3.1. Nanosuspensions and tablets
Suspensions of nanocrystals (or nanosuspensions) represent the most common, simple, representative, and precursory formulation of this family (Fig. 1A). They can be directly obtained and even be practically ready to use via wet milling since the drug is generally suspended in an aqueous vehicle. The process of size reduction generates a thermodynamically unstable system by forming additional interfaces. These particles will tend to agglomerate to minimize their total energy, also known as Gibbs free energy [13]. Therefore, the incorporation of surfactants and/or stabilizers is needed to avoid this phenomenon as well as to improve the wetting properties of the particles in the vehicle [6, 13]. A recent research example in the field was reported by Paredes et al. in 2020 [14]. They aimed to develop poloxamer 188-stabilized ricobendazole nanocrystals by wet milling and spray-drying to treat helminthiasis. In this case, no dissolution rate improvement was observed in comparison with the physical mixture of ricobendazole and the poloxamer 188, used as a control. However, their redispersed nanocrystals showed an increased oral bioavailability in dogs. When compared to the micronized drug, a higher maximum plasma concentration (Cmax) and a 1.9-fold higher AUC0−∞ was observed. The authors suggested that the better in vivo performance of the nanocrystals was due to the higher specific surface area of the drug, which promotes higher saturation concentrations. Over the last decade, several studies on nanosuspensions have explored the oral route [15–17] but also others such as the ocular [18–21], nasal [22, 23], topical [24, 25], intra-articular [26], intra-muscular delivery [27] and even the pulmonary route [28]. The cornea, for instance, might be permeable to nanosized drug particles. Based on this hypothesis, Baba et al. designed a fluorometholone nanosuspension-eye drop formulation to treat keratoconjunctivis [18]. They obtained rectangular-shaped nanocrystals around 200 nm that were stable for 6 months at 10°C. Penetration and metabolization into the aqueous humor of rabbit eyes were compared with a micronized particle suspension. Results in rabbits showed that the ocular penetration of fluorometholone was 2- to 6-fold higher after 120 min.
Incorporating nanocrystals in tablets and capsules is the alternative classic strategy to aqueous nanosuspensions (Fig. 1B) [29–31]. In fact, the majority of nanocrystals in the market are nowadays formulated as tablets. As described in the last section of this article, they emerged in the early 2000s to improve the oral bioavailability of poorly water-soluble drugs [6, 31, 32]. However, there are only a few recently published studies on tablet formulations containing nanocrystals. In one such study, Naguib et al. developed sublingual tablets containing flibanserin nanocrystals to treat pre-menopausal hypoactive sexual desire disorders [30]. This drug is known to have poor aqueous solubility and therefore low oral bioavailability. Freeze-dried nanocrystals around 450 nm in size provided a saturated solubility five times higher than the pure drug. The optimized sublingual tablets, which disintegrated in about 36 s, were then tested in vivo in rabbits. Calculated pharmacokinetic parameters showed an enhanced bioavailability with a 2-fold AUC0−∞ increase. These outcomes were achieved due to the reduced particle size of flibanserin nanocrystals together with an acidic microenvironment generated in the presence of boric acid contained in the tablets.
Overall, these studies demonstrated the versatility of nanocrystals suspensions through their applications for different routes of administration. They can be easily formulated via a fast and straightforward method to prevent solubility issues. Tablets containing dried nanocrystals are a feasible option for oral administration. Despite the potential cost-effectiveness of tableting, the transformation into solid products requires a drying step, such as freeze-drying, spray-drying or granulation before compression [34]. These procedures often require the use of other excipients, such as cryo-protectants or matrix formers, which should be carefully selected to avoid potential interaction or interference with the nanocrystals during compression. Eventually, this can lead to a higher risk of irreversible aggregation, thus limiting the effective proportion of nanocrystals in tablets [35, 36]. This hinders the redispersion of the nanocrystals in the gastrointestinal tract and subsequent dissolution, reducing their advantages [10]. Nevertheless, a drying step is sometimes favorable to improve the stability of aqueous nanosuspensions [5]. This offers the possibility to concentrate the preparation and represent an intermediate formulation step, not only for tablets but also for other drug delivery systems shown in the following sections.
3.2. Nanocrystal-loaded hydrogels
Hydrogels are cross-linked hydrophilic polymeric chains organized in a tridimensional matrix network embedding water. Nanocrystals can be easily embedded into these matrix scaffolds (Fig. 1C). Nanocrystals-containing hydrogels can be designed to improve specific characteristics such as bioadhesion, modulable viscosity, and even controlled release. This offers a high versatility and allows for topical administration on the skin, hair follicles, eyes, nasal cavity, and parenteral injections [37–39]. Together with faster drug dissolution rates and larger concentration gradients, drug diffusion through the tissue of interest can be increased via enhanced drug penetration and retention for an extended period [40–45]. Probably for these reasons, nanocrystals-containing hydrogels have become one of the most popular drug delivery systems reported in recent years. In one such study, an extended-release nanocrystal gel formulation was designed for dermal delivery to target hair follicles specifically [46]. Curcumin nanocrystals, chosen as a model drug, were incorporated in up to fourteen different gels with different viscosity and lipophilicity values. However, this did not influence nanocrystal penetration into hair follicles when tested in an ex vivo pig ear model. The authors suggested that the massage decreased the viscosity of the gels, sharing the same shear-thinning flow behavior. Still, embedding the nanocrystals into hydrogels facilitated application, adhesion on the tissue, and increased residence time of the formulation.
In situ forming hydrogels are currently gaining relevance in the field. They can be easily administered as a semi-solid/liquid form at room temperature, then gaining viscosity at body temperature to provide higher residence time or even specific functions such as joint lubrication/viscosupplementation. Tomić et al. intended to improve the efficacy and safety of topical acne treatment by formulating azelaic acid nanocrystals loaded in a hybrid poloxamer/hyaluronic acid in situ forming hydrogel [42]. This research group conducted a double-blind, randomized controlled study on patients with mild to moderate acne vulgaris comparing a 10 % dug-loaded hydrogel with a commercial cream containing 20 % o active pharmaceutical ingredient. Notwithstanding, their formulation showed better efficacy and safety after 8 weeks of daily treatment, with a significant reduction of acne-related inflammatory and non-inflammatory lesions. In another study, a higher docetaxel mucosal penetration was obtained by means of an in situ forming nanocrystal-hydrogel formulation for cervical cancer therapy [47]. For that, the surface of docetaxel nanocrystals was functionalized with the trans-activator of transcription (TAT) peptide, a common cell-penetrating peptide, using a polydopamine coating. The nanocrystals were further incorporated into a poloxamer 407-based thermosensitive gel. In vitro, TAT-coated nanocrystals per se showed higher cervical cancer cell uptake and growth inhibition compared to poly(ethylene glycol) (PEG)ylated nanocrystals. More importantly, an extended ex vivo and in vivo intravaginal retention on mice was observed with improved mucosal penetration and tumor growth inhibition when dispersed in the gel. Eventually, an injectable in situ forming hyaluronic acid hydrogel containing camptothecin nanocrystals was proposed by Yongsheng Gao and his team as a local and long-term delivery system for the treatment of rheumatoid arthritis [48]. The intra-articular injection into the joint of collagen-induced arthritis rats showed that the formulation was maintained for over four weeks. This correlated with the lowest levels of the inflammatory interleukin-1β after 60 days, compared to the controls.
All these studies demonstrate that hydrogels are potent topical or parenteral vehicles for nanocrystals. They provide a higher residence time and potentiate penetration into tissues, thus boosting drug bioavailability. Their semi-rigid polymeric network is thought to provide an extra nanocrystal physical stabilization meant to prevent re-crystallization issues. They can be easily manufactured by gelation of the aqueous nanosuspension or by simply redispersing the nanocrystals in a pre-formed gel. Hyaluronic acid is a privileged material but other biodegradable and biocompatible materials can be used for parenteral administration. In this regard, in situ forming hydrogels facilitate syringeability and injectability while providing viscosupplementation and a delivery depot on site.
3.3. Microneedle-mediated delivery of nanocrystals
Topical application of nanocrystals has been proved efficient and relatively easy to formulate as seen in the previous section. However, the use of semi-solid pharmaceutical forms or patches still presents some limitations, mainly due to the low skin permeation and the need for frequent administrations [49, 50]. This led to the investigation of novel delivery systems designed to increase drug penetration and sustained release. Among them, microneedles have gained the attention of researchers as a result to their minimally invasive administration, high targeting and ability to incorporate poorly soluble drugs [51]. Microneedles, or microarray patches, are micron-sized spikes used to deliver active compounds transdermally (Fig. 1D). They can pierce and bypass the stratum corneum by forming micron-sized pores in the skin, directly delivering the drug into the dermal tissue [52]. In addition, drug-coated, hollow, and hydrogel-forming microneedles are composed of cross-linked polymers that can swell in contact with the interstitial fluid to form a drug reservoir on site. Dissolvable microneedles are composed of a biocompatible polymeric matrix, typically sugars, natural or synthetic polymers, in which hydrophobic drugs can easily be dispersed [50, 51, 53–56].
Vora and colleagues were in the first ones to incorporate lipid nanosuspensions into dissolvable microneedles [57]. They used a sonoprecipitation method to obtain a lipophilic vitamin D3 nanosuspension. This nanosuspension was then mixed with a high molecular weight polyvinylpyrrolidone solution to obtain a gel, which was filled into a laser-engineered mold to form the microarrays. In vitro studies using Franz diffusion cells showed that the microneedles released significantly a 6.8-fold higher amount of vitamin D3 compared to the patches without microneedles. In another recent study, antiretroviral nanocrystals were loaded into a hollow microneedle delivery system [58]. Nanocrystals of rilpivirine, a second-generation non-nucleoside reverse transcriptase inhibitor, and of the integrase inhibitor cabotegravir, were loaded into separate microneedle systems for the treatment of HIV-associated neurocognitive disorder. Both types of nanocrystals were obtained by wet milling, then lyophilized, reconstituted in water, and finally incorporated into 600 µm hollow microneedle arrays. The formulations were tested ex vivo on porcine skin and then administered to rats with an intradermal injection pad. Compared to the oral control administration, results indicated that both drugs were successfully delivered to the brain with higher AUC and Cmax values after 3–4 weeks. Furthermore, rilpivirine delivery to the brain was therapeutically significant since the concentration of drug required for 90% inhibition of the reverse transcriptase (IC90) was reached after 4 weeks.
The above-mentioned studies exemplify the potential of combining nanocrystals with different types of microneedle delivery systems. Although this field has been scarcely explored so far, these minimally invasive formulations can improve the transdermal delivery of active compounds at high doses. This technology is expected to improve therapeutic efficacy and patient compliance. Even though the scalability potential remains unclear, this technology might enable the delivery of poorly soluble drugs to tissues that are unreachable with classic topical formulations.
3.4. Nanocrystal-polymer microparticles
Patients suffering from chronic diseases often need to follow a treatment for life or an extended period. Compliance may be partial, which affects clinical outcomes. The integration of nanocrystals in polymeric hydrophobic microparticles or microspheres (Fig. 1E) can provide an additional biodegradable solid shell that modulates and prolongs the release kinetics [59–61]. This can yield also an injectable formulation that confers a long-term drug/nanocrystal release on site, thus reducing the frequency of administrations.
Progressive cartilage degeneration and chronic inflammation are two factors associated with knee osteoarthritis. Current treatments are inefficient or require frequent intra-articular injections of drugs due in part to fast clearance in the joint space [62]. Based on this premise, our group proposed to combine the properties of hydrophobic polymer microparticles with nanocrystal technology to obtain high drug-loaded formulations for long-term local release. In one paper by Maudens et al. kartogenin, a very poorly soluble drug that promotes articular cartilage regeneration, was wet-milled [60]. The obtained nanocrystals of 320 nm were then freeze-dried and embedded in poly (DL-lactide) microparticles of 10–20 µm by spray-drying. The formulation showed an extended-release profile with 62 % of katogenin released over 3 months. In vivo studies in osteoarthritic mice showed higher cartilage regeneration activity compared to free kartogenin. In a similar study, celecoxib nanocrystals were embedded in poly (DL-lactide) microparticles to treat chronic inflammation associated with osteoarthritis [63]. Noteworthy, a very high drug loading of 50% w/w with an encapsulation efficiency above 80 % was obtaied for a poorly soluble drug (i.e., celecoxib). Although high drug payloads usually correlate with fast release, in this case, an extended biphasic in vitro drug release over 3 months was observed. Drug loading in this formulation approach is not limited by the solubility of the drug in the spray-drying feed solution since the drug is majorly loaded as nanosuspension. Extended drug release over several months was highly influenced by the solubility and specific surface area of the nanocrystals, rather than by the microparticle polymer type, which is the case when a drug is dissolved in a matrix.
This combined technology (wet milling + spray-drying) enabled encapsulating nanocrystals into microparticulate systems for the first time for parenteral (e.g., intra-articular) administration. The two processes are scalable and can be exploited to encapsulate a large payload of a non-soluble drug into a sustained-release formulation. In addition, the spray-drying technique allows tailoring the colloidal properties of the resuspendable and injectable powder. For instance, a particle size around 10–20 µm is thought to slow down the clearance or filtration of the drug nanocrystals towards the systemic circulation compartment. Taken together, these methods can lower injection volume, which is crucial for non-intravenous (IV) parenteral administration. For those reasons, this technological platform should be explored in the future for other non-IV parenteral administrations such as intraocular delivery.
3.5. Liposomal and surface-engineered nanocrystals
The progress of nanotechnology in recent years has also provided the possibility of engineering drug nanocrystals to ameliorate their therapeutic index. This can be crucial in diseases where systemic exposure of drugs is often associated with serious adverse effects, such is the case of cancer therapy [64]. Some studies have recently investigated the incorporation of nanocrystals into liposomes (Fig. 1F). Drug nanocrystals can be incorporated or formed in situ within the lipid layers [65–67]. Li et al. developed in situ forming nanocrystals of ciprofloxacin in liposomes for oral delivery [68]. In this case, the drug precipitates inside the vesicles following a freeze-thawing step. The release profile of the liposomes in vitro was dictated by the solid state of the drug with a steady release behavior under non-digestive conditions that increased afterward under a simulated intestinal fluid medium. The surface of liposomes can also be functionalized to provide a specific ligand-mediated targeting effect [69, 70]. Alternatively, a two-step method was proposed for a targeted liposomal delivery system model for hydrophobic antitumoral drugs in another study [71]. Nanocrystals were first obtained by wet ball-milling technique and then incorporated into PEGylated and folic acid-functionalized liposomes. These liposomes displayed enhanced colloidal stability with a drug loading of up to 20 %. In vivo studies in K56 xenograft mice showed higher tumor targeting/accumulation after parenteral administration in comparison with either the free nanocrystals or the non-targeted liposomes.
Coating or decoration of nanocrystals is gaining relevance, particularly in the field of cancer therapy and bioimaging. One of these approaches consists of grafting the nanocrystals’ surface with diverse compounds such as proteins or PEG. This usually aims at improving drug bioavailability and biocompatibility after IV injection, by reducing non-specific protein adsorption and phagocytosis [72–74]. Park et al. published in 2017 a study using albumin-coated nanocrystals of paclitaxel to improve the therapeutic outcomes for solid tumors [75]. These authors formulated paclitaxel nanocrystals by crystallization in various surfactant-containing mediums. The formulation stabilized with poloxamer 407 displayed the smallest size and a rod-shaped morphology thought to elude macrophage uptake. Then, nanocrystal surface coating was performed by incubating the nanocrystals with albumin for 24 h at room temperature, prior to centrifugation to remove the excess of unadsorbed protein. In comparison with Abraxane®, the commercial albumin-bound paclitaxel, at the equivalent dose of 15 mg/kg, this formulation showed a higher antitumor efficacy when tested in a mice model bearing subcutaneous melanoma. These results correlated with higher paclitaxel tumor accumulation (27.4 µg/g vs 13.8 µg/g for Abraxane®).
Nanocrystals were not initially specifically designed to increase drug targeting or have stealth properties via surface engineering. However, the concomitant progress of nanotechnology allows nowadays to confer these characteristics to nanocrystals, owing to their own versatility and workability. Although adding manufacturing complexity, the development of nanocrystals in liposomes might also solve some of the stability and biocompatibility constraints reported/observed in classical nanocrystal formulations. In that sense, elucidating their localization, physical state inside the liposomes is encouraged [76]. For instance, the well-known anticancer nanomedicine Doxil® is reported to display stabilized recrystallized doxorubicin inside the liposomes [77]. All these upgrades might prompt the consolidation of safe and effective parenteral administrations of nanocrystals. With similar goals as for liposomal formulations, coating of nanocrystals aimed to increase drug bioavailability holds promise due to the simplicity of the formulation process. However, further in vivo studies are still required to ratify the benefits of this surface decoration strategy.