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.

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.

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.

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.