pH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function.

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal [Formula: see text] and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid [Formula: see text] phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal [Formula: see text] transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-[Formula: see text] transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.

Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.

RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines.

Nanocrystal formulations of a poorly soluble drug. 2. Evaluation of nanocrystal liver uptake and distribution after intravenous administration to mice.

A stabilized high drug load intravenous formulation could allow compounds with less optimal pharmacokinetic profiles to be developed. Polyethylene glycol (PEG)-ylation is a frequently used strategy for particle delivery systems to avoid the liver, thereby extending blood circulation time. The present work reports the mouse in vivo distribution after i.v. administration of a series of nanocrystals prepared with the bead milling technique and PEG-ylated with DSPE-PEG2000 and Pluronic F127, with and without polyvinylpyrrolidone K30 (PVP)/Aerosol OT (AOT) as primary stabilizers. While all formulations were cleared significantly faster than expected from nanocrystal dissolution alone, purely DSPE-PEG2000 PEG-ylated particles displayed prolonged circulation time (particles elimination half-life of 9min) compared to DSPE-PEG2000/PVP/AOT formulation (half-life of 3min). The two Pluronic F127 stabilized formulations displayed similar half-lives (9min with and without PVP/AOT, respectively). Whole tissue kinetics shows that clearance of particles could be attributed to accumulation in the liver. A separate in vivo study addressed the liver cell distribution after administration. Dissolved compound accumulated in hepatocytes only, while particles were distributed between liver sinusoidal endothelial cells and Kupffer cells. More DSPE-PEG2000/PVP/AOT stabilized particles accumulated in the liver, preferably in Kupffer cells, compared to Pluronic F127/PVP/AOT stabilized particles. The present study extends the understanding of PEG-ylation and "stealth" behaviour to also include nanocrystals.

Nanocrystal formulations of a poorly soluble drug. 1. In vitro characterization of stability, stabilizer adsorption and uptake in liver cells.

In the present work, milled nanocrystals of a poorly soluble compound using different stabilizers were prepared and characterized. The aim of the study was to evaluate a fundamental set of properties of the formulations prior to i.v. injection of the particles. Two polyethylene oxide containing stabilizers; (distearoyl phosphatidylethanol amine (DSPE)) -PEG2000 and the triblock copolymer Pluronic F127, were investigated, with and without polyvinylpyrrolidone K30/Aerosol OT (PVP/AOT) present. The solubility in water was around 10nM for the compound, measured from nanocrystals, but 1000 times higher in 4% human serum albumin. The particles were physically stable during the time investigated. The zeta potential was around -30 and -10mV for DSPE-PEG2000 and Pluronic F127 stabilized particles, respectively, at the conditions selected. The dissolution rate was similar for all four formulations and similar to the theoretically predicted rate. Critical micelle concentrations were determined as 56nM and 1.4µM for DSPE-PEG2000 and Pluronic F127, respectively. The adsorption isotherms for the PEG lipid showed a maximum adsorbed amount of about 1.3mg/m2, with and without PVP/AOT. Pluronic F127 showed a higher maximum amount adsorbed, at around 3.1mg/m2, and marginally lower with PVP/AOT present. Calculated data showed that the layer of Pluronic F127 was thicker than the corresponding DSPE-PEG2000 layer. The total amount of particles distributed mainly to the liver, and the hepatocellular distribution in vitro (Liver sinusoidal endothelial cells and Kupffer cells), differed depending on the stabilizing mixture on the particles. Overall, DSPE-PEG2000 stabilized nanocrystals (with PVP/AOT) accumulated to a larger degree in the liver compared to particles with Pluronic F127 on the surface. A theoretical model was developed to interpret in vivo pharmacokinetic profiles, explaining the balance between dissolution and liver uptake. With the present, fundamental data of the nanocrystal formulations, the platform for forthcoming in vivo studies was settled.

Usefulness of a nanoparticle formulation to investigate some hemodynamic parameters of a poorly soluble compound.

Drug solubility is an important issue when progressing investigational compounds into clinical candidates. The present paper describes the development and characterization of a nanosuspension that was formulated to overcome problems with poor water solubility and possible adverse events caused by cosolvent mixtures, using ticagrelor as a model compound. A homogeneous nanosuspension of ticagrelor was formed using a wet milling approach, which yielded particle sizes around 230 nm. The nanosuspensions were chemically stable for at least 10 months at both room temperature and when refrigerated, and physically (i.e., particle size) stable for at least 10 months under refrigeration, and approximately 3 years at room temperature and when frozen. One rat model and two dog models were used to assess the pharmacokinetics and hemodynamic-related effects following intravenous administration of nanoparticles. There were no biologically consistent or dose-dependent effects of the nanoparticles on the hemodynamic parameters tested, that is, heart rate, mean aortic pressure, cardiac output, left femoral artery blood flow, or cardiac inotropy (measured as max dP/dt). In conclusion, a stable ticagrelor nanosuspension formulation was developed, suitable for intravenous administration. At the doses evaluated, this formulation was without hemodynamic effects in three sensitive preclinical models.

The metabotropic glutamate mGluR5 receptor agonist CHPG stimulates food intake.

The metabotropic glutamate receptor 5 (mGluR5) has been suggested to modulate energy balance. For example, mGluR5 antagonists inhibit food intake in rodents and mGluR5 knockout mice resist diet-induced obesity. However, nonspecific effects can reduce food intake. Thus, to further support the role of mGluR5 in feeding behaviour, we evaluated if the mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) would induce the opposite effect, i.e. increased food intake in rats. Intracerebroventricularly injected CHPG (0.5-1.5 micromol) induced a dose-dependent stimulation of food intake (349% increase at 2 h with 1.5 micromol). The mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (10 mg/kg intraperitoneally) reduced 24 h food intake, without altering CHPG-induced feeding. These findings further support a physiologically relevant role of mGluR5 in appetite regulation.

Amorphous drug nanosuspensions. 3. Particle dissolution and crystal growth.

In the present paper, we have studied particle dissolution and crystal growth of the poorly water soluble drug felodipine, using fluorescence as a probe for the amount of crystalline material. Dissolution kinetics is essentially diffusion-controlled, while the rate of crystal growth is significantly slower compared to the diffusion-controlled limit. The deviation from diffusion control was characterized by the effective length, lambda, related to the kinetics of a surface integration process. Amorphous nanoparticles may be highly unstable in the presence of small amounts of crystalline particles. This is due to the fact that the molecular solubility from the amorphous nanoparticles often is at least an order of magnitude higher than the corresponding crystalline solubility. In a mixed system where crystalline nanoparticles have been added to an amorphous nanosuspension, the bulk will have a monomer concentration intermediate between the amorphous and crystalline solubilities, and is thus supersaturated with respect to the crystalline particles while being undersaturated with respect to the amorphous particles. As a consequence, the amorphous particles spontaneously dissolve, while crystalline particles grow, in a combined process which is similar to Ostwald ripening. By knowing the parameters describing dissolution and crystal growth, respectively, it was possible to simulate the outcome of controlled seeding experiments, where a small amount of crystalline nanoparticles was added to a dispersion of amorphous nanoparticles. A good agreement between model calculations and experiments was obtained including how the crystal growth rate varied with the amounts of added crystalline seeds.

Amorphous drug nanosuspensions. 1. Inhibition of Ostwald ripening.

Amorphous drug nanosuspensions are prone to particle growth due to Ostwald ripening. By incorporating a second component of extremely low aqueous solubility, Ostwald ripening can be inhibited. These studies indicate that to inhibit ripening, the drug/inhibitor mixture (in the particles) must form a single phase. The drug/inhibitor mixture can be characterized by the interaction parameter chi using the Bragg-Williams theory, in which single phase mixtures are obtained for chi < 2. The chi parameter can be calculated from the (crystalline) solubility of the drug in the inhibitor, provided the inhibitor is a liquid, and the melting entropy and temperature of the drug.

Amorphous drug nanosuspensions. 2. Experimental determination of bulk monomer concentrations.

A simple turbidimetric method was developed to measure the bulk concentration of drug in nanosuspensions. The bulk concentrations measured were in the range from 1 microM to 1 mM. The accuracy of the method was checked by determination of the bulk concentration of crystalline nanosuspensions, i.e., the crystalline solubility, which compared favorably to solubilities measured by a conventional method. Results obtained for amorphous nanosuspensions agreed with predictions using a theory describing the relative solubility between a supercooled liquid and a crystal. Further, it was found that the bulk concentration in Ostwald ripening inhibited amorphous nanosuspensions and could be lowered by incorporation of higher amounts of the inhibitor, in agreement with predictions using the Bragg-Williams theory of nonideal solutions.