PEI-g-PEG/DNA NP systems showed higher colloidal stability and lower toxicity than their PEI/DNA NP counterparts. As expected, the performance of these PEI-g-PEG/DNA NPs are significantly influenced by the PEG chain length and PEG grafting degree.19 Recently, we have shown that PEG terminal group also plays an important role in cellular uptake and transfection efficiency of PEI-g-PEG/DNA NPs.22

Here we report a new series of PEI-g-PEG carriers with various PEG terminal groups and different PEG grafting ratios as mRNA carriers. We examined the effect of these parameters on mRNA compaction capacity, encapsulation efficiency (EE), cellular uptake, endosomal escape, and transfection efficiency in DC2.4 mouse dendritic cells and PC3 human prostate cancer cells. Furthermore, we investigated the biodistribution and gene delivery efficiency of these NPs in Balb/c mice and identified the specific cell populations transfected by the NPs using Ai14 mouse model with Cre recombinase activated tdTomato expression system. In addition, we assessed a scalable production method, flash nanocomplexation (FNC), to manufacture these PEI-g-PEG/mRNA NPs in a reproducible manner for future clinical translation.23–28

2. EXPERIMENTAL SECTION

2.1. Materials.

Linear polyethyleneimine HCl salt (Mn of PEI = 22 kDa) was kindly provided from Polymer Chemistry Innovations, Inc. (Tucson, AZ). NHS-PEG12-SPDP, dithiothreitol (DTT), AlamarBlue, eBioscience Fixable Viability Dye eFluor 780 and red blood cell lysis buffer were purchased from Thermo Fisher Scientific (Waltham, MA). Heparin sodium salt, Hoechst 33342, collagenase I, collagenase XI, DNase I and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO), unless specified otherwise. CleanCap® Firefly Luciferase mRNA was obtained from TriLink Biotechnologies (San Diego, CA). Antibodies including Alexa Fluor® 488 anti-mouse CD31 (Catalog no. 102514), APC anti-mouse CD326 (Ep-CAM) (Catalog no. 118214), Brilliant Violet 421™ anti-mouse CD45 (Catalog no. 103134), and all the isotype controls were bought from BioLegend (San Diego, CA). Human metastatic prostate cancer PC3 cells and human melanoma B16F10 cells expressing Galectin8 (Gal8)-GFP fusion protein (B16F10-Gal8-GFP) were kindly provided by Dr. M. Pomper’s lab and Dr. J. Green’s lab, respectively, at Johns Hopkins School of Medicine. Mouse dendritic cells DC2.4 were from Millipore Sigma (Burlington, MA). PC3 cells were cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 atmosphere. DC2.4 cells and B16F10-Gal8-GFP cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% FBS, 2 mM L-Glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2 atmosphere.

2.2. Synthesis and characterizations of PEI-g-PEG polymers.

The PEI-g-PEG polymers with different PEG terminal groups and PEG grafting ratios (0.5, 1.5, 3, 5 and 10%) were synthesized following our reported protocol.22 The PEG grafting ratio and the modification degree of the PEG terminal groups were determined using the methods reported previously.22 Additional details are found in the Supporting Information (SI) section.

2.3. Preparation and characterizations of PEI-g-PEG/mRNA NPs.

The PEI-g-PEG/mRNA NPs were prepared at N/P ratio 8 by mixing the luciferase mRNA solution with the same volume of PEI-g-PEG polymer solution by vortexing for 10 s. The obtained NP suspension was incubated at room temperature for 15 min before using. PEI/mRNA NPs were prepared under the same conditions by replacing PEI-g-PEG with PEI. The size distribution and zeta potential of the NPs were characterized using a dynamic light scattering (DLS) Zetasizer Nano (Malvern Instruments, Worcestershire, UK) at room temperature. Each sample was measured for three runs and the data are reported as the mean ± standard deviation.

2.4. EE measurement and heparin challenge assay.

The EE of various PEI-g-PEG/mRNA NPs was evaluated by measuring the concentration of free mRNA in the NP solutions using Quant-iT RiboGreen RNA assay kit.29 The stability of various PEI-g-PEG/mRNA NPs was evaluated using heparin assay. Briefly, the NPs were treated with heparin solutions at different concentrations and the amount of free mRNA released from the NPs was measured using Quant-iT RiboGreen RNA assay kit. The HC50 values (heparin concentration to achieved 50% mRNA release) were calculated based on the dose-responsive curves using OriginLab.

2.5. Cytotoxicity assay.

Both PC3 and DC2.4 cells were seeded onto 96-well plates at a cell density of 1 × 105 cells/mL, and 24 h later, the cells were treated with various mRNA NPs (N/P ratio 8) at a dose of 1 μg/mL. After 24 h of incubation, the cell viability was then measured using Alamarblue assay following the manufacturer’s protocol.

2.6. Transfection assay.

Both PC3 and DC2.4 cells were seeded onto 96-well plates at a cell density of 1 × 105 cells/mL. After 24 h of incubation, the cells were treated with various mRNA NPs (N/P ratio 8) at a dose of 1 μg/mL. At 24 h later, cells were treated by lysis buffer and the luciferase expression level was measured using Luciferase Assay System (Promega, WI) and normalized to the total protein level as measured by BCA analysis.

2.7. Cellular uptake assay.

The cellular uptake level of various mRNA NPs in PC3 and DC2.4 cells were measured using flow cytometry. Briefly, the cells were seeded onto 24-well plates at a cell density of 1 × 105 cells/mL. After an incubation period of 24 h, the cells were treated with various mRNA NPs (N/P ratio 8) prepared using the mixture of Cy5-labeled mRNA and unlabeled mRNA (molar ratio 1:9) at a dose of 1 μg/mL. Four hours later, the cells were collected, washed by PBS, and then subjected to flow cytometry analysis. The mean fluorescence intensity and the percentage of Cy5 positive cells were recorded.

2.8. Endosomal escape assay.

B16F10 cells expressing Gal8-GFP fusion protein were seeded onto 24 wells plate at a density of 1 × 105 cells/mL. After 24 h of incubation, the cells were treated with various mRNA NPs (N/P ratio 8) at a dose of 1 μg/mL. At 24 h post dosage, the cells were stained by Hoechst 33342, fixed by 4% paraformaldehyde and thoroughly washed by PBS. The plate was mounted onto a Cellomics Arrayscan VTI high-content screening platform and subjected to scanning of endosomal rupture events (shown as green fluorescent puncta) using the algorithm of Spot Detection V4.30

2.9. Biodistribution and in vivo gene expression assay.

All protocols for the use of animals were approved by the Johns Hopkins Institutional Animal Care and Use Committee. Female Balb/c mice (6-week old) were purchased from Jackson Laboratories and allowed to acclimate for 1 week prior to experiments. The mice were randomly divided into 9 groups (n = 3) and treated with NP1-NP8 at PEG grafting ratio 0.5% and PEI/mRNA NPs respectively at a mRNA dose of 0.5 mg/kg via intravenous (i.v.) injection. The NPs (N/P ratio = 8) were prepared using the mixture of Cy5-labeled luciferase mRNA and unlabeled luciferase mRNA (molar ratio = 1:1). At 24 h post dosing injection of NPs, the mice were treated with D-luciferin solution via intraperitoneal (i.p.) injection. The mice were then sacrificed (3 min after D-luciferin injection) and the main tissues including heart, lungs, liver, spleen and kidneys were harvested for imaging using an IVIS Spectrum Imaging System (Caliper Life Sciences, Hopkinton, MA). The distribution of Cy5-labeled mRNA in the various tissues were recorded under fluorescence module (λex = 640 nm; λem = 680 nm), and the luciferase expression levels were measured using bioluminescence module with an exposure time of 1 min.

2.10. Lung cell transfection assay in Ai14 Cre reporter mice.

Female B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14, 6-week-old) mice were purchased from Jackson Laboratories and allowed to acclimate for 1 week prior to experiments. The mice were treated with NP4 and NP7 at PEG grafting density of 0.5% via i.v. injection at a mRNA dose of 1 mg/kg. The NPs were prepared using mRNA encoding Cre Recombinase (TriLink, CleanCap® NLS-Cre, 5moU). At 24 h post injection of NPs, the mice were sacrificed, and the lungs were harvested. The expression of tdTomato in the lung were validated using an IVIS Spectrum Imaging System under fluorescence module with excitation filter 535 nm and emission filter 580 nm. After imaging, the lung was digested to single cells and stained with antibodies against epithelial (EPCAM-APC), endothelial (CD31-AF488), and immune (CD45-BV421) cell markers following the reported protocol.13 The cells were then analyzed using a Sony Biotechnology SH800 flow cytometer.

2.11. Preparation of PEI-g-PEG/mRNA NPs using FNC method.

The FNC method developed by our lab previously23 was adopted for the preparation of the PEI-g-PEG/mRNA NPs in a continuous manner. The PEI-g-PEG and mRNA solutions were independently loaded into two syringes and then mixed rapidly within a two-inlet confined impinging jet mixer (CIJM) device with an internal chamber volume of 2 μL. The flow rates of the two solutions were the same and controlled by a programmable syringe pump (New Era Pump System, model NE‐4000) at 0.5, 1, 2 and 3 mL/min in order to examine the effect of flow rate on the particle size and size distribution.

2.12. Lyophilization and storage stability assay.

The PEI-g-PEG/mRNA NPs prepared by the FNC method at a flow rate of 2 mL/min were concentrated by ultracentrifugation using a desalting tube with a molecular weight cut-off (MWCO) of 3,000 Da. After adding trehalose to a concentration of 9.5 w/v%, the NP solution was lyophilized and stored at −20 °C. Four months later, the lyophilized powders were reconstituted by adding DI water. The reconstituted NPs were characterized by particle size and size distribution. Their transfection efficiency was also measured and compared with freshly prepared NPs in DC2.4 cells.

2.13. Statistical analysis.

All data are expressed as mean ± standard deviation. The Student’s t-test and one-way ANOVA test were used to determine the significance among groups. The difference between different groups was considered to be statistically significant if the value of p < 0.05.

3. RESULTS AND DISCUSSION

3.1. PEI-g-PEG synthesis and characterizations.

The PEI-g-PEG polymers with SPDP PEG terminal group were synthesized by grafting NHS-PEG-SPDP to the backbone of linear PEI. Five polymers with different PEG grafting ratios including 0.5%, 1.5%, 3%, 5% and 10% were obtained. The PEG terminal group was then further introduced by reacting the SPDP modified polymers with functional molecules containing the desired terminal group. (Scheme 1). The modification ratios of all the five polymers with the eight functional molecules were around 100% (data not shown).

Scheme 1.

Scheme 1.

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Schematic illustration showing the preparation of PEI-g-PEG/mRNA NPs with various PEG terminal groups and PEG grafting ratios.

3.2. NP preparation and characterizations.

The PEI-g-PEG/mRNA NPs with various PEG terminal groups (Scheme 1) and different PEG grafting ratios (G = 0.5, 1.5, 3, 5 and 10% as the target average molar percent of the repeating units of PEI chain) were prepared by mixing the mRNA solution with the PEI-g-PEG polymer solution at N/P ratio 8 (Scheme 1, NPs are designated as NPR-G). The NPs showed average particle sizes ranging from 46.0 ± 0.4 to 82.2 ± 1.5 nm with polydispersity index (PDI) values below 0.30 (Figure 1A and Figure S1), indicating high complexation ability of these PEI-g-PEG polymers with mRNA. The effect of PEG terminal groups on the particle size and size distribution was not obvious. For comparison, the PEI/mRNA NPs formed at N/P ratio 8 had an average particle size of 66.3 ± 0.3 nm and a PDI of 0.24. Generally speaking, the PDI values for most of the PEI-g-PEG/mRNA NPs were lower than that of the PEI/mRNA NPs. The TEM images showed that PEI-g-PEG/mRNA NPs at PEG grafting ratio 0.5% with different PEG terminal groups were all in spherical shapes (Figure S2), indicating that PEG terminal groups had negligible effects on the morphology of the NPs. In addition, the effect of PEG grafting on the morphology of NPs was also negligible as PEI/mRNA NPs and NP3 at different PEG grafting ratios were all spheres (Figure S2). The zeta potential of the NPs was measured in PBS (pH 7.4). All the PEI-g-PEG/mRNA NPs showed positive surface charge with average zeta potential values ranging from +2 to +20 mV (Figure S3). No clear trend was found among the PEI-g-PEG/mRNA NPs with different PEG terminal groups and different PEG grafting ratios. One possible reason is that these NPs showed different particle sizes in PBS (Figure S4A). The concentration of the NPs with different sizes may also be different since they were prepared at the same mRNA concentration and the same N/P ratio. It has been shown that both particle size and concentration of NPs significantly affect the zeta potential measurement.31 In addition, since not all the secondary amine groups of PEI are reacted with PEG due to the low grafting degree, the variations of zeta potential across the formulations are expected depending on the arrangement of the NPs. The zeta potential value of PEI/mRNA NPs was +17 mV.

Figure 1.

Figure 1.

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(A) Average particle size and (B) EE of various PEI-g-PEG/mRNA NPs; (C) HC50 values to achieve 50% mRNA release of various PEI-g-PEG/mRNA NPs in heparin challenge assay. a, p < 0.05, vs PEI/mRNA NPs; b, p < 0.05, vs NPs at a PEG grafting ratio of 1.5%; c, p < 0.05, vs NPs at a PEG grafting ratio of 3%; d, p < 0.05, vs NPs at a PEG grafting ratio of 5%; e, p < 0.05, vs NPs at a PEG grafting ratio of 10%.

3.3. EE measurement.

The EE of the various PEI-g-PEG/mRNA NPs was evaluated by measuring the percentage of free mRNA in the NP solutions. As shown in Figure 1B, most PEI-g-PEG/mRNA NPs had high mRNA EE above 90% (with the exception of NP8-10%, with an mRNA EE of 88%), which were comparable to that of PEI/mRNA NPs. In general, the EE decreased with increasing PEG grafting ratio. These results may be due to the charge shielding effect of PEG, and this effect is more pronounced at higher PEG grafting ratios.19–20

3.4. Heparin challenge assay.

The intracellular release of mRNA from the NPs is a critical step towards the successful translation of mRNA.32 The stability of various PEI-g-PEG/mRNA NPs was evaluated by challenging the NPs with anionic polymer heparin sulfate at different concentrations. The heparin concentrations necessary to achieve 50% mRNA release (HC50) are summarized in Figure 1C. Overall, the HC50 values of all the PEI-g-PEG/mRNA NPs were much lower than that of PEI/mRNA NPs, indicating that the short PEG modification significantly accelerated the mRNA release from PEI/mRNA NPs. In general, the HC50 value decreased with increasing PEG grafting ratio, implying that the PEI-g-PEG/mRNA NPs at higher PEG grafting ratios might have faster mRNA release than those at lower PEG grafting ratios. These results may be due to the charge shielding effect of PEG, which leads to a less compact structure. No significant differences were found between the PEI-g-PEG/mRNA NPs at PEG grafting ratio 5% and those at PEG grafting ratio 10%. In addition, there were not clear trends of the effect of the terminal group on the HC50 values.

3.5. Transfection efficiency assay.

The transfection efficiency of various PEI-g-PEG/mRNA NPs was evaluated in DC2.4 and PC3 cells using Luciferase mRNA as a reporter. DC2.4 cells are one of the most widely used dendritic cell lines to investigate the immune activation effects of various formulations,33 and PC3 cells are human metastatic prostate cancer cells which have been extensively used for prostate cancer research and drug development.34–35 The purpose of using these two cell lines is to investigate the potential applications of PEI-g-PEG/mRNA NPs in immune system modulation or cancer treatment. In DC2.4 cells, generally the PEI-g-PEG/mRNA NPs at low PEG grafting ratios (0.5 and 1.5%) showed much higher transfection efficiencies than those at high PEG grafting ratios (3, 5 and 10%), PEI/mRNA NPs and Lipofectamine 3000/mRNA NPs (Figure S5A). The highest transfection efficiency was achieved by NP3 (R = −S(CH2)2COOH) at PEG grafting ratio 0.5%, which was 628-fold higher and 214-fold higher than that of PEI/mRNA NPs and Lipofectamine 3000/mRNA NPs respectively. The low transfection efficiency of PEI/mRNA NPs in DC2.4 cells have been reported by other researchers previously as well.36–37 Our PEI-g-PEG polymers at PEG grafting ratio 0.5% and 1.5% significantly enhanced the transgene expression level of PEI in DC2.4 cells. In PC3 cells, the PEI/mRNA NPs had higher transfection efficiency than most of the PEI-g-PEG/mRNA NPs except for NP3-0.5% (Figure S5B). NP3-0.5% and NP6-0.5% exhibited higher transfection efficiency than Lipofectamine 3000/mRNA NPs (Figure S5B). The highest transfection efficiency of PEI-g-PEG/mRNA NPs was again achieved by NP3-0.5%. No significant level of cytotoxicity was observed in these two cell lines after treated with all the PEI-g-PEG/mRNA NPs and PEI/mRNA NPs at an N/P ratio of 8 and an mRNA concentration of 0.1 μg/well (Figure S6). Nevertheless, the PEI-g-PEG/mRNA NPs are expected to have lower toxicity than PEI/mRNA NPs in vivo as demonstrated in our previous study using PEI-g-PEG as plasmid DNA carriers.20 Additional study will be performed to understand how these polymers get eliminated from the body.

3.6. Cellular uptake.

Cellular uptake is one of the limiting steps in mRNA delivery.38 The cellular uptake levels of various PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% and NP3 at different PEG grafting ratios were investigated using Cy5-labelled mRNA by flow cytometry to investigate the effect of PEG terminal groups and PEG grafting ratio on cellular uptake (Figure 2). In DC2.4 cells, the cells treated by NP1 (R = −S(CH2)2OH) at PEG grafting ratio 0.5% (NP1-0.5%) showed the highest mean fluorescence intensity compared to those treated by other PEI-g-PEG/mRNA NPs at the same PEG grafting ratio. For NP3 at different PEG grafting ratios, the mean fluorescence intensity decreased with increasing the PEG grafting ratio. Except for NP4-0.5% and NP6-0.5%, all the other PEI-g-PEG/mRNA NPs at PEG grafting ratio 0.5% and PEI/mRNA NPs induced around 90% of cells taking up NPs. For NP3 at different PEG grafting ratios, the percentage of cellular uptake did not change significantly when the grafting ratio increased from 0.5% to 1.5% but decreased with further increase of the PEG grafting ratio. The high cellular uptake level of PEI-g-PEG/mRNA NPs at low PEG grafting ratios (0.5% and 1.5%) in DC 2.4 cells may be one of the reasons leading to the high transfection efficiency (Figure S4). In PC3 cells, PEI/mRNA NPs resulted in higher mean fluorescence intensity than most of the PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% except for NP4-0.5%. The cells treated with PEI/mRNA NPs also showed a higher percentage of cellular uptake than those treated with PEI-g-PEG/mRNA NPs at PEG grafting ratio 0.5%. Both mean fluorescence intensity and the average percentage of cellular uptake of NP3 decreased with increasing PEG grafting ratio. These results may explain why PEI/mRNA NPs had higher transfection efficiency than PEI-g-PEG/mRNA NPs in general. PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% showed higher transfection efficiency than the NPs at higher PEG grafting ratios in PC3 cells. In both cell lines, although NP3-0.5% showed the highest transfection efficiency compared to other PEI-g-PEG/mRNA NPs, the highest cellular uptake levels were not achieved by the same NPs, indicating that other parameters including NP stability, intracellular release and endosomal escape could potentially be playing important roles on the efficiency of gene expression.1–2

Figure 2.

Figure 2.

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(A and B) Mean fluorescence intensity and (C and D) percentage of cellular uptake of DC2.4 cells after treatment with various PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% (A and C) and NP3 at different PEG grafting ratios (B and D); (E and F) Mean fluorescence intensity and (G and H) percentage of cellular uptake of PC3 cells after treated with various PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% (E and G) and NP3 at different PEG grafting ratios (F and H). a, p < 0.05, vs Blank; b, p < 0.05, vs Free mRNA; c, p < 0.05, vs PEI/mRNA NPs; d, p < 0.05, vs NP1-0.5%; e, p < 0.05, vs NP2-0.5%; f, p < 0.05, vs NP3-0.5%; g, p < 0.05, vs NP4-0.5%; h, p < 0.05, vs NP5-0.5%; i, p < 0.05, vs NP6-0.5%; j, p < 0.05, vs NP7-0.5%; k, p < 0.05, vs NP3-1.5%; l, p < 0.05, vs NP3-3%; m, p < 0.05, vs NP3-5%.

3.7. Endosomal escape.

Effective endosomal escape is crucial for intracellular transgene expression of mRNA NPs.39 The endosomal escape ability of various PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% and NP3 at different PEG grafting ratios were evaluated by Gal8 recruitment assay using B16F10 cells expressing Gal8-GFP fusion protein in the cytosol. The binding of the Gal8-GFP fusion protein with the glycosylation moieties located in the inner face of endosome membrane after endosome disruption causes aggregation of Gal8-GFP protein, which can be detected as distinct puncta by fluorescent microscopy.40–41 In this assay, the imaging algorism identifies stained nuclei first (Fig. S7A and B), and then defines the area of interest for each cell for the GFP channel (Fig. S7C and D) with the method of circular expansion. The GFP spots where the binding and concentration of Gal8-GFP proteins occurs were identified via comparing local light intensity against adjacent background (preset in the algorism as Isodata method, Fig. S7E,).

It has been shown that the gene knockdown activity of siRNA NPs correlated strongly with the average number of Gal8-GFP puncta per cell.40 Our results revealed that among the NPs designed at a PEG grafting ratio of 0.5%, NP3-0.5% induced the highest level of endosome disruption (Figure 3A, C and E). NP3-0.5% also showed much stronger endosomal escape ability than NP3 at high PEG grafting ratios (Figure 3B and D). In addition, endosome disruption was not detected using this method in the cells treated by NP3-5% and NP3-10%, presumably due to their low cellular uptake levels (Figure 2). Polymer 3-0.5% and polymer 4-0.5% showed comparable buffering capacity as PEI, higher than all other tested polymer carriers (Figure S8). In addition, membrane activity of the terminal group may also facilitate its interaction with endosomal membrane thus contributing to the endosomal escape ability. In a previous study with anionic polymer poly(propyl acrylic acid), it was shown that this polymer has strong endosomal escaping capacity due to the change in the protonation state of acrylic acid groups in the endosomes.41 NP3-0.5% with propionic acid PEG terminal groups may switch to a more hydrophobic, membrane-active conformation in endosome. Thus, the enhanced endosomal escaping capability of NP3-0.5% compared to other PEI-g-PEG/mRNA NPs and PEI/mRNA NPs is presumably due to its combined effect of high buffering capacity and conformation change of the surface functional group in endosomal pH. NP3-0.5% also showed higher transfection efficiency than the other PEI-g-PEG/mRNA NPs in both DC2.4 and PC3 cells, indicating that the endosomal escape ability is one of the major barriers impacting the gene expression efficacy of the PEI-g-PEG/mRNA NPs. However, the other factors may also influence the gene expression level. For example, NP3-0.5% showed higher endosomal capability than PEI/mRNA NPs, but they had similar level of transfection efficiency in PC3 cells (Figure S5B). In addition, it has been shown that same mRNA encapsulated NPs are process differently by different cell types both in vitro and in vivo.37

Figure 3.

Figure 3.

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(A and B) The average number of Gal8 spot per cell and (C and D) the percentage of Gal8 spot positive cells in various B16F10-Gal8-GFP cell groups after treated by NP1-NP8 at a PEG grafting ratio of 0.5% and NP3 at different PEG grafting ratios; (E) Representative images of B16F10-Gal8-GFP cells after treated with PBS, NP3-0.5%, NP4-0.5% and PEI NPs. a, p < 0.05, vs blank; b, p < 0.05, vs NP3-0.5%. Scale bar: 20 μm.

3.8. Biodistribution and in vivo gene expression.

Given the high transfection efficiency of PEI-g-PEG/mRNA NPs at a PEG grating ratio of 0.5%, the biodistribution and in vivo gene expression level of these NPs were further investigated in Balb/c mice using Cy5-labeled luciferase mRNA via i.v. injection. As shown in Figure 4A and B, all NPs had broad distributions in the main organs including liver, lung, kidney and spleen. NP8-0.5%, NP1-0.5%, and PEI/mRNA NPs showed higher fluorescence in the lung than other NPs. On the other hand, NP3-0.5% exhibited higher accumulation in the spleen compared to other PEI-g-PEG/mRNA NPs and PEI/mRNA NPs. The gene expression levels of these NPs in the main organs was also measured using luciferase mRNA as a reporter (Figure 4C and D). For all the NPs, robust gene expression was found in the lung with NP4-0.5% and NP7-0.5%, leading to levels of gene expression that were 5.4- and 4.6-fold higher than that observed for PEI/mRNA NPs. The higher gene expression level of these two NPs than the other NPs presumably due to their higher colloidal stability in the physiological environment (Figure S4).9 Difference between the mRNA distribution profile and the sites of gene expression have also been observed with other mRNA delivery systems.9, 43 Although there are some mRNA formulations currently under clinical trials, the delivery of mRNA to tissues beyond the liver remains a challenge.13–14 The preferential gene expression pattern of NP4-0.5% and NP7-0.5% in the lung suggests that these NPs may have the potential for pulmonary delivery. The mechanism by which these two NPs with specific PEG terminal groups selectively transfect the lung tissue needs further investigation.

Figure 4.

Figure 4.

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(A and B) biodistribution and (C and D) gene expression levels of various PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5% in Balb/c mice after i.v. injection. The dose of mRNA was 0.5 mg/kg and the tissues were collected at 24 h after injection. (A) Representative images and (B) quantitative analysis showing the biodistribution of various NPs in the main organs; (C) Representative images and (D) quantitative analysis showing the luciferase expression of various NPs in the main organs; (E) Representative image showing the expression of tdTomato in the lungs of Ai14 mice treated with NP4-0.5%; (F) Quantitative analysis showing the percentage of tdTomato positive cells in epithelial cells, endothelial cells, immune cells and other populations of cells in the lung of Ai14 mice after treated with NP4-0.5% or NP7-0.5%. a, p < 0.05, vs NP3-0.5%; b, p < 0.05, vs NP7-0.5%; c, p < 0.01, vs blank.

3.9. Lung cell transfection assay in Ai14 Cre reporter mice.

Given the high gene expression level of NP4-0.5% and NP7-0.5% in the lung, we further investigated which specific type of cells in the lung were transfected by these two NPs. We adopted Ai14 mouse model, which had lox-P flanked stop cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato). These mice express strong tdTomato signals after removing the stop cassette using Cre-recombinase mRNA, which can be detected and analyzed by flow cytometry with single cell resolution. NP4-0.5% and NP7-0.5% were prepared using Cre-recombinase mRNA and administrated to Ai14 mice via i.v. injection. The expression of tdTomato in the lungs of mice treated by NP4-0.5% was confirmed by IVIS imaging (Figure 4E). The lungs from Ai14 mice treated by NP4-0.5% and NP7-0.5% and blank mice were digested to single cells, stained by antibodies and analyzed by flow cytometry to identify the percentage of transfected cells in the populations of epithelial cells, endothelial cells, immune cells and other types of cells (e.g. mucous cells, ductal cells, smooth muscle cells, fibroblasts etc.). Both NP4-0.5% and NP7-0.5% led to gene expression primarily in the immune cells in the lung (7.2% for NP4-0.5% and 6.6% for NP7-0.5%). The delivery of mRNA to the immune cells in the lung has been reported previously, showing that around 2% of pulmonary immune cells have been transfected by a polymer-lipid NP system.13 The pulmonary immune system plays a vital role in protecting the host from threats of harmful pathogens in the inhaled air.44–45 The pulmonary immune cells are also associated with various lung diseases.46–47 The high gene expression levels of these two NPs in the pulmonary immune cells indicates that these NPs may have the potential to be used as mRNA carriers for pulmonary immunomodulation. In addition, around 3% of other types of cells in the lungs were also transfected by these two NPs.

3.10. Preparation of PEI-g-PEG/mRNA NPs using FNC method.

One of the major obstacles in the clinical translation of NPs is the lack of scalable and reproducible preparation method.48 Previously, we have developed the flash nanocomplexation (FNC) platform for the scalable production of various types of NPs. During the preparation process, the particle size, size distribution and composition of the NPs could be well-controlled by tuning the FNC formulation parameters.23, 28 In this study, we utilized the FNC method to prepare mRNA NPs for the first time. NP4-0.5% was prepared at different flow rates by rapid mixing the mRNA solution and PEI-g-PEG polymer solution through a CIJM device. As shown in Figure 5A, the particle size of the NPs decreased with increasing flow rate, which was consistent with the results that we obtained in the preparation of DNA NPs.28 All the NPs prepared by FNC method showed narrow size distribution with PDI values of around 0.20. The NPs prepared at a flow rate of 2 mL/min in four different batches exhibited the same size distribution (Figure 5B). In addition, NPs prepared at a larger scale (2 mL PEI-g-PEG solution plus 2 mL mRNA solution) showed the same average particle size and size distribution to those prepared in a standard batch mixing mode (200 μL PEI-g-PEG solution plus 200 μL mRNA solution) (Figure 5C). The NPs prepared by FNC at a flow rate of 2 mL/min showed comparable transfection efficiency level to the NPs prepared by manual batch mixing in DC2.4 cells (Figure 5D). Collectively, these results demonstrated that FNC is a suitable platform for the continuous production of PEI-g-PEG/mRNA NPs at larger scales, with the advantages of avoiding batch-to-batch variations and preserving their biological activities. It is important to note that for a small bench-scale preparation (<0.5 mL), manual mixing produces reasonably good quality nanoparticles. As shown here, the physiochemical characterization did not show notable differences between such a small-scale manual mixing and the FNC method, we expect the FNC made nanoparticles should achieve the same level of transfection efficiency in vivo as the mRNA NPs prepared by this small-scale manual mixing.

Figure 5.

Figure 5.

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(A) Average particle size and PDI values of NP4-0.5% prepared by FNC at different flow rates or by manual batch mixing; (B) DLS profiles showing the particle size distributions of NP4-0.5% prepared by FNC at a flow rate of 2 mL/min in four different batches; (C) DLS profiles showing the particle size and size distributions of NP4-0.5% prepared by FNC at a flow rate of 2 mL/min in smaller- or larger-scale; (D) transfection efficiency of NP4-0.5% prepared by FNC at a flow rate of 2 mL/min or manual mixing in DC2.4 cells; (E) Average particle sizes and PDI values and (F) transfection efficiencies in DC2.4 cells of freshly prepared NP4-0.5%, lyophilized NP4-0.5% and lyophilized NP4-0.5% following 4-month storage.

3.11. Lyophilization of NPs.

The storage stability is another important parameter for the clinical translation of NPs, particularly for NP encapsulating mRNA as a cargo. We adopted a lyophilization method developed for DNA NPs in our lab previously. The NPs prepared by FNC method at a flow rate of 2 mL/min were lyophilized in the presence of 9.5% trehalose as a cryoprotectant. Reconstitution of the obtained lyophilized NP powder can be achieved in an easy and fast manner by directly adding DI water followed by gentle shaking. The reconstituted NPs showed comparable particle size and size distribution to the NPs before lyophilization (Figure 5E). In addition, after lyophilization and reconstitution, the NPs maintained the same level of transfection efficiency in DC2.4 cells (Figure 5F). Furthermore, the lyophilized NPs also maintained their particle size distribution and transfection efficiency level following a 4-month period (Figure 5E and F). The storage stability combined with the FNC platform for scalable production of NPs will allow us to generate mRNA NPs with great potential for clinical translation.

4. CONCLUSION

We developed a group of PEI-g-PEG polymers with various PEG terminal groups and different PEG grafting ratios ranging from 0.5 to 10 % for the delivery of mRNA. These polymers compacted mRNA efficiently to form NPs. While the stability of the NPs decreased with increasing PEG grafting ratio, the highest transfection efficiency was achieved by NP3 at a PEG grafting ratio of 0.5% when the functional group is a carboxylic group in both DC2.4 and PC3 cells. However, following i.v. injection of these PEI-g-PEG/mRNA NPs at a PEG grafting ratio of 0.5%, a broad distribution in the lung, liver and kidney was observed in mice. Intriguingly, NP4-0.5% and NP7-0.5%, which carries amino terminal group and amino acid residue, respectively, showed the highest gene expression level in the lung compared to other organs and other PEI-g-PEG/mRNA NPs and PEI/mRNA NPs. In addition, these two NPs primarily transfected around 7% of pulmonary immune cells. These NPs can be successfully produced in a continuous and reproducible process using the FNC platform and can be stored in a lyophilized form for over 4 months at −20 °C without measurable changes in particle size and transfection activity. The surface-functionalized PEGylated NP system developed in this study holds great potential for preferential delivery to the lung and to immune cells for immunomodulatory treatment, with the additional advantage of ease of clinical translation.

Supplementary Material

Supplementary Information

ACKNOWLEDGEMENT

The authors would like to thank Mr. Stipe Iveljic, Mr. Rich Middlestadt and Mr. Francis Cook from Johns Hopkins University Whiting School of Engineering Manufacturing (JHU WSE machine shop) for the assistances on manufacturing the CIJ mixing blocks. The authors also would like to thank Dr. David R. Wilson (Department of Biomedical Engineering, JHMI) and Dr. Cindy Berlinicke (Department of Ophthalmology, JHMI) for their help on Cellomics quantitative fluorescence imaging of the endosomal escape assay.

Funding Sources

This work was supported by the National Institutes of Health (R21 5R21AI133533), Wilmer Microscopy Core Facility Grant (P30 EY001765) and an AstraZeneca Partnership Grant.

Footnotes

Supporting Information.

PDI values of various PEI-g-PEG/mRNA NPs; TEM images of PEI-g-PEG/mRNA NPs at PEG grafting ratio 0.5% and NP3 at different PEG grafting ratios; Zeta potential values of various PEI-g-PEG/mRNA NPs; Particle size of various PEI-g-PEG/mRNA NPs in PBS and 5% FBS solution; Transfection efficiency of various PEI-g-PEG/mRNA NPs in DC2.4 and PC3 cells; Cytotoxicity of various PEI-g-PEG/mRNA NPs in DC2.4 and PC3 cells; Image-based analysis used to identify and count Gal8-GFP spots; pH titration curve of PEI-g-PEG polymers and other experimental details.

The authors declare no competing conflicts of interest.

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