**1. Introduction**

Since the beginning of the HIV epidemic in the 1980s, 74.9 million people have been infected with the virus. In the same time period, an approximate total of 32 million people have died from AIDS-related illnesses [1].

According to a report by UNAIDS, approximately 37.9 million people globally were living with HIV at the end of 2018. Of these, 23.3 million were accessing antiretroviral therapy (ART). In addition, 1.7 million new HIV infections were reported in 2018. HIV/AIDS also accounted for 770,000 deaths from AIDS-related illnesses [1].

Co-crystals incorporate pharmaceutically acceptable gues<sup>t</sup> molecules into a crystal together with an active pharmaceutical ingredient (API). Co-crystals have gained attention as alternate solid forms for drug development [2]. For pharmaceutical applications, co-crystallization is highly promising for tailoring the properties of Active Pharmaceutical Ingredients (API) and co-former couples to improve dissolution kinetics, the rate and extent of bioavailability, and the stability of compounds [3–6].

Multi-API co-crystals, which remain relatively unexplored solid forms of API, have potential relevance in the context of combination therapy for drug and product development [7]. AZT and 3TC are antiviral agents used in combination for the suppression and prevention of HIV/AIDS [3,8–11]. The 3TC·AZT·H2O co-crystal has been synthesized by neat grinding, liquid-assisted grinding and solvent evaporation [12]. The co-crystal has the potential for co-delivery of both molecules.

Co-crystals can be produced in the nanometre range using top-down or bottom-up manufacturing approaches. The production of nanocrystals and nano co-crystals using bottom-up techniques generally involve the use of a solvent system(s) added to an anti-solvent system in the presence of ionic and/or non-ionic stabilisers. The mixing of drug solution(s) and anti-solvent is generally achieved with conventional mixing equipment such as magnetic and/or overhead stirrers fitted with an agitator blade [13]. In order to promote nucleation, sonic waves are introduced using a process now called, sonoprecipitation [13,14]. High-energy mechanical forces are involved when using top-down approaches, which are achieved using media milling (MM) (NanoCrystals ®) or High Pressure Homogenization (IDD-P ®, DissoCubes ® and Nanopure ®) to comminute large crystals [15,16].

The use of surfactants and polymers such as Tween ®, Span ®, hydroxypropyl methylcellulose (HPMC), pyrrolidone K30 and polyvinyl pyrrolidone as stabilizers has been explored in the synthesis of nanocrystals and nano co-crystals (NCC) [17–25].

Macrophages are carriers of HIV in humans [26–28] and targeting these cells by use of nanotechnological approaches has been proposed as an ideal option for more e fficient treatment of HIV and halting the progression to AIDS [29–32]. Nanocrystals and nano co-crystals o ffer a potential route for targeting macrophages. Following their administration, nanocrystals and nano co-crystals are engulfed by phagocytic cells such as macrophages after recognition as foreign bodies. In phagocytic cells, nano-dimensional crystals dissolve slowly in phagolysosomes. Consequently, any payload might pass through phagolysosomal membranes and reach the cytoplasm of cells, following which di ffusion from the cell down a concentration gradient occurs [15].

Cold-sonochemical co-crystallization as a process has been used successfully for the manufacture of nano co-crystals [14,33,34]. One-solvent systems involve dissolving the components in a single solvent and injecting the solution into an anti-solvent while applying ultrasonic energy [33], whereas in two-solvent systems, the components are dissolved in separate vehicles and injected into an anti-solvent. Preliminary studies suggested that neither of these approaches were suitable due to di fferences in the solubility of 3TC and AZT. Consequently, we developed a pseudo one-solvent system approach in which the components were dissolved in separate solvents that acted as anti-solvents for each other in situ [25].

The pseudo one-solvent cold-sonochemical approach [25] was used to synthesize and optimize co-crystals in the nanometer range with a specific Zeta potential that could potentially exploit the advantages of nanometer drug delivery systems for targeted drug delivery [35,36] as well as those of co-crystalline drug delivery systems such as enhanced solubility [37,38] and for combination therapy [39]. We have previously demonstrated the preparation of self-assembled electrosterically (both sterically and electrostatically) stabilized nano co-crystals using SDS as an electrostatic stabilizer and TPGS 1000 as a steric stabilizer using an appropriate anti-solvent [25].

A Design of Experiments (DoE) approach, specifically Central Composite Design (CCD) with the aid of Response Surface Methodology (RSM) was used to optimize formulation variables for the synthesis of 3TC and AZT nano co-crystals using cold-sonoprecipitation. Preliminary studies revealed that two independent factors viz., SDS and TPGS 1000 concentration were important.

The main purpose of these studies was to develop and optimize, using the principles of Quality by Design (QbD), a surface-modified nano co-crystal formulation, using pseudo one-solvent bottom-up cold-sonoprecipitation. The NCC will have the potential to deliver 3TC and AZT to target reservoirs of HIV in di fferent tissues, with the potential of improving the side e ffect profile of each API by reducing the dose. The surfactants, TPGS 1000 and SDS were identified and reported previously [25] and the optimization of the formulation and cytotoxicity of the nano co-crystal is reported herein.
