**4. Conclusions**

3TC and AZT nano co-crystals were successfully prepared using a sonochemical synthesis approach. The use of different surfactants during the sonication phase of preparation resulted in the formation of crystals of reduced size, a reasonably narrow particle size distribution and in the case of the anionic surfactant, SDS, surface charge reduction. The use of surfactants to coat NCC may also achieve different purposes such as stealth and targeting capabilities, as these surfactant molecules are capable of interacting with specific substrates in biological systems [66–68].

All surfactants investigated exhibited concentration-dependent stabilization, resulted in the formation of nano co-crystals, and offered further stabilization characteristics. Stabilization was established to be concentration-dependent, based on the size of the hydrophilic head group of the surfactant used and surface charge induction. Surfactants with larger hydrophilic heads and thus larger HLB values, exhibited effective stabilization at lower concentration as observed by the ability of Tween® 80 and TPGS 1000 to produce co-crystals in the nanometer size range when used at low and medium concentration.

The PDI of the NCC produced also exhibited a direct relationship to the concentration of surfactant used to achieve stabilization. It is clear that surfactants act as a steric barrier to crystal growth generated by precipitation and as a result, nucleation rate rather than growth rate increases with increasing surfactant concentration, resulting in smaller dimensions and narrow size distributions [69,70].

The use of the anionic surfactant SDS produced crystals with a low ZP. The contribution to ZP reduction appears to be an antagonistic concentration-dependent relationship with the anionic surfactant SDS. An increase in SDS concentration resulted in lower ZP which would, consequently, result in an expected increase in solution stability of the technology.

These results sugges<sup>t</sup> that the use of any of these surfactants would result in the production of co-crystals in the nanometer size range, provided that the correct concentration of surfactant is used

while only the surfactant SDS would produce nano co-crystals that meet all the CQA criteria set prior to commencing these experiments. The findings of this research could prove useful in overcoming the low bioavailability of AZT and potentially provide a dosage form capable of delivering AZT and 3TC to HIV reservoirs, thereby potentially reducing the side effect profile associated with ARV treatment. In addition, the potential to produce a long-acting and -circulating ARV regimen is a possibility.

Investigations into the use of combinations of surfactants for the production of nano co-crystals to evaluate the possibility of producing specific/targeted CQA in co-crystals is ongoing in our laboratory.

**Author Contributions:** R.B.W. conceptualized and supervised the project. B.A.W. performed the experiments, analyzed the data and wrote the article. V.J.S. contributed to the conceptualization, supervision, bibliography research and proofreading of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was not funded with an external research grant.

**Acknowledgments:** The authors acknowledge the National Research Foundation (BAW) for a bursary, the Research Committee of Rhodes University (RBW) and Rhodes University Sandisa Imbewu fund (VJS) for financial assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.
