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Despite the high potency of modern antivirals, their clinical efficacy is limited by low stability, solubility and bioavailability. Lipid-based drug delivery systems (DDS) provide a possible solution. Depending on the lipophilicity of the drug, the saturation capacity of a lipid bilayer is 0.05 – 0.2 drug-to-lipid molar ratio (mol/mol). Exceeding these limits leads to lateral separation of the drug within the bilayer plane, accompanied by 2D crystallization, or its precipitation out of the bilayer, causing bulk 3D crystallization. Hence, drug release, dissolution rate and bioavailability are attenuated.
Small- and wide-angle X-ray scattering (SAXS/WAXS) are among the most powerful methods for the analysis of crystallization in lamellar lipid systems. 2D crystallization is coupled to the physical state of the bilayer lipids, which act as a highly organized matrix. Lipids force the embedded drug molecules to orient parallel to the acyl chains, thereby lowering the activation energy for crystallization, which subsequently alters the lipid tail packing and membrane thickness. Changes in the SAXS d-spacing and splitting of the broad liquid-crystalline WAXS peak into sharp peaks representing an ordered 2D lattice appear. In contrast, 3D crystallization occurs especially for drug molecules bound to the surface of the lipid bilayer and/or for molecules with low lipid-to-water partition coefficient. Unaligned drug molecules in solution lose rotational freedom and form crystal nuclei, detected by a plateau in SAXS d-spacing and a separate, sharp set of fingerprint diffraction peaks in WAXS.
We conducted a SAXS/WAXS study to design an optimal DDS for a papain-like viral protease inhibitor (PLpro), GRL0617. Compared to the hydrophilic main protease (Mpro) inhibitor GC376, which was successfully bound within the dipalmitoylphosphatidylcholine (DPPC) lipid bilayer up to 0.5 mol/mol [1], bulk crystallization of lipophilic GRL0617 was detected already above 0.05 mol/mol in the same system. Therefore, different lipid mixtures, varying in lipid type (ester and ether lipids with phosphatidylcholine and phosphatidylethanolamine headgroups and saturated, mono- or polyunsaturated fatty acyl chains, PUFA) were prepared to alter the rigidity and propensity for non-lamellar DDS. Most of the studied lipid mixtures showed increased capacity of the bilayer for GRL0617, without detected crystallization up to 0.1 mol/mol. In the “softest” system, containing PUFA lipids with ester bonds, no crystallization was present even at 0.3 mol/mol. Results suggest that optimizing the lipid composition of the DDS may help to prevent crystallization of the transported drug and maintain the favorable biopharmaceutical performance.
Research was supported by VEGA 1/0305/24 and APVV-17-0250 grants. SAXS experiments were performed on the BL11-NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff.
[1] M. Klacsová et al., Coll Surf B: Biointerfaces 220, 112918, 2022
