*5.2. In Vitro Dissolution and Kinetics of Drug Release*

When NCM is used for delivery of API, the release of the API is evaluated over time in order to determine the availability of API for absorption and ultimately at target sites thereby influencing therapeutic outcomes [72]. Three possible mechanisms of release from NCM can be envisaged viz., desorption of adsorbed API (A), API diffusion from a polymer/surfactant matrix (B), and/or release following polymer/surfactant erosion (C) as depicted in Figure 1. *Crystals* **2021**, *11*, x FOR PEER REVIEW 17 of 26

**Figure 1.** Possible mechanisms of drug release from NCM; desorption-controlled drug release (**A**), diffusion-controlled drug release (**B**) and erosion-controlled drug release (**C**). **Figure 1.** Possible mechanisms of drug release from NCM; desorption-controlled drug release (**A**), diffusion-controlled drug release (**B**) and erosion-controlled drug release (**C**).

In the case of matrix-type polymer/surfactant NCM in which the crystalline API is uniformly distributed or embedded in the matrix, drug release would be predominantly controlled by diffusion through the matrix and/or erosion of the matrix. Depending on the API and matrix physicochemical properties, either one of these mechanisms can be the dominant one. If diffusion occurs more rapidly than matrix/surfactant degradation, diffusion is likely to be the main mechanism of release. In many instances, an initial rapid initial release is noticed and attributable to the crystalline material fraction adsorbed or weakly bound to the surface(s) or that is not entirely embedded in the system (Figure 1 A) In the case of matrix-type polymer/surfactant NCM in which the crystalline API is uniformly distributed or embedded in the matrix, drug release would be predominantly controlled by diffusion through the matrix and/or erosion of the matrix. Depending on the API and matrix physicochemical properties, either one of these mechanisms can be the dominant one. If diffusion occurs more rapidly than matrix/surfactant degradation, diffusion is likely to be the main mechanism of release. In many instances, an initial rapid initial release is noticed and attributable to the crystalline material fraction adsorbed or weakly bound to the surface(s) or that is not entirely embedded in the system (Figure 1A) [171].

[171]. API release from NCM is investigated in a number of ways. These are by dialysis membrane diffusion, membrane-less diffusion, sampling and separation, or the use of an in situ analytical technique [172]. When using the sampling and separation technique, API API release from NCM is investigated in a number of ways. These are by dialysis membrane diffusion, membrane-less diffusion, sampling and separation, or the use of an in situ analytical technique [172]. When using the sampling and separation technique, API release is determined by separating the released API from the sample by filtration,

release is determined by separating the released API from the sample by filtration, centrifugation, or centrifugal filtration, and subsequent quantification using an appropriate

this method having the capability of being performed with a small sample size and simple analytical equipment, several drawbacks exist: the separation methods is slow, tedious, and inefficient, making it inappropriate for studying rapid/immediate release NCM. In addition, the force of centrifugation or shear stress during filtration required for NCM separation tends to increase with a reduction in NCM PS. This can ultimately alter the release kinetics of the API. The main advantage of the dialysis membrane diffusion technique is that he diffusion of the NCM is continuous across the dialysis membrane and not subject to destructive separation processes, making sample acquisition simple and rapid

The use of dialysis membranes may attenuate API release as it is a diffusion barrier that may behave as an adsorptive surface. Thus, this approach should be conducted with control experiments in which free API is used to assess membrane effects. The dialysis membrane diffusion method typically makes use of large volumes of dissolution media. While large volumes maintain sink conditions for release, API analysis may be hindered due to the low concentrations to be tested. An in situ analytical technique is useful for studying NCM, which are prepared almost exclusively of an API. This technique is used to analyse the properties of NCM in situ to indirectly determine the quantity of API released. Various analytical techniques, including electrochemical analysis, solution calo-

[163,173,174].

centrifugation, or centrifugal filtration, and subsequent quantification using an appropriate analytical method such as (U)HPLC or UV spectroscopy.

The NCM are augmented with fresh release medium, preferably simulated bodily fluids and resuspended and incubated further until the next sampling interval. Despite this method having the capability of being performed with a small sample size and simple analytical equipment, several drawbacks exist: the separation methods is slow, tedious, and inefficient, making it inappropriate for studying rapid/immediate release NCM. In addition, the force of centrifugation or shear stress during filtration required for NCM separation tends to increase with a reduction in NCM PS. This can ultimately alter the release kinetics of the API. The main advantage of the dialysis membrane diffusion technique is that he diffusion of the NCM is continuous across the dialysis membrane and not subject to destructive separation processes, making sample acquisition simple and rapid [163,173,174].

The use of dialysis membranes may attenuate API release as it is a diffusion barrier that may behave as an adsorptive surface. Thus, this approach should be conducted with control experiments in which free API is used to assess membrane effects. The dialysis membrane diffusion method typically makes use of large volumes of dissolution media. While large volumes maintain sink conditions for release, API analysis may be hindered due to the low concentrations to be tested. An in situ analytical technique is useful for studying NCM, which are prepared almost exclusively of an API. This technique is used to analyse the properties of NCM in situ to indirectly determine the quantity of API released. Various analytical techniques, including electrochemical analysis, solution calorimetry, or turbidometric and the light scattering techniques have been used for this purpose [172]. These approaches do not need separation of NCM and enable real-time assessment of the release kinetics of API.

While the aforementioned characterizations may be generally applicable, it may not always be applicable to use all of them. The role of PS, PDI, ZP, and polymorphism on NCM performance with regards to processability, content uniformity, and stability of a drug product is recognizable. The possible effect of PS and polymorphism on solubility, dissolution, and bioavailability parameters being strictly related to each other is one of the primary concerns. Definitions of specifications are required particularly when the product performance is affected by PS and polymorphism. We provide an appropriate decision tree in Figure 2, that is closely adapted to that in the ICH Q6A guidelines [175], on what characterization is required and to which performance parameter it relates.

characterization is required and to which performance parameter it relates.

rimetry, or turbidometric and the light scattering techniques have been used for this purpose [172]. These approaches do not need separation of NCM and enable real-time assess-

While the aforementioned characterizations may be generally applicable, it may not always be applicable to use all of them. The role of PS, PDI, ZP, and polymorphism on NCM performance with regards to processability, content uniformity, and stability of a drug product is recognizable. The possible effect of PS and polymorphism on solubility, dissolution, and bioavailability parameters being strictly related to each other is one of the primary concerns. Definitions of specifications are required particularly when the product performance is affected by PS and polymorphism. We provide an appropriate decision tree in Figure 2, that is closely adapted to that in the ICH Q6A guidelines [175], on what

ment of the release kinetics of API.

**Figure 2.** Decision tree relating to NCM characterization. **Figure 2.** Decision tree relating to NCM characterization.

#### **6. Conclusions 6. Conclusions**

NCM are non-toxic crystalline carriers composed almost entirely of API and very little excipients. NCM are non-toxic crystalline carriers composed almost entirely of API and very little excipients.

Unlike other nanoparticle technologies such as solid lipid nanoparticles (SLN), nano lipid carriers (NLC), and nanocapsules, NCM are suitable for the delivery of hydrophilic and hydrophobic compounds with very high efficiency. NCM are also relatively easy to prepare and synthesize when compared to other nanoparticle technologies such as liposomes and nanocapsules that require multiple steps and use of organic solvents. NCM have the added advantage of exhibiting better stability than other nanoparticles due to the crystalline state of the particles. In addition, NCM formulations prevent the accumulation of the payload in cells and tissues of healthy organs and often exhibit improved bioavailability for some API. The suitability of the carriers has been proven since they are already on the market (Rapamune® and Emend®). NCM fulfil the key prerequisites for the introduction of the technology to the clinic trial phases and the market, that is, they are in line with regulatory requirements and provide the possibility of qualified industrial large-scale production. Furthermore, NCM exhibit flexibility in terms of the route of ad-

ministration and have been used for oral, parenteral, ophthalmic, and topical delivery of a variety of compounds.

The characterization of NCM delivery systems is an area that requires the formulation development and production of a products of high quality. Ideally, NCM should be in the nano range with a PDI < 0.500, determined using DLS and Photon Correlation Spectroscopy (PCS) in combination with Laser Diffraction (LD). However, PCS and DLS cannot be used to characterize the shape, surface morphology, and elemental composition of NCM and additional analytical tools are required. Therefore, TEM, SEM, and EDX should be used to assess the shape and surface morphology of NCM in the solid-state and dispersions. The crystalline nature and polymorphic transition of NCM are characterised using DSC, TGA, PXRD, and FTIR/Raman spectroscopy to investigate potential interactions between the API and excipients to be used. Laser Doppler Anemometry (LDA) is used to establish electrophoretic mobility and the ZP of NCM, which should be > +30 mV or < −30 mV in dispersion.

Other characterisations perhaps not considered the most fundamental but worth additional exploration include two-dimensional (2D) nuclear magnetic resonance (NMR) techniques such as correlation spectroscopy (COSY), diffusion-ordered spectroscopy (DOSY), and cross-polarization magic-angle spinning (CPMAS). These are used to generate additional information in terms of the stability of the nanocarriers in solution and solid-state in addition to the presence of hydrogen bonding.

**Author Contributions:** Conceptualization, B.A.W.; writing—original draft preparation, B.A.W. and M.A.; writing—review and editing, B.A.W., M.A., L.L.M. and P.A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was not funded with an external research grant.

**Institutional Review Board Statement:** Not applicable

**Informed Consent Statement:** Not applicable

**Acknowledgments:** The authors acknowledge the Research Committee of Rhodes University (P.A.M.).

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