**1. Introduction**

More than 7000 people worldwide die of HIV-related causes daily. Many people are not benefiting fully from the use of orally administered antiretroviral (ARV) drugs, which provide the only effective means of halting the progression of HIV disease and AIDS [1]. Eight million of the estimated 37 million HIV-positive people should be treated, but only two million are currently receiving ARV therapy. This unmet need is expected to increase on an annual basis [1].

Crystal engineering is described as the exploitation of non-covalent interactions between molecular or ionic components for the rational design of solid-state materials [2,3]. The application of crystal engineering in pharmaceutics is usually related to understanding polymorphism and its associated properties.

Co-crystals are single-phase crystalline solids that are composed of two or more different molecules, which generally associate in a stoichiometric ratio [4]. Co-crystals can be constructed using several types of molecular interactions such as hydrogen bonds, halogen bonds, π–π stacking, van der Waal's forces, amongs<sup>t</sup> others [5–8]. They are thermodynamically more stable than crystals of the pristine compounds, while for pharmaceutical applications they are highly promising for tailoring the properties of the active pharmaceutical ingredient (API) [9]. Co-crystals are known to exhibit different properties from the parent compounds including enhanced solubility, improved dissolution kinetics, improved bioavailability as well as increased phase stability when compared to amorphous forms, which tend to spontaneously crystallize on standing. Co-crystal formation does not involve or require covalent bond formation or breaking and usually requires rather mild conditions during synthesis. Solid-state synthetic methods such as neat grinding, liquid-assisted grinding, and other mechanochemical methods have recently come into prominence as reliable methods for co-crystal synthesis and because they are inherently green methods capable of producing high yields without the need for large or excessive quantities of solvent [10].

Co-formers are molecules that are selected to co-crystallize with an API and are chosen from the, "generally regarded as safe" list (GRAS) or the, "everything added to food in the United States" list (EAFUS) [11]. They include but are not limited to food additives, preservatives, pharmaceutical excipients, and other API molecules [7,9].

Finally, co-crystallization of important API molecules may lead to patents or intellectual property protection emanating from their development [12].

Despite the advantages of co-crystallization, further benefit can be derived by combining different technologies to ensure targeted drug delivery, enhanced bioavailability, flexibility in respect of administration and stealth delivery. Combining co-crystallization with nano-sizing to yield nano co-crystals presents such an opportunity. Several techniques can be used to develop co-crystals with nano-scale dimensions, many of which are derived from techniques used in nanocrystal manufacture. Nanocrystals can be manufactured using two approaches, namely: a top-down technique that uses shear forces to reduce the particle size from micrometer to nanometer dimensions [13,14] and a bottom-up approach that involves nucleation and crystal growth. The growth of individual crystals can be arrested in the nanometer range by using a suitable stabilizer [15,16].

The use of surfactants as stabilizers has previously been explored in the synthesis of nanocrystals [17–19] and nano co-crystals [20–22]. Nano co-crystals are co-crystals of nano-scale dimensions which exhibit properties that are superior to those generally associated with co-crystals and nanocrystals [20,23,24]. Stabilizers are primarily used as growth prevention agents and function by the adsorption of surfactant/polymer molecules onto nucleated nanocrystals or co-crystals, lowering the surface free energy and consequently particle reactivity [25]. Known stabilizers include surfactants such as sodium dodecyl sulfate (SDS) [26–28], Tween® [29–31], Span® [20,32], α-tocopheryl polyethylene glycol succinate 1000 (TPGS 1000) [33,34], Pluronic® [35,36] and polymers such as hydroxypropyl methylcellulose (HPMC) [19,37], pyrrolidone K30 [37] and polyvinyl pyrrolidone [19]. Different stabilizers impart different properties to the resultant nano co-crystals. For instance, TPGS 1000 is known to inhibit P-glycoprotein efflux and stealth properties to formulations in which it has been incorporated, while Tween® 80 facilitates brain targeting [31,34,38,39] and Span® is effective in reducing the size of nano co-crystals [20].

The use of a combination of techniques in the manufacture of nano co-crystals has been applied with success on a few occasions. For example, a top-down high-pressure homogenization technique (HPH) was used to produce nano co-crystals of the flavonoid, baicalein with nicotinamide. The resultant nano co-crystal exhibited a marked improvement in the rate and extent of dissolution [22]. Similarly, a bottom-up approach was used to develop myricetin-nicotinamide nano co-crystals. The nano co-crystal product also displayed an increased rate and extent for dissolution [40].

Sonochemical co-crystallization is a bottom-up process that has been successfully used for nano co-crystal synthesis [20,40–42]. One solvent systems involve dissolving all the co-crystal components in one solvent and injecting the solution into an anti-solvent while simultaneously sonicating the solution [41]. Two solvent systems involve dissolving the components of the co-crystal separately in different solvents followed by injecting each solution into the same anti-solvent at the same time. Top-down approaches such as wet media milling [40] and high-pressure homogenization [22], which are not covered in this work, have also been used with success.

Attempts have been made to produce co-crystals with nano-scale dimensions with varying success. The pharmaceutical compound ca ffeine and the co-former 2,4-dihydroxybenzoic acid were co-crystallized via sonochemical synthesis and stabilized with the surfactant Span ® 85. The resultant co-crystal dimensions for the smallest particles were 190 × 200 nm while the largest particles had dimensions of 200 × 800 nm [20]. The presence of surfactant was found to promote nucleation and moderate crystal growth. Myricetin-nicotinamide nano co-crystals were synthesised via both bottom-up and top-down approaches using Tween ® 80 as the stabiliser. The smallest particles had dimensions of 100 × 200 nm, whereas the largest particles were 200 × 800 nm [40].

Baicalein-nicotinamide (BE-NCT) nano co-crystals were successfully prepared via a top-down approach. The BE-NCT nano co-crystals were compared with BE coarse powder, BE-NCT co-crystals and BE nanocrystals, BE-NCT nano co-crystals exhibited a significantly enhanced performance both in in vitro and in vivo evaluations, suggesting that the nano co-crystals could be proposed as an advanced strategy for dissolution rate and bioavailability enhancement of poor soluble natural products such as BE [22].

Bhatt et al. synthesized a co-crystal of the ARV compounds 3TC and AZT using slow evaporation from a variety of solvents and several other methods including liquid assisted grinding [42]. The resultant co-crystal (3TC.AZT. H2O) contains a molecule of water, one molecule of 3TC and one molecule of AZT. Each AZT molecule hydrogen bonds via N–H...O and O–H...O interactions to three di fferent 3TC molecules as well as to two di fferent H2O molecules while each 3TC molecule hydrogen bonds to a single H2O molecule via an O–H...N interaction [43].

A preliminary investigation into the possibility of using a one or two solvent approach to producing nano co-crystals of 3TC and AZT proved unsuccessful. This was probably owing to the vastly di fferent solubilities of the API molecules. This led to the development of a pseudo one solvent approach in which both components were dissolved separately in di fferent solvents so that each solvent serves as an anti-solvent for the other, in situ.

Nano co-crystals are considerably easy to produce but stabilizing them against continual growth after forming and stabilizer(s) selection, are critical [44]. Herein, we report the use of surfactants in combination with sonochemical methods to synthesize nanometer sized co-crystals and to investigate the impact of di fferent surfactants on the critical quality attributes (CQA) of the resultant nano co-crystal particles. To the best of our knowledge, this is the first comparative investigation of four stabilizers, viz., Tween ® 80, Span ® 80, SDS and TPGS 1000 and their e ffect on the three CQA parameters: particle size (PS), polydispersity index (PDI) and Zeta potential (ZP) for the reported co-crystals.
