RNA-based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self-degradable lipid-like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self-degradability is based on a unique reaction "Hydrolysis accelerated by intra-Particle Enrichment of Reactant (HyPER)." In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid-scaffold lipid-like material that mounts all of these units (ssPalmO-Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.
In
the present study, the molecular state of drug-rich amorphous
nanodroplets was evaluated using NMR techniques to reveal the mechanism
underlying the crystallization inhibition of drug-rich amorphous nanodroplets
by a polymer. Ibuprofen (IBP) with a low glass transition temperature
was used for direct characterization of drug-rich amorphous nanodroplets.
Highly supersaturated IBP formed IBP-rich amorphous nanodroplets through
phase separation from aqueous solution. Increasing the concentration
of hypromellose (HPMC) in the aqueous solution contributed to the
inhibition of IBP crystallization and maintenance of supersaturation
at IBP amorphous solubility. Solution 1H NMR measurements
of IBP supersaturated solution containing IBP-rich amorphous nanodroplets
clearly showed two kinds of 1H peaks derived from the dissolved
IBP in bulk water phase and phase-separated IBP in IBP-rich amorphous
nanodroplets. NMR spectral analysis indicated that HPMC did not affect
the chemical environment and mobility of the dissolved IBP. However, 1H spin–spin relaxation time measurements clarified
that the dissolved IBP in the bulk water phase was exchanged with
the IBP-rich amorphous nanodroplets with an exchange lifetime of more
than 10 ms. Moreover, the 1H peaks of HPMC partially disappeared
due to the formation of IBP-rich amorphous nanodroplets, suggesting
that a part of HPMC distributed into the IBP-rich amorphous nanodroplets
from the bulk water phase. The incorporation of HPMC significantly
changed the chemical environment of the phase-separated IBP in the
IBP-rich amorphous nanodroplets and strongly suppressed molecular
mobility. The resulting molecular mobility suppression effectively
inhibited IBP crystallization from the IBP-rich amorphous nanodroplets.
Thus, direct investigation of drug-rich amorphous nanodroplets using
NMR can be a promising approach for selecting appropriate pharmaceutical
excipients to suppress drug crystallization in supersaturated drug
solutions.
We examined the inhibitory effect of hydroxypropyl methylcellulose acetate succinate (HPMC-AS) on drug recrystallization from a supersaturated solution using carbamazepine (CBZ) and phenytoin (PHT) as model drugs. HPMC-AS HF grade (HF) inhibited the recrystallization of CBZ more strongly than that by HPMC-AS LF grade (LF). 1D-1H NMR measurements showed that the molecular mobility of CBZ was clearly suppressed in the HF solution compared to that in the LF solution. Interaction between CBZ and HF in a supersaturated solution was directly detected using nuclear Overhauser effect spectroscopy (NOESY). The cross-peak intensity obtained using NOESY of HF protons with CBZ aromatic protons was greater than that with the amide proton, which indicated that CBZ had hydrophobic interactions with HF in a supersaturated solution. In contrast, no interaction was observed between CBZ and LF in the LF solution. Saturation transfer difference NMR measurement was used to determine the interaction sites between CBZ and HF. Strong interaction with CBZ was observed with the acetyl substituent of HPMC-AS although the interaction with the succinoyl substituent was quite small. The acetyl groups played an important role in the hydrophobic interaction between HF and CBZ. In addition, HF appeared to be more hydrophobic than LF because of the smaller ratio of the succinoyl substituent. This might be responsible for the strong hydrophobic interaction between HF and CBZ. The intermolecular interactions between CBZ and HPMC-AS shown by using NMR spectroscopy clearly explained the strength of inhibition of HPMC-AS on drug recrystallization.
The
polymer used in an amorphous solid dispersion (ASD) formulation
plays a critical role in dosage form performance. Herein, drug–polymer
interactions in aqueous solution were evaluated in order to better
understand the dispersion stability of the colloidal drug-rich phase
generated when the amorphous solubility is exceeded. The amorphous
solubility (S
a,IBP) of ibuprofen (IBP)
decreased when hypromellose (HPMC) or polyvinylpyrrolidone/vinyl acetate
(PVP-VA) were present in solution. Solution nuclear magnetic resonance
(NMR) spectroscopy revealed that a large amount of HPMC and PVP-VA
distributed into the IBP-rich phase. The mixing of HPMC and PVP-VA
with the IBP-rich phase led to the decreased S
a,IBP. In contrast, the charged amino methacrylate copolymer
(Eudragit E PO, EUD-E) showed minimal mixing with the IBP-rich phase;
however, this polymer did lead to solubilization of IBP in the bulk
aqueous phase. Although the IBP-rich phase generated by dissolving
IBP at concentrations above S
a,IBP rapidly
coarsened followed by creaming in the absence of polymer, all of the
polymers stabilized the IBP dispersion to some extent. The droplet
size of the IBP-rich phase immediately after formation was around
300 nm in HPMC and PVP-VA solutions, and around 800 nm in the EUD-E
solution. The mixing of the former two polymers with the drug-rich
phase is thought to account for the smaller droplet size. Despite
a smaller initial size, the dispersion stability of the IBP-rich droplets
was relatively poor in the presence of PVP-VA. In contrast, the coalescence
of the IBP-rich droplets was effectively suppressed by the steric
repulsion and electrostatic repulsion derived from adsorbed HPMC and
EUD-E, respectively. The present study highlights the complex effects
of a polymer on the drug amorphous solubility and colloidal stability,
which should be considered when optimizing ASD formulations with the
goal of maximizing drug absorption.
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