In this project we will develop a new niosome-based multi-drug delivery system loading simultaneously two anti Mycobacterium tuberculosis-drugs, formed by different liposomes glued together in multi-compartment clusters. The innovation is connected both with the modular structure of the carrier and with the possibility to transport and deliver different drugs simultaneously towards the same target, encapsulating them in the different niosomes forming the aggregates. It is recognized that inhalation of drug-loaded liposomes offers a potential value in antituberculosis(TB)-therapy. Multicompartment niosomal clusters, with size larger than a single vesicles, have been demonstrated to possess an intrinsic selectivity towards macrophages, thus representing an emerging platform for the intracellular delivery of antiTB drugs to the primary site of infection. The opportunity of combining an increased efficacy of intracellular delivery with the ability of carrying several different active molecules, simultaneously and with a controlled stoichiometry, to the same target could represent a significant breakthrough. Nowadays, the increasing spread of biocompatible niosome-based drug nanocarriers is a result of the unique properties of surfactants and of the vesicular structure. Here we will tackle advantage of the large variety of surfactant to optimize the formulation of niosomes in terms of physico-chemical properties of vesicle bilayer, stability of the vesicles and drug encapsulation efficiency, with the aim to improve their biocompatibility and ability to entrap drugs, optimizing also their pharmacokinetics and therapeutic index.
Some studies explored the possibility of drugs co-encapsulation even if with little resonance [Gursoy 2004 Int J of Pharm 2004; Pandey, Int. J. Antimicrob. Ag 2004]. The first attempt has been performed by Gursoy et al with unilamellar DPPC-Chol liposomes, which have been proved to co-encapsulate IS and RIF better than eggPC-Chol liposomes [Gursoy Int J Pharm 2004], but no further development of this work has been reported. Pandey and coworkers developed a respirable multilamellar liposome encapsulating both RIF and IS, containing PC and Chol as bilayer lipid-forming components. In the AM their accumulation rates were high, proving the ability of aerosolized liposomal drug to reach the target. In contrast to the free drugs that were unable to be detected in the lungs or AM beyond 48 h, nebulized liposomal drugs remained for 5 days after nebulisation.
To obtain a specific macrophage targeting, specific ligands [Vias Int Pharm 2004, 269:37] or mannose-surface modification of anionic liposomes [ Chono et al., J. Cont. Rel. 2009,127:50] has been explored. Liposomes have been prepared with mixture of HSPC, DOPC, CH, DCP lipids in different molar ratio and used to incorporate ciprofloxacin (CPFX). It has been shown that 1000 nm is the most effective particle size of liposomes for CPFX targeting to AMs by pulmonary administration, and uptake of liposomes by AMs is increased by surface mannose modification.
Recently, new multimodal PEGylated liposomes with specific AM targeting encapsulating four clinically commonly used anti-TB drugs, have been studied [Niu, Drug Design, Devel. and Therapy, 2015,9:4441 ]. The strategy is rather complex, considering that DMSE-PEG is chemically conjugated to Streptomycin, then it is used in mixture with the cationic lipid DOTAP and Cholesterol to prepare liposomes, which entrap, in the same vesicles three drugs (IS, RIF and pyrazinamide). Specific AM targeting is obtained by electrostatic linkage of cationic vesicles to small interfering RNA (siRNA) against transforming growth factor-ß1 (TGF-ß1). This novel multimodal siRNA-liposomes have shown good selectivity and minimal cytotoxicity and open the way for anti TB multi-drug delivery.
The most recent advances on multi-drug delivery nanocarriers for combinatorial therapy are discussed in a recent review of Gadde [Gadde Med. Chem. Comm.,2015, 6:1916 ]. Different strategies are reviewed, for example the co-encapsulation of two drugs in the same liposomal vesicle, with the hydrophilic drug in the aqueous compartment and hydrophobic drug in the bilayer, or the co-loading in the core of polymeric particles, or metallic nanoparticles, dendrimers, and carbon-based materials chemically linked to different drugs. As a general feature, sophisticated carriers can load different drugs in the same structure, however batch reproducibility and combinations with controlled delivery and precise ratiometric drug loading are still far from being reached.
This short overview indicates that the use of vesicular carriers, both large multilamellar and unilamellar, for antiTB therapy is a viable approach. However success of AM targeting critically depends on the physico-chemical characteristics of the nanocarriers, such as size, shape, surface charge, structure, which can be modulated by lipid composition, and presence of specific targeting molecules.
At the present, the designing of multi-drug delivery vectors based on aggregates of different unilamellar vesicles, each vesicle entrapping (separately) a single drug, for controlled co-encapsulation of antiTB drug within the same vector has not been tried yet, although this simple route would apparently provide an optimal control of the ratiometric drug loading. The main obstacle is the lack of a simple and reliable procedure to "glue together" the vesicles carrying the different drugs into one single multi-compartment vector.
As a further advantage, being the drugs distributed in the different compartments, a more gradual drug release can be hypothesized.
At the best of our knowledge, no niosomal carriers has been investigated for combinatorial therapy.
The key-element of our proposal is represented by the purely electrostatic basic mechanism governing the aggregation and the stability of the vesicular aggregate, which confers to this system modularity and adaptability to different requirements. Thanks to that, we will consider to use different surfactant and we will compare the physico-chemical properties to understand how the specific detail of the formulation will affect the capability to stably entrap and release high amount of antiTB drugs and to interact selectively with macrophages. This study could also open the way for further development of research on amphiphilic molecules with improved features as vesicle builders, able to improve niosomal formulations for pharmaceutical use.