**1. Introduction**

There has been an increasing interest in developing drug delivery systems that circumvent the challenges associated with conventional drug delivery [1]. Among those challenges are poor drug solubility and formulation stability, low bioavailability, and undesirable side effect profiles [1]. Solutions to some of these shortcomings have been suggested and have included the use of nanotechnology. Nanotechnology is defined as the engineering and manufacture of materials at an atomic or molecular scale resulting in nanoparticles [2]. The definition of nanoparticles regarding particle size is constantly under deliberation. This has resulted in different disciplines adopting different definitions. As an example, colloid chemistry describes nanoparticles as having particle sizes below 100 nm or and in some instances 20 nm. In the pharmaceutical domain, nanoparticles are identified as having a size ≤ 1000 nm [3]. Therefore, in this review, we define nanocrystalline (NCM) as crystalline materials composed of nanoparticles having dimensions <1000 nm [4,5]. Some of the desirable properties of nanoparticles are that they often maintain crystallinity after their manufacturing process. Crystallinity relates to the extent of structural order in a solid and is characterized by atomic or molecular arrangement being regular and periodic—the production of nanoparticles that exhibit crystallinity results in the formation of NCM. The production of NCM requires a combination of crystal engineering and nanotechnological approaches.

**Citation:** Witika, B.A.; Aucamp, M.; Mweetwa, L.L.; Makoni, P.A. Application of Fundamental Techniques for Physicochemical Characterizations to Understand Post-Formulation Performance of Pharmaceutical Nanocrystalline Materials. *Crystals* **2021**, *11*, 310. https://doi.org/10.3390/ cryst11030310

Academic Editor: Etsuo Yonemochi

Received: 28 February 2021 Accepted: 17 March 2021 Published: 21 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Crystal engineering is the manipulation of non-covalent interactions between molecular or ionic components for the rational design of solid-state structures that may exhibit desirable electrical, magnetic, and optical properties [6]. Intermolecular hydrogen bonds can be used to assemble supramolecular structures that, at the very minimum, control or influence dimensionality [6,7]. Crystal engineering can also be described as the knowledge of intermolecular interactions in the context of crystal packing and the use of such understanding in the design of new solid materials with desirable physicochemical properties [8,9].

Co-crystals are single-phase crystalline solids that are composed of two or more different molecular or ionic compounds, generally in a stoichiometric ratio [10]. Co-crystals can be constructed using several types of molecular interactions such as hydrogen bonds, ionic interactions, π–π stacking, and van der Waal's forces [11–14].

Two general methods are used to manufacture NCM. The first utilizes collision forces to cause particle size reduction to nanometer dimensions and is called the top-down approach [15,16], while the second approach makes use of nucleation and crystal growth. A suitable stabilizer is utilized to prevent crystal growth into the micrometre range and is termed the bottom-up approach [17,18]. In the broadest sense, NCM used in drug delivery can be subdivided into organic and inorganic NCM. In this review, we focus on the characterization of organic NCM for the delivery of active pharmaceutical ingredients (API). More specifically, we highlight the characterization techniques most applicable to NC and NCC as models NCM for the delivery of API.

Drug NC are crystals with a size in the nanometre range. This means that they are nanoparticles that exhibit a high degree of crystallinity [3]. Similarly, drug NCC are crystalline solids existing as a single phase that is composed of different molecules that have nanometric dimensions [19]. In the case of both types of the aforementioned NCM, crystal growth from the nanometer to the micrometre range is inhibited by stabilizers [20–22].

In drug delivery, these NCM have found a broad range of applications in circumventing the shortcomings of conventional drug delivery while exhibiting flexibility regarding the routes of administration.

When applied in transdermal delivery, NCM are expected to pack tightly and form a dense layer that hydrates the skin and improves drug penetration and permeation. Dissolved NCM may be topically retained for a sufficient period and offer sustained API release [23]. As a way of example, a formulation of L-ascorbic acid demonstrated longterm stability as NC dispersed in an oil base. The NC oil dispersion exhibited improved penetration and stability when compared to conventional technologies [24].

Oral administration remains the most preferred route and is generally considered a safe and suitable drug delivery route [25]. Dissolution is often the rate-determining step for absorption, and because NCM generally provide a larger surface area for dissolution, increase saturation solubility, and ultimately increase the dissolution extent, they have been shown to enhance API absorption [26]. Rapamune®, a formulation composed of sirolimus NC blended with additional excipients and directly compressed into tablets, was the first US FDA-approved nanocrystalline drug launched in 2000 by Wyeth Pharmaceuticals (Madison, NJ, USA) for oral use. The oral bioavailability of the API from the nanocrystalline tablets was 21% higher than that of sirolimus delivered in aqueous solution [27]. Similarly, the advantages of nano-drug delivery have been applied to modulate the pharmacokinetic profile of aprepitant (Emend®), which requires delivery at an absorption window in the gastrointestinal tract [28].

Drug delivery to the eye is hindered by pharmacokinetic, physiological, and in some instance's environmental factors. Conventional formulations are rapidly cleared from the site of administration due to rapid eye movement such as blinking and/or lacrimation, resulting in low ocular bioavailability. Consequently, repeated dosing and subsequent reduction in adherence results in poor clinical outcomes. Frequent dosing may also lead to an increase in dose-dependent side effects [29,30]. NCM technologies can play a critical role in drug delivery to the eye by improving the solubility of poorly soluble API. This was

explored and showed favourable outcomes when utilized in formulations containing budesonide, dexamethasone, hydrocortisone, prednisolone [31], and fluorometholone [31,32]. A technique based on combining microfluidic and milling technologies resulted in the production of NC of hydrocortisone. The ocular bioavailability of the NC was evaluated in vivo using albino rabbits. Extended duration of action and a significant improvement in the area under the curve (AUC) for hydrocortisone delivered using the NC were observed when compared to coarse hydrocortisone [33].

Parenteral drug delivery ensures a shorter onset of action, higher bioavailability, and use of reduced doses when compared to oral drug delivery. These benefits are ideal target parameters for drug delivery; however, the use of the intravenous route is challenging as only a limited number of solvents and excipients can be used during formulation development. This is due to an increased possibility of adverse outcomes in addition to those caused by the API. NC of ascularine [34], melarsoprol [35], oridonin [36], itraconazole [37], and curcumin [38] have been successfully developed and resulted in increases in their Cmax and AUC0–∞.

Targeted delivery approaches have been implemented in combination with NC and NCC technology for drugs that exhibit low bioavailability, poor aqueous solubility and stability, and limited in vitro–in vivo correlations (IVIVC) [39,40]. Buparvaquone NC suspended in a mucoadhesive system of Carbopol® 934, 971, 974, 980 or 0.5 % *w*/*w* Noveon® AA-1 was used for targeting the gastrointestinal parasite, *Cryptosporidium parvum*, and resulted in better targeting and greater stability than pure buparvaquone [41].

Surface modification of NCM can also be used to reduce the potential toxicity of API to selected cell lines. For instance, surface modification of lamivudine-zidovudine NCC with sodium dodecyl sulphate (SDS) and α-tocopheryl polyethylene glycol succinate 1000 (TPGS 1000) was reported to have reduced cytotoxic effect on HeLa cells [42].

Despite their success in drug delivery, NCM have to undergo rigorous characterization prior to their use in humans. Many of these techniques are centred on developing quality into the product and ensuring that critical process parameters (CPP) do not have a significant impact on the critical quality attributes (CQA) of the product [43]. Consequently, the resultant NC or NCC meets the quality target product profile (QTPP) that could lead to inclusion into suitable dosage forms and subsequent human use.

Formulation and morphology of drug NCM, including fundamental characterizations and their implications on post-formulation performance are described below.
