The oral administration of drugs is the most used method of drug therapy because it is considered the safest and preferred route by patients. However, some challenges are associated with oral drug delivery, such as the hepatic first pass metabolism, the presence of enzymes and an unfavorable pH in the stomach that can inactivate the drug as well as inter-individual characteristics due to oral diseases, gastrointestinal damage, the phenomena of malabsorption, etc., that can affect the absorption and bioavailability of the API. Some of these issues could be overcome by means of the oral administration of the drugs as nanocrystals (NCs). An example is given by the fact that NCs can overcome the local gastrointestinal (GI) damages caused by many anti-inflammatory drugs [28]^[1]. Compounds, such as meloxicam, can produce gastric lesions when administered orally due to the high concentration or repeated doses of the drug. The oral administration of meloxicam in the form of NCs could improve drug bioavailability, reducing or avoiding the repeated drug administration with the consequently reduction in drug toxicity in the GIT [29]^[2]. Xu et al. demonstrated the improved efficiency of cinacalcet NC capsules compared to conventional cinacalcet hydrochloride tablets, evaluating the dissolution rate in four media (pH 1.2 hydrochloric acid buffer solution, pH 4.5 acetic acid buffer solution, pH 6.8 PBS and water). The values of accumulated dissolution were all more than 95% without the effect of pH compared with the commercial tablet Sensipar^® and raw material [30]^[3]. One of the main advantages of NCs is that they can be formulated in a great variety of pharmaceutical forms, including easy-to-administer ones, such as NC-based oral film strips. The NC strips are also useful when it is necessary to formulate an API that must act quickly, releasing the drug either by chewing or in contact with salivary fluid in a short time [31]^[4]. This strategy could be very useful to treat patients affected by oral pathologies, with difficulties in swallowing or for pediatric patients. Studies showed that formulations based on NCs (tablets and capsules) exhibit a better drug-kinetic profile compared to the raw drug and conventional forms on the market. A pharmacokinetic study performed by Li et al. on nimodipine NCs demonstrated that this antiarrhythmic drug, when administered orally in the form of NCs, had a bioavailability that was 397% greater than that of conventional tablets (Nimotop^®). Specifically, the conventional form of nimodipine dissolved quickly and underwent supersaturation, precipitating in the GI fluid and reducing the overall bioavailability of the drug. NCs, on the other hand, adhere to the wall of the GIT releasing the drug gradually and dissolving slowly, giving a more controlled release of the drug [32]^[5]. NCs of the beta-blocker carvedilol stabilized by alpha-tocopherol succinate (VES) showed increased dissolution rate and bioavailability compared to the commercial tablets [33]^[6]. The shape of NCs can also influence parameters, such as mucus permeation, transport through epithelial cells and bioavailability. NCs with rod-shape characteristics showed a significant cellular absorption and a greater epithelial transport than NCs with spherical shape, probably due to the larger surface area of elongated particles that increases the contact area between NCs and the cellular membrane [34]^[7]. Particle size is an influencing parameter in the bioavailability of orally administered drugs as reported by several studies; a twenty-fold increase in bioavailability between raw drug and NCs and a five-to-six-fold increase between micrometric and nanometric particles has been reported. An optimal size for increased oral absorption could be between 200 and 600 nm. The dissolution rate, the biodistribution and the oral bioavailability of coenzyme Q10 NCs was clearly affected by particle size. For NCs in a 120 to 700 nm size range, higher C_max and AUC_0–48 values were registered in the smallest Q10 NCs [35]^[8]. In any case, the reduction in size below the micrometer range offers numerous advantages in terms of greater bioavailability [36]^[9].

In addition to particle shape and size, another factor that can influence the bioavailability of oral drug-loaded NCs is the stabilizer agent. It has been widely demonstrated that the type and concentration of stabilizer influence the size and stability of NCs, but it is also true that they can positively or negatively affect the in vivo drug performance. Mu et al. prepared spironolactone NCs with four different stabilizers, showing that the use of ionic stabilizers individually is not recommended. The use of a charged stabilizer alone increases the possibility of agglomeration between the NCs in the GIT, mainly due to the different pH variations. Sodium deoxycholate (NaDC) was used as stabilizer agent for spironolactone NCs: it is converted into insoluble deoxycholic acid at the acidic pH of the stomach, leading to the formation of aggregates that were not redispersed even in the intestinal pH 6.8. On the other hand, the in vivo performance was enhanced by spironolactone nanonization using stabilizers, such as Pluronic^® F127 and F68, and HPMC-C5. All of them gave similar results in terms of improved dissolution and bioavailability compared to the raw drug [37]^[10]. In other studies, the use of stabilizers, such as PVP, ethylcellulose or the combination of HPMC and PVA, produced an improvement in oral drug bioavailability [38]^[11]. Additionally, in the study of Tian et al., focused on the preparation of NCs with a multicomponent inartificial compound (Bufadienolides) with antitumour activity, it was demonstrated that different stabilisers caused multiple different mechanisms of NC endocytosis, intracellular transport, and transmembrane transport. The choice of an anticancer molecule as model drug is due to the fact that the bioavailability of orally used chemotherapeutic drugs is hampered by the difficulty of effectively penetrating through the mucus layer and transport layer of intestinal epithelial cells [39]^[12].

The combination of two types of innovative formulations (emulsion and NCs) into one is of great interest for improving the oral bioavailability of a drug. In the study by Zhang et al., NCs were incorporated into a self-stabilizing Pickering emulsion for oral administration. A Pickering emulsion is a special emulsion that uses ultrafine solid particles as emulsifier [40]^[13]. In this study, NCs were the solid particles that acted as emulsifier. The study showed that the relative bioavailability of a puerarin NC emulsion was significantly higher than of the crude puerarin suspension (262.43%), the NC suspension (155.92%) and the surfactant emulsion (223.65%), proving that the combination of these two types of formulation (NCs and Pickering emulsion) increased the oral bioavailability of the drug and, at the same time, the NCs are able to stabilize the emulsion [41]^[14]. Two recent studies focused on the preparation of NCs of irbesartan for oral administration. The first study by Deguchi et al. focused on the preparation by media milling of NCs coated with methylcellulose with a size less than 200 nm. To overcome stability issues, a second study by Nagai et al. was conducted to obtain a solid oral pharmaceutical form. Indeed, the NCs were then incorporated into tablets through drying approaches, resulting in a particle size of 118 nm after redispersion of the tablet [42,43]^[15][16]. NCs of naringenin, a molecule recognized for its anti-inflammatory activity, have been studied for the oral treatment of rheumatoid arthritis. Compared to the crude drug, the NCs showed dissolution behaviour, increased cellular uptake, and improved transcellular diffusion in comparison to the bulk drug naringenin. In vivo tests in rats demonstrated an improvement in rheumatoid arthritis in collagen-induced arthritic rats by reducing the infiltration of inflammatory cells and synovial damage [44]^[17].

Ocular drug delivery is a major challenge due to the unique anatomy and physiology of the eye. The amount of drug that enters the anterior chamber is about 5% of the administered dose. There are numerous obstacles that have to be considered for this route, including the small volume that can be administered due to the conjunctival sac, which holds a volume of about 7 microliters, nasolacrimal drainage, eye barriers, blinking and lacrimal reflexes, irritation, and the tolerability of an instilled formulation. All these issues could be improved by exploiting NC technology [45]^[18]. Studies related to the ocular delivery of drug NCs are mainly directed to the treatment of inflammatory diseases and ~15% to glaucoma [46,47,48]^[19][20][21]. The most widely used ocular dosage form is the eye drops. As mentioned, one of the main limits is the small volume that the conjunctival sac can hold, which leads to the expulsion of most of the administered formulation. Researchers have tried to overcome this issue by producing formulations capable of increasing the residence time of the eye drops. The choice falls on increasing the mucoadhesive properties of the formulation, by using suitable stabilizers or by manufacturing positively charged NCs in order to increase their adhesiveness on the negatively charged ocular surface. This strategy was made possible by the use of stabilizers, such as cetylpyridinium chloride and benzalkonium chloride [49]^[22]. Another approach is to increase drug residence time by producing viscous solutions that adhere better on the corneal surface. The increase in viscosity, in fact, leads to a decrease in drug transport with the tear fluid and therefore to a lower drainage speed. One strategy is to prepare in situ gelling systems, which are normally administered as a solution that becomes a gel due to a change in electrolyte concentration, pH or temperature at the application site. In the study of Nagai et al., an in situ gelling system was prepared based on indomethacin NCs stabilized by Pluronic^® F127, characterized by a specific sol–gel transition temperature. This behavior could increase the residence time of the drug on the ocular surface increasing its permeability and bioavailability. The reseauthorchers found that the concentration of the stabilizers played a crucial role since F127 at concentrations of 5% and 10% improved the drug permeability through the corneal epithelium, and this result was not achieved at higher (15%) stabilizer concentration [50]^[23]. Overall, it has been demonstrated that the use of NC technology can exert a double benefit to improve drug ocular administration, A short-term advantage is created due to the immediate increase in the drug mechanism of action, which is obtained by increasing the dissolution of the drug, with a consequent increase in the amount of drug available for absorption and the possible reduction in ocular toxicity. The long-term advantage consists of the prolonged release of the drug over time, given by the mucoadhesive NC properties that improve the NC ocular retention time and prolong drug action [51]^[24]. Therefore, the selection of suitable stabilizers and an optimal particle size is considered essential for ophthalmic NC administration since particles with size less than 200 nm are preferred to reach the retina district, and safe and well-tolerated stabilizers, such as PVP, PVA, HPMC, and poloxamers, are required to avoid ocular irritation [52,53]^[25][26]. Particle size exerts a significant impact on drug concentration in the tear fluid and on the ocular bioavailability of drugs from topical suspension. A nanosuspension of indomethacin with small particle sizes showed approximately a two times higher drug release into the aqueous humor than suspensions with larger particles (>1000 nm). Particle size affected the drug absorption of formulations with the same viscosity [54,55]^[27][28].

Two main issues are generally associated with drug administration via parenteral routes: toxicity and target site achievement. NCs have been studied for this purpose, and the choice of the stabilizer agent is crucial since not all stabilizers are suitable due to their toxicity. Ahire et al. showed a list of stabilizers allowed for parenteral administration: in addition to poloxamers, D-α-tocopheryl polyethylene glycol succinate (TPGS), amino acid derivatives, such as albumin and leucine, at concentrations ranging from 2% to 52.6%, respectively, or others, such as arginine and proline, can be found. NC mean size is another relevant point for parenteral administration, since particles should possess a diameter in the range of 100–300 nm [56]^[29]. As reported in the literature, it has been demonstrated that, after parenteral administration, curcumin NCs with a size <100 nm were poorly captured by cells, while particles with a size >500 nm were phagocytosed by macrophages. In fact, after injection, NCs can be recognized and captured by the mononuclear phagocyte system (MPS) and passively deposited in the liver, lung and spleen (organs rich in MPS) [57]^[30]. Changes in the NC surface can negatively or positively affect the attack by the endothelial reticulum system (RES): coating with serum albumin or dextran leads to increased recognition by the immune system and subsequent deposition in the liver and spleen. Instead, a PEG coating limits this event, justifying the fact that, in some cases, the fabrication of “stealth” NCs is relevant [58]^[31]. NC coating with cationic lipids, such as DOTAP or IGg, leads to greater recognition by the immune system and by monocytes of the CD14 and CD16 line. By exploiting this mechanism, it is possible to design NCs that can be transported by macrophages into cancer cells [59]^[32]. NCs, generally constituted by 100% of the drug, allow a high drug bioavailability from the first administration. Furthermore, the low concentration of stabilizers used avoids toxicity problems related to the parenteral administration of formulations containing higher quantities of surfactants [60]^[33]. The FDA approved Tween^® 80 (up to 10%) and Poloxamer^® F188 as stabilizers for injectable nanosuspensions, whereas TPGS is considered safe, but is more expensive [56]^[29].

Another strategy to improve drug targeting is to design pH-responsive NCs. This approach is useful to target the drug to an inflamed tissue or, for example, to a cancer cell in which the environment is acid. In this case, the NCs can be coated, for example, with calcium carbonate, which releases the drug in a pH-dependent manner. In this way, a low release at physiological pH and a rapid release at acid pH can be established, simulating the tumor environment [61]^[34]. NCs have been investigated also for peritoneal route, which consists of the administration of drugs directly into the peritoneal fluid. Intraperitoneal NCs are beneficial for the administration of chemotherapy drugs also known as PIPAC (pressurized intraperitoneal aerosol chemotherapy). It is carried out by administering a highly concentrated liquid NC formulation that is transformed in the form of aerosol and coats the abdominal cavity by increasing the diffusion coefficient and the final efficacy of the drug [62]^[35]. NCs were also studied for the hyperthermic intraperitoneal chemotherapy (HIPEC) strategy of paclitaxel [63]^[36]. In another study, a hyaluronic-acid-based hydrogel, containing paclitaxel NCs, was produced. The goal was to create a chemotherapy depot system, which would allow the slow and localized release of the molecule. In this way, the severe effects associated with conventional anticancer therapy could be avoided [64]^[37].

A recent study by Ancìc et al. focused on the design of resveratrol NCs for intraperitoneal administration, for a potential anticancer treatment of Ehrlich ascites tumor (EAT). In vivo studies in EAT-bearing mice showed that the administration, having a low systemic toxicity, led to a significant reduction in tumor cell proliferation in the abdominal cavity, and a reduction in angiogenesis in the peritoneum, demonstrating low risk and the high beneficial effects associated with resveratrol NCs [65]^[38].

The studies on NCs for pulmonary drug delivery have grown over the past decade. The main advantages deriving from this pathway are the large pulmonary surface, the thin barriers, the low enzymatic activity, the high vascularization, and the possibility of avoiding the first pass hepatic metabolism. NCs can overcome many issues typical of conventional pulmonary formulations, including poor release profiles of hydrophobic drugs, different particle size and heterogeneity in the conventional aerosolized formulations and the intense drug clearance by the alveolar macrophages that eliminate particles larger than 1 µm. Thus, NC technology becomes advantageous, providing a minimal use of excipients in the formulation, the reduction in particle size with a major surface area exposed to lung lining fluids and the possibility to obtain prolonged drug release. The ability of NCs to settle in the lung and escape from mucus entrapment is mainly due to the particle size, which is sufficiently small to evade steric obstruction by the dense environment of mucin fibers, the shape (rod-like) and the type of stabilizer used that should be sufficiently muco-inert to evade association to mucins. In the work of Costabile et al., NCs coated with PEG improved particles diffusion in the mucus layer since the presence of a PEG moiety formed a brush layer on the NC shell, which reduces their adhesion to mucins [66,67]^[39][40]. As mentioned, for the pulmonary route, particle size plays a pivotal role, affecting the lung deposition of an aerosol and influencing the clinical effectiveness of a drug. A recent study demonstrated the correlation between the size and bioavailability of drug NCs. In particular, curcumin NCs with different sizes were analyzed. Small-sized NCs with dimensions up to 250 nm showed a great diffusion profile, through a model of mucus layer by Franz cells, compared to NCs with a medium (around 500 nm) or large size (1089 nm). The reason was attributed to the pores of the respiratory mucus layer with a diameter of ~200 nm, as particles with a diameter less than 200 nm penetrated the mucus layer, while the others were trapped in it. Similar findings were obtained with Calu-3 cell lines selected as bronchial epithelial cells [68]^[41]. Furthermore, it has been shown that crystals with a particle size below 300 nm are able to escape mucociliary clearance and alveolar macrophages and present a better lung retention compared to microparticles. For these reasons, the ideal particle size should be in the range below 300 nm to avoid elimination through mucociliary clearance and improve penetration through mucus [69]^[42]. Curcumin-based NCs have been designed to be delivered as dry inhalation powder (PPE). They are prepared by the wet milling method followed by spray drying, obtaining a useful formulation for the deposit of a good amount of drug in the lung, avoiding the problems of toxicity derived from the deposit of the drug in other sites. The final formulation was characterized by good flow properties due to small particle size and spherical shape, as shown in the SEM analysis, as well as good aerodynamic properties to ensure maximum bioavailability by inhalation, according to the fact that the ideal aerodynamic particle size of less than 5 μm improves the deposition in the lung [70]^[43]. NCs prepared with hyaluronic acid (HA) directed through the pulmonary pathway to lung cancer cells were obtained to target breast cancer cells through the CD44 receptor that has a binding domain for HA overexpressed in cancer cells [71]^[44]. Another study was focused on the design of NCs coated with mannose as a fluorescent probe with the aim of detecting the cancer cells that overexpressed the mannose receptors [72]^[45]. Other strategies are well summarized in the work of Kumar et al. [73]^[46].

NCs administered via the skin are an advantageous strategy for water-insoluble molecules because they increase the degree of dissolution of the drug. This involves an increase in the concentration of the drug in solution on the side opposite to the targeting site and therefore an increase in its passage through the skin by a concentration gradient. This is advantageous to limit the application of the drug only to the affected area, thus reducing possible side effects. This is the case, for example, of the cortisone drug since the formulation through NCs improves its bioavailability and can be considered an alternative to the oral route. For example, in the work of Lohan et al., the authors compared a conventional cream formulation of dexamethasone with a dexamethasone NC-based formulation that promoted drug penetration through the stratum corneum, increasing drug bioavailability and efficiency [74]^[47]. The preparation of antioxidant-based nanosuspensions, incorporated into creams or gels, is a valid alternative in the field of cosmetics or aesthetic medicine. The topical administration of flavonoid-based NCs and antioxidant molecules, such as lutein, quercetin or hexperitine, preserves their biological properties, increasing their poor bioavailability [75,76,77]^[48][49][50]. Diosmin NCs were the constituents of wafers designed for the treatment of diabetic ulcers, combining the advantages of the adhesion of the pharmaceutical form (based on gelatin and alginate) with the increase in bioavailability given by the formulation of NCs [78]^[51]. Slow-release formulations can be formulated with NC-based patches and, as an innovation strategy, suspensions that can be administered follicularly can be used. The hair follicle has, in fact, proved to be an excellent reservoir for the containment and slow release of drugs, when dimensions that vary in the range between 400 and 700 nm. In particular, a study revealed that the increase in penetration through this path is due to the moisturizing characteristics that are provided on the skin by the excipient used for the preparation [79]^[52]. NC technology allows the preparation of dermal formulations with low viscosity and with a good release profile, making the active ingredient easily absorbable even at concentrations below 0.2% [80]^[53]. Thus, they became very useful in the case of anti-inflammatory drugs, such as highly gastrolesive diclofenac, increasing its topical bioavailability, as alternative to the oral administration.

The synergy between the use of micro-needles and nanosystems has enormous potential, as reported by several studies. Indeed, micro-needles promote the penetration of systems by creating microchannels on the skin surface. For example, in a study performed by Pireddu et al., the administration of diclofenac NCs by micro-needles was investigated. This association was found to be synergistic and valid to increase the bioavailability of the drug at the target site without obtaining the side effects of gastric injury typical of a nonsteroidal anti-inflammatory drugs (NSAIDs) [81]^[54].

In recent years, the intranasal route has emerged as an attractive approach in the treatment of various diseases due to its potential for multiple actions. Indeed, a drug deposited on the nasal mucosa can exert a local effect and/or be absorbed into the bloodstream, performing a systemic action. Once the molecule dissolves into the nasal mucus layer, absorption is facilitated by a large, highly vascularized surface area with relatively low enzymatic activity. The nasal route is used for the topical administration of molecules for local treatment, typically with anti-allergens and nasal decongestants. In fact, the application of the drug to the site of action carries a lower risk of systemic side effects, such as the typical drowsiness associated with the oral administration of antihistamines [82,83]^[55][56].

In addition to the local and systemic action, intranasal administration has been widely investigated for the possibility to serve as a direct transport route to the brain (“nose-to-brain delivery”) since the nasal cavity is the only portion of the body in direct contact with both the central nervous system (CNS) and the external environment [84]^[57]. Examples of the application of intranasal administration for local and systemic delivery are discussed in the following subsections. Considering the emerging interest in the nose-to-brain delivery, this topic is detailed in a dedicated section (Section 4).