*2.2. Methods*

2.2.1. Preparation of Micro and Nano Co-Crystals Using a Pseudo One Solvent Bottom-Up Method

Micro co-crystals of AZT and 3TC have been reported and were synthesized according to previously described methods [44]. The micro co-crystals produced were used as reference material in the characterization experiments in order to elucidate the characteristics of nano co-crystal formation in this investigation. A quantity of 3TC and AZT equivalent to 2 mmol of each ARV was accurately weighed using a model AG 135 Mettler Toledo (Greifensee, Switzerland) analytical balance and dissolved in 10 mL of water and 5 mL of ethanol (EtOH) respectively. The two solutions were mixed and gently stirred at 50 ◦C for an hour. The solution was allowed to cool to ambient temperature (22 ◦C) for 48 h to allow micro co-crystals to grow.

Supersaturation studies were conducted by adding 1 mL of MeOH to the 2 mmol of AZT and similarly, 1 mL of water was added to 2 mmol of 3TC. The individual solutions were then sonicated for 5 min using a Branson® 8510E-MT ultrasonic bath (Danbury, CT, USA). Subsequently, 1 mL aliquots of solvent were added and further sonicated for 5 min until a clear solution resulted.

Nano co-crystals (NCC) of AZT and 3TC were prepared using a cold-sonochemical precipitation bottom-up technique [16,20]. The batch size was approximately 1 g, specifically 534 mg of AZT and 458 mg of 3TC amounting to the use of 2 mmol of each API. The 3TC was dissolved in 7 mL of water and AZT was dissolved in 6 mL of MeOH. The solutions were rapidly injected into a pre-cooled conical flask incubated at 4 ◦C ± 2 ◦C in an ice bath. A sonication output of 50 kHz ± 6 kHz was applied to the solution for 20 min using a Branson® 8510E-MT ultrasonic bath.
