Formulation and Intracellular Trafficking of Lipid–Drug Conjugate Nanoparticles Containing a Hydrophilic Antitubercular Drug for Improved Intracellular Delivery to Human Macrophages

[Image: see text] Isoniazid is an important first-line antitubercular drug used in the treatment of all major clinical manifestations of tuberculosis, including both pulmonary and cerebral diseases. However, it is associated with significant drawbacks due to its inherent hydrophilic nature, including poor gut permeability and an inability to cross the lipophilic blood–brain barrier, which, in turn, limit its clinical efficacy. We hypothesized that the addition of a hydrophobic moiety to this molecule would help overcome these limitations and improve its bioavailability in the bloodstream. Therefore, we designed a stable, covalently linked lipid–drug conjugate of isoniazid with a short lipid chain of stearoyl chloride. Further, lipid–drug conjugate nanoparticles were synthesized from the bulk lipid–drug conjugate by a cold high-pressure homogenization method enabled by the optimized use of aqueous surfactants. The nanoparticle formulation was characterized systematically using in vitro physicochemical analytical methods, including atomic force microscopy, transmission electron microscopy, differential scanning calorimetry, X-ray diffraction, attenuated total reflectance, particle size, ζ-potential, and drug release studies, and the mechanism of drug release kinetics. These investigations revealed that the lipid–drug conjugate nanoparticles were loaded with an appreciable amount of isoniazid conjugate (92.73 ± 6.31% w/w). The prepared lipid–drug conjugate nanoparticles displayed a uniform shape with a smooth surface having a particle size of 124.60 ± 5.56 nm. In vitro drug release studies showed sustained release up to 72 h in a phosphate-buffered solution at pH 7.4. The release profile fitted to various known models of release kinetics revealed that the Higuchi model of diffusion kinetics was the best-fitting model (R(2) = 0.9929). In addition, confocal studies showed efficient uptake of lipid–drug conjugate nanoparticles by THP-1 macrophages presumably because of increased lipophilicity and anionic surface charge. This was followed by progressive intracellular trafficking into endosomal and lysosomal vesicles and colocalization with intravesicular compartmental proteins associated with mycobacterium tuberculosis pathogenesis, including CD63, LAMP-2, EEA1, and Rab11. The developed lipid–drug conjugate nanoparticles, therefore, displayed significant ability to improve the intracellular delivery of a highly water-soluble drug such as isoniazid.