**4. Conclusions**

The formulation optimization for production of electrosterically stabilized 3TC and AZT NCC using a pseudo one-solvent bottom-up approach was successful. The PS and PDI were affected by the amount of surfactant used in the formulation. The presence of surfactants reduces the energy at the surface of nucleating crystals preventing crystal growth [64]. The PS was not as affected by the concentration of TPGS 1000 used than when SDS was used suggesting that electrostatic stabilisation was more effective in the synthesis of the NCC using a bottom-up approach. The PDI was significantly affected by the concentration of SDS used, due to the combined effect of increasing SDS and TPGS 1000 concentrations in the composition.

The ZP is primarily dependent on the presence of SDS and a linear relationship exists between SDS concentration and ZP. The data reveal that an increase in SDS content results in a proportional decrease in ZP for the NCC suspensions produced.

In order to achieve the development of a suitable technology for the delivery of AZT and 3TC, the optimization objective was to minimize the ZP, increase the stability of the formulation [65,66] and with particle size reduction, potentially target macrophages [61–63,67].

Characterisation of the OPT-NCC formulation using TEM revealed that the NCC were <500 nm. PXRD and DSC data indicate the OPT-NCC are crystalline, which could potentially increase stability, and co-crystal formation was confirmed using FTIR. SEM-EDX was used to determine the elemental surface composition of the NCC. The spectra confirmed the presence of stabilizer on the surface of crystals in addition to the presence of molecular peaks associated with the API.

In vitro cytotoxicity studies revealed the OPT-NCC were less cytotoxic to HeLa cells than the individual API and a physical mixture of the API. The presence of hydrophilic PEG heads in TPGS 1000 may be the reason for increased HeLa cell viability.

The use of DoE and cold sonoprecipitation to manufacture optimized NCC is an inexpensive, reproducible and precise method of manufacturing NCC with specific pre-defined desirable PS, PDI and ZP, while maintaining crystallinity of the individual compounds. The NCC produced in this study exhibit less toxicity to HeLa cells than individual components, which in turn, may result in reduced side e ffects of each API. The improvement of the API side e ffect profile may well improve patient adherence to therapy and lead to better treatment outcomes.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/4/367/s1, Table S1: Summary of melting temperatures of AZT, 3TC, reported co-crystal value and the OPT-NCC. Figure S1: DSC thermogram of 3TC (Black), AZT (Orange) and OPT-NCC (Blue). Table S2: Summary of the comparison FTIR for the reported co-crystal and the OPT-NCC. Figure S2: A plot of the values reported in Table S2 reflecting a one-to-one agreemen<sup>t</sup> between the FTIR wavenumbers reported in the literature [68] (blue) and those recorded for the NCC (orange). Figure S3: FTIR spectra of the OPT-NCC (orange), 3TC (blue) and AZT (black). Figure S4: PXRD di ffractograms of OPT-NCC and Co-crystal (Di ffractogram obtained from CSD [69]). Figure S5: TEM of uncoated bottom-up micro co-crystal. Figure S6: TEM depicting the mean particle size of the OPT-NCC, Figure S7: TEM images of the smallest product obtained (OPT-NCC). Figure S8: DLS intensity size distribution curve of the OPT-NCC.

**Author Contributions:** R.B.W. conceptualized, supervised and contributed to writing and editing of the manuscript. B.A.W. performed the experiments, analyzed the data and wrote the manuscript. V.J.S. contributed to the conceptualization, supervision, writing, editing, 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 (B.A.W.) and the Research Committee of Rhodes University (R.B.W.) and Rhodes University Sandisa Imbewu fund (V.J.S.) for financial assistance. The contribution of Pascal Ntemi for running some samples is gratefully acknowledged.

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