*3.9. In Vitro Efavirenz Release*

In vitro release testing revealed burst release of EFV for batches F1, F2, F4, F5 and for pure EFV within the first two hours of commencement of testing. Dissolution testing was conducted for the formulations listed in Table 6 and the % EFV released was based on actual drug loading data generated following assay of the nanoemulsion formulations. The nanoemulsion for batch F5 exhibited the greatest extent of release at 12 h that decreased for batches F1, F4, F5 and F3 which exhibited a sustained release effect and the release profiles are depicted in Figure 11. The reduction in amount of EFV released from the nanoemulsions may be attributed to solubilization of EFV in droplets of the nanoemulsion and/or formation of the EFV solvate on crystallization. Drugs may precipitate in vitro and in vivo due to a rapid change in pH, dilution with body fluids or digestion of solubilizing excipients subsequently resulting in lower EFV concentrations within the aqueous phase and better entrapment of EFV within the crystal lattice structure. As the saturation method of manufacture was used to produce the nanoemulsions, following supersaturation the process of nucleation continues and promotes crystallization [62,63]. As the proportion of ethanol is increased, the percent efavirenz released at 12 h decreases. On interaction with 1% *m*/*v* SLS in 0.1-M HCl dissolution fluid, the nanoemulsion formulations crystallized and formed a white edged crystalline semisolid that sank to the bottom of the dissolution vessel. Nanoemulsions of batch F4 and F5 were prepared with the same amount of ethanol and different proportions of Tween® 80 and Span® 20 and batch F5 released a larger amount of EFV than batch F4 at 12 h possibly due to the different hydrophilic lipophilic balance (HLB) value of the surfactants used. Tween® 80 is soluble in an aqueous environment, whereas Span® 20 is not and therefore Tween® 80 interacts with the dissolution medium and contributed to better dissolution of EFV. Crystallization was observed for all batches and was greatest for batch F3. EFV precipitation is an undesirable outcome following administration of SNEDDS formulations. The loss of ethanol as observed with FT–IR analysis following dissolution testing may be the largest contributing factor to a reduced solubilization capacity and increased rate of nucleation in the supersaturated nanoemulsion formulations and hybridization of the C–H bond stretch that reduces the hydrophobicity of the formulation thereby affecting the interaction of EFV to form a solution.

form a solution.

formulations listed in Table 6 and the % EFV released was based on actual drug loading data generated following assay of the nanoemulsion formulations. The nanoemulsion for batch F5 exhibited the greatest extent of release at 12 h that decreased for batches F1, F4, F5 and F3 which exhibited a sustained release effect and the release profiles are depicted in Figure 11. The reduction in amount of EFV released from the nanoemulsions may be attributed to solubilization of EFV in droplets of the nanoemulsion and/or formation of the EFV solvate on crystallization. Drugs may precipitate in vitro and in vivo due to a rapid change in pH, dilution with body fluids or digestion of solubilizing excipients subsequently resulting in lower EFV concentrations within the aqueous phase and better entrapment of EFV within the crystal lattice structure. As the saturation method of manufacture was used to produce the nanoemulsions, following supersaturation the process of nucleation continues and promotes crystallization [62,63]. As the proportion of ethanol is increased, the percent efavirenz released at 12 h decreases. On interaction with 1% *m/v* SLS in 0.1-M HCl dissolution fluid, the nanoemulsion formulations crystallized and formed a white edged crystalline semisolid that sank to the bottom of the dissolution vessel. Nanoemulsions of batch F4 and F5 were prepared with the same amount of ethanol and different proportions of Tween® 80 and Span® 20 and batch F5 released a larger amount of EFV than batch F4 at 12 h possibly due to the different hydrophilic lipophilic balance (HLB) value of the surfactants used. Tween® 80 is soluble in an aqueous environment, whereas Span® 20 is not and therefore Tween® 80 interacts with the dissolution medium and contributed to better dissolution of EFV. Crystallization was observed for all batches and was greatest for batch F3. EFV precipitation is an undesirable outcome following administration of SNEDDS formulations. The loss of ethanol as observed with FT–IR analysis following dissolution testing may be the largest contributing factor to a reduced solubilization capacity and increased rate of nucleation in the supersaturated nanoemulsion formulations and hybridization of the C–H bond

**Figure 11.** Dissolution profiles (mean ± SD (*n* = 3) for batches F1, F2, F3, F4 and F5 and EFV powder. **Figure 11.** Dissolution profiles (mean ± SD (*n* = 3) for batches F1, F2, F3, F4 and F5 and EFV powder.

#### **4. Conclusions 4. Conclusions**

The recommended dose of efavirenz for adults is 600 mg taken once daily and the most common dosage form available is a tablet, which is inconveniently large in size that may negatively affect patient adherence. The design of LBDDS focused on making the release characteristics independent of the gastrointestinal physiology and the fed/fasted state of the patient [64,65] and in a dosage form size that is small and convenient for the patient to use would be an advantage. The administration of EFV using these SNEDD technology would require a small unit size of the dosage form due to the high drug loading capacities exhibited which may produce a more convenient sized dosage form. The recommended dose of efavirenz for adults is 600 mg taken once daily and the most common dosage form available is a tablet, which is inconveniently large in size that may negatively affect patient adherence. The design of LBDDS focused on making the release characteristics independent of the gastrointestinal physiology and the fed/fasted state of the patient [64,65] and in a dosage form size that is small and convenient for the patient to use would be an advantage. The administration of EFV using these SNEDD technology would require a small unit size of the dosage form due to the high drug loading capacities exhibited which may produce a more convenient sized dosage form. Co-solvents and co-surfactants maybe used to improve the thermodynamic stability of formulations that exhibit an increased solubilization capacity resulting in enhanced therapeutic performance. The negative ZP of nanoemulsion droplets would be useful for macrophage targeting since macrophages identify and take up negatively charged particles [52]. Macrophages are key cells in HIV infection and are significant reservoirs of the virus [53].

Investigation of phase behavior of LBDDS components is useful for optimization of formulations and pre-formulation studies assist in defining appropriate proportions of each component to use, in addition to facilitation of decisions in relation to manufacturing processes such as whether high pressure or high shear homogenization can be used. Such decisions are required to ensure that an optimum product with predefined quality attributes is produced. The phase behavior of crude cold pressed flaxseed oil with non-ionic surfactants revealed an area within pseudo-ternary phase diagrams for surfactant-mixtures S1 and S2 that formed gels/semisolid structures which can be exploited for other drug delivery strategies such as topical application. This research identified that optimization of mixture compositions to produce a product with the required characteristics through estimation of the effects of the formulation components. However, accurate optimization using a D-optimal design may not be possible to only a relatively good predictability in droplet size alone and therefore other designs should be explored for future optimization.

Kinetically stable low energy nanoemulsions of flaxseed oil, Tween® 80 and Span® 20 surfactant and co-surfactant with ethanol were successfully manufactured. On visual observation, different release profiles for efavirenz were observed from different nanoemulsions. These can be exploited for further optimization to produce formulations suitable for undertaking in vivo and pharmacokinetic studies. The side effects of EFV associated with dose dumping may be reduced by using nanoemulsions to modulate release. The nanoemulsion approach is promising however, stability of formulations in gelatin or other encapsulated forms in which crystallization of EFV from solution is minimized, should be explored. The use of flaxseed oil in dosage forms intended for oral delivery may be beneficial to patients as there are health benefits associated with use of polyunsaturated fatty acids in addition to flaxseed oil being a cheap renewable raw material for dosage forms. Flaxseed oil contains an abundant source of, viscous fiber components and phytochemicals, such as lignans and protein that have demonstrated clinical activity as one of the six plant materials in the study of cancer-preventive foods [66].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/12/9/797/s1, Table S1: Scan images for investigation of turbidity and transparency during phase identification studies and results for selected critical quality attributes (CQA) viz., droplet size (DS), polydispersity index (PDI) and Zeta Potential (ZP), Table S2: PRESS statistic values and sum of squares for all models used to analyze the data, Table S3: ANOVA data for special quartic model for droplet size, df = degrees of freedom, Table S4: ANOVA data for special quartic model for polydispersity index, df = degrees of freedom, Table S5: ANOVA data for a linear model of Zeta Potential, df = degrees of freedom, Table S6: Constraints used for the target optimization criteria, Table S7: Solutions from Design Expert software for specified optimization criteria, Table S8: Theoretical (reported) and experimental wavenumbers characterized by FT-IR and Raman spectroscopy of efavirenz. Figure S1: Transmission electron micrograph of nanoemulsion F2, Figure S2: Transmission electron micrograph of nanoemulsion F4, Figure S3: (A) Diagnostic Box-Cox plot for power transforms and (B) predicted vs. actual plot for droplet size, Figure S4: (A) Diagnostic Box-Cox plot for power transforms and (B) predicted vs. actual plot for polydispersity index, Figure S5: (A) Diagnostic Box-Cox plot for power transforms and (B) predicted vs. actual plot for Zeta Potential, Figure S6: Overlay plot of the desirable area (yellow) derived using the specified criteria listed in Table S6, Figure S7: Raman Spectra of pure EFV, the control and EFV loaded nanoemulsions, Figure S8: FT-IR spectra of, pure EFV, Tween® 80, Span® 20, the control nanoemulsion, EFV loaded nanoemulsion and the nanoemulsion harvested following dissolution studies.

**Author Contributions:** R.B.W. conceptualized, supervised and contributed to writing and editing of the manuscript. P.M. performed the experiments, analyzed the data and wrote the article. S.M.M.K. contributed to the conceptualization, supervision, bibliographical research and proof reading of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors acknowledge the National Research Foundation and the Research Committee of Rhodes University (R.B.W.). The authors acknowledge Bronwyn Tweedie for graphic assistance and Bwalya A. Witika for helpful research and experimental discussion.

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