Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution.

Nanoparticle (NP) formulations are inherently polydisperse making their structural characterization and justification of specifications complex. It is essential, however, to gain an understanding of the physico-chemical properties that drive performance in vivo. To elucidate these properties, drug-containing poly(lactic acid) (PLA)-poly(ethylene glycol) (PEG) block polymeric NP formulations (or PNPs) were sub-divided into discrete size fractions and analyzed using a combination of advanced techniques, namely cryogenic transmission electron microscopy, small-angle neutron and X-ray scattering, nuclear magnetic resonance, and hard-energy X-ray photoelectron spectroscopy. Together, these techniques revealed a uniquely detailed picture of PNP size, surface structure, internal molecular architecture and the preferred site(s) of incorporation of the hydrophobic drug, AZD5991, properties which cannot be accessed via conventional characterization methodologies. Within the PNP size distribution, it was shown that the smallest PNPs contained significantly less drug than their larger sized counterparts, reducing overall drug loading, while PNP molecular architecture was critical in understanding the nature of in vitro drug release. The effect of PNP size and structure on drug biodistribution was determined by administrating selected PNP size fractions to mice, with the smaller sized NP fractions increasing the total drug-plasma concentration area under the curve and reducing drug concentrations in liver and spleen, due to greater avoidance of the reticuloendothelial system. In contrast, administration of unfractionated PNPs, containing a large population of NPs with extremely low drug load, did not significantly impact the drug's pharmacokinetic behavior - a significant result for nanomedicine development where a uniform formulation is usually an important driver. We also demonstrate how, in this study, it is not practicable to validate the bioanalytical methodology for drug released in vivo due to the NP formulation properties, a process which is applicable for most small molecule-releasing nanomedicines. In conclusion, this work details a strategy for determining the effect of formulation variability on in vivo performance, thereby informing the translation of PNPs, and other NPs, from the laboratory to the clinic.

Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery.

mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.

Characterization and demonstration of drug compound ring-chain tautomer formation and its impacts on quality control.

Unknown chromatographic peaks, potential impurities, were observed in a series of related compounds. This led to the identification and characterization of tautomeric equilibria. Structural elucidation was required to understand the potential impurity profile, thus impacting method development for quality control. In this work, characterization of the chemical structures, AZ13581258 and AZD5718, and equilibria of the tautomeric forms was performed using a range of advanced analytical techniques such as preparative chromatography, nuclear magnetic resonance (NMR), chromatographic detection by mass spectrometry (MS), MSMS, and ultraviolet spectroscopy (UV). Predictions using density functional theory (DFT) further explains and confirms the tautomer equilibria through predictions of reaction barrier energies, UV-spectra and NMR data. These investigations led to fully understand the impurity profile and to the development of a quality control method for AZD5718 drug substance and drug product. In conclusion, ring-chain tautomeric structures are predominately formed under acidic conditions, and the additional peaks observed in LC during organic impurity determination were found to originate from ring-chain closed tautomers in equilibria with the parent open form compound. Hence, the closed and open tautomer forms should all be considered as the same compound.