Lipid nanoparticle formulations for enhanced co-delivery of siRNA and

Apr 25, 2018 - While mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitat...
1 downloads 0 Views 930KB Size
Download PDF
Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/NanoLett

Lipid Nanoparticle Formulations for Enhanced Co-delivery of siRNA and mRNA Rebecca L. Ball,† Khalid A. Hajj,† Jamie Vizelman,†,‡ Palak Bajaj,†,‡ and Kathryn A. Whitehead*,†,‡ †

Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213, United States S Supporting Information *

ABSTRACT: Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Coformulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the “helper” RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2−3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged “helper polymer” enhances the efficacy of LNP delivery systems. KEYWORDS: Lipid nanoparticle, lipidoid, mRNA delivery, siRNA delivery, nanoparticle formulation, polystyrenesulfonate

R

Given the considerably different molecular characteristics of siRNA and mRNA, successful co-formulation will require nanoparticle chemistry that accommodates both therapeutic molecules. For example, siRNA and mRNA have drastically different molecular weights (104 vs 106 g/mol), stability, and molecular conformation.19,20 Ionizable lipid nanoparticles (LNPs) have shown significant translational promise in the delivery of siRNA21−23 and, separately, mRNA. 24−27 Typically, the LNPs used to encapsulate siRNA and mRNA consist of the same four primary components: an ionizable lipid or lipidoid compound, cholesterol, a helper lipid, and polyethylene glycol (PEG)-lipid. However, the ratio of these components, together with the ratio of the ionizable lipid to RNA can significantly alter delivery efficacy in vitro and in vivo. 28−30 Previous work has demonstrated that siRNA and mRNA are most effectively delivered in distinct LNP formulations.31 We asked whether we could identify a single LNP formulation capable of simultaneous delivery of siRNA and mRNA. For the purposes of this study, we chose to work with the ionizable, biodegradable, amine-containing lipidoid 306Oi10, which we have previously identified as a potent RNA delivery material.32

NA drugs, including short interfering RNA (siRNA) and messenger RNA (mRNA), can theoretically treat any disease caused by gene dysregulation.1,2 Conditions associated with protein overexpression may benefit from siRNA drugs, which inhibit protein production by cleaving mRNA. On the other hand, diseases caused by insufficient protein production are candidates for mRNA therapy. Both types of RNA therapy have made significant translational progress over the past several years, often being delivered in ionizable polymer or lipid nanoparticles.3−10 The clinical translation of siRNA therapy, in particular, hit a major milestone in 2017 with the completion of the first successful Phase 3 clinical trial by Alnylam Pharmaceuticals.11 Although there is significant literature describing the delivery of siRNA or mRNA, to our knowledge there are no reports of their co-delivery. Delivery of both RNAs would enable simultaneous knockdown of undesirable protein(s) and expression of desirable protein(s). Such an approach would apply to many diseases, including liver cancer, which is characterized by the upregulation of oncogenes12−15 and downregulation of tumor suppressor genes.16−18 Encapsulation of siRNA and mRNA in a single particle guarantees that transfected cells receive both drugs, maximizing the intended therapeutic effect. A single formulation would also reduce production costs and regulatory hurdles compared to a therapy comprising two separate siRNA and mRNA formulations. © XXXX American Chemical Society

Received: March 19, 2018 Revised: April 24, 2018 Published: April 25, 2018 A

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters Table 1. Five LNP Formulations Were Investigated for Co-delivery of siRNA and mRNAa siRNA original 1

2

3

4

5

lipidoid:RNA weight ratio lipidoid helper lipid

5:1 50.0% DSPC 10%

6.25:1 46.3% DSPC 8.6% DOPE 2.9% 40.5% 1.75%

7.5:1 42.5% DSPC 6.5 DOPE 6.5% 42.5% 2.0%

8.75:1 38.8% DSPC 3.6 DOPE 10.9% 44.5% 2.25%

10:1 35.0%

cholesterol C14-PEG2000 a

mRNA original

formulation #

38.5% 1.5%

DOPE 16% 46.5% 2.5%

The percentages shown are molar. DSPC = 1,2-distearoyl-sn-glycero-3phosphocholine. DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

Figure 1. Lipid nanoparticles co-delivered siRNA and mRNA in vitro. HeLa cells that stably expressed firefly luciferase were incubated with LNPs containing 10 nM of siRNA against firefly luciferase and/or 100 ng of mRNA encoding mCHERRY. Expression of both proteins was assessed 24 h post-transfection. (A) LNPs co-formulated with siRNA and mRNA resulted in greater gene silencing than LNPs formulated only with siRNA. (n = 12−18) (B) LNP formulations 3−5 delivered mRNA to HeLa cells regardless of whether siRNA was included in the formulation. (n = 12−18). The same cells generated the data in panels A and B. (C) Total RNA entrapment increased at higher formulation numbers. (D) The RNA cargo, but not the LNP formulation number, affected LNP size. (n = 2−3).

We tested the efficacy of the five formulations for co-delivery of siRNA and mRNA in HeLa cells that stably expressed firefly and Renilla luciferase (Figure 1). For these experiments, siRNA targeted the firefly luciferase gene and mRNA encoded the fluorescent protein, mCHERRY. The nanoparticles were incubated with HeLa cells for 24 h at an siRNA dose of 10 nM (27 ng) and mRNA dose of 1.5 nM (100 ng). All five formulations were tested with cargos of siRNA only (blue circles), mRNA only (red squares), and a mixture of siRNA with mRNA (purple triangles). Figure 1A shows resultant luciferase gene silencing. As expected, control particles containing only mRNA induced no gene knockdown. For particles loaded with siRNA, gene silencing increased with increasing formulation number. We were surprised to note that nanoparticles co-formulated with siRNA and mRNA (purple triangles) mediated significantly higher levels of gene silencing compared to LNPs containing siRNA only (blue circles). When the same set of cells were assessed for mRNA delivery, higher formulation numbers again produced better results (Figure 1B). The co-formulated LNPs did not produce statistically significant mCherry production compared to the mRNA-only

Given the vast formulation space available for testing, we began with two LNP formulations previously reported for potent siRNA32−34 or mRNA25,31 delivery. We will refer to these preparations as Formulations 1 and 5, respectively (Table 1). Compared to Formulation 1, Formulation 5 features a higher lipidoid to RNA ratio and a lower molar percentage of lipidoid in relation to helper lipid, cholesterol, and PEG-lipid. Additionally, the helper lipid shifts from DSPC in Formulation 1 to DOPE in Formulation 5. DSPC contains two saturated aliphatic tails, while DOPE contains a cis-double bond in each of its two aliphatic tails. This structural difference changes the packing within the lipid nanoparticle and can affect endosomal escape,35−39 which is important given the significant molecular differences between siRNA and mRNA. In addition to Formulations 1 and 5, which had been previously designed for siRNA and mRNA delivery, respectively, we evaluated three intermediate formulations. These preparations, which we refer to as Formulations 2, 3, and 4, spanned the molar and weight ratios of Formulations 1 and 5 (Table 1). Notably, each intermediate formulation contained a blend of the helper lipids DSPC and DOPE. B

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 2. Lipid nanoparticles co-formulated with siRNA and mRNA resulted in enhanced efficacy in mice. All animals received siRNA specific for Factor VII (FVII) at a dose of 0.03 mg/kg and/or mRNA encoding firefly luciferase at a dose of 0.5 mg/kg. (A) LNPs co-formulated with both RNAs (purple circles) using Formulations 3−5 induced higher levels of gene silencing than LNPs formulated with only siRNA (blue triangle) and only mRNA (control, red square) (n = 3). (B) Co-formulated LNPs resulted in more luciferase expression than LNPs formulated with only mRNA. The same animals were used to generate the data in panels A and B (n = 3−4). (C) Total RNA entrapment increased with formulation number (n = 3 technical replicates). (D) Co-formulation resulted in enhanced RNA delivery. “Form’d Separately” mice received an injection containing LNPs formulated with siFVII combined with LNPs formulated with mLuc. “Co-formulated” mice received an injection of LNPs co-formulated with siFVII and mLuc. All LNPs were generated using Formulation 4 (n = 3).

mRNA or only siRNA (Figures 2B and S1A). Data in Figure 2A,B were collected from the same animals. No weight loss was observed for any of the groups (Figure S1B). As with in vitro studies, RNA entrapment improved with increasing formulation number (Figure 2C). Size was relatively consistent and nanoparticle surface charge remained approximately neutral across formulations (Figure S1C and Table S1). On the basis of these efficacy data, Formulation 4 was chosen for the remainder of experiments. Next, we confirmed that LNPs co-formulated with mRNA and siRNA were more potent than LNPs containing a single RNA species (Figure 2D). We compared efficacy in two groups of mice: one group received a mixture of siRNA-formulated LNPs and mRNA-formulated LNPs (i.e., each particle contained only one type of RNA). The second group received LNPs that had been co-formulated (i.e., each particle contained both RNAs). All LNPs were made using Formulation 4. Both treated groups received an siFVII dose of 0.03 mg/kg and an mLuc dose of 0.5 mg/kg. Remarkably, co-formulated LNPs improved Factor VII silencing from 50 to 90% (purple open circles) and tripled luciferase expression (red squares and Figure S1D). The two treated animal groups received the same total amount of each LNP ingredient, including the lipidoid. These results suggest there is a substantial efficacy boost imparted through the co-formulation of siRNA and mRNA in LNPs compared to single RNA species formulations.

nanoparticles. The improved delivery observed at higher formulation numbers was at least partially due to increases in RNA entrapment (Figure 1C). LNP size corresponded to the size of the RNA cargo but not the formulation number (Figure 1D). Given the successful co-delivery of siRNA and mRNA in HeLa cells, the five formulations from Table 1 were also tested for co-delivery efficacy in mice. Because 306Oi10 LNPs deliver RNA to the liver,32 we formulated LNPs with siRNA specific for the protein Factor VII (FVII). Factor VII, a blood clotting factor produced by hepatocytes, is readily measured from a blood sample using a commercially available assay. The mRNA chosen for in vivo studies encoded firefly luciferase (mLuc). Mice were injected by tail vein with LNPs loaded with RNA doses of 0.03 mg/kg siFVII and/or 0.5 mg/kg mLuc. Six hours post-injection, mRNA delivery-mediated luciferase expression was assessed using whole body luminescence imaging. Efficacy of siRNA delivery was measured 48 h after injection by quantifying serum Factor VII protein levels. As shown in Figure 2A, Formulations 3−5 loaded with both RNAs produced the highest levels of Factor VII silencing (∼90%, purple circles). This degree of knockdown was markedly better than when siRNA was delivered in Formulation 1 (∼50% silencing, blue triangle) and the mRNA control (0% silencing, red square). Co-delivery of siRNA and mRNA in Formulations 4 and 5 also improved mRNA delivery compared to LNPs containing only C

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 3. Enhanced delivery from co-formulation depended on the total RNA concentration. (A) The addition of “helper” mRNA to an LNP intended to deliver siRNA improved gene silencing efficacy for intermediate total RNA concentrations. All mice were dosed with 0.03 mg/kg siRNA specific for Factor VII (FVII) (n = 3). (B) The addition of “helper” siRNA to an LNP intended to deliver mRNA significantly improved luciferase protein expression at the intermediate total RNA concentration of 0.106 mg/mL. All mice were dosed with 0.5 mg/kg mRNA encoding firefly luciferase (n = 3−4). (C) LNP efficacy depended on the total RNA and lipidoid concentrations. LNPs containing siFVII were formulated with and without helper mRNA and with a low or high amount of lipidoid. All mice were dosed with 0.03 mg/kg siFVII (n = 3).

These results underscore the importance of total RNA concentration during LNP formulation on efficacy and suggest that a total RNA concentration of ∼0.106 mg/mL is optimal. The RNA entrapment of co-formulated LNPs was excellent (>85%) except at the highest RNA formulation concentration tested (Figure S3C,D). The z-average size of LNPs remained relatively consistent across formulations (100−150 nm) and did not correlate with potency (Figure S3E,F). These differences in entrapment and size for the highest RNA formulation concentration may explain why it was less efficacious than lower concentrations. We also calculated the nitrogen to phosphate ratio (N/P), which stayed fairly constant at approximately 8.4 (Table S2). As such, the differences in formulation efficacy cannot be attributed to N/P. It is possible that LNPs formulated with increased RNA content and, therefore, more negative charge, were more tightly and stably formed, leading to enhanced LNP efficacy. We noticed that, when measuring RNA entrapment, an unusually high concentration of surfactant was required to disrupt any LNPs formulated at a total RNA concentration greater than 0.1 mg/mL. While some increase in particle density appears to confer efficacy, it is possible that at some increased RNA concentration, intraparticle molecular attractions are strong enough to prevent release of the RNA cargo. This may explain the trends observed in Figure 3A,B in which efficacy improves only until the total RNA concentration reaches 0.106 mg/mL.

To better understand how co-formulation enhanced the delivery efficacy of both siRNA and mRNA, we investigated the role of total RNA concentration during the co-formulation process. The RNA concentration used for formulation does not necessarily go hand in hand with in vivo dose. In one experiment (Figure 3A), a set of five LNPs were formulated in which the siRNA concentration was held constant at 0.006 mg/ mL while the concentration of mRNA was gradually increased from 0 to 0.2 mg/mL. All LNPs were dosed in mice at 0.03 mg/kg of siRNA. The addition of mRNA improved FVII silencing, but only up to a total RNA concentration of 0.106 mg/mL. Beyond that, the benefit of co-formulation diminished and finally disappeared at a total RNA concentration of 0.206 mg/mL. The influence of total RNA concentration on mRNA delivery was also investigated (Figure 3B). In this case, LNPs were formulated with a constant concentration of mRNA (0.1 mg/ mL) and an increasing concentration of siRNA (0−0.08 mg/ mL). These formulations were dosed in mice at an mLuc dose of 0.5 mg/kg, and resultant luciferase protein expression was assessed 6 h post-injection (Figure S2). Here, we observed that adding 0.006 mg/mL of siRNA to the mRNA formulation improved efficacy. Beyond a total RNA concentration of 0.12 mg/mL, mRNA delivery efficacy decreased back to “baseline” (mRNA alone) levels. No toxicity, as determined by weight change, was observed for any animal groups (Figure S3A,B). D

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

RNA, we added each of them to the LNP formulation at a concentration that provided the same total negative charge as in co-formulated LNPs. When testing for siRNA efficacy, we found that co-formulation with 70 kDa PSS provided the same degree of potency enhancement as helper mRNA (Figure 4A).

We also considered that the increase in efficacy observed with co-formulated LNPs may be due to the increasing amount of lipid. LNPs co-formulated included higher amounts of lipidoid and other lipid content, as we believed this would be necessary to provide enough material to encapsulate the higher RNA amount. Because an increase in lipidoid concentration can sometimes confer LNP efficacy, we asked if this was the reason for enhanced efficacy of both RNA species upon coformulation. To investigate this, we prepared four LNP formulations that all contained the same amount of siRNA. Two of the four formulations contained “helper” mRNA. We evaluated two lipidoid concentrations: the lower concentration (0.05 mg/mL) represented the standard lipidoid concentration used when formulating LNPs encapsulating only siRNA. The higher concentration (0.93 mg/mL) represented the lipidoid concentration we had been using to co-formulate both RNAs. Figure 3C shows Factor VII levels following injection of the four formulations into mice, all at a dose of 0.03 mg/kg siFVII. LNPs formulated with high lipidoid content and without “helper” mRNA only modestly improved FVII protein silencing from 37 to 49% compared to LNPs formulated at a low lipidoid concentration. We also found no correlation between lipidoid concentration and efficacy when we globally considered all of the LNPs formulated for this manuscript and delivered in vivo (Figure S4). Together, these data confirmed that higher amounts of lipidoid were not primarily responsible for the increased efficacy observed with co-formulation. It was only once helper mRNA was included in the formulation along with the increased lipidoid that a marked increase in protein silencing to 87% occurred. Finally, efficacy disappeared if LNPs were formulated with both RNA species at a lower lipidoid concentration (rightmost bar in Figure 3C). In this case, RNA entrapment was close to zero (Figure S5A), suggesting that there was not enough lipid to promote LNP formation. Therefore, both a higher lipidoid concentration and increased total RNA content are needed for enhanced LNP efficacy with co-formulation. There was no relationship between LNP diameter and efficacy for the formulations tested in Figure 3C, and none of the formulations induced weight loss in mice (Figure S5B,C). Although the improvements to both mRNA and siRNA efficacy upon co-formulation are exciting, there are many instances in which treatment will require the delivery of only one type of RNA. Although the addition of a second RNA species to these LNP formulations would increase potency, it would also add cost, regulatory complexity, and the potential for unwanted biological effects. Therefore, we sought an alternative to “helper” RNA that would improve the effectiveness of single RNA formulations. We reasoned that because RNA is a negatively charged biopolymer, it may be possible to replace it with a negatively charged synthetic polymer. We evaluated several inexpensive options: uridine homopolymer (Poly(U)), poly(sodium 4-sytrenesulfonate) (PSS), and poly(acrylic acid) (PAA). Poly(U), which is a polymer of the RNA base uracil, is available at a much lower cost than sequence specific mRNA given the increased simplicity of synthesis. PSS is a synthetic, biocompatible polymer that is used clinically to treat hyperkalemia and lithium poisoning in humans.40−43 PAA, another synthetic polymer, has also been used in biomedical applications.44 To assess whether these polymers could replace the efficacy-enhancing effect of helper

Figure 4. Inclusion of a negatively charged “helper” polymer in single RNA LNP formulations improved efficacy. (A) All LNPs were dosed in mice at 0.03 mg/kg siRNA. Only the LNPs loaded with siFVII induced FVII gene silencing. LNPs formulated with helper mRNA, 70 kDa PSS, or Poly(U) improved knockdown compared to LNPs formulated without helper polymer (n = 3−4). (B) All LNPs were dosed at 0.5 mg/kg mRNA with only mLuc treatments resulting in gene expression. Compared to LNPs containing no helper material, the addition of siRNA or 6.8 kDa PSS to the formulation enhanced mLuc delivery (n = 3−4). (C) LNPs formulated with PSS facilitated comparable FVII silencing to LNPs without PSS using three times less drug. PSS-formulated LNPs induced dose-dependent gene knockdown (n = 3).

Poly(U) also increased protein knockdown compared to LNPs formulated without helper polymer, although not to the same degree as PSS. LNPs formulated with PAA lost efficacy. LNPs formulated with control siRNA did not alter Factor VII activity regardless of PSS content (gray bars). We also tested the effect of each polymer on mRNA formulations (Figure 4B). Although 70 kDa PSS did not improve efficacy (data not shown), we found that reducing the molecular weight to a size that more closely approximated E

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters siRNA (6.8 kDa) resulted in a 2-fold increase in protein expression compared to LNPs formulated without helper polymer (Figure S6). LNPs encapsulating control mRNA did not induce luciferase expression with or without PSS. Neither RNA entrapment nor LNP size correlated with efficacy (Figures S7A−D). None of the formulations induced weight loss in mice (Figures S7E,F). The surface charge of LNPs formulated with a helper polymer remained slightly negative and did not correlate with efficacy (Table S3). One potential reason why PSS best mimics the effect of helper RNA is that it is negatively charged at all pH values relevant to LNP formulation and delivery. Both the PSS monomer and the phosphodiester group in the RNA backbone have pKa values of one.45,46 LNPs are formulated under acidic conditions so that the lipidoid amines protonate and subsequently attract the negatively charged RNA. It is possible that the additional negative charge provided by the polyanion promotes the formation of a more stable and/or compact nanoparticle by increasing the electrostatic attraction inside the particle. PAA with a higher pKa of four47 may not mediate the same electrostatic effect. Poly(U), imparting some benefit to siRNA-loaded LNPs, may not facilitate the same intraparticle molecular interactions as mRNA because of its polydispersity (Mw 100−1000 kDa). Because the addition of 70 kDa PSS to siRNA-loaded LNPs was particularly successful, we wanted to understand how much siRNA drug can be saved by adding this helper polymer to the formulation. We first established that LNPs formulated without PSS and dosed at 0.03 mg/kg siRNA resulted in 49% Factor VII protein silencing (leftmost blue bar, Figure 4C). We then compared this result to when PSS was added to the LNP formulation and dosed from 0.005−0.03 mg/kg siRNA. Silencing was dose responsive with a dose of 0.03 mg/kg now inducing 90% silencing. LNPs formulated with PSS and dosed at 0.01 mg/kg achieved the same level of silencing as those formulated without PSS and dosed at 0.03 mg/kg. In other words, the incorporation of PSS into an LNP formulation facilitated a 3-fold reduction in the amount of siRNA required. Importantly, the total amount of lipidoid dosed to mice was the same for each group in Figure 4C. As such, the efficacy “savings” afforded by PSS will not be offset by increased toxicity due to increased lipid content. In these experiments, PSS did not improve RNA entrapment, and nanoparticle size did not correlate with efficacy (Figure S8A,B). None of the treatments resulted in significant weight loss compared to the untreated mice (Figure S8C). Dual gene therapy that enables simultaneous gene expression and gene silencing through the delivery of mRNA and siRNA has the potential to benefit countless patients. Together, the data presented here show that not only is the co-formulation of the two RNA drugs in the same particle possible but that it substantially enhances efficacy compared to particles formulated with individual RNAs. This observation inspired the development of an improved LNP formulation for the delivery of single RNA species. Specifically, we propose the incorporation of the biocompatible helper polymer, PSS, into lipid nanoparticle formulations for siRNA and mRNA delivery (Table 2). Our results suggest that the additional negative charge provided by the helper polymer may promote electrostatic attraction in the particle and facilitate stability. LNPs containing PSS mediate the same degree of in vivo efficacy with only one-half to one-third of the drug dose, which

Table 2. Recommended Incorporation of the Helper Polymer, PSS, into Lipid Nanoparticle Formulationsa lipidoid/(RNA + PSS) weight ratio PSS/RNA weight ratio PSS mol. weight (kDa) lipidoid helper lipid cholesterol C14-PEG2000

siRNA Formulation

mRNA Formulation

8.75:1

8.75:1

10.7:1 70 38.8% DSPC 3.6% DOPE 10.9% 44.5% 2.25%

0.04:1 6.8 38.8% DSPC 3.6% DOPE 10.9% 44.5% 2.25%

a

The percentages shown are molar. DSPC = 1,2-distearoyl-sn-glycero3phosphocholine. DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

we anticipate will reduce the cost and potentially the offtargeting events associated with RNA treatment. Materials and Methods. Materials. Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO), and distearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoylsn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000] (14:0 PEG2000-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). HeLa cells were purchased from American Type Culture Collection (Manassas, VA). Dulbecco’s Modified Eagles Media (DMEM), trypsin, penicillin/streptomycin, phosphate buffered saline (PBS), and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Waltham, MA). Anti-firefly luciferase siRNA for Luciferase (siLuc) was purchased from Dharmacon (Lafayette, CO). Anti-Factor VII siRNA was custom ordered from Sigma-Aldrich with a sense sequence of 5′-GGAucAucucAAGucuuAcT*T-3′ and an antisense sequence of 5′GuAAGAcuuGAGAuGAuccT*T-3′, where lower case nucleotides are 2′-fluoro-modified and asterisks indicate phosphorothioate linkages. Clean Cap mCHERRY and firefly luciferase mRNA were purchased from TriLink Biotechnologies (San Diego, CA). Polysodium 4-styrenesulfonate (PSS Mw 70,000 and 6,800), poly(acrylic acid) (PAA Mv ∼ 450,000), and polyuridylic acid potassium salt (Poly(U) Mw 100−1,000 kDa) were purchased from Sigma-Aldrich. Lipid Nanoparticle Formulation and Characterization. LNPs were prepared as previously described.48 The lipidoid 306Oi10 was synthesized by Michael addition of isodecyl acrylate to 3,3′-diamino-N-methyldipropylamine. The fully substituted version, which was isolated using a Teledyne Isco chromatography system, was used for all experiments.32 LNPs were formulated by first dissolving the lipidoid, cholesterol, DSPC, DOPE, and PEG2000 in ethanol. A lipid solution was made by mixing these components according to the molar ratios in Table 1 in 90% ethanol and 10% 10 nM sodium citrate (by volume). The RNA (siRNA and/or mRNA) was diluted in 10 nM sodium citrate to achieve a final lipidoid/RNA weight ratio between 5:1 and 10:1. Rapid pipet mixing was used for spontaneous formation of the LNPs. For in vivo experiments, LNPs were dialyzed for 1 h in PBS to remove ethanol. Finally, LNPs were diluted in PBS to achieve the desired final concentration. For all LNP characterization studies, the nanoparticles were diluted to 1 μg/mL total RNA. LNPs were characterized for RNA entrapment using a Quant-iT Ribogreen Assay (Invitrogen, Carlsbad, CA) according to the manufacture’s F

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

and RNA solutions were prepared in ethanol and 10 nM sodium citrate, respectively, as described in Lipid Nanoparticle Formulation and Characterization. Negatively charged polymer (Poly(U), PSS, or PAA) was dissolved in PBS and added to the RNA solution. Finally, the lipid solution was added to the RNA-polymer solution and rapidly pipetted for nanoparticle formation. LNPs containing siRNA as the active drug were formulated at 0.006 mg/mL siFVII and with either mLuc (0.1 mg/mL), PSS (Mw 70 kDa, 0.064 mg/mL), Poly(U) (Mw 100−1,000 kDa, 0.1 mg/mL), or PAA (Mv 450 kDa, 0.1 mg/ mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.03 mg/kg siFVII. LNPs containing mRNA as the active drug were formulated at 0.1 mg/mL mLuc and with either siFVII (0.006 mg/mL), PSS (Mw 6.8 kDa, 0.004 mg/ mL), Poly(U) (Mw 100−1,000 kDa, 0.005 mg/mL), or PAA (Mv 450 kDa, 0.0013 mg/mL) while keeping the lipidoid concentration constant at 0.93 mg/mL. All mice were dosed with LNP formulations containing 0.5 mg/kg mLuc. The negative polymer concentrations were decided based on maintaining the same amount of total negative charge as when the LNPs are made with 0.006 mg/mL siRNA and 0.1 mg/mL mRNA. For experiments in Figure 4C, siFVII was used as the active drug and formulated at either 0.004 mg/mL siFVII and PSS (70 kDa, 0.066 mg/mL), 0.002 mg/mL and PSS (70 kDa, 0.067 mg/mL) or 0.001 mg/mL siFVII and PSS (70 kDa, 0.068 mg/ mL) and IV-injected to mice at doses of 0.005−0.3 mg/kg. The negative polymer concentrations for these experiments were selected to maintain the same total negative charge as when the LNPs were formulated with 0.006 mg/mL siRNA and 0.1 mg/ mL mRNA. Statistical Analysis. All statistical analysis was performed using GraphPad Prism (La Jolla, CA) software. Error bars represent standard deviation (sample sizes provided in each figure). To compare two groups, unpaired Student’s t tests were performed, assuming Gaussian distribution. Groups of three or more were compared by one-way ANOVA. Statistical significance is indicated by * p < 0.05, ** p < 0.01 and, **** p < 0.0001.

protocol. Dynamic light scattering (DLS) was used to characterize LNP size and polydispersity (PDI) on a Malvern Zetasizer Nano (Malvern Instruments, U.K.). Transfection of Co-delivered siRNA and mRNA in Vitro. HeLa cells stably modified to express firefly and Renilla luciferase were grown in DMEM supplemented with 100 mL/L of FBS, 10 IU/mL of penicillin, and 10 mg/mL of streptomycin. The cells were incubated at 37 °C in a 5% CO2 environment and subcultured by partial digestion with 0.25% trypsin and ethylenediaminetetraacetic acid. Passages 10−30 were used for experiments. HeLa cells were seeded at 15,000 cells per well in a black 96-well plate. LNPs were made according to the formulations in Table 1 with either siRNA, mRNA, or siRNA + mRNA at RNA concentrations of 0.0125 mg/mL siRNA and 0.05 mg/mL mRNA. For in vitro experiments, siRNA was specific for firefly luciferase and mRNA encoded mCherry. LNPs were delivered to luciferaseexpressing HeLa cells at a dose of 10 nM siRNA and/or 100 ng (∼1.6 nM) mRNA for 24 h. Resultant mCHERRY fluorescence (ex:587 nm/em:615 nm) was measured 24 h after transfection. Then, luciferase activity was quantified using a Dual-Glo Luciferase Assay Kit (Promega, Madison, WI) according to the manufacture’s protocol. After the first Luciferase Assay Kit reagent was added, the lysed cells were transferred to a white plate to measure luminescence. Renilla luciferase activity served as a control. Animal Studies. Animal protocols were approved by the Institutional Animal Care and Use Committee at Carnegie Mellon University (Pittsburgh, PA). C57BL/6 mice (female and male) of at least 6 weeks of age were purchased from Charles River Laboratories. Mice were housed under controlled temperature (25 °C) in 12 h light-dark cycles. Animals were given access to standard diet and water. LNPs for in vivo experiments were formulated with either anti-Factor VII siRNA (siFVII), mRNA encoding luciferase (mLuc) or a combination of the two and administered to mice via tail-vein injection. Six hours later, luciferase activity was assessed by administering an intraperitoneal injection of DLuciferin substrate (130 μL at 30 mg/mL in PBS). Fifteen minutes later, luminescence was measured by whole mouse imaging using an IVIS (PerkinElmer, MA) and quantified using Living Image software (PerkinElmer). Factor VII expression was measured 48 h after LNP injection from a submandibular blood sample. A BIOPHEN FVII assay was used according to the manufacture’s protocol (Aniara, OH). Mice were also weighed 48 h post-injection, as significant weight loss may indicate LNP toxicity. For the experiments in Figure 2, LNPs were formulated according to the specifications in Table 1 at concentrations of 0.006 mg/mL siFVII and/or 0.1 mg/mL mLuc. Mice were dosed with 0.03 mg/kg siRNA and/or 0.5 mg/kg mRNA. For the experiments in Figures 2D, 3, and 4, all LNPs were made using Formulation 4. For experiments in Figure 3A, the siRNA formulation concentration was kept constant at 0.006 mg/mL while mRNA concentration varied from 0−0.2 mg/mL. This corresponded to a final dose in mice of 0.03 mg/kg siRNA and 0−1 mg/kg mRNA. For Figure 3B, the LNP mRNA formulation concentration was kept constant at 0.1 mg/mL and the siRNA concentration varied from 0−0.08 mg/mL. Mice were dosed with 0.5 mg/kg mRNA and 0−0.4 mg/kg siRNA. LNP Formulation with Negatively Charged Polymers. For LNP formulation with negatively charged polymers, the lipid

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b01101. Size and RNA entrapment data for all LNPs formulated in the manuscript, raw IVIS images of luminescence in mice, and data related to mouse weight change, N/P ratio for LNPs in Figure 3A,B, and correlation between efficacy and lipidoid formulation concentration (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kathryn A. Whitehead: 0000-0002-0100-7824 Author Contributions

R.B. and K.W. conceived of the idea. R.B., K.H., J.V., and P.B. conducted the experiments, and R.B. and K.H. analyzed results. R.B., K.H., and K.W. wrote the manuscript. All authors have given approval to the final version of the manuscript. G

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters Notes

(15) Liu, J. Y.; Chiang, T.; Liu, C. H.; Chern, G. G.; Lin, T. T.; Gao, D. Y.; Chen, Y. Delivery of siRNA Using CXCR4-Targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer. Mol. Ther. 2015, 23 (11), 1772−1782. (16) Fujimoto, A.; Furuta, M.; Totoki, Y.; Tsunoda, T.; Kato, M.; Shiraishi, Y.; Tanaka, H.; Taniguchi, H.; Kawakami, Y.; Ueno, M.; et al. Whole-Genome Mutational Landscape and Characterization of Noncoding and Structural Mutations in Liver Cancer. Nat. Genet. 2016, 48 (5), 500−509. (17) Lu, L.; Li, Y.; Kim, S. M.; Bossuyt, W.; Liu, P.; Qiu, Q.; Wang, Y.; Halder, G.; Finegold, M. J.; Lee, J.-S.; et al. Hippo Signaling Is a Potent in Vivo Growth and Tumor Suppressor Pathway in the Mammalian Liver. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (4), 1437− 1442. (18) Xue, W.; Zender, L.; Miething, C.; Dickins, R. A.; Hernando, E.; Krizhanovsky, V.; Cordon-Cardo, C.; Lowe, S. W. Senescence and Tumour Clearance Is Triggered by p53 Restoration in Murine Liver Carcinomas. Nature 2007, 445 (7128), 656−660. (19) Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down Barriers: Advances in siRNA Delivery. Nat. Rev. Drug Discovery 2009, 8 (2), 129−138. (20) Sahin, U.; Karikó, K.; Türeci, Ö . mRNA-Based Therapeutics  Developing a New Class of Drugs. Nat. Rev. Drug Discovery 2014, 13 (10), 759−780. (21) Katakowski, J. A.; Mukherjee, G.; Wilner, S. E.; Maier, K. E.; Harrison, M. T.; Di Lorenzo, T. P.; Levy, M.; Palliser, D. Delivery of siRNAs to Dendritic Cells Using DEC205-Targeted Lipid Nanoparticles to Inhibit Immune Responses. Mol. Ther. 2016, 24 (1), 146− 155. (22) Wang, H.; Tam, Y. Y. C.; Chen, S.; Zaifman, J.; Van Der Meel, R.; Ciufolini, M. A.; Cullis, P. R. The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA. Mol. Ther. 2016, 24 (12), 2100−2108. (23) Kedmi, R.; Veiga, N.; Ramishetti, S.; Goldsmith, M.; Rosenblum, D.; Dammes, N.; Hazan-Halevy, I.; Nahary, L.; Leviatan-Ben-Arye, S.; Harlev, M.; et al. A Modular Platform for Targeted RNAi Therapeutics. Nat. Nanotechnol. 2018, 13, 214−219. (24) Pardi, N.; Hogan, M. J.; Pelc, R. S.; Muramatsu, H.; Andersen, H.; DeMaso, C. R.; Dowd, K. A.; Sutherland, L. L.; Scearce, R. M.; Parks, R.; et al. Zika Virus Protection by a Single Low-Dose Nucleoside-Modified mRNA Vaccination. Nature 2017, 543 (7644), 248−251. (25) Oberli, M. A.; Reichmuth, A. M.; Dorkin, J. R.; Mitchell, M. J.; Fenton, O. S.; Jaklenec, A.; Anderson, D. G.; Langer, R.; Blankschtein, D. Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy. Nano Lett. 2017, 17 (3), 1326−1335. (26) Liang, F.; Lindgren, G.; Lin, A.; Thompson, E. A.; Ols, S.; Röhss, J.; John, S.; Hassett, K.; Yuzhakov, O.; Bahl, K.; et al. Efficient Targeting and Activation of Antigen-Presenting Cells In Vivo after Modified mRNA Vaccine Administration in Rhesus Macaques. Mol. Ther. 2017, 25 (12), 2635−2647. (27) Patel, S.; Ashwanikumar, N.; Robinson, E.; Duross, A.; Sun, C.; Murphy-Benenato, K. E.; Mihai, C.; Almarsson, Ö .; Sahay, G. Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA. Nano Lett. 2017, 17 (9), 5711−5718. (28) Akinc, A.; Goldberg, M.; Qin, J.; Dorkin, J. R.; Gamba-Vitalo, C.; Maier, M.; Jayaprakash, K. N.; Jayaraman, M.; Rajeev, K. G.; Manoharan, M.; et al. Development of Lipidoid-Sirna Formulations for Systemic Delivery to the Liver. Mol. Ther. 2009, 17 (5), 872−879. (29) Chen, S.; Tam, Y. Y. C.; Lin, P. J. C.; Leung, A. K. K.; Tam, Y. K.; Cullis, P. R. Development of Lipid Nanoparticle Formulations of siRNA for Hepatocyte Gene Silencing Following Subcutaneous Administration. J. Controlled Release 2014, 196, 106−112. (30) Miller, J. B.; Kos, P.; Tieu, V.; Zhou, K.; Siegwart, D. J. Development of Cationic Quaternary Ammonium Sulfonamide Amino Lipids for Nucleic Acid Delivery. ACS Appl. Mater. Interfaces 2018, 10, 2303.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank Christopher Knapp for technical assistance, Daniel Siegwart for helpful discussions, and Kyle Cochran for his feedback on the manuscript. Funding was provided by the Center for Nucleic Acids Science and Technology, the DSF Charitable Foundation, and the Defense Advanced Research Projects Agency (DARPA), Grant number D16AP00143.

■

REFERENCES

(1) Wittrup, A.; Lieberman, J. Knocking down Disease: A Progress Report on siRNA Therapeutics. Nat. Rev. Genet. 2015, 16 (9), 543− 552. (2) Hajj, K. A.; Whitehead, K. A. Tools for Translation: Non-Viral Materials for Therapeutic mRNA Delivery. Nat. Rev. Mater. 2017, 2 (10), 17056. (3) Dahlman, J. E.; Barnes, C.; Khan, O. F.; Thiriot, A.; Jhunjunwala, S.; Shaw, T. E.; Xing, Y.; Sager, H. B.; Sahay, G.; Speciner, L.; et al. Vivo Endothelial siRNA Delivery Using Polymeric Nanoparticles with Low Molecular Weight. Nat. Nanotechnol. 2014, 9 (8), 648−655. (4) Thi, E. P.; Mire, C. E.; Lee, A. C. H.; Geisbert, J. B.; Zhou, J. Z.; Agans, K. N.; Snead, N. M.; Deer, D. J.; Barnard, T. R.; Fenton, K. A.; et al. Lipid Nanoparticle siRNA Treatment of Ebola-Virus-MakonaInfected Nonhuman Primates. Nature 2015, 521 (7552), 362−365. (5) Zhao, Y.; Wang, W.; Guo, S.; Wang, Y.; Miao, L.; Xiong, Y.; Huang, L. PolyMetformin Combines Carrier and Anticancer Activities for in Vivo siRNA Delivery. Nat. Commun. 2016, 7 (11822), 1−9. (6) Kaczmarek, J. C.; Patel, A. K.; Kauffman, K. J.; Fenton, O. S.; Webber, M. J.; Heartlein, M. W.; DeRosa, F.; Anderson, D. G. Polymer−Lipid Nanoparticles for Systemic Delivery of mRNA to the Lungs. Angew. Angew. Chem. 2016, 128, 14012−14016. (7) Stadler, C. R.; Bähr-Mahmud, H.; Celik, L.; Hebich, B.; Roth, A. S.; Roth, R. P.; Karikó, K.; Türeci, Ö .; Sahin, U. Elimination of Large Tumors in Mice by mRNA-Encoded Bispecific Antibodies. Nat. Med. 2017, 23 (7), 815−817. (8) Richner, J. M.; Jagger, B. W.; Shan, C.; Fontes, C. R.; Dowd, K. A.; Cao, B.; Himansu, S.; Caine, E. A.; Nunes, B. T. D.; Medeiros, D. B. A.; et al. Vaccine Mediated Protection Against Zika Virus-Induced Congenital Disease. Cell 2017, 170, 273−283. (9) Jackson, M. A.; Werfel, T. A.; Curvino, E. J.; Yu, F.; Kavanaugh, T. E.; Sarett, S. M.; Dockery, M. D.; Kilchrist, K. V.; Jackson, A. N.; Giorgio, T. D.; et al. Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol. ACS Nano 2017, 11 (6), 5680−5696. (10) Miller, J. B.; Zhang, S.; Kos, P.; Xiong, H.; Zhou, K.; Perelman, S. S.; Zhu, H.; Siegwart, D. J. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. Angew. Chem., Int. Ed. 2017, 56 (4), 1059− 1063. (11) Sheridan, C. With Alnylam’s Amyloidosis Success, RNAi Approval Hopes Soar. Nat. Biotechnol. 2017, 35 (11), 995−997. (12) Makita, Y.; Murata, S.; Katou, Y.; Kikuchi, K.; Uejima, H.; Teratani, M.; Hoashi, Y.; Kenjo, E.; Matsumoto, S.; Nogami, M.; et al. Anti-Tumor Activity of KNTC2 siRNA in Orthotopic Tumor Model Mice of Hepatocellular Carcinoma. Biochem. Biophys. Res. Commun. 2017, 493 (1), 800−806. (13) Lin, F.; Marcelo, K. L.; Rajapakshe, K.; Coarfa, C.; Dean, A.; Wilganowski, N.; Robinson, H.; Sevick, E.; Bissig, K. D.; Goldie, L. C.; et al. The CaMKK2/CaMKIV Relay Is an Essential Regulator of Hepatic Cancer. Hepatology 2015, 62 (2), 505−520. (14) Dudek, H.; Wong, D. H.; Arvan, R.; Shah, A.; Wortham, K.; Ying, B.; Diwanji, R.; Zhou, W.; Holmes, B.; Yang, H.; et al. Knockdown of β-Catenin with Dicer-Substrate siRNAs Reduces Liver Tumor Burden in Vivo. Mol. Ther. 2014, 22 (1), 92−101. H

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters (31) Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; Derosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015, 15 (11), 7300−7306. (32) Knapp, C. M.; Guo, P.; Whitehead, K. A. Lipidoid Tail Structure Strongly Influences siRNA Delivery Activity. Cell. Mol. Bioeng. 2016, 9, 305. (33) Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, V.; Hossain, N.; Bacallado, S. a.; Nguyen, D. N.; Fuller, J.; Alvarez, R.; et al. A Combinatorial Library of Lipid-like Materials for Delivery of RNAi Therapeutics. Nat. Biotechnol. 2008, 26 (5), 561− 569. (34) Dong, Y.; Dorkin, J. R.; Wang, W.; Chang, P. H.; Webber, M. J.; Tang, B. C.; Yang, J.; Abutbul-Ionita, I.; Danino, D.; Derosa, F.; et al. Poly(glycoamidoamine) Brushes Formulated Nanomaterials for Systemic siRNA and mRNA Delivery in Vivo. Nano Lett. 2016, 16 (2), 842−848. (35) Litzinger, D. C.; Huang, L. Phosphatodylethanolamine Liposomes: Drug Delivery, Gene Transfer and Immunodiagnostic Applications. Biochim. Biophys. Acta, Rev. Biomembr. 1992, 1113 (2), 201−227. (36) Brown, P. M.; Steers, J.; Hui, S. W.; Yeagle, P. L.; Silvius, J. R. Role of Head Group Structure in the Phase Behavior of Amino Phospholipids. 2. Lamellar and Nonlamellar Phases of Unsaturated Phosphatidylethanolamine Analogues. Biochemistry 1986, 25 (15), 4259−4267. (37) Fasbender, A.; Marshall, J.; Moninger, T. O.; Grunst, T.; Cheng, S.; Welsh, M. J. Effect of Co-Lipids in Enhancing Cationic LipidMediated Gene Transfer in Vitro and in Vivo. Gene Ther. 1997, 4 (7), 716−725. (38) Harvey, R. D.; Ara, N.; Heenan, R. K.; Barlow, D. J.; Quinn, P. J.; Lawrence, M. J. Stabilization of Distearoylphosphatidylcholine Lamellar Phases in Propylene Glycol Using Cholesterol. Mol. Pharmaceutics 2013, 10 (12), 4408−4417. (39) Farhood, H.; Serbina, N.; Huang, L. The Role of Dioeoyl Phosphatidylethanolamine in Cationic Liposome Mediated Gene Transfer. Biochim. Biophys. Acta, Biomembr. 1995, 1235, 289−295. (40) Chernin, G.; Gal-Oz, A.; Ben-Assa, E.; Schwartz, I. F.; Weinstein, T.; Schwartz, D.; Silverberg, D. S. Secondary Prevention of Hyperkalemia with Sodium Polystyrene Sulfonate in Cardiac and Kidney Patients on Renin-Angiotensin-Aldosterone System Inhibition Therapy. Clin. Cardiol. 2012, 35 (1), 32−36. (41) Sterns, R. H.; Rojas, M.; Bernstein, P.; Chennupati, S. IonExchange Resins for the Treatment of Hyperkalemia: Are They Safe and Effective? J. Am. Soc. Nephrol. 2010, 21 (5), 733−735. (42) Linakis, J. G.; Lacouture, P. G.; Eisenberg, M. S.; Maher, T. J.; Lewander, W. J.; Driscoll, J. L.; Woolf, A. D. Administration of Activated Charcoal or Sodium Polystyrene Sulfonate (KayexalateTM) as Gastric Decontamination for Lithium Intoxication: An Animal Model. Pharmacol. Toxicol. 1989, 65 (5), 387−389. (43) Lee, Y. C.; Lin, J. L.; Lee, S. Y.; Hsu, C. W.; Weng, C. H.; Chen, Y. H.; Yang, C. W.; Yen, T. H. Outcome of Patients with Lithium Poisoning at a Far-East Poison Center. Hum. Exp. Toxicol. 2011, 30 (7), 528−534. (44) Liu, J.; Huang, Y.; Kumar, A.; Tan, A.; Jin, S.; Mozhi, A.; Liang, X.-J. pH-Sensitive Nano-Systems for Drug Delivery in Cancer Therapy. Biotechnol. Adv. 2014, 32 (4), 693−710. (45) Li, L.; Ferng, L.; Wei, Y.; Yang, C.; Ji, H. F. Effects of Acidity on the Size of Polyaniline-Poly(sodium 4-Styrenesulfonate) Composite Particles and the Stability of Corresponding Colloids in Water. J. Colloid Interface Sci. 2012, 381 (1), 11−16. (46) Thaplyal, P.; Bevilacqua, P. C. Experimental Approaches for Measuring pKas in RNA and DNA. Methods Enzymol. 2014, 4 (549), 189−219. (47) Lee, J. W.; Kim, S. Y.; Kim, S. S.; Lee, Y. M.; Lee, K. H.; Kim, S. J. Synthesis and Characteristics of Interpenetrating Polymer Network Hydrogel Composed of Chitosan and Poly (Acrylic Acid). J. Appl. Polym. Sci. 1999, 73, 113−120.

(48) Whitehead, K. A.; Dorkin, J. R.; Vegas, A. J.; Chang, P. H.; Veiseh, O.; Matthews, J.; Fenton, O. S.; Zhang, Y.; Olejnik, K. T.; Yesilyurt, V.; et al. Degradable Lipid Nanoparticles with Predictable in Vivo siRNA Delivery Activity. Nat. Commun. 2014, 5, 4277.

I

DOI: 10.1021/acs.nanolett.8b01101 Nano Lett. XXXX, XXX, XXX−XXX