Nanocrystals: A perspective on translational research and clinical studies

Abstract Poorly soluble small molecules typically pose translational hurdles owing to their low solubility, low bioavailability, and formulation challenges. Nanocrystallization is a versatile method for salvaging poorly soluble drugs with the added benefit of a carrier‐free delivery system. In this review, we provide a comprehensive analysis of nanocrystals with emphasis on their clinical translation. Additionally, the review sheds light on clinically approved nanocrystal drug products as well as those in development.


| INTRODUCTION
Over the years, nanoparticles (NPs) made of both organic and inorganic materials have been engineered to circumvent the biological barriers and deliver drugs for a variety of indications. 1,2 Waterinsoluble or hydrophobic drugs, pose a challenge in terms of achieving optimal bioavailability and thereby, adequate efficacy. 3 As reported in 2015, 40% of drugs on the market and 90% of drugs within the discovery pipeline face solubility issues. 4 Other statistics, cite 40% of all potential drug candidates were shelved as a result of intrinsic aqueous solubility issues. 5 Thus, a number of hydrophobic drugs, which could potentially be useful for treatments are in need of clinically acceptable carriers. 6 For the purpose of this review article, drug nanocrystals may be defined as pure solid particles with a mean diameter <1 μm and a crystalline character. The platform offers an exceptional opportunity to deliver hydrophobic drugs ( Figure 1). Its uniqueness originates from the fact that nanocrystals are composed entirely of 100% drug or the payload thereby eliminating the ancillary role of a carrier. 7 In addition, surfactants or stabilizers are commonly used to stabilize the crystalline dispersions in liquid media.
Nanocrystalline drug technology improves the solubility of hydrophobic drugs due to an increased surface area to volume ratio and improved dissolution rates (i.e., dissolution velocity) associated with nanosizing. 8 The drug crystals are singularly well-suited for the rehabilitation of previously unsuccessful Biopharmaceutics Classification System (BCS) Class II and IV drugs (low solubility drugs). 9 The BCS classification system is an experimental model that measures permeability and solubility under prescribed conditions. The system divides the drugs into four classes. While Class I drugs have high solubility and high permeability, Class II molecules have low solubility and high permeability, Class III identifies with high solubility and low permeability, and drugs in Class IV have low solubility and low permeability. 4 Nanocrystal drug formulations have also been shown to be stable in suspensions and are often referred to as nanocrystal colloidal dispersions (NCD's). The dispersions provide a platform for easy scale-up and manufacturing of highly stable and marketable products. Their synthesis and scale-up considerations have been described at length elsewhere. 10,11 Commonly used synthesis techniques include the use of microfluidic based platforms or the milling method, which, among others, is both flexible and tunable. 7,[12][13][14][15][16][17] Taken together, the nanocrystal drug technology has been studied extensively and is well positioned for further exploration in the field of drug delivery.
Several hydrophobic drugs have been salvaged via the nanocrystal formulation method. The drugs were successfully developed, and approved by the FDA to treat a variety of indications ranging from dental disorders to cancer in the clinic. 14,[18][19][20][21][22][23][24][25][26][27][28] Depending on the disease, the approved formulations can be administered via different routes including oral, dermal, and parenteral. This highlights the versatility of a nanocrystal drug platform. Pharmacokinetic, biodistribution, and bioavailability data for organs involved in delivery routes tested using nanocrystal technology have been addressed at length previously. 10,13,18,24,25,[29][30][31][32][33][34] Specifically, the reviews of Lu et al. 2016 and 2017 delve into the biodistribution pattern of nanocrystal drugs in the blood, heart, liver, spleen, lung, kidney, tumor, and thymus (i.e., the organs involved in clearance/circulation and host immune responses). 24,35 Several articles have been published, discussing the techniques used to synthesize nanocrystal drugs; the type of stabilizers or surfactants involved; and the methods adopted for physicochemical and biological characterization. 10,19,36,37 However, a wide translational gap exists between this highly promising platform and its clinical approval. In this review, we discuss the nanocrystal drug technology and its development from a translational perspective. We speak to the paucity of FDA approved products despite the platform's obvious strengths. We discuss the challenges involved in their successful translation to the clinic.

| PREPARATION AND CHARACTERIZATION OF NANOCRYSTALS
Properties such as crystallinity, size, shape, surface charge, and the type of stabilizers or polymer coatings used during formulation influence the therapeutic outcome of nanocrystal drug products.
Other physicochemical properties currently under investigation for their influence on preclinical (i.e., in vivo/in vitro) performance assays include stiffness and surface texture.

| Nanocrystal drug dissolution: Concept and theory
Although often overlooked, crystallinity is a foundational parameter for drug nanocrystals. It can provide insights into the structure of the final formulation. Assessing crystallinity is critical in verifying the successful integration of stabilizers, surface polymers (chemically conjugated or physically adsorbed), and targeting ligands. Further, nanocrystals with an amorphous crystalline substructure have an increased dissolution rate and are better suited for delivering multiple hydrophobic drugs. 21,38,39 This is better explained using the "spring and parachute" concept adapted to describe dissolution rates of amorphous, crystalline, or co-crystalline drugs.
Co-crystalline drugs are often composed of multiple components, including a hydrophobic drug and a stabilizer. Stabilizers are supplementary molecules which when added during formulation, can control the nanocrystal size, agglomeration, and its overall biodistribution in vivo. 8,14 Stabilizers are generally 50-500 fold more soluble in water than the drug in its free powder form. When exposed to an aqueous environment, the stabilizer first begins to leach into solution and leaves behind the drug particles. Subsequently, the loosely selfaggregated drug particles form supramolecular aggregates. The cocrystal at this point is in an amorphous crystalline state resulting in a high peak concentration ( Figure 2). The highly soluble amorphous cocrystal slowly transforms to a stable species following Ostwald's law that promotes dissolution into a free form of the drug (Curve 1). The time required for the drug to move through the high-energy aggregate stage to a low-energy solubilized state exhibit a "parachute effect." Here, a high dissolution rate is achieved over an extended period of time. In absence of the stabilizer, the drug precipitates rapidly to a stable polymorph via the so-called "spring effect." This results in a modest improvement in solubility which is short-lived and not sustained for long-term dose regimens. The stable crystalline drug is almost insoluble as shown by Curve 1.

| Physical analytical methods to characterize nanocrystal drug products
Stabilizers that are widely used in nanocrystal drug formulations are presented in Table 1. These are amphiphilic molecules that increase the nanocrystal's surface wettability, and when used at optimal concentrations, do not interfere with the crystal growth. Its successful integration into the final formulation can be confirmed simultaneously with the crystal's substructure using X-Ray spectroscopy methods such as Small-angle X-ray scattering, X-ray diffraction, and X-ray photoelectron spectroscopy.
Nanocrystal drug sample preparation for X-Ray spectroscopy analysis often involves freeze-drying. The effects of freeze-drying on the agglomeration of nanocrystals and its subsequent re-dispersibility have been studied and reported previously. 51 Liquid nanocrystal dispersions designed for dosing in their solid form must be freezedried above the critical freezing rates. Using lower rates would increase particle aggregation and affect formulation redispersability. 52 Critical freezing rates may be determined by treating re-dispersibility as a result of competition between the freezing speed and the particle collision frequency. With increase in drug concentrations, the average inter-particle distance decreases, thereby increasing the frequency of collisions. This results in an overall increase in the critical freezing rate. 52 Thus, understanding the role of freeze-drying in determining nanocrystal drug stability is critical during translation.
Methods commonly used to confirm the incorporation of stabilizer, polymer coating, or targeting ligands during co-crystallization include Fourier Transform Infrared spectroscopy, Raman spectroscopy, and Nuclear Magnetic Resonance spectroscopy. The aforementioned techniques are among the most preferred methods for characterizing nanocrystal compositions. In a review by Luykx et al., 53 analytical techniques that may be used to characterize size, shape, charge, and the composition of NP drugs have been described. Some of these methods are deemed as facile and are not as widely used in nanocrystal drug development. These include field flow fractionation for size, desorption electrospray ionization-mass spectrometry, and ion-mobility spectrometry-mass spectrometry for mass and composition, photon correlation spectroscopy for size and distribution, and analytical ultracentrifugation for size, shape, and structural analysis.
Previous studies have probed the effects of NP size, shape, surface morphology, and charge on therapeutic outcome. It has been shown that these parameters influence phagocytosis, immune responses, endothelial targeting, adhesion under flow, transport mechanisms, and intracellular delivery. [54][55][56][57][58][59][60][61][62][63] As an example, Mitragotri and co-workers described the differences in internalization rate and the pathways of NPs differing in shape, size, and aspect ratio in mouse peritoneal macrophages. 58 Large particles (>100 nm) are usually internalized via non-specific pathways such as phagocytosis and macropinocytosis. However, nanocrystal drug surfaces can be modified to minimize non-specific uptake and facilitate entry via specific pathways such as receptor-mediated endocytosis. This can be achieved by coating the surface with polymers or surfactants such as PEG, PEG derivatives, polydopamine, and Pluronic F127 or with antibody coatings.
Chung et al. showed that coating iron oxide NPs with positively charged multi-arm PEG derivatives could reduce mass aggregates and used to uniquely label mesenchymal stem cells. 64 66 On the contrary, negatively charged nanocrystals exhibited significant uptake via clathrin-and caveolaemediated uptake mechanisms. 83 Additionally, excessive positive and negative surface charges on NPs have been shown to induce higher rates of opsonization and capture by the immune cells in vivo. 83 A vastly diverse array of nanocrystalline material fabricated with various surface charges for drug delivery purposes has been explored and reported in the literature (Table 3). Finally, a majority of nanocrystal drug products approved for use in the clinic and or in clinical trials are delivered via oral or intravenous administration. Figure 3 [98][99][100][101][102] The suspensions are then exposed to a series of collisions, strong cavitation, and high shear forces generated by passing through a narrow gap. This causes it to boil due to change in    Table 5 and media milling is the most widely accepted method used to produce a majority of the marketed products.

| Nanocrystal drug-products in clinical trials
As seen in Table 5, a majority of nanocrystal drug products are currently approved for oral ingestion and treating diseases other than FIGURE 3 A schematic depicting the in vivo barriers and properties that influence in vivo biodistribution and site-specific delivery of nanocrystal drug products administered orally or intravenously (IV)  Table 6. Semapimod ® nanocrystals from Cytokine Pharamsciences (CPSI) is a synthetic guanylhydrazone and was found to act as an immunomodulator, preventing the production of TNF-α, a proinflammatory cytokine, involved in inflammation-associated carcinogenesis during a Phase I study in cancer patients. 111  One such issue is determining the most appropriate conditions for testing in vitro dissolution rates of newly formulated products.
where Dx/dt is dissolution rate, A is surface area of the dissolving particle, D is the diffusion rate constant, δ is thickness of the stagnant layer surrounding the particle, Cs is saturation solubility of the drug, concern would be identifying the ideal technique to validate particle size. To ensure reliability, at least two analytical methods that support each other should be used to determine particle size and distribution.
Dependence on pH may also be taken into account since nanocrystal size and stability are often affected by pH of the dispersion media. In terms of crystallinity, change in structure to amorphous or polymorphic variants could affect its dissolution, stability, and bioavailability. It is therefore important to ensure that the change in crystalline structure is monitored and controlled during manufacture and shelf life of the final product. [122][123][124][125][126][127][128][129] Drug product is referred to as the prototype or the marketed dosage form of the drug substance formulated with excipients. Its control refers to factors that affect the quality and in vivo performance of the final product. This is often affected by changes in viscosity, dissolution rate, specific gravity, content uniformity, and redispersability. The factors are also influenced by the presence of impurities formed during manufacturing. Hence, due consideration must be given for assays that test the purity and continuously monitor degradation products formed during manufacture or shelf life of the final product.
Factors critical during the product's manufacturing process include determining the most appropriate tests or controls to monitor particle size distribution, agglomeration, and presence of contaminants at various steps during the process. It is therefore important to continuously track impurities generated during the process. 29,130 In case of a top-down approach as in wet milling, impurities depend on the milling media used, the milling material that comes in contact with the drug, the milling mechanism and the number of milling cycles used for the process. Further, other factors such as product and chiller temperature as a function of time, the drive motor speed, shape, aspect ratio, viscosity of product dispersion, and so forth may affect the process and particle size distribution. 27,29,130 In case of a bottom-up approach, care should be taken to ensure that (a) a uniform dispersion of the drug is maintained and agglomeration is prevented while adding the drug slowly in to the melt, (b) an optimal viscosity is maintained for the molten material, (c) consistency is sustained during sampling and the solidification/cooling procedure and finally, (d) solvent residues and other impurities in the drug substance/product are tracked and isolated on a constant basis. 131 Deviations in any of the above steps could significantly impact product quality, and affect its in vivo performance.
Since crystallinity of the final product could significantly impact its quality and in vivo performance, it is important to ensure that structural stability is retained post-manufacturing and throughout its shelf life. As stated previously, techniques such as X-ray powder diffraction, differential scanning calorimetry, or spectroscopic methods, and so forth can be used to study and compare the structure of the initial molecule with the final product and at the end of its shelf life.
Additional studies may be designed concerning short or long-term stability, or its dosage form since particles tend to aggregate due to sedimentation, redispersion, or caking, and so forth.

| NANOCRYSTAL DRUGS: A CARRIER-FREE THERAPEUTIC PLATFORM FOR CANCER AND OTHER DISEASES?
By the year 2021, nanocrystal drug products are estimated to account for 60% of the total NP-based drug delivery market. 132 This is valued to be at~$82 billion. While nanocrystal technology is attractive due to its ease of formulation, uniform composition, and attractive pharmacoeconomic values, it also has the potential to overcome some of the biggest challenges for drug development. Poor solubility could result in abysmal bioavailability, thereby affecting optimal delivery of the drug. By formulating poorly soluble drugs into nanocrystals, the resulting increase in surface/volume ratio, saturation solubility, and the rate of dissolution can ensure an enhanced bioavailability of most insoluble drugs irrespective of its route of administration. Clinical efficacy of nanocrystal drugs depend on several factors including the size, morphology, surface charge, amount of drug loaded, the type of excipient used, the degree of redispersability, and site-specific targeting.
Also, multimodal theranostic nanocrystal drug products would be vital to assess and monitor in vivo bioavailability. A majority of nanocrystal drug products are currently approved for oral ingestion and for treating diseases other than cancer. The manufacturing process for such oral products is consistent and form crystals at sizes well above the 100-200 nm range. Since nanocrystals are not expected to undergo rapid dissolution in the blood due to a minimum volume of distribution; they can be injected intravenously. However, crystals with dimensions >100-200 nm could promote macrophage-mediated phagocytosis, rapid blood clearance, and minimal efficacy compared to the drugs' conventional form. Efforts must, therefore, be directed ACKNOWLEDGMENT Image templates made freely available by Servier Medical Art (http:// smart.servier.com) were used for the preparation of Figure 3.