Article pubs.acs.org/Langmuir

Mechanism of Macromolecular Structure Evolution in SelfAssembled Lipid Nanoparticles for siRNA Delivery Marian E. Gindy,*,†,‡ Katherine DiFelice,†,‡ Varun Kumar,§,† Robert K. Prud’homme,§ Robert Celano,†,‡ R. Matthew Haas,†,‡ Jeffrey S. Smith,†,‡ and David Boardman†,‡ †

Department of Pharmaceutical Sciences and ‡Department of RNA Therapeutics, Merck Research Laboratories, Merck and Co., Inc., West Point, Pennsylvania 19486, United States § Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08540, United States S Supporting Information *

ABSTRACT: Lipid nanoparticles (LNPs) are a leading platform for therapeutic delivery of small interfering RNAs (siRNAs). Optimization of LNPs as therapeutic products is enabled by the development of structure−activity relationships (SAR) linking LNP physiochemical and macromolecular properties to bioperformance. Methods by which LNP properties can be rationally manipulated are thus critical enablers of this fundamental knowledge build. In this work, we present a mechanistic study of LNP self-assembly via a rapid antisolvent precipitation process and identify critical physiochemical and kinetic parameters governing the evolution of LNP three-dimensional macromolecular structure as a biorelevant SAR feature. Using small-angle X-ray scattering, LNPs are shown to undergo a temporal evolution in macromolecular structure during self-assembly, rearranging from initially disordered phases after precipitation into well-ordered structures following a necessary annealing stage of the assembly sequence. The ability of LNPs to undergo structural reorganization is shown to be effected by the chemical nature of the aqueous antisolvent used for precipitation. Antisolvents of varying buffering species differentially influence LNP macromolecular features, revealing a new participatory role of buffer ions in LNP self-assembly. Furthermore, the formation of macromolecular structure in LNPs is shown to improve the efficiency of siRNA encapsulation, thereby offering a simple, nonchemical route for preparation of high-payload LNPs that minimize the dose of lipid excipients. The developed LNP precipitation process and mechanistic understanding of self-assembly are shown to be generalizable, enabling the production of LNPs with a tunable range of macromolecular features, as evidenced by the cubic, hexagonal, and oligo-lamellar phase LNPs exemplarily generated.



INTRODUCTION RNA interference (RNAi) is an endogenous mechanism for post-transcriptional gene silencing.1 The discovery that exogenously delivered, small interfering RNAs (siRNAs) could effectively trigger RNAi in mammalian cells was a ground-breaking advance, enabling the development of new research tools to interrogate gene function and presenting a promising strategy for design of novel therapeutics.2−4 However, the single most critical factor limiting broad utility of siRNA as a therapeutic modality is the difficulty of delivering siRNA molecules in vivo via systemic administration.5 The unfavorable physiochemical properties of siRNA present challenges in the unaided bypass of extracellular and intracellular barriers against effective delivery. © 2014 American Chemical Society

Lipid nanoparticles (LNPs) currently represent the most advanced clinical strategy for vehicle-aided, nonviral delivery of therapeutic siRNA.6,7 LNPs are typically composed of ionizable amino lipids, neutral lipids, and poly(ethylene glycol) (PEG)lipid conjugateseach component contributing to physiochemical properties and biological performance of the delivery system.8,9 The amino lipids serve to effectively encapsulate siRNA during LNP formation and play a key role in facilitating siRNA escape from endosomes following endocytosis.10 Neutral lipids, such as cholesterol and phospholipids, are Received: February 18, 2014 Revised: March 30, 2014 Published: March 31, 2014 4613

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used to selectively modulate the fluidity and phase behavior of the LNP and impact interactions of the particle with biological membranes.11 The PEG-lipid component is used to confer physical stability during LNP manufacture and to prolong particle circulation upon intravenous administration in vivo.12 The development of LNPs as therapeutic products has been advanced through a structure−activity relationship (SAR) approach to particle design. For example, LNP potency improvements have been realized through SAR-guided design of novel amino lipids directed by putative mechanisms of in vivo cellular internalization and endosomal escape processes.13−15 Similarly, the physical and macromolecular structure properties of LNPs present opportunities for SAR-guided optimization. For instance, different stages in the pathway of transfection are reported to be influenced by the macromolecular structure of the LNP, with various structures (e.g., lamellar, hexagonal, cubic, etc.) leading to differentiated mechanisms of cellular uptake, endosomal escape, and intracellular disassembly.16−18 Thus, the ability to tune both physiochemical and macromolecular properties of LNPs should allow for a comprehensive interrogation of SAR guiding product optimization. Production methods by which LNP properties can be reproducibly tuned are thus critical to robust SAR development. We previously reported the development of a rapid precipitation process for the production of LNPs.19 The process utilizes a confined volume micromixing device to generate LNPs with precisely controlled nanometer size, narrow polydispersity, and high siRNA encapsulation efficiencies. The control of LNP physical properties afforded by the rapid precipitation presents improvements over alternative LNP synthesis techniques employing macroscopic mixing technologies, such as the preformed vesicle method20 and the spontaneous vesicle formation process.21 In addition, the design simplicity of the mixing device, coupled with the capacity for continuous flow operation, makes the rapid precipitation process suitable to commercial scale-up, presenting an operational advantage over alternative micromixing techniques such as microfluidics.22 In this paper, we extend our prior work and present a detailed study of LNP self-assembly via rapid precipitation, with focus on developing a mechanistic understanding of LNP macromolecular structure formation during particle assembly. We use small-angle X-ray scattering (SAXS) to quantitatively characterize structural features of LNPs and identify key physiochemical and kinetic parameters governing the formation of ordered lamellar phases for a prototype class of particles. The developed mechanistic insight into LNP self-assembly is expected to support the rational design of LNP products by enabling facile optimization of LNP physiochemical and structural properties relevant to in vivo bioperformance.

Figure 1. Lipid components and nanoparticle assembly process. (a) Chemical structures of lipids used in this study and (b) process flowchart for preparation of LNPs by rapid precipitation. Process steps detailed in this work are highlighted in bold.

siRNA using an ionizable lipid amine (N) to siRNA phosphate (P) molar ratio (N/P) ranging from 1 to 3. A flow diagram detailing LNP preparation by rapid precipitation is shown in Figure 1b. The assembly process is conceptually similar to the spontaneous vesicle formation method first described by Jeffs and co-workers.21 Formation of LNPs is initiated when a solution of lipids dissolved in ethanol is mixed with an acidic aqueous solution containing siRNA. Upon mixing, the ethanol falls below a critical concentration required to support solubilization of lipids, triggering precipitation and self-assembly of lipid particles. During particle precipitation, encapsulation of siRNA is facilitated by electrostatic interactions between the anionic oligonucleotide and the positively charged amino lipid. Because particle assembly relies on molecular interactions between initially segregated siRNA and lipid solutes, the process is highly sensitive to mixing conditions, with suboptimal mixing efficiency yielding LNPs with heterogeneous physical and chemical properties.19,22,25 To ensure particle precipitation commences from a state of molecular homogeneity, rapid micromixing is required. In the rapid precipitation process, we employ a confined volume mixing apparatus to achieve molecular mixing on the millisecond time scale. The collision of opposing fluid streams in the small mixing volume of the device creates a region of high-energy dissipation that enables highly efficient micromixing of siRNA and lipid reagents.19 Under the conditions of this study, siRNA-lipid complexes are further “annealed” postprecipitation. Annealing was performed in a solution containing 50% v/v ethanol, resulting from the equal volume micromixing of ethanol and aqueous reagent solutions. The incubation was conducted for 20 h at a temperature of 20−23 °C under quiescent conditions. Following incubation, the LNP solution was diluted with excess buffer and the solution pH neutralized. Nonentrapped oligonucleotide was removed via anion exchange chromatog-



RESULTS AND DISCUSSION LNP Preparation by Rapid Precipitation Process. The lipid components and assembly process used for generation of LNPs of this study are shown in Figure 1. The lipid composition (Figure 1a) consisted of CLinDMA, an ionizable amino lipid, cholesterol, and a poly(ethylene glycol) conjugate having a dimyristoyl lipid anchor (PEG-DMG). The selection of lipids was made to facilitate comparison with similar LNPs previously reported.23,24 The ionizable lipid content of the nanoparticles ranged from 52 to 60 mol % and the PEG-lipid content from 2 to 14 mol %. LNPs were formulated with 4614

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Figure 2. Effects of annealing on macromolecular structure of LNPs. SAXS data for LNPs composed of CLinDMA:Chol:PEG-DMG (60:38:2 mol:mol) at N/P = 1.5. LNPs were prepared by rapid precipitation in (a) acetate buffer or (b) citrate buffer and annealed for 0 h (black) or 20 h (red). Inset: the log of the scattering intensity plotted as a function of the momentum transfer q.

raphy for in vivo studies only. Residual ethanol removal and exchange into a buffer suitable for in vivo administration were accomplished via dialysis or ultrafiltration and followed by concentration and sterile filtration. Small-Angle X-ray Scattering Studies Show Evolution of LNP Macromolecular Structure Occurs during Annealing. In order to determine the influence of assembly conditions on the formation of macromolecular structures in LNPs prepared via rapid precipitation, we first investigated the influence of the added annealing stage on particle properties. A prototype LNP formulation with a lipids composition of CLinDMA:Chol:PEG-DMG (60:38:2 mol:mol) and N/P ratio of 1.5 was studied. After initial mixing of lipids and siRNA reagent solutions, precipitated complexes were either immediately quenched via dilution with excess buffer or annealed, as earlier detailed, prior to quench. The macromolecular structure of the final LNP product was quantitatively characterized using small-angle X-ray (SAXS) diffraction. The SAXS profiles of LNPs generated with or without annealing are compared in Figure 2. X-ray diffraction patterns for particles prepared without annealing (black curves) indicate the formation of a loosely organized phase with a characteristic broad SAXS correlation profile. This loosely organized structure is observed for LNPs prepared under two different conditions of assembly: one using acetate buffer as the aqueous siRNA feed solution (Figure 2a) and the other using citrate buffer (Figure 2b). The significance of these different buffers in particle assembly will be discussed subsequently. In contrast, LNPs that were annealed after initial mixing display SAXS patterns characteristic of an organized lamellar phase (Lα) (red curves). As highlighted by the arrows in the insets of Figure 2, the lamellar ordering gives rise to a series of well-defined peaks in the momentum transfer (q). These peaks, labeled as d, d/2, and d/3, correspond to the first-, second-, and third-order reflections and are positioned at a relative ratio of 1:2:3. The lamellar repeat distance (a), calculated as a = 2π/d, is estimated at 66.1 Å for LNPs prepared in citrate buffer and 69.8 Å for those prepared in acetate buffer. These values are consistent with the sum of the thickness of a lipid bilayer and the diameter of the double-stranded siRNA molecules.18 For each of the LNPs studied, the respective repeat distances are unchanged by addition of an annealing stage, as evidenced by coincidence of the first-order reflections in the diffraction patterns of Figure

2a,b. The emergence of ordered lamellar structures in annealed LNPs was not correlated with increases in particle size or size polydispersity. Representative dynamic light scattering (DLS) data for LNPs generated using acetate buffer are shown in Figure S1 of the Supporting Information. The intensityaveraged particle size distribution remains equivalent for formulations generated with or without annealing. The SAXS data indicate that LNPs with lipid components and composition as defined are thermodynamically prone to form ordered lamellar structures. However, the observation that annealing is required to generate particles with well-defined lamellar ordering suggests that structure evolution is kinetically arrested. To bypass this kinetic arrest and facilitate lipid rearrangement, ethanol was used as a membrane-destabilizing agent during annealing. Several previous studies have shown ethanol, and other short-chain alcohols, to influence the hydration, acyl chain order, permeability, curvature, and phase transitions of lipid membranes.26,27 These perturbations are detectable even in the presence of low alcohol concentrations. Thus, annealing of siRNA-lipid complexes in a solution containing 50% v/v ethanol was expected to facilitate lipid fluidity and enhance membrane fusion interactions. These interactions ultimately enable the rearrangement of macromolecular structure from initially disorganized phases into ordered lamellar structures in the final LNP product. Macromolecular Structure Rearrangement Is Impeded by Increasing PEG-Lipid in the LNP. We next studied the influence of composition on macromolecular structure of LNPs with lipid components as defined in Figure 1a. In particular, the effects of the PEG-lipid on LNP physical and macromolecular properties were studied. The PEG-lipid is a critical compositional parameter often manipulated for bioperformance optimization of lipid-based drug delivery vehicles. Appropriate selection of PEG-lipid and its relative molar ratio within a formulation can favorably alter the pharmacokinetics, biodistribution, cellular uptake, intracellular unpackaging, endosomal escape, and apparent transfection efficiency of oligonucleotide-lipid nanoparticles in vivo.9,28,29 Consequently, we generated LNPs with a high loading of PEG-lipid and characterized the influence of increased PEGlipid on the developed macromolecular structure. LNPs containing 14 mol % PEG-DMG were prepared and their structural properties compared to LNPs containing 2 mol % 4615

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Figure 3. Increasing PEG-lipid composition in LNPs inhibits macromolecular structure rearrangement during annealing. SAXS data for LNPs prepared with variable N/P ratio as a function of PEG-DMG in formulation. LNPs were prepared in acetate buffer at (a) N/P = 1.5 and (b) N/P = 3 and composed of CLinDMA:Chol:PEG-DMG at molar ratios of 60:38:2 (dark blue) or 52:34:14 (light blue). Inset: log of the scattering intensity as a function of momentum transfer q.

Figure 4. Critical micelle concentration of PEG-DMG. (a) Fluorescence intensity ratios of pyrene excitation bands (I336 nm/I333 nm) as a function of PEG-DMG concentration in (●) 50% v/v citrate/ethanol and (○) 50% v/v acetate/ethanol solutions. The inflection points of the curves (dashed lines) are taken as values of the CMC. (b) Proposed mechanism of cooperative hydrogen bonding between PEG ethers and acidic carboxylic acids of citric acid.

PEG-DMG. The molar fraction of PEG-DMG was selected to ensure complete saturation of LNP bilayers by the PEG-lipid during particle assembly. It is acknowledged that at this concentration a concomitant population of PEG-DMG micelles exists [unpublished work]. The molar ratio of cationic lipid to cholesterol was held equal between the high and low PEGDMG formulations, yielding LNP compositions of 52:34:14 and 60:38:2 CLinDMA:Chol:PEG-DMG, respectively. LNPs were also prepared at two different N/P ratios: a low N/P of 1.5 and high N/P of 3. For all preparations, LNPs were generated using acetate buffer as the aqueous siRNA reagent solution, annealed following initial mixing, and subsequently processed as previously described. Figure 3 shows the SAXS data for the various LNPs studied. As seen, LNPs containing 14 mol % PEG-DMG lack the SAXS diffraction pattern characteristic of an ordered lamellar structure (light blue curves). This is in distinct contrast to LNPs prepared with 2 mol % PEG-DMG (dark blue curves), where clearly distinguished diffraction peaks indicate the

existence of an ordered lamellar phase. The SAXS data are similar for LNP formulations prepared at low N/P = 1.5 (Figure 3a) and high N/P = 3 (Figure 3b). The diffraction data provide direct evidence for PEG-lipid interference with macromolecular structure rearrangement during the annealing stage of LNP assembly. This can be understood in context of PEG-lipid interactions in the spontaneously assembling complexes. At low concentrations of PEG-DMG, the PEG polymer is expected to exist in a random coil or mushroom conformation tethered to the surfaces forming lipid bilayers. As the concentration of PEGDMG increases above a threshold density, the polymer chains begin to interact with one another and undergo a transition from the globular state to an extended or brushlike configuration.29 The dense surface coverage of PEG in this configuration results in an inhibition of bilayer fusion interactions required for reorganization of lipids into ordered lamellar structures. 4616

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Figure 5. Inhibition of macromolecular structure rearrangement by PEG-lipid is mitigated by assembly buffer species. SAXS data for LNPs prepared in citrate buffer at (a) N/P = 1.5 and (b) N/P = 3. LNPs with molar compositions of 60:38:2 (dark green) or 52:34:14 (light green) CLinDMA:Chol:PEG-DMG. Inset: log of the scattering intensity plotted as a function of the momentum transfer q. Ordered lamellar structures are formed for LNPs with both low (2%) and high (14%) PEG-DMG at N/P ratios tested. This is in contrast to highly PEGylated LNPs generated with acetate buffer (Figure 3).

Buffer Species Are Active Participants in LNP Assembly. Rational Species Selection Mitigates PEGLipid Interference with Structure Rearrangement. The interdependency between two principal chemical and physical features of the LNP, namely PEG-lipid composition and macromolecular structure, is not ideal as it can confound the ability to clearly identify critical attributes relevant to optimal design of the therapeutic product. In order to mitigate the observed interdependency, we hypothesized that highly PEGylated LNPs with ordered lamellar structures could be generated through tailored modification of the assembly conditions used in the rapid precipitation process. We postulated that enhancing the solvation of the PEG-lipid during the critical annealing stage would lessen the affinity for lipopolymer interactions with forming lipid bilayers and mitigate interference in macromolecular. Once structure rearrangement is completed, the solvated PEG-lipid could then be precipitated onto the structured particles through a postannealing stabilization process. This proposed strategy is analogous to an in situ postinsertion of the PEG-lipid, where PEG protection is afforded through deposition of PEG-lipid molecules onto the surfaces of preformed lipid nanoparticles.30 To test this hypothesis, we elected to prepare highly PEGylated LNPs using two different types of aqueous buffers for the siRNA reagent solution. The choice of aqueous buffer was predicated on the identification of buffer species yielding differentiated solubility of PEG-DMG in 50% v/v buffer/ ethanol as per conditions of the annealing stage. As a measure of PEG-DMG molecular (unimer) solubility, we determined the critical micelle concentration (CMC) of PEG-DMG in the various mixed aqueous-solvent systems used. The CMC was measured via fluorescence spectroscopy using pyrene as hydrophobic probe. Figure 4a displays the results obtained for PEG-DMG micellization in 50% v/v ethanol solutions of acetate buffer (pH 3.9) or citrate buffer (pH 3.9). The pyrene fluorescence intensity ratio (I336/I333) is plotted against the logarithm of the PEG-DMG concentration. The CMC value is determined as the point of intersection of two tangents drawn to the curve at high and low concentrations. CMC values for PEG-DMG are estimated at approximately 1 and 0.3 mM for

ethanol solutions of citrate and acetate buffers, respectively. The measured CMCs are ∼1000-fold higher than CMC values reported for similar PEG-lipids in pure aqueous systems.31 These higher CMC values are attributed to the presence of ethanol in our solutions, which causes disruption of PEG-DMG micelles through lowering of the hydrocarbon−solvent interfacial tension and reduction of the solvophobic effect. Reducing the ethanol concentration to 25% v/v resulted in a decrease of PEG-DMG CMC in both citrate and acetate mixed solutions, yielding measured values of approximately 0.02 mM for each (Figure S2). The greater than 3-fold increase in PEG-DMG CMC with change in buffer species, from acetate to citrate, is noteworthy. Figure 4b proposes a mechanism for the observed increase in PEG-lipid CMC in the presence of citrate ions. The formation of cooperative, intermolecular hydrogen bonds between a proton donor (diacids of citrate) and an acceptor (PEG ethers) is thought to enhance the solubility of PEG-DMG unimers in solution, necessitating higher concentrations of the lipopolymer to effect micelle formation. Intermolecular interactions between PEG and acetic acid are not permitted, and as such micelle formation occurs more readily in the acetate buffer system. Similar cooperative hydrogen-bonding interactions between PEG or poly(ethylene oxide) (PEO) and polyacidic moieties, such as poly(acrylic acid) or poly(methacrylic acid), have previously been reported.32 Supported by the CMC data, we next investigated the impact of changing the assembly buffer species, from acetate to citrate, on the generation ordered lamellar structures in LNPs with high PEG-lipid content. LNPs with lipids molar compositions of 52:34:14 and 60:38:2 CLinDMA:Chol:PEG-DMG were generated at N/P ratios of 1.5 and 3, as per earlier conditions. The structural features of citrate-generated formulations, as characterized by SAXS, are shown in Figure 5. The SAXS patterns for LNPs with PEG-DMG molar compositions of 2% or 14% and N/P = 1.5 are compared in Figure 5a. As evidenced by the diffraction data, highly PEGylated LNPs assembled in citrate buffer are able to form ordered lamellar structures. The well-defined lamellar ordering gives rise to a series of distinct reflections, as indicated by the arrows in the inset of the figure. 4617

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Figure 6. Schematic of PEG-lipid interaction with lipid bilayers. When PEG-lipid concentration exceeds the PEG-lipid CMC, adsorption of the lipopolymer onto lipid bilayers is favored. The high density of adsorbed PEG frustrates lipid rearrangement into ordered structures (panel 1). When the PEG-lipid concentration falls below the CMC, the low density of adsorbed polymer poses little hindrance to rearrangement (panel 2). A change in solvent quality induces lowering of CMC and adsorption of solubilized PEG-lipid onto formed particles (panel 3).

Figure 7. Effects of LNP composition and assembly conditions on siRNA encapsulation efficiency. (a) LNPs (2 mol % PEG-DMG) prepared in citrate or acetate buffers as a function of annealing time: 0 h (black) or 20 h (gray). (b) Effect of N/P ratio on siRNA encapsulation for highly PEGylated LNPs (14 mol % PEG-DMG). LNPs generated in citrate buffer were more efficient at trapping siRNA relative to acetate-generated formulations.

The SAXS scans of Figure 5b similarly indicate the presence of the ordered lamellar phase for LNPs prepared at N/P 3, although structural features are slightly less defined for this composition. The reduced propensity for formation of ordered lamellar structures with decreasing siRNA loading is consistent with earlier experiments to this effect [unpublished data]. These results are in distinct contrast to the macromolecular structure of acetate-generated highly PEGylated LNPs as is shown in Figure 3, where particles lacked the diffraction pattern reflective of ordered lamellar structure formation, irrespective of the N/P ratio as tested. The data point to a mechanism of PEG-lipid induced interference with macromolecular restructuring. For high PEGDMG (14 mol %) siRNA-LNPs, the concentration of lipopolymer during annealing is 0.7 mM under the conditions used in this work. When LNPs are prepared using acetate buffer, the lipopolymer concentration during annealing is more than twice the CMC of PEG-DMG. Under these conditions,

PEG-DMG assembly with lipid bilayers is favored, and the resulting dense corona of PEG chains on bilayer surfaces inhibits the fusion interactions required for reorganization of precipitated complexes into multilamellar structures (Figure 6, panel 1). When the PEG-DMG composition in the siRNA-LNP formulation is lowered to 2 mol %, the concentration of lipopolymer during annealing is reduced to 0.1 mM, which now falls well below the CMC of PEG-DMG in 50% v/v ethanol solutions of both acetate and citrate buffers. Consequently, the majority of PEG-DMG remains dissolved as unimers. The resulting lower-density PEG coverage poses a lower hindrance to lipid rearrangement and enables restructuring into multilamellar LNPs (Figure 6, panel 2). Rather than reduce the PEG-lipid concentration through a physical reduction in the lipopolymer composition of the LNP, the same effect can be achieved chemically through suitable selection of the aqueous buffer species in particle assembly. When citrate buffer is used for assembly of LNPs containing 14 4618

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observed with a lowering of the N/P ratio. Acetate generated LNPs with N/P = 1 had the lowest siRNA loading, with encapsulation efficiency of approximately 40%. Oligonucleotide incorporation was improved by increasing the N/P ratio, reaching nearly 80% for N/P = 3 formulations. In contrast, LNPs generated with citrate buffer were more efficient at trapping siRNA, with the lowest N/P formulation tested (N/P = 1) yielding a siRNA loading efficiency of nearly 75%. Nonetheless, all of the 14 mol % PEG-DMG LNP preparations studied yielded reduced siRNA encapsulation efficiencies relative to their 2 mol % PEG-DMG counterparts. The collective physical and structural data point to several significant features of the developed rapid precipitation process. First, LNPs with low N/P ratio can be generated with high siRNA encapsulation efficiency through simple tailoring of the buffer species used for particle assembly. Second, the formation of ordered lamellar phases is not a prerequisite for high siRNA encapsulation but can improve payload loading under conditions where encapsulation is less favored. This is evidenced by the high siRNA encapsulation efficiency for low N/P LNPs prepared in acetate buffer, irrespective of the formation of ordered lamellar structure as achieved through annealing (Figure 2a). In contrast, for low N/P LNPs prepared in citrate buffer, structure rearrangement, as achieved through annealing (Figure 2b), resulted in a significant increase in siRNA encapsulation efficiency. The observed differences in siRNA encapsulation efficiencies for particles with low N/P and low PEG-DMG content can again be understood in context of the micellization behavior of the lipopolymer. The PEG-lipid plays an active role in particle assembly by inhibiting excessive aggregation and fusion during the initial precipitation and growth phase, when cationic lipids associate with anionic siRNA to form “nuclei” that serve as hydrophobic sites for deposition of colipids from solution. These nuclei continue to grow until solute concentrations reach their respective equilibrium solubilities or until they are kinetically arrested through adsorption of a protective polymer layer.19 Sufficient polymer adsorption can only occur once the concentration of lipopolymer in solution exceeds the lipopolymer CMC. The low CMC of PEG-DMG in 50% v/v ethanol/acetate facilitates lipopolymer adsorption at low concentrations, permitting stabilization of initially precipitated complexes and enabling high encapsulation of the siRNA cargo. For LNPs prepared in citrate buffer, the lipopolymer concentration exceeds the CMC only when further reduction in the ethanol concentration is achieved through a secondary dilution with aqueous buffer (Figure 1b). If dilution occurs immediately after mixing (no annealing), low siRNA encapsulation results, suggesting that steric stabilization provided by the PEG-lipid is insufficient to trap surface-associated siRNA within the formed complexes once electrostatic interactions are neutralized. If the complexes are instead allowed to anneal, rearrangement into ordered lamellar structures enables the improved incorporation of siRNA between lipid bilayers. In this case, the protection afforded to siRNA through bilayer encapsulation reduces losses upon electrostatic neutralization. LNP Macromolecular Structure Can Be Tuned through Assembly and Composition Design. Macromolecular features of LNPs result from a balance of kinetic and thermodynamic processes that occur during particle assembly. As shown in this work, these processes can be viewed in context of four distinctive stages: an initial micromixing stage that

mol % PEG-DMG, the lipopolymer concentration (0.7 mM) falls below the lipopolymer CMC in the 50% v/v ethanol solution during annealing (1 mM). This selective exchange in buffer species serves to effectively reduce the PEG-DMG available for interaction with siRNA-lipid bilayers. The reduction in PEG bilayer coverage diminishes interference with structural rearrangement in a manner comparable to that achieved through physical reduction in PEG-DMG composition. In this way, we are able to generate highly PEGylated LNPs that retain multilamellar structure. Once macromolecular structure has evolved, subsequent dilution with excess buffer results in further lowering of the PEG-DMG CMC and induces adsorption of the lipopolymer onto the assembled particles (Figure 6, panel 3). siRNA Encapsulation Is Influenced by the Formation of Ordered Lamellar Structures. A common strategy to increase siRNA loading is to enhance the affinity between siRNA and the lipid matrix through increased loading of the cationic lipid, effectively increasing the N/P ratio. However, most cationic lipids are associated with toxicity, and their reduction in the therapeutic product is often desired.33 It is therefore appropriate to evaluate opportunities for optimization of siRNA loading in low N/P LNP compositions through alternative means, for example, via the manipulation of LNP macromolecular structure. Accordingly, we next investigated the impact of assembly conditions relevant to macromolecular structure formation on the siRNA encapsulation efficiency of LNPs prepared via rapid precipitation. Oligonucleotide encapsulation efficiencies were determined prior to anion exchange chromatography, thus providing an unadulterated representation of oligonucleotide encapsulation and assembly process yield. In Figure 7a, the effects of annealing on the siRNA encapsulation efficiency for LNPs comprising 2 mol % PEGDMG are examined. Particles were prepared at N/P ratios of 1.5 and 3 in both citrate and acetate aqueous buffers, as indicated. As can be seen, both annealed and nonannealed formulations generated at N/P = 3 were highly efficient at encapsulating siRNA, with trapping efficiencies between 84 and 91%, irrespective of the assembly buffer used. When the N/P ratio is lowered to 1.5, effectively halving the cationic lipid to siRNA ratio, the encapsulation efficiency becomes highly dependent on inclusion of the annealing stage as well as on the type of buffer species used for assembly. LNPs with low N/ P ratio prepared in acetate buffer retained their high siRNA encapsulation efficiency, with annealing adding only a minor improvement in oligonucleotide loading, within experiment-toexperiment and assay variability. In contrast, addition of the annealing stage was critical to improving the siRNA encapsulation efficiency of low N/P LNPs prepared in citrate buffer, where an approximately 15% increase in siRNA loading was observed for annealed particles. The concentration of PEG-DMG during particle assembly also influenced the siRNA encapsulation efficiency. Oligonucleotide trapping efficiencies for LNPs containing 14 mol % PEG-DMG with N/P ratios ranging from 1 to 3 are shown in Figure 7b. All particle preparations included an annealing step as previously defined. Consistent with the developed mechanistic understanding of particle assembly, the effect of increasing PEG-DMG was different for LNPs prepared in citrate versus acetate buffers. The influence of increasing PEGlipid was more pronounced for acetate-prepared LNPs, where significant reductions in encapsulation efficiencies were 4619

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Figure 8. Cryo-TEM micrographs of LNPs with varying macromolecular structures. LNPs with diversity of macromolecular features generated through a coupled manipulation of rapid precipitation conditions and formulation composition. See Figure S3 for chemical structures of lipids used to generate LNPs shown. DMG) was manufactured by NOF Corporation (White Plains, NY), and cholesterol was obtained from Sigma-Aldrich (St. Louis, MO). Ethanol was obtained from Sigma-Aldrich (St. Louis, MO), and SYBR gold was obtained from Molecular Probes (Eugene, OR). Dulbecco’s phosphate buffered saline solution (PBS) was obtained from HyClone laboratories (Logan, UT). All other buffers used in the study were prepared from the acid or sodium salt form of the components. Lipid Nanoparticle Preparation via Rapid Precipitation. Lipid nanoparticles (LNPs) encapsulating luciferase siRNA were prepared via the rapid precipitation assembly process as previously described,19 with modifications as follows. Particles were prepared by mixing appropriate volumes of an organic lipids solution with an aqueous solution containing siRNA duplexes using a confined volume T-mixer device. The lipids stock solution was prepared by dissolving lipids, in molar ratios of 60:38:2 or 52:34:14 CLinDMA:cholesterol:PEG-DMG as specified, in ethanol. The total concentration of lipids for the two compositions was 6.6 and 10.1 mg/mL, respectively. siRNA duplexes were dissolved in either 25 mM citrate buffer (100 mM NaCl, pH 3.9) or 25 mM acetate buffer (100 mM NaCl, pH 3.9) at amino lipid nitrogen (N) to siRNA phosphate (P) ratios of 1, 1.5, or 3, as specified. The concentration of siRNA in buffer solution ranged between 0.67 and 2 mg/mL for the N/P ratios studied. Equal volumes of the lipids in ethanol and the siRNA in buffer were combined at flow rates of 50 mL/min each in a T-mixer device using syringe pumps (Harvard Apparatus PHD 2000, Holliston, MA). Where specified, the mixed material was annealed via incubation at a temperature of 20−23 °C under quiescent conditions for a period of 20 h. After annealing, the material was diluted 20-fold by volume into PBS. In the absence of annealing, the initially mixed material is directly diluted 20-fold by volume into PBS using a two-stage, in-line T-mixing process. Following dilution, residual ethanol was removed and buffer exchanged into PBS via dialysis using 6−8 kDa Spectra/Por dialysis membranes (Spectrum Laboratories, Rancho Dominguez, CA). Finally, LNPs were prepared for analytical characterization as specified. Small-Angle X-ray Scattering Characterization of LNPs. Small-angle X-ray scattering (SAXS) experiments were performed on a Bruker Nanostar SAXS instrument. siRNA-LNPs were concentrated by centrifugal filtration (Amicon Ultra, RC 100 kDa membrane) by a factor of 8−10 by mass. Samples were scanned at room temperature for 60 min. Silver behenate was used to calibrate the detector-tosample distance, and Datasqueeze v. 2.1.5 was used to analyze the 2D X-ray data. The intensity of scattering was integrated azimuthally on the 2D diffraction pattern and plotted as a function of the scattering vector, q = 4π sin(θ/2)/λ, where θ is the scattering angle. PEG-DMG Critical Micelle Concentration Determination. The critical micelle concentration (CMC) of PEG-DMG in mixed ethanol−buffer solutions was determined with fluorescent spectroscopy using pyrene as a hydrophobic fluorescent probe. Briefly, stock solutions of pyrene in ethanol (2 × 10−3 M) and PEG-DMG (1 × 10−3) in ethanol were prepared and thoroughly mixed with either aqueous citrate buffer (pH 3.9) or aqueous acetate buffer (pH 3.9) at volume ratios yielding 50% v/v ethanol or 25% v/v ethanol in the final solutions, as specified per experiment. For each of the four conditions tested (e.g., 50% v/v or 25% v/v ethanol/citrate buffer or ethanol/

ensures homogeneous comingling of siRNA and lipid solutes over a millisecond time scale. A secondary binding and adsorption step, where electrostatic interactions of siRNA and cationic lipids lead to nucleation and growth of LNP precursor complexes. This step is also rapid, occurring on the time scales of milliseconds to seconds and is dependent on the supersaturation of siRNA and cationic lipids in solution.19 In a third stage, that of internal rearrangement, local reorganization of siRNA and lipids within complexes takes place. This process occurs over significantly longer time scales, on the order of hours, and is sensitive to LNP composition as well as solution-phase conditions employed for particle assembly. The final step is that of particle stabilization, where the macromolecular and physical properties of the LNP are “locked”, preventing further changes in LNP properties on relevant experimental time scales. The developed mechanistic understanding of particle selfassembly, coupled with the flexibility afforded by the rapid precipitation process, enables the rational manipulation of macromolecular properties. To test the generalizability of this approach, we elected to produce LNPs accessing a broad macromolecular structure space. All formulations were generated using the same lipid components (Figure S3), were of fixed N/P, and varied only in lipid molar ratios and conditions used for particle assembly. Representative cryogenic transmission electron microscopy (cryo-TEM) micrographs of select formulations are shown in Figure 8. As evidenced, wellformed nanometer-sized particles with hexagonal, cubic, unilamellar, and bilamellar macromolecular structures were effectively generated.



CONCLUSIONS Successful development of siRNA LNP therapeutics will require the elucidation of meaningful structure−activity relationships (SAR) that enable the rational design of chemical, physical, and macromolecular LNP features targeted to biological performance. Our work represents a first step in enabling the controllable manipulation of desirable physical and macromolecular features of LNPs. It is anticipated that the developed mechanistic understanding of LNP assembly, when coupled with lipid chemistry optimization, will enable a more comprehensive interrogation of SAR that advance increasingly more potent, less toxic LNP therapeutic products.



MATERIALS AND METHODS

Materials. (2-{4-[(3β)-Cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine) (CLinDMA) was synthesized at Merck (West Point, PA) as previously described.24 Poly(ethylene glycol)2000−dimyristoylglycerol (PEG4620

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acetate buffer), the concentration of PEG-DMG in the final solution ranged from 4 × 10−11 to 1 × 10−5 M. The fluorescent intensity of pyrene was recorded using a SpectraMax M5e multimode microplate fluorescence spectrometer (Molecular Devices, Sunnyvale, CA) with excitation wavelengths of 336 nm (I3) and 333 nm (I1) and emission wavelength of 390 nm. The intensity ratio (I336/I333) was plotted against the logarithm of polymer concentration. The CMC value was estimated as the point of intersection of two tangents drawn to the curve at high and low concentrations, respectively. siRNA Encapsulation in LNPs. The fraction of siRNA encapsulated within LNPs was quantified using a SYBR Gold fluorescent method. Briefly, the fluorescence intensity of LNP solutions in the presence of SYBR Gold indicator was measured and compared to the fluorescence intensity of LNP solutions in the presence of both 0.5% Triton X-100 and SYBR Gold reagents. The difference between measured intensity values in the absence of Triton X-100 (free siRNA) and in its presence (total siRNA) is indicative of the amount of siRNA encapsulated within LNPs. The assay was performed using a SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with excitation wavelength of 485 nm and emission wavelength of 530 nm. Cryo-Transmission Electron Microscopy of LNPs. Cryotransmission electron microscopy (Cryo-TEM) was conducted on a FEI Tecnai Spirit BioTWIN (Hillsboro, OR) equipped with a Gatan ultrascan 1000 CCD camera. Undiluted samples of siRNA-LNP solution were suspended on a plasma-treated Quantifoil holey carbon film supported on a 300 mesh copper grid (Quantifoil Micro Tools GmbH, Germany). Samples were vitrified using an FEI Vitrobot Mark IV operating at 4 °C and 95% relative humidity and then subjected to imaging at 30 000× (0.35 nm/pixel) magnification.



Palmer, L. R.; Racie, T.; Rohl, I.; Seiffert, S.; Shanmugam, S.; Sood, V.; Soutschek, J.; Toudjarska, I.; Wheat, A. J.; Yaworski, E.; Zedalis, W.; Koteliansky, V.; Manoharan, M.; Vornlocher, H. P.; MacLachlan, I. RNAi-mediated gene silencing in non-human primates. Nature 2006, 441 (7089), 111−114. (4) Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464 (7291), 1067−1070. (5) Whitehead, K. A.; Langer, R.; Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discovery 2009, 8 (2), 129−138. (6) Mintzer, M. A.; Simanek, E. E. Nonviral vectors for gene delivery. Chem. Rev. 2009, 109 (2), 259−302. (7) Davidson, B. L.; McCray, P. B. Current prospects for RNA interference-based therapies. Nat. Rev. Genet. 2011, 12 (5), 329−340. (8) Tseng, Y. C.; Mozumdar, S.; Huang, L. Lipid-based systemic delivery of siRNA. Adv. Drug Delivery Rev. 2009, 61 (9), 721−731. (9) Li, W.; Szoka, F. C., Jr. Lipid-based nanoparticles for nucleic acid delivery. Pharm. Res. 2007, 24 (3), 438−449. (10) Wasungu, L.; Hoekstra, D. Cationic lipids, lipoplexes and intracellular delivery of genes. J. Controlled Release 2006, 116 (2), 255−264. (11) Senior, J.; Gregoriadis, G. Stability of small unilamellar liposomes in serum and clearance from the circulation - the effect of the phospholipid and cholesterol components. Life Sci. 1982, 30 (24), 2123−2136. (12) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 1990, 268 (1), 235−237. (13) Akinc, A.; Querbes, W.; De, S. M.; Qin, J.; Frank-Kamenetsky, M.; Jayaprakash, K. N.; Jayaraman, M.; Rajeev, K. G.; Cantley, W. L.; Dorkin, J. R.; Butler, J. S.; Qin, L. L.; Racie, T.; Sprague, A.; Fava, E.; Zeigerer, A.; Hope, M. J.; Zerial, M.; Sah, D. W. Y.; Fitzgerald, K.; Tracy, M. A.; Manoharan, M.; Koteliansky, V.; de Fougerolles, A.; Maier, M. A. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther. 2010, 18 (7), 1357−1364. (14) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez, I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.; Jeffs, L. B.; Nechev, L.; Eisenhardt, M. L.; Jayaraman, M.; Kazem, M.; Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, R.; Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.; Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy, M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T. D.; Hope, M. J. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010 , 28 (2), 172−176. (15) Stanton, M. G.; Budzik, B. W.; Beutner, B. W.; Liao, H. Novel Low Molecular Weight Cationic Lipids for Oligonucleotide Delivery, 2012. (16) Ewert, K.; Slack, N. L.; Ahmad, A.; Evans, H. M.; Lin, A. J.; Samuel, C. E.; Safinya, C. R. Cationic lipid-DNA complexes for gene therapy: Understanding the relationship between complex structure and gene delivery pathways at the molecular level. Curr. Med. Chem. 2004, 11 (2), 133−149. (17) Hoekstra, D.; Rejman, J.; Wasungu, L.; Shi, F.; Zuhorn, I. Gene delivery by cationic lipids: in and out of an endosome. Biochem. Soc. Trans. 2007, 35, 68−71. (18) Leal, C.; Bouxsein, N. F.; Ewert, K. K.; Safinya, C. R. Highly efficient gene silencing activity of siRNA embedded in a nanostructured gyroid cubic lipid matrix. J. Am. Chem. Soc. 2010, 132 (47), 16841−16847. (19) Gindy, M. E.; Leone, A. M.; Cunningham, J. J. Challenges in the pharmaceutical development of lipid-based short interfering ribonucleic acid therapeutics. Expert Opin. Drug Delivery 2012, 9 (2), 171− 182. (20) Maurer, N.; Wong, K. F.; Stark, H.; Louie, L.; McIntosh, D.; Wong, T.; Scherrer, P.; Semple, S. C.; Cullis, P. R. Spontaneous

ASSOCIATED CONTENT

S Supporting Information *

Figure S1: particle size distributions of LNPs as in Figure 2A; Figure S2: critical micelle concentrations of PEG-DMG in 25% v/v ethanol−buffer solutions; Figure S3: chemical structures of lipids for LNPs in Figure 8. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 215-652-7004; Fax 215-652-5299 (M.E.G.). Notes

The authors declare the following competing financial interest(s): All research was funded by Merck Research Laboratories, Merck and Co., Inc. R. K. Prud’homme received a sponsored research grant from Merck and Co., Inc.



ACKNOWLEDGMENTS V. Kumar acknowledges support from Merck and Co., Inc., through an internship in the department of Pharmaceutical Sciences.



REFERENCES

(1) Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specific genetic interference by doublestranded RNA in Caenorhabditis elegans. Nature 1998, 391 (6669), 806−811. (2) Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411 (6836), 494−498. (3) Zimmermann, T. S.; Lee, A. C. H.; Akinc, A.; Bramlage, B.; Bumcrot, D.; Fedoruk, M. N.; Harborth, J.; Heyes, J. A.; Jeffs, L. B.; John, M.; Judge, A. D.; Lam, K.; McClintock, K.; Nechev, L. V.; 4621

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entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes. Biophys. J. 2001, 80 (5), 2310−2326. (21) Jeffs, L. B.; Palmer, L. R.; Ambegia, E. G.; Giesbrecht, C.; Ewanick, S.; MacLachlan, I. A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm. Res. 2005, 22 (3), 362−372. (22) Jahn, A.; Stavis, S. M.; Hong, J. S.; Vreeland, W. N.; Devoe, D. L.; Gaitan, M. Microfluidic mixing and the formation of nanoscale lipid vesicles. ACS Nano 2010 , 4 (4), 2077−2087. (23) Abrams, M. T.; Koser, M. L.; Seitzer, J.; Williams, S. C.; DiPietro, M. A.; Wang, W.; Shaw, A. W.; Mao, X.; Jadhav, V.; Davide, J. P.; Burke, P. A.; Sachs, A. B.; Stirdivant, S. M.; Sepp-Lorenzino, L. Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: Effect of dexamethasone co-treatment. Mol. Therapy 2010, 18 (1), 171−180. (24) Tao, W.; Davide, J. P.; Cai, M.; Zhang, G.-J.; South, V. J.; Matter, A.; Ng, B.; Zhang, Y.; Sepp-Lorenzino, L., Noninvasive imaging of lipid nanoparticle-mediated systemic delivery of smallinterfering RNA to the liver. Mol. Therapy 201018, (9), 1657−1666. (25) Gindy, M. E.; Panagiotopoulos, A. Z.; Prud’homme, R. K. Composite block copolymer stabilized nanoparticles: Simultaneous encapsulation of organic actives and inorganic nanostructures. Langmuir 2008, 24 (1), 83−90. (26) Ly, H. V.; Longo, M. L. The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers. Biophys. J. 2004, 87 (2), 1013−1033. (27) Patra, M.; Salonen, E.; Terama, E.; Vattulainen, I.; Faller, R.; Lee, B. W.; Holopainen, J.; Karttunen, M. Under the influence of alcohol: The effect of ethanol and methanol on lipid bilayers. Biophys. J. 2006, 90 (4), 1121−1135. (28) Torchilin, V. P. Micellar nanocarriers: Pharmaceutical perspectives. Pharm. Res. 2007, 24 (1), 1−16. (29) Vonarbourg, A.; Passirani, C.; Saulnier, P.; Simard, P.; Leroux, J. C.; Benoit, J. P. Evaluation of pegylated lipid nanocapsules versus complement system activation and macrophage uptake. J. Biomed. Mater. Res., Part A 2006, 78A (3), 620−628. (30) Iden, D. L.; Allen, T. M. In vitro and in vivo comparison of immunoliposomes made by conventional coupling techniques with those made by a new post-insertion approach. Biochim. Biophys. Acta, Biomembr. 2001, 1513 (2), 207−216. (31) Ashok, B.; Arleth, L.; Hjelm, R. P.; Rubinstein, I.; Onyuksel, H. In vitro characterization of PEGylated phospholipid micelles for improved drug solubilization: Effects of PEG chain length and PC incorporation. J. Pharm. Sci. 2004, 93 (10), 2476−2487. (32) Hao, J.; Yuan, G.; He, W.; Cheng, H.; Han, C. C.; Wu, C. Interchain Hydrogen-Bonding-Induced Association of Poly(acrylic acid)-graft-poly(ethylene oxide) in Water. Macromolecules 2010, 43 (4), 2002−2008. (33) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controlled Release 2006, 114 (1), 100−109.



NOTE ADDED AFTER ASAP PUBLICATION After this paper was published ASAP on April 9, 2014, a correction was made to the TOC graphic and abstract graphic. The corrected version was reposted April 14, 2014.

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Mechanism of macromolecular structure evolution in self-assembled lipid nanoparticles for siRNA delivery.

Lipid nanoparticles (LNPs) are a leading platform for therapeutic delivery of small interfering RNAs (siRNAs). Optimization of LNPs as therapeutic pro...
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