In Vivo Introduction of mRNA Encapsulated in Lipid Nanoparticles to

Apr 11, 2018 - In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As...
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Communication Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

In Vivo Introduction of mRNA Encapsulated in Lipid Nanoparticles to Brain Neuronal Cells and Astrocytes via Intracerebroventricular Administration Hiroki Tanaka,† Taichi Nakatani,‡ Tomomi Furihata,§ Kota Tange,∥ Yuta Nakai,∥ Hiroki Yoshioka,∥ Hideyoshi Harashima,‡ and Hidetaka Akita*,† †

Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba City, Chiba 260-8675, Japan Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Sapporo City, Hokkaido 060-0812, Japan § Department of Pharmacology, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba City, Chiba 260-8670, Japan ∥ NOF CORPORATION, 3-3 Chidori-cho, Kawasaki-ku, Kawasaki City, Kanagawa 210-0865, Japan ‡

S Supporting Information *

treatment improved the motor symptoms of Parkinson’s disease, and the expression of the protein was maintained for over five years. More recently, a clinical trial for treating Alzheimer’s disease was reported.5 The introduction of the AAV coding nerve growth factor (NGF) succeeded in preventing of neuronal deaths for periods of up to 10 years. These successful outcomes clearly indicate the potential feasibility of gene therapy for curing brain disorders which are currently considered to be intractable. However, although the therapeutic efficiency of viral carriers is well-known, there are technological limitations for the currently used vectors, including immunogenicity and/or limited size of the encoding gene.6 Thus, a safe and highly efficient nonviral gene carrier would be highly desirable. As a material for nonviral gene therapy, we developed a series of materials that we refer as to an SS-cleavable proton-activated lipid-like material (ssPalm, Figure 1a).7 The ssPalm contains three structural units; 1. hydrophobic scaffolds for the formation of a bilayer structure, 2. tertiary amines for proton sponging ability, and 3. disulfide bonding for cytoplasmic cleavage. The gene-encapsulating lipid nanoparticle (LNP) composed of ssPalm (LNPssPalm) carries a neutral charge at physiological conditions. The neutrally charged surface is advantageous for controlling the pharmacokinetics and intracellular trafficking in comparison with cationic materials that were commonly used in gene delivery. The neutral carrier flows through the systemic circulation in a highly dispersed form since it can avoid nonspecific interactions with endogenous macromolecules.8 Furthermore, neutral gene carriers are typically less toxic.9,10 Once taken up by cells, the LNPssPalm develops a positive charge by the protonation of tertiary amines as the environment becomes acidic during endosome maturation. The positive charge then triggers an interaction between the carriers and the endosomal membrane for endosomal escape.11,12 In the reducing cellular environment

ABSTRACT: Gene therapy is a promising strategy for curing certain types of brain diseases. Supplementation of therapeutic proteins such as aromatic amino acid decarboxylase (AADC) or nerve growth factor (NGF) have been reported to be successful examples of such treatments. However, there are safety concerns because these systems are based on virus-based gene vectors. A safe and efficient artificial carrier is thus urgently needed as an alternative. In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As a carrier, a lipid nanoparticle (LNP) composed of environmentally sensitive lipid-like materials called an SS-cleavable protonactivated lipid-like material is used. The apolipoprotein E mediated cellular uptake of the lipid nanoparticles is one of the key features for its superior and homogeneous transfection activity compared to commercially available transfection reagents in both in vitro and in vivo situations. Immunostaining of brain specimens suggested that exogenous proteins can be introduced into neuronal cells as well as astrocytes using the mRNA-based gene carrier. This cannot be achieved using DNA-based artificial gene carriers. The findings suggest that a combination of an mRNA and a lipid based delivery system have great promise as a platform for the treatment of brain disorders. KEYWORDS: brain, mRNA delivery, nanoparticles, lipid

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INTRODUCTION Therapeutics based on genetic material (pDNA and mRNA) are promising approach to the treatment for brain disorders caused by genetic defects.1 Supplementation of therapeutic proteins by the introduction of exogenous nucleic acids is one of the strategies available for rescuing the innate functions of cells in the central nervous systems (CNS). Gene therapy for a brain disorder was reported to be successful in treating Parkinson’s disease. In that treatment, an adeno-associated virus (AAV) vector coding aromatic amino acid decarboxylase (AADC) was introduced into the brain putamen.2−4 The © XXXX American Chemical Society

Received: December 4, 2017 Revised: March 29, 2018 Accepted: April 2, 2018

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DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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drastic decrease of hydrophobicity results in the dissociation of ssPalm from the particle, and the envelope structure spontaneously collapses, thus enabling the cargo to be efficiently released into the cytoplasm. It has been reported that intravenously administrated LNPs composed of ionizable lipids accumulate into the liver in an apolipoprotein E (ApoE)-dependent manner via members of the low-density lipoprotein receptor (LDLR) family.15,16 In addition to the liver, the brain is another ApoE abundant tissue, in that astrocytes produce high-density lipoprotein (HDL)-like particles with ApoE.17 The particles in the brain are taken up by astrocytes and neuronal cells for anti-inflammatory and antiapoptotic functions, respectively. In addition to lipid metabolism, the exclusion of amyloid beta also take places in an ApoE-dependent process.18−20 It is thus assumed that the ApoE-LDLR mediated uptake of LNPs would also occur in the fluid in brain, similar to the liver.21,22 Based on this assumption, we previously reported on the intracerebroventricular administration of a plasmid DNA (pDNA)-encapsulating LNPssPalm. The administered LNP was taken up by astrocytes and neuronal cells in the corpus callosum. However, gene expression of reporter genes (LacZ and mCherry) was only observed in astrocytes.23 Protein expression was not observed in neuronal cells, probably due to its essential nonmitotic nature; the encapsulated DNA could not enter the nucleus of neuronal cells. An increasing number of reports point to the therapeutic potential of in vitro transcribed mRNA (IVT-mRNA) for nonviral gene therapy.24−26 From the viewpoint of safety, mRNA would be more feasible because of the lack of genomic integration, one of the major safety concerns of pDNA-based gene therapy. The IVT-mRNA also requires carriers to protect it from degradative enzymes and RNA sensors. Since the siteof-action of IVT-mRNA is the cytoplasm, the carriers need to be taken up by cells, overcome the endosomal membrane, and release the IVT-mRNA cargo into the cytoplasm. In this case, the ssPalm, a material equipped with dual functions for endosomal escape and spontaneous cytoplasmic collapse, would be suitable for delivering mRNA, especially in the case of nonmitotic cells. ApoE-dependent cellular uptake would also be a key feature in delivering IVT-mRNA into brain cells. In this study, we verified the hypothesis that the intracerebroventricular administration of IVT-mRNA encapsulating LNPssPalm could introduce exogenous proteins into neuronal cells as well as into astrocytes.

Figure 1. (a) Chemical structure of the ssPalm. Myristic acid (ssPalmM), retinoic acid (ssPalmA), and tocopherol succinate (ssPalmE) were employed as hydrophobic scaffolds. The pH sensitive units (tertiary amine) of the ssPalmE were further modified by attaching them to a piperizine ring (P4C2) to elevate the pKa of the preparation. (b) Transfection activity of ssPalm with different hydrophobic scaffolds. An astrocyte (KT-5 cell line) was transfected with the LNPssPalm containing luciferase mRNA. The cumulative luciferase activity was calculated as a sum of luciferase activity at every 1 h. Each bar indicates average of three independent experiments ± SD. Statistical analyses were performed by one-way ANOVA followed by SNK test (**p < 0.01). (c) Transfection activity of the ssPalmE with different pH sensitive units. The luciferase activity was measured after 24 h. Luciferase activity was normalized by the protein amount. Each bar indicates the average of three independent experiments ± SD. Statistical analyses were performed by Student’s t test (**p < 0.01).

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EXPERIMENTAL SECTION Materials and Antibodies are listed in the Supporting Information. In Vitro Transcription (IVT) of mRNA. pDNA containing Luciferase or eGFP expression cassette under the T7 promoter (pcDNA3.1-(+)-Luc(0) and pcDNA3.1-(+)-eGFP) were used as templates for in vitro transcription. pDNA was linearized using EcoRV-HF (for Luciferase) or NotI-HF (for eGFP), respectively. One microgram of the linearized DNA was transcribed using a mMESSAGE mMACHINE T7 Ultra Kit. The length of the transcript was 2115 nt for Luciferase and 849 nt for eGFP. The mRNA was further polyadenylated according to the manufacturer’s protocol. In the preparation of chemically modified mRNA, pseudouridine-5′-triphosphate (10%), 5methylcytidine-5′-triphosphate (20%), and 2-thiouridine-5′triphosphate (10%) were added to the NTP mixture.27

such as endosomes and cytoplasm,13,14 the disulfide bonding in the ssPalm is cleaved. The cleavage results in a structural transition of the ssPalm from two hydrophobic chains to a single hydrophobic chain. This structural transition reduces the index of hydrophobicity, the logarithm of estimated octanol− water partition coefficient (cLogP), of the ssPalm by half. This B

DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Animal Experiments. Protocols of animal experiments were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the “Guide for Care and Use of Laboratory Animals”. ICV Injection and in Vivo Luciferase Assay. ICR mice (male, 10 weeks of age) were anesthetized with isoflurane. LNPs (1.0 μg of total mRNA) were injected to mouse brain intracerebroventricularly (ICV). Ten microliters of the LNP solution in PBS or LFN working solution (0.1 μg/μL mRNA) was administrated over a period of 30 s. After injection, the syringe was left as it was for 1 min, and then carefully removed. Twenty-four hours after injection, the brains were collected. Quantification of luciferase activity and protein amount was described previously.23 Quantification of Interleukin-6 Production. ICR mice (aged 10 weeks) were administered with LNPs encapsulating luciferase mRNA by ICV at a dose of 1 μg/mouse. After 6 h, the brains were collected as two sections. Each brain half was homogenized by a Micro Smash MS-100R in 400 μL of extraction buffer (100 mM Tris-HCl, 2 mM ethylenediamine tetraacetic acid, 151 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, pH 7.4). The lysate was rotated for 2 h at 4 °C and centrifuged (12,000g, 20 min, 4 °C). The supernatant from each brain half was mixed. Interleukin-6 was quantified by using Mouse IL-6 ELISA Kit. Analysis of Gene Expressing Cells after ICV Injection. ICR mice (10 weeks, of age, male) were administered with LNPs encapsulating eGFP mRNA (1 μg/per mouse). The brain specimen was prepared as reported previously.23 Primary antibodies used in this report were as follows: GFP (1:500 dilution), GFAP (1:600 dilution), or MAP-2 (1:1000 dilution). The stained samples were observed by Nikon A1 confocal laser scanning microscopy (Nikon Co Ltd., Tokyo, Japan).

Evaluation of Encapsulation Efficiency. The encapsulation efficiency of the mRNA was evaluated by a Ribogreen assay. Ribogreen was diluted 200-fold in PBS with or without 0.4% w/v TritonX-100. The LNPssPalm, corresponding to 50 ng of mRNA (in 50 μL), was mixed with an equal volume of the Ribogreen solution in a 96-well black plate. The plate was incubated for 5 min under shaking (100 rpm). The fluorescence (Ex, 484; Em, 535) was evaluated by a plate reader (Infinite 200 PRO, Tecan, Switzerland). A calibration curve was prepared by sequential dilution from 0 to 2000 ng/ mL of mRNA. Encapsulation efficiency was calculated from the concentration of mRNA that was not encapsulated (without TritonX-100) and total mRNA (with TritonX-100). Preparation of LNPssPalm. mRNA solutions were prepared in malic acid buffer (pH 3.0, 20 mM with 30 mM NaCl) at a concentration of 0.067 μg/μL (3 μg of mRNA/45 μL). The lipid composition of the LNPssPalm was ssPalm/DOPE/ cholesterol = 3/3/4 with additional DMG-PEG2000 (3 mol % for in vitro experiments or 1 mol % for in vivo experiments). The mRNA solution was rapidly mixed with the lipids ethanol solution (131 nmol/30 μL) at room temperature under vortexing. MES buffer (925 μL, pH 5.5, 20 mM) was then added to the mixture. Subsequent buffer exchange was conducted by using Amicon Ultra-4-100 K centrifugal units (Merck Millipore) as described previously.23 Luciferase Assay (Time-Dependent Monitoring or One Time Point Assay). Astrocyte-derived KT-5 cells were transfected with mRNA encapsulating LNPssPalm. Cell number was 1 × 105 and 5 × 104 for time-dependent monitoring and one time point assay, respectively. LNP containing 1.6 μg of mRNA (time-dependent monitoring) or 0.4 μg of mRNA (one time point assay) was transfected. The detailed procedure was described previously.7,28 Flow Cytometry Analysis of EGFP Expressing Cells. Cells (1 × 105) were seeded on a 6-well plate 1 day before transfection. Cells were incubated with a LNP containing 1.6 μg of mRNA in 2 mL of culture medium with or without human apolipoprotein E3 (ApoE). The final concentration of the ApoE in culture medium was 3 μg/mL. After 24 h, the cells were washed with 1 mL of PBS and then trypsinized for 3 min. After incubation, 1 mL of culture medium was added, and the cellular suspension was collected. Cells were precipitated by centrifugation (400g, 4 °C, 5 min) and washed with 1 mL of FACS buffer. The cell suspension was filtered through a nylon mesh (45 μm). Fluorescence from the cells was determined by using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Flow Cytometry Analysis of Cellular Uptake of Fluorescent Labeled mRNA. mRNA encoding luciferase was labeled with Cy5 fluorescent dye using a LabelIT Cy5 Labeling Kit. The protocol for the transfection was the same as described above (Flow Ctyometry Analysis of EGFP Expressing Cells). Calculation of Coefficient of Variance (CV). Coefficient of variance of eGFP expression and cellular uptake was calculated as follows:

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RESULTS In Vitro Transgene Activity of LNPssPalm Encapsulating mRNA. To investigate the effect of the hydrophobic scaffold on the transfection efficiency of IVT-mRNA, the transfection activity of ssPalms with a myristic acid scaffold (ssPalmM), a vitamin A scaffold (ssPalmA), and a vitamin E scaffold (ssPalmE) were compared (Figure 1a). mRNA coding luciferase was prepared using an mMESSAGE mMACHINE T7 ULTRA Transcription kit.7 The diameters of the LNPssPalm encapsulating IVT-mRNA particles were 100−120 nm and carried a neutral charge, as summarized in Table 1. The encapsulation efficiency, as determined by a Ribogreen assay, was 54−81%. The LNPssPalm was transfected to an astrocyte (KT-5 cell line). Luciferase activity was measured using a Table 1. Particle Properties of the LNPssPalm lipids ssPalmM ssPalmA ssPalmE ssPalmEP4C2 (3% PEG) ssPalmEP4C2 (1% PEG)

CV = standard deviation (SD)/mean fluorescence intensity (MFI)

where SD and MFI were calculated from the fluorescence intensity of each cell.

a

C

a

size [nm] 125 128 115 101

± ± ± ±

4 5 8 1

121 ± 11

PdI 0.23 0.21 0.22 0.15

± ± ± ±

a

0.03 0.02 0.01 0.02

0.16 ± 0.01

zeta potentiala [mV] −4.4 −9.2 −7.1 −2.5

± ± ± ±

1.1 3.6 4.5 0.9

−14.9 ± 4.3

encapsulation [%] 63 54 56 81

± ± ± ±

3 13 1 1

73 ± 6

Particle properties were obtained by dynamic light scattering. DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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quantified by flow cytometry (Figure 3a). The histogram plot shows that the protein expression of LNPssPalmE‑P4C2 was

Kronos Luminometer. Luciferase activity that accumulated after 24 h (Figure 1b) indicated that LNPssPalmE showed a significantly superior mRNA transfection efficiency in comparison with LNPssPalmA and LNPssPalmM. For further improvement of mRNA transfection activity, the chemical structures of the tertiary amine groups were modified so as to elevate the pKa value for the preparation: the flexible tertiary amine of ssPalmE was fixed into the piperizine ring (ssPalmE-P4C2, Figure 1a).12 This modification resulted in a 2.7-fold increase in luciferase activity at 24 h after transfection (Figure 1c). To mimic the environment in the brain, luciferase activity was further evaluated in the presence of recombinant human apolipoprotein E3 (Figure 2). The luciferase activity

Figure 2. Time dependency for luciferase expression. KT-5 cells were transfected with the LNPssPalmE‑P4C2 encapsulating luciferase mRNA in the presence or absence of human recombinant apolipoprotein E3. As a control, a commercially available transfection reagent (lipofectamine messenger max; LFN) was used. Luciferase activity was monitored at 2 h intervals and plotted against time. Each data point indicates average of three independent experiments ± SD. Statistical analyses were performed by one-way ANOVA followed by Bonferroni’s multicomparison test vs LFN (*p < 0.05, **p < 0.01). Figure 3. (a) Heterogeneity of gene expression. KT-5 cells were transfected with the LNPssPalmE‑P4C2 encapsulating eGFP mRNA in the presence or absence of human recombinant apolipoprotein E3. Representative histogram was shown. LFN was used as a control. The expression of eGFP was measured by using flow cytometry. The coefficient of variance (CV) was calculated as described in Experimental Section. Each bar indicates mean ± SD of triplicate experiments. (b) Heterogeneity of mRNA cellular uptake. Cy5-labeled mRNA was encapsulated in LNPssPalmE‑P4C2. Cellular uptake was measured by using flow cytometry. Statistical analyses were performed by one-way ANOVA followed by the SNK test (*p < 0.05, **p < 0.01, ## p < 0.01 vs LFN).

was monitored at 2 h intervals, starting immediately after transfection. The expression of functional protein was observed from 2 h post-transfection. The luciferase activity reached a maximum at 8 h post-transfection for LNPssPalmE‑P4C2 in the absence of ApoE. In the presence of ApoE, the peak for luciferase activity moved forward to 6 h, as observed in lipofectamine messenger max (LFN), a commercially available transfection reagent optimized for delivering mRNA. ApoE supplementation increased the transfection efficiency of LNPssPalmE‑P4C2 by approximately 2.5-fold. The transfection activity was higher than that of LFN by 1.5-fold. Homogeneous Transgene Activity of LNPssPalmE‑P4C2. In the case of conventional gene delivery using DNA, heterogeneity of protein expression is a major drawback. For protein expression from pDNA, disruption of the nuclear membrane during mitosis is required to allow DNA to be internalized into the nucleus. It is therefore considered that heterogeneity of the transfection efficiency of DNA was partially cell cycle dependent. We expected that mRNA-based gene delivery would overcome this heterogeneity since its site-of-action is cytoplasm. To verify this hypothesis, KT-5 cells were transfected with the IVT-mRNA coding green fluorescent protein (eGFP). The expression of eGFP in individual cells was

homogeneous, with or without ApoE. Unexpectedly, we found that LFN showed heterogeneous protein expression compared to the LNPssPalmE‑P4C2. The eGFP positive cells transfected by the LNPssPalmE‑P4C2 (+ApoE) and LFN were 81.9 ± 1.9% and 47.2 ± 4.8%, respectively. To investigate the cause of this difference in the heterogeneity between LFN and LNPssPalmE‑P4C2, the cellular uptake of Cy5-labeled mRNA was investigated. The histogram plot for cellular uptake (Figure 3b) indicates that the homogeneous expression of LNPssPalmE‑P4C2 can mainly be attributed to the homogeneous cellular uptake: nearly all the cells (99.5 ± 0.1%) took up the LNPssPalmE‑P4C2 with a narrow unimodal distribution, while the uptake of LFN D

DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics showed a bimodal distribution. To quantify the heterogeneity of the transgene activity, the coefficient of variance (CV), that is denoted as a standard deviation of the GFP-fluorescence intensity in individual cells divided by the mean GFPfluorescence intensity, was calculated for each sample (Figure 3a). A lower CV value is indicative of a more homogeneous protein expression in the cell population. The results confirmed that the expression of LNPssPalmE‑P4C2, the neutral gene carrier, was significantly more homogeneous than cationic LFN, regardless of the presence or absence of ApoE. This trend was further confirmed by the CV for the cellular uptake; the value of LNPssPalmE‑P4C2 was significantly lower than that of the LFN. It was also suggested that the improvement in transfection efficiency in the presence of ApoE (Figures 2 and 3a) is due to the increased amount of LNPs taken up. Collectively, LNPssPalmE‑P4C2 was a potent mRNA carrier with homogeneous transfection efficiency by virtue of homogeneous cellular uptake. Intracerebroventricular Administration of LNPs Encapsulating mRNA. The LNPssPalmE‑P4C2, LFN, and naked mRNA were intracerebroventricularly administered and luciferase expression was measured after 24 h. Prior to the intracerebroventricular administration, the amount of surface PEGylation was optimized by monitoring the protein expression in KT-5 cells. As a result, we found that a smaller PEG density (1 mol %) resulted in a higher transgene activity. Thus, in the following in vivo experiments, the PEG density was fixed at 1 mol % (Table 1). In the case of naked mRNA, no luciferase activity was observed, probably due to the vulnerability of mRNA (Figure 4a). The luciferase activity for LNPssPalmE‑P4C2 (9.9 × 106 RLU/mg protein) was 7.3-fold higher than that of LFN (1.3 × 106 RLU/mg protein). This trend is consistent with the results of in vitro transfection experiments (Figure 2). To identify protein expressing cells, brain specimens were subjected to immunostaining (Figure 4c). mRNA coding eGFP was administrated intracerebroventricularly. Thereafter, the eGFP was stained with an anti-GFP antibody (green) as well as astrocytes and neuronal cells (antiGFAP antibody or anti-MAP-2 antibody, respectively (red)). Cell nuclei were counter-stained with Hoechst33342. The immunostaining revealed that eGFP was expressed in cells that were stained with both the neuronal marker MAP-2 and the astrocyte marker GFAP. These data indicate that using LNP systems to administer mRNA conferred protein expression in nonmitotic neuronal cells as well as astrocytes. Because of the immune stimulating nature of exogenous mRNA, interleukin-6 (IL-6) was slightly induced by the intracerebroventricular administration. The level of IL-6 in naked mRNA, LFN, and LNPssPalmE‑P4C2 was 73 ± 27, 78 ± 46, and 232 ± 66 pg/mL, respectively. To attenuate the immune activation, IVT-mRNA was prepared using the chemically modified nucleotides, 2-thiouridine, pseudouridine, and 5methylcytidine. Incorporation of the chemically modified nucleotides successfully suppressed the production of IL-6 to the level of naked mRNA. In addition to the avoidance of immune activation, the incorporation of modified nucleotides resulted in a 1.8-fold increase in luciferase activity.

Figure 4. (a) In vivo transfection activity in the brain. The LNPssPalmE‑P4C2 or LFN encapsulating luciferase mRNA was introduced to mouse brains via intracerebroventricular administration. The luciferase activity from whole brain tissue was measured by means of a luminometer at 24 h after transfection. Statistical analysis was performed by Student’s t test (*p < 0.05). (b) IL-6 production after the administration of the mRNAs. The concentration of interleukin-6 in brain lysates was measured by ELISA at 6 h after transfection. As a control, LPS was introduced to the brain intracerebroventricularly. Statistical analyses were performed by one-way ANOVA followed by SNK test (**p < 0.01). (c) Identification of the expressing cells. Fixed brain sample was sectioned and immunologically stained. The eGFP expression was visualized by using FITC-conjugated anti-GFP antibody. MAP-2 was used as a marker for neuronal cells. GFAP was used as a marker for astrocytes. Cell nuclei were counter-stained by Hoechst 33342. Bars indicate 50 μm.

succinyl (ssPalmE) moieties were compared. ssPalmM contains a simple fatty acid chain scaffold and was used as a control sample. ssPalmA possesses nuclear homing ability derived from its vitamin A scaffold.28 ssPalmE contains the most hydrophobic scaffold. In vitro transfection experiments using KT-5 cells revealed that LNPssPalmE was more potent in mRNA delivery. This result is consistent with the findings that ssPalmE showed a higher transfection efficiency for pDNA delivery to the astrocyte cells.23 Transfection efficiency of mRNA was further improved by the modification of amine groups to the piperazine ring. The improvement in transfection efficiency by the modification is attributed to the elevation of the pKa value from 6.08 to 6.18, which facilitates endosomal escape of LNPs. These results support the widely accepted hypothesis that endosomal escape is the rate limiting step in mRNA delivery as in the case of siRNA delivery. The hypothesis was further supported by the finding that the LNPssPalm prepared with a phosphatidyl choline group (DOPC), which forms a stable lipid membrane compared to DOPE, shows a 200-fold lower transfection efficiency (Figure S1). These data indicate that protonation of the tertiary amines in ssPalm and the overall conical shape of the phosphatidyl ethanolamine is a major factor in the successful cytoplasmic delivery of encapsulated mRNA. This observation is also consistent with recent reports indicating that the incorporation of DOPE is one of the determinants for the efficient mRNA delivery in other types of LNP preparations.29,30 The encapsulation efficiency of the mRNA was also improved by the elevation of the pKa value (56

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DISCUSSION Prior to the intrabrain administration, the chemical structure of LNPssPalm was optimized by using astrocyte cell line. To compare the effects of hydrophobic scaffold, ssPalm with myristoyl (ssPalmM), retinoyl (ssPalmA), and tocopherol E

DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics ± 1% for the LNPssPalmE and 81 ± 1% for the LNPssPalmE‑P4C2, respectively). We consider that the enhanced protonation of the piperizine ring in ssPalmE−P4C2 probably enhanced the interaction between the ssPalm and the mRNA during ethanol dilution process. Observation of time dependency of luciferase activity showed that expression of the protein begins as early as 2 h post-transfection. The faster protein expression of mRNA compared to pDNA (maximum expression at 20 h post transfection23) can be explained by the nature of the ratelimiting process: endosomal escape, nuclear entry, transcription, and translation are needed for pDNA, but only endosomal escape and translation are needed for mRNA. The order of the protein production, as evaluated by luciferase activity, was LNPssPalmE‑P4C2 + ApoE > LFN > LNPssPalmE‑P4C2. However, the order of homogeneity of protein production was LNPssPalmE‑P4C2 + ApoE > LNPssPalmE‑P4C2 > LFN. These results indicate that LNP with a neutral surface can deliver mRNA more homogeneously to cells, while the cationic transfection reagent results in a very high protein expression, but only in a limited population of cells. Furthermore, the gene expression in LFN was limited to 47.2 ± 4.8%, while it was taken up by 71.1 ± 1.9% of the cells. We previously demonstrated that cationic materials inhibited the translation process via electrostatic interactions with mRNA.31 Thus, the translation process might partially contribute to the heterogeneous expression in LFN. In previous study, intracerebroventricularly administrated LNPs encapsulating pDNA were taken up by astrocytes and neuronal cells in the corpus callosum. However, gene expression of reporter genes (LacZ and mCherry) was limited to astrocytes in brain. Because of its neutral surface in a physiological environment, the interaction of LNPssPalm with biomaterials including the nuclear membrane in the cytoplasm is considered to be poor. Thus, the transfection of pDNA to intrinsically nonmitotic neuronal cells is significantly hampered by the nuclear membrane. In the case of LNP encapsulating mRNA, transfected eGFP and astrocyte marker GFAP were observed to be colocalized. This indicates that the LNPssPalmE‑P4C2 was successfully taken up by astrocytes regardless of the nucleic acid cargo, probably in an ApoEdependent manner. The most notable observation in this study was that the eGFP was also colocalized with neuronal cells (anti-MAP-2 antibody). This observation highlights the superiority of mRNA-encapsulating LNPssPalm, a vector specialized in the cytoplasmic delivery of nucleic acids, over conventional pDNA delivery systems, especially in a nonmitotic cell population. In the case of in vivo application of mRNA, immune stimulation was inevitable safety concerns. Actually, LNPssPalm containing mRNA caused the release of IL-6, a proinflammatory cytokine. The production of IL-6 indicates activation of NFkB pathway by the administered mRNA in LNPssPalmE‑P4C2. It is thus assumed that endosomal RNA sensors such as Toll-like receptors 3, 7, and 8, and cytosolic RNA sensors like RIG-I were involved.32 The production of the cytokine was successfully suppressed by the introduction of chemically modified nucleotides (Figure 5) since these modifications have been reported to protect mRNA from being recognized by TLRs and cytosolic RNA sensors.33 In the intracerebroventricular administration of the LNPssPalmE‑P4C2, the chemical modification of the mRNA contributes to the elevated transgene activity, probably due to the avoidance of degradation by endogenous RNases. However, recent literature

Figure 5. (a) Structures of chemically modified nucleotides. 2Thiouridine, pseudouridine, and 5-methylcytidine were incorporated into luciferase mRNA during in vitro transcription reactions by adding each nucleotide to the NTP mixture. (b) Luciferase activity in brain with chemically modified mRNA. LNPssPalmE‑P4C2 and LFN encapsulating chemically modified mRNA were administrated to the mouse brain intracerebroventricularly. Luciferase activity was measured at 24 h after transfection. Statistical analysis was performed by Student’s t test (**p < 0.01). (c) Reduced immunogenicity of the chemically modified mRNA. The interleukin-6 production from mouse brain administered with naked mRNA, LFN, or LNPssPalmE‑P4C2, as measured by ELISA. Statistical analyses were performed by one-way ANOVA followed by the SNK test (N.S., not significant).

reports indicate that protein expression was decreased by using the chemically modified mRNA.34 It is evident that the effects of chemical modification on transgene efficiency depends on the target tissues/organs, the therapeutic proteins being used, RNA sequences, and the nature of the carriers employed. Thus, the development of an alternate strategy for avoiding immune activation is also necessary. Another concern regarding nanoparticle administration is the activation of the complement system. The possibility of complement activation-related pseudoallergy (CARPA)-like effects cannot be excluded because the abnormal activation of the complement system in the brain is one of the triggers for producing neurotoxic substances in the brain. An appropriate experimental model for evaluating complement activation would be needed for further investigation.35 One strategy for avoiding complement activation is the induction of tachyphylaxis as observed in the case of liposomal doxorubicin.36 The contribution of complement activation and the effectiveness of a tachyphylaxis strategy remains to be clarified. The major challenge to the current gene therapy for CNS is targeting the brain parenchyma for both viral and artificial carriers.37 In the case of viral vectors, recent literature reports that AAV9 can transit brain endothelial cells via an energydependent transcytosis process.38,39 However, details regarding this intracellular trafficking remains to be fully characterized. In F

DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics the case of artificial vectors, a similar transcytosis strategy would be a solution for crossing the blood−brain barrier. One of the ligands that participates in both uptake and transcytosis in brain endothelial cells is glucose, a ligand for a glucose transporter that is present in brain endothelial cells.40 A combination of such a ligand and LNPssPalm would be a rational strategy for developing a more practical artificial carrier in the future. It was recently reported that Huntington’s disease was ameliorated by the deletion of a mutant Huntingtin gene in adult mouse brain by delivering the virus based CRISPR/ Cas9.41 Such a genomic editing approach is a promising strategy for the permanent cure of neurodegenerative diseases. An increasing number of reports point to the feasibility using an artificial mRNA carrier to mediate in vivo gene editing.42,43 As to the chronic disease, the application of the mRNA delivery is hampered by the short-term gene expression. However, in the case of gene editing, the transient gene expression is beneficial for avoiding undesired off-targeting effects. An artificial carrier encapsulating a gene editing-related mRNA might be a key technology for the radical treatment of brain disorders.

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ASSOCIATED CONTENT

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REFERENCES

AADC, aromatic amino acid decarboxylase; AAV, adenoassociated virus; ApoE, apolipoprotein E; CNS, central nervous systems; CV, coefficiency of variance; DMG-PEG2000, 1(monomethoxy polyethylene glycol2000)2,3-dimyristoylglycerol; DOPC, dioleoyl-sn-glycero phosphatidyl choline; DOPE, dioleoyl-sn-glycero phosphatidyl ethanolamine; HDL, highdensity lipoprotein; ICV, intracerebroventricular injection; IVT, in vitro transcription; LDLR, low-density lipoprotein receptor; LFN, lipofectamine messenger max; LNP, lipid nanoparticle; NGF, nerve growth factor

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In this report, we demonstrate the successful mRNA delivery to neuronal cells, as well as astrocytes, by using neutral lipid nanoparticles. It is noteworthy that ApoE mediated uptake was one of the key features of these particles that make it suitable for the targeting neuronal cells. Immune activation can be avoided by using chemically modified nucleotides such as 2thiouridine, pseudouridine, and 5-methyl cytidine. These results clearly indicate that LNPs represent a potentially useful platform for mRNA-based therapeutics for the treatment of brain disorders.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b01084.

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Communication

Materials and antibodies and the effect of phosphatidyl choline on transgene efficiency (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Hidetaka Akita: 0000-0001-6262-2436 Notes

The authors declare the following competing financial interest(s): Hokkaido University and the NOF CORPORATION hold patent-pending (PCT/JP2012/079160) on the ssPalm chemicals. H.T., K.T., H.H., and H.A. are the inventors of the patent.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI (15H01806). This work is also supported in part by Asahi Glass Foundation and by JST CREST (Grant Number, JPMJCR17H1). The authors would like to thank Dr. M. S. Feather for his helpful advice in writing the English manuscript. G

DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Communication

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DOI: 10.1021/acs.molpharmaceut.7b01084 Mol. Pharmaceutics XXXX, XXX, XXX−XXX