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Formulation Strategies of Nanosuspensions for Various Administration Routes

Sıla gülbağ pınar.

1 Department of Pharmaceutical Technology, Faculty of Pharmacy, Süleyman Demirel University, Isparta 32260, Turkey; rt.ude.uds@gablugalis

Ayşe Nur Oktay

2 Department of Pharmaceutical Technology, Gülhane Faculty of Pharmacy, University of Health Sciences, Ankara 06018, Turkey; [email protected]

Alptuğ Eren Karaküçük

3 Department of Pharmaceutical Technology, Faculty of Pharmacy, Ankara Medipol University, Ankara 06050, Turkey; [email protected]

Nevin Çelebi

4 Department of Pharmaceutical Technology, Faculty of Pharmacy, Başkent University, Ankara 06790, Turkey

Associated Data

Not applicable.

Nanosuspensions (NSs), which are nanosized colloidal particle systems, have recently become one of the most interesting substances in nanopharmaceuticals. NSs have high commercial potential because they provide the enhanced solubility and dissolution of low-water-soluble drugs by means of their small particle sizes and large surface areas. In addition, they can alter the pharmacokinetics of the drug and, thus, improve its efficacy and safety. These advantages can be used to enhance the bioavailability of poorly soluble drugs in oral, dermal, parenteral, pulmonary, ocular, or nasal routes for systemic or local effects. Although NSs often consist mainly of pure drugs in aqueous media, they can also contain stabilizers, organic solvents, surfactants, co-surfactants, cryoprotectants, osmogents, and other components. The selection of stabilizer types, such as surfactants or/and polymers, and their ratio are the most critical factors in NS formulations. NSs can be prepared both with top-down methods (wet milling, dry milling, high-pressure homogenization, and co-grinding) and with bottom-up methods (anti-solvent precipitation, liquid emulsion, and sono-precipitation) by research laboratories and pharmaceutical professionals. Nowadays, techniques combining these two technologies are also frequently encountered. NSs can be presented to patients in liquid dosage forms, or post-production processes (freeze drying, spray drying, or spray freezing) can also be applied to transform the liquid state into the solid state for the preparation of different dosage forms such as powders, pellets, tablets, capsules, films, or gels. Thus, in the development of NS formulations, the components/amounts, preparation methods, process parameters/levels, administration routes, and dosage forms must be defined. Moreover, those factors that are the most effective for the intended use should be determined and optimized. This review discusses the effect of the formulation and process parameters on the properties of NSs and highlights the recent advances, novel strategies, and practical considerations relevant to the application of NSs to various administration routes.

1. Introduction

In recent years, one of the most challenging issues encountered in both the pharmaceutical industry and pharmaceutical studies has been drug candidates with low water solubility [ 1 ]. The main challenges are that the dose-response linearity of drugs with low water solubility may decrease, and unexpected collapse of the drug may be encountered after administration, leading to decreased patient compliance and decreased bioavailability. In addition, due to the low solubility of active substances in water, variations may occur as a result of changing the absorption of the drug in fasted and fed states [ 2 ]. There have been very promising developments in the studies carried out in the last century to overcome these problems, and the most important of these is the development of nanosized drug delivery systems. The basis of these approaches is related to the increase in solubility when surface area is increased, depending on the Noyes–Whitney equation as a result of reducing the particle size of the active substance, thus increasing the dissolution and bioavailability [ 3 ]. To increase solubility and thus bioavailability, drug delivery systems such as liposomes [ 4 ], nanoparticles [ 5 ], solid lipid nanoparticles [ 6 ], polymeric micelles [ 7 ], dendrimers [ 8 ], quantum dots [ 9 ], nanoemulsions [ 10 ], and nanosuspensions [ 11 , 12 ] are the most widely used. Many studies on nanosuspensions using poorly soluble drugs have been conducted since nanosuspensions were first reported by Müller et al. in 1994 [ 13 ].

Nanosuspensions (NSs) are colloidal dispersions of submicron drug particles and are generally defined as very finely dispersed and biphasic colloids containing solid drug particles smaller than 1 μm [ 14 ]. Although there are some differences in the literature on the definition of nanosuspensions, and the words nanosuspensions and nanocrystals are used interchangeably in these studies, they are characterized as “pure active pharmaceutical ingredients (APIs) between 10–1000 nm stabilized with surfactant or polymer” [ 15 ], as well as “particles with a particle size of approximately 200–600 nm below 1 micrometer, formed by 100% pure active substance” [ 16 ]. An NS is expressed when prepared with stabilizers in the form of nanosized drug crystals.

NSs have many advantages over other drug delivery systems. These advantages can be summarized as follows:

• NSs provide enhanced oral bioavailability of drugs by increasing the saturation solubility and dissolution of the active substance and by increasing adhesion to the cell surface membranes [ 17 ].
• NSs can also allow passive targeting because the particle is of nanometer size [ 18 ].
• They are simple, easy, and inexpensive to produce, and they themselves produce rapid and reproducible formulations [ 19 ].
• Production costs are very low because of the low excipient requirements during their preparation. Moreover, their production can be scaled up [ 20 ].
• They reduce the bioavailability differences in fasted/fed states caused by the effects of food [ 21 ].
• They reduce inter-subject variability in bioavailability [ 17 ].
• They have a high drug content (accepted as 100%), so the dose used is reduced in therapy [ 22 ].
• Physical stability is increased in solidified nanosuspensions, and solidified formulations can be presented to patients in solid dosage forms such as tablets or capsules [ 17 ].
• NSs can be formulated for parenteral, pulmonary, topical, and ophthalmic routes of administration, in addition to the oral route [ 14 ].
• They can be sterilized by various methods such as filtration, dry heat, steam, and radiation [ 23 ].

The many advantages of NSs (or nanocrystals) have led to the development of many commercial products produced with nanocrystal technology in the pharmaceutical industry. Nanocrystal-based formulations are widely used to treat cancers, pains, nausea, asthma, hypertension, hypercholesterol, inflammatory diseases, cardiovascular diseases, bronchial dilatation, depression, dermal diseases, and other diseases [ 24 , 25 ]. There are many clinical trials related to NSs in different phases (such as Phase II and III) [ 26 ].

In spite of these many advantages and numerous commercially available products, NSs also have several disadvantages:

• The formulation may not be suitable for some pharmaceutical active ingredients, and difficulties may be encountered in choosing the stabilizer type and stabilizer ratio used in the formulation.
• There is a potential for physical stability problems in liquid form during preparation in nanosuspension form [ 19 ].
• Particle growth may occur in the drying step because of insufficient cryoprotectant power [ 27 ].
• Undesirable polymorphic changes may be encountered because of the need to use devices (high-pressure homogenizers or wet bead mills) in the preparation of NSs and because of the high pressure and temperature increase and of the mechanical power applied accordingly [ 19 ].

In the first part of this review, an overview of the preparation methods of NSs, stabilizers used, characterization studies, and solidification techniques will be given. The second part addresses the routes of administration of nanosuspensions for systemic or local effects, and each route of administration is summarized in tables.

2. Preparation Methods for Nanosuspensions

Many methods have been developed by research laboratories and pharmaceutical experts for the preparation of NSs, and these methods are broadly divided into three categories: bottom-up technology, top-down technology, and a combination of these two ( Figure 1 ). Apart from these methods, other preparation techniques, such as supercritical fluid technology, an emulsification–solvent evaporation method, and a melt emulsification method, have also been successfully developed in line with advanced studies [ 24 ].

Nanosuspension preparation methods (conventional and combination technologies).

2.1. Bottom-Up Technology

The bottom-up technology, which is also referred to as “nanoprecipitation”, was first used in 1987 by List and Sucker [ 28 ]. This bottom-up technology is based on the principle of obtaining nanosized particles by precipitating dissolved molecules with the addition of another insoluble substance. For this method to be applicable, the active substance must be soluble in at least one solvent and suitable stabilizers must be used to prevent the growth of particles after precipitation [ 17 ]. In Figure 2 , the parameters that affect particle size and particle size distribution in NS formulations obtained by the bottom-up method are shown by the fishbone diagram. The advantages and limitations of the bottom-up method are summarized in Table 1 .

Schematic representation of the critical parameters of bottom-up technology by the fishbone diagram.

Comparison of advantages and disadvantages of nanosuspension preparation methods.

Newly developed bottom-up methods such as liquid antisolvent precipitation (LAS), precipitation assisted by the acid-base method, high-gravity-controlled precipitation (HGCP), the supercritical fluid method (SCF), and the emulsion polymerization method are also available in the literature [ 24 ].

2.2. Top-Down Technology

Top-down technologies are based on the reduction of large particles down to the nanoscale. The main methods used include the high-energy process called high-pressure homogenization and the low-energy process called media milling [ 14 ]. These methods are more suitable for industrial production than bottom-up technology, and they are applied to currently marketed products [ 16 ].

2.2.1. High-Pressure Homogenization Method

The high-pressure homogenization (HPH) method relies on excessive shear forces and possibly cavitation, which is performed by pressing a suspension from voids or crevices and applying it to the drug crystals to disperse them. The two homogenization principles applied and the type of homogenizer used in line with these principles are microfluidization and piston-gap homogenization. Microfluidization is based on a jet-stream principle in which the coarse suspension accelerates and passes through the homogenizing chamber, especially under the influence of high-speed collision, shear, and cavitation forces, and the particle size becomes smaller as a result of these forces [ 29 ]. There are two types of chambers used in this method, the “Z” type and the “Y” type. When in the “Z”-type chamber, the suspension changes several times in the direction of flow, causing particle collision and shear forces; in the “Y” type, the suspension current is split into two streams, which then collide from the front [ 16 ]. In the second homogenizer type, the piston gap homogenizer, the coarse suspension passes through a very fine gap at an extremely high speed. The pressures applied in all these processes can vary from 500 bar to 350 Mpa [ 30 , 31 ]. Increasing the pressure and number of cycles generally allows for the preparation of NSs with smaller particles [ 32 , 33 ]. In Figure 3 , the parameters that affect particle size and distribution in nanosuspension formulations obtained by the high-pressure homogenization method are shown by the fishbone diagram. The advantages and limitations of this method are summarized in Table 1 .

Schematic representation of the critical parameters of the high-pressure homogenization method by the fishbone diagram.

2.2.2. Wet Media Milling Method

The media milling method was discovered by Liversidge et al. in 1992 [ 34 ]. The most widely used is ball mills, which are used in the preparation of nanosuspensions by a grinding method, although jet mills or colloid mills are also used. The ball mill method can be expressed in different ways: bead milling, wet media milling, and pearl milling. In this method, the substance and stabilizer solution are put into a chamber and mechanical grinding is achieved with the help of balls (beads) hitting it [ 1 ]. The wet media milling method involves a milling chamber, milling beads, a suitable stabilizer, and a dispersion medium, usually distilled water. The active substance is dispersed in this dispersion medium, and this coarse suspension is added to the milling chamber [ 35 ]. An average of one-third of the chamber is filled with dispersion medium and one-third with milling beads; the remaining one-third is left empty to provide the necessary space for milling [ 36 ]. Beads (zirconium, stainless steel, etc.) that are suitable for the process, of the desired number (amount of beads in mL or weight), and size (different bead diameter) are added to this chamber; the rotation speed of the device is adjusted, and the milling process begins with the milling time. The most common problem in this method is the wear caused by the milling chamber or the impact of the beads. It is necessary to use a chamber made of a material such as stainless steel or porcelain and beads made of porcelain, glass, agate, zirconium oxide, or chrome [ 37 ]. In Figure 4 , the parameters that affect particle size and distribution in NS formulations obtained by the wet milling method are shown by the fishbone diagram. The advantages and limitations of the wet milling method are summarized in Table 1 .

Schematic representation of the critical parameters of the wet milling method by the fishbone diagram.

A schematic representation of all the preparation methods described above is summarized in Figure 5 .

Schematic representation of the nanosuspension preparation methods.

2.3. Combination Technology

In addition to these two technologies (bottom-up and top-down), it is possible to use several techniques together in the preparation of NSs and to obtain NSs with desired properties by making some modifications [ 20 , 38 ]. There are studies in the literature regarding the use of more than one method in combination in Table 1 , and these studies also provide an evaluation of the advantages of the above-mentioned methods. With combined methods, it is possible to prepare NSs of the obtained formulation using bottom-up technology and then top-down technology or vice versa.

3. Selection of Stabilizers

Stability is crucial for NSs as with all other drug delivery systems. During the preparation of NSs, problems such as attraction or agglomeration may be encountered as a result of the reduction in size of the particles [ 39 ]. Because the particles are small and have high energy, it is usual for the particles to grow because of recrystallization or the Ostwald ripening effect [ 40 ]. Stabilizers are used in formulations to prevent the particle growth that causes instability in NSs [ 12 , 41 ].

The development of successful NSs is mainly based on the selection of suitable stabilizers. Several stabilizers such as surfactants or polymeric excipients were evaluated for the optimization of the NS. Parameters of stabilizers such as type, ratio, and molecular weight must be evaluated for the stability of prepared NS. In addition, the optimum parameters of stabilizers are dependent on the formulation preparation method and on the active pharmaceutical ingredients. For these critical properties, researchers make individual evaluations on the basis of active substances in the screening of stabilizers and decide on optimum stabilizers by a simple trial-and-error approach [ 39 ].

Some excipients used for the stabilization of NSs, as discussed in several studies, are summarized below ( Table 2 ).

Stabilizers used in nanosuspension formulations.

Stabilization in NSs can be steric and/or electrostatic. In steric stabilization, a steric barrier is created by the adsorption of a polymer on the particle surface of the nanocrystals, and aggregation of particles is prevented. In electrostatic stabilization, which is the other mechanism, the NS is stabilized by reducing the surface tension at the interphase interface thanks to electrostatic repulsion from the ionic surfactants added to the particle surface [ 24 ]. Stabilizers commonly used in NSs are surfactants such as poloxamer 188, poloxamer 407, vitamin E TPGS, polysorbate 80, sodium lauryl sulfate and polymeric substances such as polyvinyl pyrrolidone, polyvinyl alcohol, and cellulose derivatives (hydroxypropyl methyl cellulose—HPMC, hydroxypropyl cellulose—HPC, hydroxyethyl cellulose—HEC, and Methyl cellulose—MC, etc.) [ 39 ].

In NS formulation, the drug:stabilizer ratio ( w/w ) can vary widely, from 1:20 to 20:1 [ 39 , 47 ]. Stabilizers can be used in NS formulations using different preparation methods after the optimum ratio is determined by preliminary studies. In the use of stabilizers, specific choices are not made for drug administration routes. The stabilizers and results obtained in NSs prepared using different preparation methods for different routes of administration are summarized in the following sections of this review.

4. Characterization Studies for Nanosuspensions

Physical, chemical, physicochemical, and biological tests are performed for the characterization of the prepared NSs before and/or after solidification. Mean particle size and particle size distribution (polydispersity index), crystalline state and particle morphology, surface charge, saturation solubility, dissolution rate, stability, and in vivo biological performance studies are some of the basic characterizations for NSs. Various characterization methods of nanocrystalline formulations are summarized in Table 3 .

Characterization studies for nanosuspensions.

5. Solidification of Nanosuspension and Stability

Despite the individual and even combined use of the many stabilizers shown in Table 2 , it is impossible to completely inhibit the crystal growth of nanosized particles. Thermodynamically, the presence of NSs in a liquid dispersion medium accelerates crystal growth. Therefore, to obtain long-term stability and avoid aggregation, hydrolysis, and other stability problems, the use of appropriate stabilizers, as well as the drying of the formulation, is necessary. The drying process in NSs is undertaken by freeze-drying or spray-drying methods. These dried nanocrystals thus obtained can be presented to patients in solid dosage forms such as powder, tablet, or capsule [ 40 ].

To solidify the liquid suspensions, drying methods such as either lyophilization or spray drying are preferred. In cases where the active substance is likely to be affected by heat, the drying process is by lyophilization; the drying method may be selected by spray drying with effective substances that are not affected by heat and in cases where the drying particles should be more spherical.

Freeze drying, the lyophilization method, is the most common method of drying NSs. After a sudden freezing step, primary and secondary drying is performed under a vacuum. In this method, the segregation of nanocrystals as unfrozen small liquid packages and particle aggregation are prevented by using cryoprotectant material. Water-soluble matrix-forming sugars such as mannitol, sucrose, glucose, dextran, and trehalose are used for cryoprotectant purposes [ 49 , 58 , 60 ].

In the spray-drying method, temperature and pressure are rapidly applied to the NS formulation [ 47 ]. The dried particles are spherical, and the flow properties are quite good.

6. Administration Routes of Nanosuspensions

NSs can be administered by dermal, parenteral, ocular, and pulmonary routes, as well as by the oral route, which is the most common route for NS administration (constituting more than 60%) [ 19 , 49 ].

6.1. Oral Administration

The oral route is the preferred route of administration of drugs in terms of patient compliance, non-invasiveness, ease of use, dose flexibility, and safety. Beside patient benefits, oral dosage forms have advantages regarding cost-effectiveness, feasibility, and suitability for large-scale production. Due to these advantages, it is estimated that 90% of commercial drugs are for oral use [ 62 ].

When a drug is given orally, bioavailability and efficacy depend on solubility and absorption in the gastrointestinal tract. Poor aqueous solubility, poor permeability, the effects of being fed or fasted, enzymatic degradation, and the high first-pass effect are challenges that the development of oral medications encounter; they may result in inadequate in vivo absorption and the formulation not reaching an effective therapeutic concentration [ 63 ]. In addition, 40–70% of newly discovered drugs emerge with low solubility properties with limited oral bioavailability [ 64 ]. Due to the low bioavailability, a drug candidate may have to be administered in larger doses than usual, increasing the cost of treatment [ 65 ].

Currently, the Biopharmaceutical Classification System (BCS) is used to identify the physicochemical limitations for oral bioavailability based on the drug solubility properties of the drug throughout the upper gastrointestinal tract. BCS Class II and IV drugs limit both the rate and the extent of drug absorption. The rate of dissolution of the drug is the principal limitation of oral absorption [ 66 ]. This is why the dissolution rate can be considered the primary effective parameter for drug pharmacokinetics, which is related to drug solubility and particle size. Increased saturation solubility results in an increased concentration gradient between the gastrointestinal tract and blood and in an increased dissolution rate of the drug. In this way, the adhesiveness of drug particles provides enhanced bioavailability.

Particle size reduction is one of the most common approaches to increasing the saturation solubility and dissolution rate. The micronization process is widely used for this purpose, using colloid mills or jet mills. Micron-sized particles above 1 μm increase the dissolution rate because of the increased surface area. However, this does not change the saturation solubility of a drug, and low oral bioavailability still poses a problem [ 67 ]. Although it is known that the saturation solubility of an active substance is a compound-specific property depending on crystalline form, the lipophilicity of the drug, fed/fasted state, pKa, temperature, and properties of dissolution medium, at a nanometer range of saturation solubility, are also functions of particle size, according to the Ostwald–Freundlich and Kelvin equations [ 16 , 68 , 69 ]. NSs provide a tremendous increase in the surface-area-to-volume ratio and this leads to a higher solubility. Particle size reduction from 10 microns to 200 nm generates a 50-fold increase in the surface-area-to-volume ratio [ 70 ]. Higher solubility results in higher Ct and improved dissolution rate. This phenomenon can be explained by the Noyes–Whitney equation [ 71 ].

The mechanical properties, surface area, and surface morphology of drug substances affect their properties of adhesion to biological surfaces [ 72 ]. With respect to the increased surface area of drugs, NSs form a high concentration gradient between the gastrointestinal tract and blood vessels. A decrease in diffusion layer thickness is provided and this results in the high saturation solubility and dissolution of the drug [ 73 ].

In the first studies on the increase in in vivo bioavailability of NSs, the active substance danazol was administered to beagle dogs in nanocrystalline form, and it was determined that oral bioavailability increased approximately 16 times [ 74 ]. With the success of NS technology in the research area, various commercial oral products began to appear on the market in 2000. These included Rapamune ® (Sirolimus tablets—Company: Wyeth), Tricor ® (fenofibrate tablets—Company: Abbott), Focalin ® XR (dexmethylphenidate HCl capsules—Company: Novartis), Emend ® (aprepitant capsules—Company: Merck), Zanaflex ® (tizanidine HCl capsules—Company: Acorda) and Megace ® ES (megestrol acetate oral suspension—Company: PAR Pharmaceutical) [ 75 ]. The reasons for the preparation of NSs for oral administration and the main problems associated with it are presented in Table 4 [ 76 , 77 , 78 ].

Features of nanosuspensions in oral drug delivery.

NSs are obtained as nanocrystal drug particles in an aqueous medium with surfactants or polymers as stabilizers. It is possible to use NSs in liquid dosage form for oral use. Thus, the general advantages of liquid dosage forms such as higher flexibility of dosing, rapid absorption, higher bioavailability, and suitability for patients who suffer from swallowing difficulties can be achieved. While conventional oral suspensions have some excipients as suspending agents or carriers, drug NS systems do not contain any carriers, and they are prepared completely with the parent drug [ 26 ]. As a disadvantage, however, NS formulations in liquid have lower physical and chemical stability.

After solidification, NSs can be used as oral powders. Oral powders have better long-term physical and chemical stability. It is also possible to easily wet and redisperse the powders into a suitable liquid. Pellets can be obtained from dry powders by extrusion–spheronization or directly from liquid NSs by the fluid-bed coating method [ 14 ]. The pellets can reduce variations in gastric emptying rates and, hence, reduce the intra and inter-subject variability. They disperse freely in the gastrointestinal tract, avoiding high local concentrations that may irritate and drug absorption increases, with minimized potential side effects [ 79 ]. In addition, wet granulation or spray drying can be used to obtain granules. Powders and granules can be blended with appropriate excipients and put in capsules or compressed as tablets to achieve patient-compliant dosage forms.

NSs are feasible systems for oral film formulations. Liquid NSs are dispersed in polymer solutions with a plasticizer, and oral films can be obtained by the conventional casting method. The NS-loaded oral film formulations offer ease of preparation, rapid disintegration, no need for water intake, easy administration in the mouth or under the tongue, avoiding first-pass metabolism, and enhanced bioavailability [ 80 ]. The most recently published studies on oral NS are summarized in Table 5 .

Recent studies on the oral administration of nanosuspensions.

PS: particle size, PDI: particle size distribution, ZP: zeta potential, NS: nanosuspension, SDS: sodium dodecyl sulfate, PVA: polyvinyl alcohol, PVP: polyvinyl pyrolidone, FeSSIF: fed-state simulated intestinal fluid, FaSSIF: fasted-state simulated intestinal fluid, HPMC: hydroxypropyl methylcellulose, SLS: sodium lauryl sulfate.

6.2. Parenteral Administration

The parenteral route of drug administration is a widely used route of drug administration in clinical practice, with the advantage of reduced dosing, approximately 100% bioavailability, rapid onset of action, independence from the gastrointestinal tract, and avoidance of hepatic first-pass metabolism [ 90 ].

NSs for parenteral administration are nanometer size and are frequently prepared because of their ease of permeability, high drug loading capacity, and small volume of administration. In addition, the risks of toxicity and allergic reactions are prevented as a result of the low amount of excipients used in these formulations. While developing a new drug delivery system for parenteral delivery, it should be kept in mind that the delivery system should not be phagocytosed by Kupffer cells in the reticuloendothelial system and liver. Therefore, a size range of ≤100 nm is crucial for parenteral NSs [ 40 ].

In addition, during the parenteral administration of nanocrystals in vivo, the duration of blood circulation can be increased by surface modification with substances such as PEG in NSs to prevent opsonization.

Some studies on the parenteral administration of NSs are shown in Table 6 .

Some studies on the parenteral administration of nanosuspensions.

6.3. Pulmonary Administration

While the local effect can be achieved through the pulmonary route, the systemic effect can also be achieved because of the large surface area of the lung, thin alveolar epithelium, and low enzymatic activity [ 94 ]. At the same time, the anatomical structure of the respiratory tract provides an appropriate site for the immune response. Particle sizes, shapes, densities, and loads of inhaled drug particles are the leading factors affecting the retention (deposition) of aerosols in the lungs. In addition, the physicochemical properties of the active substance such as solubility, partition coefficient, permeability, molecular weight, enzymatic stability and formulation form, biophysical parameters, and the tools used affect the bioavailability of the inhaled drug. With recent advances in nanotechnology, there has been an increase in research for the development of new pulmonary drug delivery systems for the treatment of various diseases such as chronic obstructive pulmonary disease and asthma [ 95 , 96 ].

Nanocrystalline technology can significantly increase the bioavailability of poorly soluble drugs by reducing particle size and prolonging lung residence time. It provides a potential formulation development strategy for the delivery of drugs to the lungs [ 97 , 98 , 99 ]. In addition, nanocrystals—as a free-carrier nanotechnology—have gained increasing interest in the pulmonary administration of poorly soluble drugs because of their improved dissolution rate and saturation solubility, biological properties, and the low toxicity of poorly soluble drugs. Problems seen in conventional pulmonary delivery systems such as rapid drug release, poor residence time, and lack of selectivity can be solved with NSs. Furthermore, NSs increase bioavailability by improving drug diffusion and dissolution rate and preventing unwanted drug accumulation in the mouth and pharynx.

For pulmonary administration, NSs can be nebulized using jet or mesh nebulizers or aerosolized via metered dose inhalers [ 100 ] and dry powder inhalers [ 101 ]. For pulmonary administration, itraconazole [ 102 , 103 ], budesonide [ 104 , 105 , 106 ], and fluticasone NSs have been developed [ 107 ]. Previous studies have shown that nebulized NSs have acceptable aerodynamic performance and several advantages over conventional micronized drugs, including the ability to shorten nebulization time, improve patient compliance, and promote uniform distribution of drugs in the lungs by rapid diffusion [ 100 , 105 ].

Inhalable aerodynamic properties are an important factor affecting the pulmonary inhalation of drugs. The size distribution of respirable particles is usually expressed by the aerodynamic diameter, which varies with the shape, size, and density of the objects. The aerodynamic diameter of respirable particles determines whether they can accumulate in the lungs. Nevertheless, regardless of the method of aerosol administration, strict control of particle size to within the aerodynamic diameter (dA) range of 1–5 µm is necessary for optimal pulmonary delivery. Particles with dA > 5 µm are mostly deposited on the walls of the upper respiratory tract by inertial impaction, while particles with dA < 1 µm tend to remain airborne in the airways and are exhaled during the normal breathing cycle [ 108 ].

In addition, there are studies of pulmonary applications of NSs as nanocrystal-based inhalation systems, aerosol, adhesive microparticles, composite microparticles, and mucus-penetrating nanocrystals [ 109 ].

Selected pulmonary-route-administered NS study examples are summarized in Table 7 .

Example studies on the pulmonary administration of nanosuspensions.

PS: particle size, PDI: particle size distribution, ZP: zeta potential, HPMC: hydroxypropyl methylcellulose, SLS: sodium lauryl sulfate, EDTA: ethylenediaminetetraacetic acid, PVP: polyvinyl pyrolidone, DPPC: dipalmitoylphosphatidylcholine, COVID: coronavirus disease.

6.4. Ocular Administration

The eye is the most particular organ of the body, and various drug delivery systems were employed to deliver the drugs into the eye. The design of drug delivery systems for ocular administration has become a challenge in the pharmaceutical field [ 117 ]. Ocular drug delivery is needed in the treatment of some diseases such as glaucoma, dry eyes, diabetes retinopathy, proliferative vitreoretinopathy, keratoconus, macular degeneration, conjunctivitis, blepharitis, and uveitis. Systemic application used in the treatment of these diseases might have a limited effect because of blood–aqueous and blood–retinal barriers after ocular administration. These barriers can limit the amount of drug that reaches the extravascular retinal space and the aqueous and vitreous humors of the eye. For this reason, local or ocular application of drugs presents a higher drug concentration to the specific site of the ocular region [ 118 , 119 ]. Thus, the main purpose of ocular drug administration is to enhance the number of drugs reaching the specific ocular site and, thus, to improve the therapeutic effect. Although 90% of the marketed ophthalmic formulations are conventional eye drops, the low bioavailability related to the precorneal loss factors (static and dynamic barriers) became a major limitation for their usage. About 5% of the drug can pass through the cornea and reach the intraocular tissue because of vast and quick precorneal drop loss caused by high tear fluid output or blinking. While some ocular ointments have managed to overcome this problem, they also cause a blurring of vision. Controlled drug delivery systems and nanotechnological drug delivery systems have shown promise in tackling these problems. The ocular application of NS is an especially valuable approach to delivering both highly hydrophobic and hydrophilic drugs across the ocular mucosa. The main mechanism of the increase in ocular bioavailability via NS is the increment of dissolution velocity along with saturation solubility of poorly water-soluble drugs. Moreover, NSs can be prepared with various surfactants, viscosity enhancers, or charge modifiers. There is a possibility of a wide range of NS formulation designs that can gain mucoadhesive properties and the controlled release profile and enhance the retention time, permeation, and tolerability on the ocular site [ 117 , 120 , 121 ]. NS has a low risk of ocular irritation because of using nanosized particles, and the charge on the surface of NS facilitates their adhesion to the cornea. Based on all these advantages, the NSs can solve major issues such as the low contact time and poor ocular bioavailability related to the drainage of drug solution, tear turnover, and dilution or lacrimation [ 122 ]. The advantages of ocular NSs are also given in Table 8 . NS has been explored for ocular drug delivery by various researchers, and Table 9 shows the application of various NSs in ocular drug delivery.

Features of nanosuspensions in ocular drug delivery.

Recent studies on the ocular administration of nanosuspensions.

PS: particle size, PDI: particle size distribution, ZP: zeta potential, EDTA: ethylenediaminetetraacetic acid, NS: nanosuspension, PVP: polyvinyl pyrolidone, HPMC: hydroxypropyl methylcellulose, PVA: polyvinyl alcohol, HY: hyaluronic acid, PG: poly-γ-glutamic acid, HPC: hydroxypropyl cellulose, HEC: hydroxyethyl cellulose.

6.5. Dermal and Transdermal Administration

Dermal drug application has many advantages such as reducing side effects, ensuring drug accumulation in the specific area, controlled administration of the drug to the organism, self-administration of the patient, high patient compliance, and providing a specific effect [ 130 , 131 ]. There are two basic approaches to the dermal application of drugs: transdermal and dermal. While the applied formulation is localized in the dermal layers in the dermal application, it passes through the carrier to the lower layer of the skin and then enters the systemic circulation in the transdermal application [ 132 , 133 ]. Dermal and transdermal drug application has advantages as well as disadvantages. Because of the barrier effect of the stratum corneum layer, it is not possible to administer all drugs by this route. Crossing the stratum corneum barrier is only suitable for low-dose/high-permeability drugs. For an active substance to reach the lower layers of the skin, it needs to be small (molecular weight ≤ 500 Da), lipophilic (log p value ≤ 1–3), and compatible [ 134 ]. If these conditions are not met, sufficient blood concentration may not be reached because of the skin barrier. Adhesive structures used for transdermal purposes may not be suitable for all skin types. Since drugs and drug formulations may cause skin irritation and sensitivity, this situation should be evaluated in the drug development process. The advantages and challenges of dermal/transdermal NSs are presented in Table 10 .

Features of nanosuspensions in dermal drug delivery.

In recent years, many nanotechnological systems have been investigated to enhance the effectiveness of drugs after dermal/transdermal application. NSs are an especially promising system among the nanosystems for the dermal/transdermal application of drugs. By decreasing the particle size of the active substance to nanosize with NS technology, increasing the surface area and solubility, and thus the bioavailability, provides superiority in terms of dermal use. With the increase in saturation solubility, the concentration of the active substance on the skin surface increases, and depending on the increase in the concentration gradient, the passage of the active substance through the skin by passive diffusion accelerates [ 12 , 14 ]. In addition, with the increase in the surface area, the spreadability and adhesion of the particles to the skin surface also increase. By choosing positively charged polymers in the structure of NSs, penetration into the negatively charged stratum corneum layer can be increased. Therefore, in recent years, the development of NS formulations has gained importance in increasing the dermal bioavailability of active substances with low or medium water solubility. In 2007, NS formulations of low-soluble antioxidant-effective rutin and hesperetin-active ingredients have been developed, and the first effective NS-based anti-aging cosmetic preparation has been introduced to the market [ 135 , 136 ].

Table 11 shows NS formulations developed for dermal use.

Recent studies on dermal administration of nanosuspensions.

PS: particle size, PDI: particle size distribution, ZP: zeta potential, NS: nanosuspension, HPH: high-pressure homogenization, HPMC: hydroxypropyl methylcellulose, PVP: polyvinyl pyrolidone, SDS: sodium dodecyl sulfate, TPGS: vitamin E polyethylene glycol succinate, DoE: design of experiment, HEC: hydroxyethyl cellulose.

When NSs are applied dermally, they can have a local effect by penetrating the skin surface, or they can have a systemic effect by passing under the skin through intercellular hydrophilic routes, depending on the increase in saturation solubility. However, it is thought that the depot effect formed by the accumulation of particles in the hair follicles is more effective in the passage of NSs through the skin [ 145 ]. Carrier systems such as creams, anhydrous ointments, or gels are used to facilitate the dermal application of NSs and to increase their effectiveness. Thus, the residence time on the skin surface can be extended or the release of the active substance can be controlled depending on changes in viscosity. On the basis of all these advantages and findings in new studies, NSs are now understood to be a very promising system for dermal application.

7. Challenges and Future Perspective

Many newly discovered drug molecules are in BCS Class II and have very low water solubility. With the increasing number of these low-soluble drugs, which are not able to be formulated via traditional approaches, NSs have recently gained more importance. The advantages of NSs, such as applicability to a broad range of drugs, ease of scale-up, minimum use of excipients, and increased solubility followed by increased dissolution rate and bioavailability, lead to their broad acceptance in the development of formulations. NSs that allow drug administration by the dermal, pulmonary, parenteral, and ocular routes, especially the oral route, can be used successfully in various diseases for therapeutic purposes. These advantages are mainly reflected in the increasing number of NS-based commercial products. In addition to existing commercial products, further commercialization of NSs is likely with the future conclusion of clinical studies into various administration routes. At present, even though the NS formulations have progressed significantly, there are limited in vivo studies and clinical trials and also many problems in the selection of stabilizers, maintenance of stability, and other aspects. There is, therefore, a need to increase the number of clinical trials, to enrich the pharmacokinetic data after the administration of various NSs, and to establish theoretical models to identify the formulation development and optimization process of NSs. Moreover, some supporting equipment and technologies that are additionally providing high stability and providing easy scaling up will be more important in the future. The value of the technology and principle of NS formulations can be assessed by considering the number of products in clinical phases and in the market, paying attention also to the dates of entry into the market.

Funding Statement

This research received no external funding.

Author Contributions

Conceptualization, S.G.P., A.N.O., A.E.K. and N.Ç.; methodology, S.G.P., A.N.O., A.E.K. and N.Ç.; investigation, S.G.P., A.N.O., A.E.K. and N.Ç.; resources, S.G.P., A.N.O., A.E.K. and N.Ç.; writing—review and editing, S.G.P., A.N.O., A.E.K. and N.Ç.; visualization, S.G.P., A.N.O., A.E.K. and N.Ç.; supervision, N.Ç.; project administration, N.Ç. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Informed consent statement, data availability statement, conflicts of interest.

The authors declare no conflict of interest.

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• Open access
• Published: 15 January 2020

Emerging role of nanosuspensions in drug delivery systems

• Shery Jacob   ORCID: orcid.org/0000-0003-1851-6673 1 ,
• Anroop B. Nair 2 &
• Jigar Shah 3  

Biomaterials Research volume  24 , Article number:  3 ( 2020 ) Cite this article

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Rapid advancement in drug discovery process is leading to a number of potential new drug candidates having excellent drug efficacy but limited aqueous solubility. By virtue of the submicron particle size and distinct physicochemical properties, nanosuspension has the potential ability to tackle many formulation and drug delivery issues typically associated with poorly water and lipid soluble drugs. Conventional size reduction equipment such as media mill and high-pressure homogenizers and formulation approaches such as precipitation, emulsion-solvent evaporation, solvent diffusion and microemulsion techniques can be successfully implemented to prepare and scale-up nanosuspensions. Maintaining the stability in solution as well as in solid state, resuspendability without aggregation are the key factors to be considered for the successful production and scale-up of nanosuspensions. Due to the considerable enhancement of bioavailability, adaptability for surface modification and mucoadhesion for drug targeting have significantly expanded the scope of this novel formulation strategy. The application of nanosuspensions in different drug delivery systems such as oral, ocular, brain, topical, buccal, nasal and transdermal routes are currently undergoing extensive research. Oral drug delivery of nanosuspension with receptor mediated endocytosis has the promising ability to resolve most permeability limited absorption and hepatic first-pass metabolism related issues adversely affecting bioavailability. Advancement of enabling technologies such as nanosuspension can solve many formulation challenges currently faced among protein and peptide-based pharmaceuticals.

Introduction

The advancement in combinatorial chemistry and high throughput screening during the last few decades have generated number of potential drug candidates, which possess excellent target receptor binding. But due to large molecular weight and high log P values, these candidates have inherently low aqueous solubility thus restricts further development as a successful dosage form. It is a well-established fact that large surface area offered by particle size reduction can significantly enhance dissolution rate and bioavailability according to classical Noyes-Whitney equation [ 64 ]. Pharmaceutical nanosuspensions of drugs are nanosized, heterogeneous aqueous dispersions of insoluble drug particles stabilized by surfactants. In contrast, nanoparticles are either polymeric or lipid colloidal carriers of drugs. Nanosuspension technique is the only option available, when a drug molecule has many disadvantages such as inability to form salt, large molecular weight and dose, high log P and melting point that hinder them in developing suitable formulations. A major limitation of molecular complexation using cyclodextrin in pharmaceutical formulations is their inherent nature to increase the formulation bulk because of large molecular weight of complexing agent [ 31 ]. Nanosuspensions can solve such unique drug delivery issues associated with the active pharmaceutical ingredients (API) by retaining it in a crystalline state while enable them with increased drug loading during formulation development. Accommodating large drug amount with minimum dose volume has additional benefits in parenteral and ophthalmic drug delivery system owing to the minimization of excessive use of harmful non-aqueous solvents and extreme pH. Other advantages include increased stability, sustained release of drug, increased efficacy through tissue targeting, minimum first pass metabolism and deep lung deposition. The method of preparation, dosage forms, components and applications of nanosuspensions in drug delivery systems are schematically represented in Fig.  1 . Currently, many nanosuspension products of poorly soluble drugs are marketed or under development (Table  1 ). These advantages have driven towards faster development of nanosuspension technology during last few decades. Despite the intricacies associated manufacturing, selecting appropriate unit operation, equipment and process optimization can counteract these complexities to larger extent.

Schematic representation of method of preparation, dosage forms, components and applications of nanosuspensions in drug delivery systems

The stability of the submicron particles achieved in the nanosuspension is mainly attributed to the uniform particle size, which is formed by different manufacturing techniques. Particles of nanosuspensions must remain unchanged in size throughout its shelf-life otherwise it can initiate spontaneous crystal growth. Therefore, maintaining the uniform particle size distribution can hinders the presence of varying saturation solubility and thereby inhibit any crystal growth due to Oswald ripening effect [ 66 ].

Fundamental principle

Nanoparticles are frequently prepared by reduction/dispersion of large particles to nanosized range like in milling process (Scale down technology) or condensation/aggregation of particles from molecular dispersion to nanosized particles, as in precipitation method (bottom up technique). Since the particle size of nanoparticle is less, it possesses an enormously enhanced surface area compared to its original surface area. Consequently, it can increase saturation solubility determined by Ostwald-Freundlich’s equation as given below:

where S is the saturation solubility of small particles of radius (r), S 0 is the solubility of the large particles (normal solubility), γ is the interfacial tension between solid and liquid, M is the molecular weight of the solid, R is the gas constant, T is the absolute temperature, and ρ is the density of the solid.

The equation is significant when the particle size of the compound is submicron. This makes nanosizing more effective than micronization. Another theory explaining the increased saturation solubility is the formation of high-surface energy surfaces during nanosizing. This disrupt the ideal crystal lattice, thereby exposing the internal hydrophobic surface of the crystal to the aqueous medium. Significant effect of interfacial energy on the saturation solubility between different polymorphic forms of the drug was demonstrated. Similar explanation might be valid true for highly soluble metastable nanosuspension having high surface energy in comparison to more stable coarse suspension that possess low surface free energy and low saturation solubility [ 36 ].

The solid drug dissolution rate is also directly proportional to surface area available to dissolution, which can be described by Nernst-Brunner/Noyes-Whitney equation [ 64 ];

where, dx/dt is the dissolution velocity, D is the diffusion coefficient, A is the surface area of the particle exposed to the dissolution media, h is the diffusion layer thickness, Cs is the saturation solubility of the solute at defined temperature, X d is the concentration of the solute in the media at time, t and V is the volume of the dissolution media. It is apparent that size reduction to nano-size range will substantially increase dissolution rate due to increase of effective surface area.

A system is said to be thermodynamically stable, if it has low surface free energy (ΔG). The relationship is as follows: ΔG = γ S/L ΔA, where γ S/L is the interfacial tension between solid and liquid. The nanoparticulate system tends to decrease the increased surface area by either preferential solubility of crystal nuclei or regrouping of small particles. Irrespective of the preparation methods, this can cause increase in particle size subsequently results in reduction of surface free energy.

Optimum concentrations of surfactants lower the surface free energy by decreasing the interfacial tension exists between solid and liquid medium. Electrostatic and steric repulsion are facilitated by adding both polymers and ionic surfactants as stabilizers during nanosuspension preparation. Neutral polymers added to the system adsorb onto the particle surface and cause steric repulsion. Further, polymer can inhibit crystal growth and result increase in the particle size. Addition of ionic surfactant to a particle surface previously stabilized with non-ionic polymer allows superior surface coverage than using surfactant alone [ 95 ]. Therefore, charged nanoparticles exists in minimum repulsion region of potential energy barrier versus interparticle distance plot. The hydrophobic portion of stabilizers will coat the lyophobic crystal surface and consequently establish stable nanoparticles. Particles in nanosuspension do not settle due to Brownian motion and thereby improves the physical stability. When nanoparticles are introduced into the biological environment, many intermolecular interactions with biological environment can occur resulting in undesired effects such as aggregation, coagulation and precipitation [ 29 ]. Therefore, physical characterization tests and methods should be applied to various physical states of nanosuspension as given in Table  2 .

Manufacturing techniques

Precipitation techniques.

Materials of sub colloidal dimensions are caused to aggregate into particles of colloidal size range. The method involves rapid production of nuclei by preparing supersaturated solution of drug in water miscible organic solvent at optimum temperature and dispersing as small metered amount in non-solvent, water, under rapid stirring [ 65 ]. Change in solvent causes high supersaturation conditions, which results in rapid nucleation and at the same time not allowing supersaturation near the nucleating crystals. Rapid nucleation and slow growth rate are the key decisive factors for successful thermodynamically stable crystal form according to classical Ostwald law of nucleation [ 79 ]. Generation of finely dispersed uniform sized drugs, simple and economical process and ease of scale-up are the main advantages of precipitation method.

High supersaturation condition can create acicular or needle like crystal habit which can be broken down quite easily to generate additional nuclei formation at the expense of crystal growth. Secondly, non-introduction of impurities can cause imperfections or defects in crystal lattice that can be homogenized to decrease the particle to nanometer range. Crystal growth must be suppressed by appropriate concentration of surfactant or selective crystallization inhibitors. A patented technology (Lucarotin® (BASF) based on precipitation method to produce amorphous drug nanoparticles is utilized for pharmaceuticals is named as Nanomorph™ (Soligs/ Abbott/ Patent No. D 19637517),

Homogenization

In homogenization, size reduction of the particles is accomplished by driving suspension under high pressure (100–1000 bars) through a valve that has a small aperture [ 49 ]. The static pressure reduction due to sudden drop in fluid velocity causes cavitation induced implosion forces and shock waves in the liquid medium break down the microparticles (< 25 μm) to nano-size range. Further, shear forces due to the collision of particles and high velocity helps to fracture particles with inherent crystal defects. Viscosity enhancers can increase particle density within dispersion region, inhibit crystal growth hence improves the process of nanosizing [ 10 ]. Homogenization can transform metastable amorphous particle prepared by the precipitation method to stable crystal form. Generally, multiple cycles are required to produce particles of desired size ranges. Ease of scale-up, adaptability of the process to dilute or concentrate suspensions, low risk of contamination and feasibility for aseptic manufacturing are the key benefits of this method. The drawbacks of the techniques are pre-requirement for micronized particles, repeated cycles, high energy technique and possibility of contamination that could occur from the metal wall of the container.

Many homogenization technologies has been either patented or patent application are pending such as Hydrosol (Novartis/ Patent No. GB 2269536), Nanocrystal™ (Elan Nanosystems/ Patent No. US 5145684), Dissocubes® (Skye Pharma/ Patent No. US 5858410), Nanopure (Pharma Sol/ Patent Application No. PCT/EP00/0635), NANOEDGE™ (Baxter/ Patent No. US 6884436).

Wet milling or media milling

In this method, nanosuspension are prepared by high shear ball mill or media mill. The size reduction within the chamber charged with drug, stabilizer (s) and water is carried out by both impact and attrition of particles. Low energy utilization, ease of scale-up, minimum batch to batch variation and capability to handle large quantities of material, and four approved Food and Drug Administration (FDA) drugs makes this process more attractive. The amount of material in the mill is of considerable importance since too much feed produces a cushioning effect and too little causes loss of efficiency and abrasive wear of the mill parts. The wear and tear of milling media may occasionally introduce residues in the finished product. However, this problem has been minimized by using highly crosslinked polystyrene resin milling medium. Extended milling process time can introduce more amorphous fraction into the materials, which may lead to instability. Wet-milling process to manufacture uniform sized nanosuspension using small hard zirconium dioxide beads have been described [ 63 ]. The X-ray diffraction analysis showed that the initial crystal nature as well as crystallinity of the active remained same even after the wet-milling process.

Dry co-grinding

Stable nanosuspension prepared by dry co-grinding technique using various polymers and copolymers such as polyvinyl pyrrolidone, hydroxypropyl methylcellulose (HPMC), polyethylene glycol, sodium dodecyl sulphate and cyclodextrin derivatives [ 88 ] have been described. Unlike wet grinding process, dry grinding methods are more economical and can be carried out without addition of any toxic solvents. The most significant effect of this method is the improvement of surface polarity and modification of major portion of crystalline state of the drug to amorphous form. Controlled stability of the amorphous phase could significantly improve the saturation solubility and hence dissolution rate of poorly soluble drug nanosuspensions [ 86 ].

Emulsion-solvent evaporation technique

An emulsion is formed by first dissolving drug in an organic solvent or cosolvents and subsequently dispersing in an aqueous phase containing a surfactant, which act as a stabilizer. Rapid evaporation of solvent under reduced pressure instantaneously produce nanosuspension. Key factors that should be considered during emulsification method are globule size and concentration of stabilizer.

Ultrasound assisted sonocrystallization method

It is a novel approach used for the preparation of stable nanosuspension. Ultrasound employed at the frequency range of 20–100 khz enhances particle size reduction and controls the size distribution of pharmaceutical active ingredient. It is also considered as an effective technique for minimizing the nucleation and crystallization process [ 80 ].

Miscellaneous methods

Nanosuspension of the drug can also be achieved by dilution of emulsion, thereby causing full diffusion of dispersed phase into the continuous phase resulting in the production of nanosuspension. Microemulsion can be treated in similar manner for the production of nanosuspensions. The effect of globule size and amount of surfactant (s) on the drug uptake of internal phase should be examined to produce optimum drug loading. Nanosuspension developed by such methods must be cleared from adhering solvents and other ingredients by means of ultrafiltration technique to make it convenient for administration. Lyophilization of the nanosuspensions shall be done to improve the physical and chemical stability and to overcome the incompatibilities between the various formulation components. Sterilization of the nanosuspensions can be done either by membrane filtration (< 0.22 μm), steam heat sterilization or gamma irradiation. Literature suggests that optimization of bottom up nanosuspension approach requires appropriate selection and setting suitable concentration of excipients such as surfactant and polymer [ 79 ].

• Formulation components

The most frequently used excipients in nanosuspensions are stabilizers, polymers, surfactants, osmotic agents, organic solvents, cryoprotectants, buffers, complexing agent, buffers, organoleptic agents and preservatives.

Stabilizers

It was reported in the literature that APIs carrying high log P and high enthalpy values have more practicability of developing a stable nanosuspension by both steric and electrostatic stabilization [ 19 ]. Further, physical properties such as molecular weight did not demonstrate to have a direct influence on the particle size or stabilization step. The zeta potential (ζ) measured at the shear plane determines the degree of repulsion between similarly charged adjacent particles in the system. Though, the magnitude of zeta potential typically signifies the physical stability of the nanosuspensions, it may not be a good indicator for stability achieved through electrostatic stabilization. This is because electrical properties at the interface is also due to the dissociation of charged functional groups on the particle surface influenced by both pH of the medium and pKa of the drug. The dynamic state of nanosuspension formation is a complex interaction involving various factors particularly surface free energy and functional groups. Faster surface adsorption rate of stabilizer in comparison to newly created surface during various preparation methods may dictates the stability of the nanosuspensions as well as efficiency of the selected technique.

The polymorphic and amorphous-crystalline transformation in nanosuspension can significantly change the solubility and clinical effect. Solid state stability involving crystal defects of API during milling process, percentage crystalline/amorphous content can be studied in comparison with physical mixtures by powder X-Ray diffractometer (XRD) supported by other physical characterization methods [ 25 ]. In contrast to XRD, small angle X-ray scattering can provide information about the size, shape, orientation, and crystalline and amorphous state of a variety of polymers, proteins, and nanomaterial bioconjugate systems in solution [ 51 ]. Thermal characteristics of the air/freeze dried nanosuspension powder is typically evaluated using modulated differential scanning calorimetry (Table 2 ). Maintaining the in vivo stability is critical to establish long-circulating characteristics and to achieve passive drug targeting through enhanced permeation and retention (EPR) effect. Since nanosuspensions are frequently prepared with aqueous medium, common chemical stability issues such as oxidation and hydrolysis must be addressed. Thus, the important function of stabilizer to prevent agglomeration or aggregation due to high surface energy of the nanoparticles. Any variation in particle size distribution and polydispersity index at different stages of nanosuspension such as during production, storage and stability conditions can be examined using various types of particle size analyzers technique (Table 2 ). Other key roles of stabilizer are wetting of hydrophobic drug particles, prevent Ostwald’s ripening and provide steric or ionic repulsion to make physically stable product. The concentration of stabilizer has significant impact on the physical stability and in vivo performance of nanosuspensions. Various types of stabilizers have been investigated such as cellulose derivatives, phospholipids, poloxamers, non-ionic surfactants and polyvinylpyrrolidone.

These synthetic polymers are generally regarded as safe (GRAS) by FDA for oral, parenteral and topical pharmaceutical applications. Most frequently used Poloxamer in nanosuspension is Poloxamer 188 and Poloxamer 407. Hydrophilic-lipophilic ratio, morphology, functional groups and molecular weight are the key factors, which determine the crystal growth and stability of nanosuspension [ 47 ].

Amino acid-based stabilizers

Leucine copolymers have been demonstrated to successfully produce stable drug nanocrystals in aqueous medium [ 45 ]. Lecithin is preferred as stabilizing agent for sterile, steam heat sterilizable parenteral nanosuspensions. Albumin has been employed as a surface stabilization and drug targeting at various concentrations as low as 0.003% up to 5% in nanosuspension [ 92 ]. Other pharmaceutically acceptable amino acid co-polymers used for the physical stability of nanocrystals were arginine, proline, and transferrin.

Cellulose based derivatives

HPMC, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC) have been widely used as stabilizing agent in nanosuspensions. The underlying mechanism of steric stabilization provided by these polymers is due to surface adsorbed hydrophobic groups [ 56 ].

D-α-Tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS)

Vitamin E polyethylene glycol succinate is an esterified, water soluble vitamin E (tocopherol) derivative used in many nanosuspension formulation as a stabilizing and solubilizing agent [ 21 ]. High physical stability, and low toxicity profile consider it as a most effective excipient for oral, ophthalmic and parenteral applications.

Miscellaneous

Soluplus® is a novel excipient made of polyvinyl caprolactam-polyvinyl acetate-PEG copolymer developed by BASF industries. It has been used as a stabilizer in many nanosuspension with enhanced stability and increased dissolution rate and bioavailability [ 74 ]. Water soluble polymers such as polyvinyl alcohol (PVA), polyvinyl pyrrolidone, and PEGylated chitosan used as stabilizers was found to significantly increase the dissolution rate and bioavailability of nanosuspensions. Functionalized surface coating on low soluble drug, beclomethasone dipropionate was carried out with hydrophobin, a protein-based surfactant. Adaptability for surface modification by genetic engineering make it suitable for different drug delivery applications [ 81 ].

Surfactant and co-surfactants

The selection of surfactant and co-surfactant are important, when nanosuspensions are prepared using microemulsions as template. It can influence the phase behavior, solubility of drug as well as drug loading in the internal phase. Different types of surfactants such as Tweens [ 37 ], sodium dodecyl sulphate [ 78 ] and co-surfactants such as bile salts, transcutol, glycofurol, ethyl alcohol and isopropyl alcohol were successfully used in the stabilization and development of nanosuspensions.

Organic solvents

Class three organic solvents with less toxic potential to humans such as ethyl alcohol, acetone, butanol, ethyl formate, ethyl acetate, ethyl ether, methyl acetate, methyl ethyl ketone, triacetin are preferred over conventional hazardous residual solvents. Water miscible organic solvents can be used as internal phase to solubilize drug substance, when nanosuspensions are prepared based on emulsion solvent evaporation technique.

Other ingredients

Complexing agent such as cyclodextrin derivatives have been explored for the improvement of dissolution and bioavailability of actives in nanosuspensions [ 31 ]. Nanosuspensions like coarse suspension might contain buffers, preservatives, osmotic agents, cryoprotectants, organoleptic agents, depending on the type, route and physicochemical nature of drug.

Nanosuspensions in drug delivery systems

Oral drug delivery.

Oral suspension ensures chemical stability while allowing liquid medication, which is preferred in geriatric and pediatric age groups. Other advantages are masking the bitterness of drugs, extending the duration of action of drugs, improving the aqueous solubility of poorly water-soluble drugs, dissolution and bioavailability enhancement of drugs. Further, if the drug is insoluble in water and other solvents are not allowed, then suspension is the preferred choice.

Nanosizing of drug can cause significant improvement in dissolution rate because of large increment of surface area and subsequent rise in saturation solubility. This leads to an increased dissolution velocity and concentration gradient across gastrointestinal tract causing increased absorption and significant enhancement of bioavailability. Thus, nanosuspensions are most beneficial for drug candidates belongs to class II and class IV as per the Biopharmaceutical Classification System (BCS). The bioavailability studies of poorly water soluble drug, fenofibrate formulated as nanosupension type, Dissocube® showed two-fold enhancement in terms of rate and extent compared to the reference formulation of micronized fenofibrate suspension [ 23 ].

The in vivo test of nitrendipine nanosuspension (~ 290 nm) in rats showed that the maximum concentration of drug ( C max ) and area under plasma-drug concentration time profile (AUC) were approximately 6 folds higher in comparison to marketed tablets [ 89 ]. Pharmacokinetic study demonstrated significant improvement in the oral bioavailability in rabbits by naringenin nanosuspension in comparison to naringenin solution [ 70 ]. Pharmacokinetic evaluation of fluvastatin nanosuspensions in rats showed 2.4 folds improvement in bioavailability in comparison to fluvastatin capsule control group [ 48 ]. Faster absorption from naproxen nanosuspension decreased time to reach maximum concentration (T max ) by nearly 50% and increased AUC to about 5 folds, when compared to conventional tablet and suspension [ 55 ]. The C max and T max of the prepared nanosuspension incorporated mucoadhesive buccal films of carvedilol was increased due to both enhanced surface area and by avoiding hepatic metabolism [ 71 ].

Selection and optimizing the concentration of stabilizers are important for the successful formulation and stabilization of nanosuspension. A combination of co-processed nanocrystalline cellulose-carboxy methyl cellulose was successfully used as a steric stabilizer in the preparation of baicalin nanosuspension [ 90 ]. Similarly, spray dried nanosuspension of itraconazole for bioavailability enhancement was stabilized by including poloxamer 407 or low viscosity (50 cp) HPMC. In vivo studies in rats revealed that AUC 0–36 calculated from dried itraconazole nanosuspensions was significantly higher (~ 2 folds) than the commercial Sporanox® beads in both fed and fasted conditions ( p  < 0.05).

Though, particle size reduction increases dissolution rate, enormous surface free energy possessed by nanoparticles can sometimes results in reduced uptake of drug. The aqueous solubility of the drug was enhanced with considerable improvement of bioavailability was exhibited after oral administration of amphotericin B nanosuspension [ 35 ]. Large surface area of nanoparticles can also lead to mucoadhesion, which can prolong gastrointestinal residence time and improve bioavailability. But the penetration to underlying mucosa can be affected by both surface charge and the nature of the surfactant [ 4 ]. Transcytosis of nanoparticles is adversely affected by the enteric coating after the initial adsorption by salivary proteins [ 17 ]. Nanoparticle uptake by rat gastrointestinal mucosa indicated that M-Cells in Peyers patches involves in lymphatic transport of drug to systemic circulation bypassing first pass metabolism by liver. This mechanism is beneficial for targeting lymphatic diseases or lymph mediated diseases. Lower inter-individual variability indicated that nanosuspension enhance the absorption from the targeted site of small intestine irrespective of food intake [ 60 ]. Despite the promising advantages of nanosuspensions, it is not applicable in situations, where bioavailability is compromised by biotransformation and/or permeation related problems particularly observed in class II and class IV drugs. Nanoengineering these crystals using various agent may minimize both permeation [ 3 ] and gut related metabolic issues [ 40 ].

Nanosuspensions offer potential advantages such as reduction of dose as well as cost of therapy, avoiding dose dumping in the body, minimizing fast/fed state plasma level fluctuation and intersubject variability. Sustained release technology has evolved remarkably during last several decades, but it is restricted to few hydrophobic drugs. Nanocrystals in nanosuspension have the capability to incorporate potentially all hydrophobic drugs in various sustained release methodologies. In addition, drug nanoparticles can be included in tablets, capsules and pellets using conventional manufacturing method.

Modification of liquid nanosuspensions to solid intermediate

Powders are the starting process material for most solid dosage forms such as granules, tablets and capsules. Nanosuspensions of poorly soluble drugs are often converted into different powder dosage forms for oral administration as shown in Table  3 . Nanosuspensions can be converted to dry powders either by freeze drying, spray granulation, vacuum drying, or spray drying techniques. The key challenge is in preserving the redispersibility of nanoparticles upon reconstitution of powder form with water or in gastric fluids. Powders are both the simplest dosage forms and the basis of many other solid dosage forms, such as tablets, capsules, and so on. Many drugs or ingredients are also in powder form before processing. A good redispersants should rapidly disperse agglomerates formed during the drying so that the original particle size is regained within a short span of time. Redispersants must be added previously to the nanosuspensions before drying step. Typical redispersants used are sucrose, trehalose, maltodextrin, lactose and mannitol, which is also used as cryoprotectant during lyophilization. In vivo pharmacokinetic evaluation of ritonavir nanosuspension in rats demonstrated a significant rise of C max and AUC 0 − t values, when compared to coarse powder and marketed product (Norvir®) evaluated among fed group human volunteers [ 27 ].

As a multiparticulate dosage forms, pellets offer number of advantages such as controlled release of drugs, release independent of gastric emptying rate, less chance of dose dumping, minimum local irritation, adaptability to accommodate different release profile, ability to combine different drugs and patient compliance.

Dried indomethacin nanosuspensions were prepared by spraying the nanosuspensions onto pellets by fluid bed coating method. Similar dissolution profiles were demonstrated between dried pellets with nanosuspension and drug nanosuspensions [ 24 ]. Pellets incorporating nanosuspension of ketoprofen for sustained release of drugs up to 24 h duration has been disclosed [ 82 ]. Mucoadhesive hydrocortisone nanosuspension was produced by high pressure homogenization method. Pellets were initially spray coated with nanosuspensions and further coated with enteric polymers to maintain a controlled drug release. In vitro dissolution studies demonstrated an enhanced dissolution rate and drug release from the nanocrystals present in the pellets [ 59 ]. It was suggested that spray coating of nanosuspension containing water soluble binder to pellets followed by enteric coating is most likely to control the release rate for high dose drugs. Similarly, pellets prepared by spheronization-extrusion technology with same methodology to make matrix cores can be applied for low dose drugs [ 58 ].

Pharmaceutical tablets are solid unit dosage form containing drug substances usually prepared with the aid of suitable pharmaceutical excipients by compression. In most of the investigations, granules were prepared by either freeze drying or spray drying techniques. Nanosuspension after conversion to dry powder by these methods can be compressed as tablets by molding or compression. Nanosuspension can also be converted to tablet form by directly freeze drying the nanosuspension in a blister pack. Since the stresses during freezing and drying cycles are diverse, multiple stabilizers such as sugars, sugar alcohols, polymers and amino acid are often used to contribute adequate protection and stability which are essential for the drug (s). These excipients are frequently combined to build either amorphous or crystalline structure of freeze dried nanosuspension.

Unlike in case of electrostatic stabilization, steric stabilizers are comparatively unaffected in presence of electrolytes and other excipients added during the formulation of tablets. Thus, the selection of appropriate steric stabilizers based on the properties of API can produce a stable nanosuspension at varying pH of gastrointestinal tract after oral administration. A zeta potential value of around − 20 mV can be considered as ideal for a nanosuspension system stabilized by both steric and electrostatic methods [ 85 ]. Nanocrystals in a tablet above certain limit can aggregate to a larger extent under the compressive force during tableting process. This can decrease the dissolution velocity and bioavailability particularly poorly water soluble BCS II drugs. Formulation of low dose drugs with total nanoparticle content less than 1% relative to total tablet weight is effective in releasing it as fine nanosuspension to the solution [ 9 ].

Naproxen granules were prepared from nanodispersion by spray drying technique and tablets were prepared by compressing the same with a bulking and stabilizing agent, mannitol and a disintegrating agent. It was found that the dissolution of the nanodispersion was finished within 1 min under sink and non-sink conditions [ 7 ]. Spray drying technology has also been used to prepare nanocrystals of lovastatin, a poorly soluble drug from nanosuspension using 20% polyvinyl pyrrolidone K17 and 5% sodium lauryl sulphate as stabilizers. Optimized sustained release tablets were prepared using lactose (diluent), Avicel PH101 (compressing agent) and Ac-Di-Sol (superdisintegrant) [ 93 ]. Increased dissolution has been reported with oral nanosuspension tablets of nebivolol hydrochloride, a poorly water-soluble drug. Enhanced dissolution rate is due to the transformation of crystalline form of the drug to amorphous state, which was later proved by X-ray diffraction studies. In a similar study, orally disintegrating tablets piroxicam tablets were prepared using nanosuspensions to which poloxamer 188 was used as stabilizer, showed a superior dissolution rate compared with the orally disintegrating tablets prepared from coarse piroxicam [ 42 ]. Increased dissolution rate is owing to increased surface area associated with nanosized drug particles. In another study, spray dried nanosuspension of risperidone orally disintegrating tablets showed improved dissolution rate in comparison to marketed orally disintegrating tablets [ 62 ]. In a continuation of the study, the effect of different excipients on the piroxicam dissolution properties was investigated. It was established that gelatin or croscarmellose as excipient exhibited a faster piroxicam dissolution rate, when compared with the marketed formulation and orally disintegrating tablets formulated using xanthan gum. This study additionally implies the significance of different excipients used in the formulation.

Including polymers like Poly (DL-lactide- co -glycolide) and complexing agent such as cyclodextrin are a well-known approach to enhance the biopharmaceutical performance of poorly soluble drugs. It was confirmed that concentration of nanosuspension, spraying rate and atomization air pressures are the key factors that influence the various physicochemical properties of granules such as redispersibility and particle size distribution [ 28 ].

Novartis compound A and itraconazole nanosuspension dried as powders and subsequently filled in capsules was found to enhance bioavailability after oral administration in beagle dogs [ 6 ] and rats [ 39 ], respectively. In vitro dissolution test and in vivo studies in rats demonstrated a marked improvement in bioavailability of glimepiride nanocrystal-loaded capsules compared to the marketed formulation [ 15 ].

Oral films or orodispersible films have many advantages than other oral dosage forms since it undergoes quick disintegration and dissolution in the oral cavity, rapidly delivers the drug across oral mucosa bypassing hepatic metabolism and resulting enhancement of bioavailability.

Buspirone fast dissolving oral films was prepared from nanosuspension by solvent evaporation method using film forming agents HPMCE5 and PVA. Buspirone oral film showed excellent physicomechanical characteristics, good stability and in vitro studies exhibited burst release followed by sustained drug release [ 5 ]. It was anticipated that incorporating nanoparticles in fast dissolving oral films can increase both dissolution and permeability characteristics of many poorly aqueous soluble drugs. Fast dissolving oral films incorporating nanoparticles of lercanidipine, a poorly aqueous soluble and low bioavailable drug were prepared via antisolvent evaporation method. Significant enhancement of i n vitro dissolution rate and ex vivo steady state flux was noticed from the formulations [ 11 ].

Feasibility studies of low bioavailable, quercetin fast dissolving oral films prepared using maltodextrins as film forming material and glycerin as plasticizer indicated that the inclusion of nanocrystals did not influence the elasticity and ductility. The dissolution rate was found to be better than that of bulk drug [ 41 ]. Pharmacokinetic studies of lutein nanocrystals fast dissolving oral films in rats indicated a major reduction of T max and considerable increase of C max compared to oral solution. Further, the AUC 0-24h of nanocrystal fast dissolving oral films was ~ 2-folds larger than that of the oral solution thereby confirming the drastic improvement of both rate and extent of bioavailability [ 50 ].

Recently, nanosuspension based mucoadhesive film was prepared with carvedilol nanosuspension containing layer held between mucoadhesive and backing layers. Nanosuspension was incorporated into hydrogel prepared from HPMC and Carbopol 934P using PEG400 as plasticizer. In vivo studies performed in rabbits displayed significant rise in the relative bioavailability, when compared to commercial tablets [ 71 ].

Parenteral drug delivery

Intravenous and intraspinal preparations are seldom given in a form other than aqueous solutions. The danger of capillary blockage, particularly in the brain deter the use of other forms via intravenous administration. Though, microemulsions have been used such as total parenteral nutrition, particle size of the dispersed phase is rigidly controlled below 5 μm. Parenteral products can be given as solutions, suspensions, or emulsions through either intramuscular, subcutaneous or transdermal route of administration.

Injections of poorly aqueous soluble is challenging and often formulated with cosolvents, solubilizing agent, selecting suitable salt forms and complexing agents such as cyclodextrin. Due to the limitation associated with the excessive use of toxic cosolvents, solubilizing agent and complexing agents, these techniques suffer from lack of solubilizing power and parenteral acceptability. In this context, nanosuspension can be best alternative in such conditions since all hydrophobic drugs can be nanosized while circumvent all the problems frequently encountered during formulation of parenteral products. Improvement in bioavailability facilitate dose reduction, cost effectiveness of therapy without affecting therapeutic outcome of the drug.

Safety profile of the paclitaxel nanosuspension was increased many folds higher than the commercial taxol injection, which employs cosolvent mixtures to solubilize the drug. Paclitaxel nanosuspension resulted no death at maximum dose of 100 mg/kg whereas taxol at dose of 30 mg/kg demonstrated a death rate of 22% in human lung xenograft murine tumor. Therapeutic efficacy of paclitaxel nanosuspension was enhanced in comparison to taxol employing transplantable mouse 16/c mammary adenocarcinoma as a model [ 55 ].

Cytotoxicity studies indicated that superior cytotoxicity in Hela and MCF-7 cells with curcumin nanosuspension than curcumin solution. Small particle size (~ 250 nm) has increased dissolution rate and retention of crystalline nature improved physical stability of curcumin. Further, the safety evaluation studies with nanosuspension demonstrated minimum local irritation, allergic reaction, phlebitis and decreased rate of red blood cell lysis [ 18 ].

Improvement in stability and efficacy was noticed with aphidicolin, a low aqueous soluble antiparasitic drug [ 34 ] and hydrophobic antileprotic drug, clofazimine, when formulated as nanosuspension [ 67 ].

It is important to note that soon after parenteral intake, nanosuspension undergoes opsonization and phagocytosis due to uptake by Kupffer-Bowcisz cells located in liver [ 57 ]. The natural uptake by these cells serve as reservoir or depot and thereby control or prolong the duration of action of the drug [ 76 ]. Targeting to macrophages using antibiotic nanosuspension is beneficial since many pathogens subvert the process of phagocytosis and replicate inside macrophages. In contrast, various research investigations are in progress to avoid uptake by macrophages by varying the size, shape and surface properties of nanosuspensions.

Surface properties of the nanosuspension can be modulated in order to change the protein adsorption patter. Factors like physicochemical characteristics of the drug particles, dose, duration of administration, drug-protein binding properties, solubility in body fluid pH affect the biodistribution and pharmacokinetic evaluation of the nanosuspension after parenteral administration.

Pharmaceutical composition for the intravenous administration of sparingly soluble staurosporine derivative N-benzoyl- staurosporine nanaosupension having particle size 5–20 nm has been patented [ 87 ]. The key excipients included in the composition are polyoxyethylene/ polyoxypropylene block copolymer, Poloxamer 188, phospholipid surfactant, lecithin, water soluble osmotic agent, mannitol and co-solvents, water and ethanol.

Sustained release natural progesterone nanosuspension was developed and then successfully dispersed in a thermosensitive gel matrix. It was demonstrated that sustained release action after intramuscular injection in rats was continued up to 36 h and thus anticipated to provide a much safer alternative for semi-synthetic progesterone [ 73 ].

Safety and efficacy of long acting GSK1265744 (744) and rilpivirine (TMC278) nanosuspension were evaluated after multiple intramuscular dosing in healthy subjects. All volunteers achieved the steady state plasma concentration within 3 days and clinical data proved the potential use of long acting 744 and rilpivirine injection in HIV-1 treatment [ 77 ].

Significant reduction in the viability of undifferentiated/anaplastic thyroid cancer cell line was demonstrated, when tested with docetaxel (100 and 1000 μM/ml) loaded nanoparticles stabilized by 5% sodium deoxycholate [ 30 ].

Ophthalmic drug delivery

Low ocular bioavailability of many drugs from conventional ophthalmic drug delivery system is largely due to the major anatomical, physiological and physicochemical barriers of the eye. Nanosuspensions offer a means of administering increased concentrations of poorly soluble drugs and extended residence time to targeted site of cul-de-sac. Nanosuspension can address many problems of conventional suspensions such as poor intrinsic and saturation solubility in lachrymal fluids, low ocular bioavailability and irritation due to the large particle size. Further, it can avoid high osmolarity generated by ophthalmic solution dosage forms. Nanosuspension due to its peculiar nature, can improve the saturation and inherent solubility of hydrophobic drugs in lachrymal fluids while allowing sustained release and prolonged residence time in cul-de-sac.

Poor ocular bioavailability of many drugs from conventional eye drops is chiefly due to the physiological barriers of the eye. Investigations of poly (lactic-co-glycolic acid) based sparfloxacin ophthalmic nanosuspension demonstrated an improvement in precorneal retention time and ocular permeation. In addition, the developed lyophilized nanosuspension was found to be more stable than conventional marketed formulations [ 22 ]. Nanosuspensions shall be dispersed further in selected ointment, hydrogel or mucoadhesive base to control the release and residence time depends on the physicochemical nature of the drug.

Long term disease management of fungal infections in the eye is a challenging task because of the incapability of the delivery system to provide adequate drug distribution without affecting intraocular structures and/or systemic drug exposure. In vitro release studies of antifungal drug, amphotericin loaded Eudragit RS-100 nanosuspension (150–290 nm) is proposed as an efficient vehicle for delivery for 24 h and no ocular irritation was observed after topical instillation into rabbit’s eye [ 13 ]. Poly (methyl methacrylate) polymers like Eudragit® RS100 based nanosuspensions containing piroxicam has been successfully tested for ocular delivery in endotoxin-induced uveitis [ 1 ] and Eudragit RL100 polymeric nanosuspension loaded with sulfacetamide was successfully evaluated for ocular delivery [ 53 ]. Polymethacrylate polymer such as Eudragit RS 100 and RL 100 was used for the preparation of ophthalmic nanosuspensions of flurbiprofen and ibuprofen [ 8 ]. Nanosuspensions were evaluated for drug content, particle surface charge, particle size distribution, in vitro drug release and ocular irritation. Comparison between commercial ophthalmic suspensions demonstrated far superior in vivo performance of prepared ophthalmic nanosuspension.

Application of conventional dosage forms such as ointment, solutions and suspension are limited due to poor ocular bioavailability and different anatomical and pathophysiological barriers existing in the eye. Recent developments and findings of various nanoparticulate systems like nanosuspensions can be appropriately exploited to apply in ocular drug delivery system.

Pulmonary drug delivery

Potentially, nanosuspensions can minimize many problems associated with conventional dry powder inhalers and suspension type inhalation aerosols. Conventional aerosols have many limitations such as limited diffusion and dissolution in the alveolar fluids, rapid clearance and short residence time due to ciliary movement, deposition in pharynx and upper respiratory tract due to agglomeration and aggregation of the particles [ 32 ]. Nanoparticulate nature of the drug can offer quick onset of action due to rapid diffusion and dissolution in the alveolar fluids. Furthermore, it can sustain the release of drug because of its increased affinity to the mucosal surfaces. Antioxidant coenzyme Q10 nanosuspension stabilized with PEG32 stearate demonstrated maximum respirable fraction (70.6%) having smallest mass median aerodynamic diameter (3.02 μm) in comparison to nanosuspension stabilized with lecithin and Vitamin E TPGS. In vitro cellular toxicity carried out utilizing A549 human lung cells showed no noticeable cytotoxicity for the nanosuspension [ 72 ]. Due to the unique physicochemical characteristics, uniform and narrow size distribution of the nanoparticles, it is unlikely to cause uneven drug distribution and drug delivery in lung, when compared to microparticulate inhalation aerosols. The lung distribution rate of hydrophobic budenoside nanosuspension was found to be very high (872.9 ng/g) and exceptionally different ( p  < 0.05), when compared to coarse drug particles ( p  < 0.01) and micronized drug particles [ 94 ].

Conversion of nanosuspensions to solid oral and inhalable dosage forms lessens the physical instability associated with their liquid state and enables targeted drug delivery. Most frequently used solidification methods include spray and freeze- drying techniques. Redispersibility of solid nanocrystalline formulations is essential for potential oral and pulmonary clinical application [ 52 ].

Targeted drug delivery

Nanosuspensions have the potential capacity to investigate as targeted drug delivery system by surface modification in order to circumvent the phagocytosis by macrophages. Active or passive targeting using stealth nanosuspensions using various surface coatings is an interesting area to be explored. Significant difference in the efficacy of buparvaquone, an antiparasitic nanosuspension has been noticed, when administered in presence or absence of mucoadhesive polymer [ 61 ]. Because of the prolonged accumulation of drug at the targeted site, drastic decline in the infectivity episode was exhibited by nanosuspension with mucoadhesive polymers. A comparative bioadhesion study was carried out between micro and nano carriers to the mucosa of colon [ 44 ]. It was found that nanosized carriers has the potential capability for targeted delivery of drugs to the inflamed colonic mucosa case of inflammatory bowel disease. Studies carried out with budenoside nanosuspension coated with Pluronic F-127 showed significant reduction of local colorectal tissue inflammation as confirmed by decreased inflammatory macrophages and IL-β producing CD11b + cells [ 14 ]. Recently, mucus permeating budenoside nanosuspension enema for targeted delivery to inflammatory bowel disease was developed [ 14 ]. Mucosal penetration was aided by surface coating with water soluble or hydrophilic polymers, most typically polyalkylene oxide polymers or copolymers such as Poloxamer F127.

Pulmonary targeting of amphoteric B as nanosuspension is reported to be more effective than stealth liposome in conditions of aspergillosis [ 38 ]. Most probably, similar strategy using nanosuspensions can be utilized to target gastrointestinal sites such as duodenum, colon, rectum that are susceptible to bacterial infection such as Helicobacter pylori or fungal infections due to Candida albicans . Two types of biodegradable, lipid based nanosuspensions was developed, a poly (ethylene glycol)- modified docetaxel-lipid-based-nanosuspensions to enhance the cycle time of the drug inside the tumor site and docetaxel-lipid-based-nanosuspensions employing folate as the target ligand. Data from the in vivo antitumor efficacy and biodistribution studies indicated that docetaxel-lipid-based-nanosuspensions demonstrated higher antitumor efficacy significantly by subsiding the tumor volume ( P  < 0.01) as a result of increased residence time of the drug within the tumor [ 84 ].

Though, nanosuspension technology is undergoing rapid expansion during last few decades, the in vivo safety and health implications of nanosuspension should be seriously considered during development and scale-up [ 83 ]. Nanosuspension has the potential ability to cross blood-brain barrier due to nanosize range and hydrophobicity of the drug. Donepezil loaded nanosuspension as a potential brain targeted drug delivery system for Alzheimer’s disease was tested in Sprague–Dawley rats. The nanosuspension with particle size ranges between 150 and 200 nm was administered to brain via intranasal administration. The C max showed significant ( p  < 0.05) concentration of donepezil in brain (147.54 ± 25.08 ng/ml) in comparison to suspension (7.2 ± 0.86 ng/ml), at an equal dose of 0.5 mg/ml [ 54 ]. In another study, amphotericin B nanosuspension was developed as a brain delivery system. Results indicate that nanosuspensions coated with Tween-80 and sodium cholate enhanced penetration to brain and remarkably inhibited encephalitis causing amoeba, Balamuthia mandrillaris, in vitro, however showed low in vivo inhibitory activity [ 46 ].

Targeted drug delivery into the vaginal tract to prevent pre-term birth rate exploiting mucoinert progesterone nanosuspension was developed. Characteristic plasma progesterone double peak noticed in humans was also observed in pregnant mice after vaginal administration thus confirming uterine recirculation [ 26 ]. Passive targeting through PEGylation and coating with polysorbate-80 can cause deposition of apolipoprotein E on the polymeric nanoparticles, which will promote brain uptake by endothelial cells.

Transdermal drug delivery

Diclofenac sodium, a non-steroidal anti-inflammatory drug was successfully dispersed into isopropyl as a nanosized solid-in-liquid suspension via complex formation using surfactant, sucrose erucate. The resultant formulation enhanced the steady state flux of the drug up to 3.8-fold when compared with the control in Yucatan micropig skin model. The size of the nanosuspension was found to depend on the weight ratio of the surfactant and the average diameter of the nanoparticles was 14.4 nm [ 69 ].

Tretinoin nanosuspension and nanoemulsion was investigated to check the feasibility for dermal and transdermal targeting. In vitro diffusion studies and in vivo studies in pigs showed that tretinoin transdermal permeation was superior in nano-emulsion formulation and photostability was considerably improved in nanosuspension [ 43 ].

Transdermal delivery of methotrexate coated with non-ionic surfactant and stabilized by l-arginine was tested by solid-in-oil technique. The transdermal efficiency for the solid-in-oil nanosuspension was 2–3 folds than the control aqueous solution. On account of smaller particle size (< 100 nm), the oil-based nanosuspension is more efficient in permeating the stratum corneum [ 91 ]. The performance of ibuprofen nanosuspension and effect of varying concentrations of the solubilizer, Vitamin E TPGS to increase the permeation rate across the skin has been investigated. It was found that the entire transdermal permeation across the skin was mainly dictated by the size of nanoparticles and concentration of solubilizer [ 20 ].

Future perspectives and challenges

The future prospects of nanosuspension are encouraging since they can contribute as a valuable tool for product development scientists to overcome various formulation and drug delivery challenges particularly with intractable drugs. Regardless of the several published research in the area of nanosuspension, the critical aspects of stability issue pertaining to nanosuspension is still unresolved. The stabilization capability of the electrostatic and steric stabilizers and its relationship with the properties APIs, attainable maximum particle size and resulting physical stability are the critical factors need to be further investigated. Advancement in biotechnology and aid of modification tools such as antibody-drug conjugate and nanobodies will probably create subcutaneously administered, high concentration monoclonal (mAb) and biosimilar products with enhanced biopharmaceutical and safety characteristics. Recently, Johnston et al. [ 33 ] have developed biologically active, nanocluster dispersions of antibodies in solution (~ 250 mg/ml) using carbohydrate stabilizers like trehalose. Future development of enabling technologies like nanosuspension will provide technical solutions to many formulation challenges currently faced by protein and peptide based drugs.

Conclusions

Nanosuspension can be considered as the best formulation option for inflexible hydrophobic drugs restricted by high log P, molecular weight, melting point and dose. Conventional size reduction operations such as wet milling and homogenization and formulation approaches such as precipitation, emulsion-solvent evaporation, solvent diffusion and microemulsion techniques can be successfully utilized to prepare and scale-up nanosuspensions. Substantial improvement of bioavailability due to increased saturation and intrinsic solubility, appreciable mucoadhesivity, adaptability for surface modification in drug targeting have broadly expanded the scope of this novel formulation. Nanosuspension has the potential to consider as a valuable tool for formulation scientist to overcome many formulation and drug delivery challenges pertaining to various drug entities. The application of nanosuspensions in oral, ocular and pulmonary drug delivery systems have been extensively researched during the last few decades. Further, utilization of nanosuspensions in other drug delivery systems such as brain, topical, buccal, nasal and transdermal routes are under extensive investigation. Though nanosuspension has received serious consideration from pharmaceutical scientists, the exact mechanisms of stabilization, solidifications and redispersibility of dried nanosuspension are yet to be explored.

Availability of data and materials

Please contact author for data requests.

Abbreviations

Area under plasma-drug concentration time profile

Biopharmaceutical Classification System

Maximum concentration of drug

Enhanced Permeation and Retention Effects

Food and Drug Administration

Generally regarded as safe

Hydroxyethyl cellulose

Hydroxypropyl cellulose

Hydroxypropyl methyl cellulose

Polyvinyl alcohol

D-α-Tocopheryl polyethylene glycol 1000 succinate

X-Ray diffractometer

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Jacob, S., Nair, A.B. & Shah, J. Emerging role of nanosuspensions in drug delivery systems. Biomater Res 24 , 3 (2020). https://doi.org/10.1186/s40824-020-0184-8

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Nanosuspension: Principles, Perspectives and Practices

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• 1 School of Pharmaceutical Sciences, Lovely Professional University, Phagwara-144411, Punjab, India. [email protected].
• PMID: 26721266
• DOI: 10.2174/1567201813666160101120452

In the last three decades, nano-sizing of hydrophobic drugs has emerged as one of the most commonly used strategies to overcome their solubility and bioavailability related issues. Nanosuspensions offer versatile features and unique advantages over other approaches that have been utilized for this purpose. The unique inherent properties of nanosuspensions have been explored for a wide variety of applications. Commercial production of stable nanosuspensions has been made possible by the use of techniques such as media milling and high pressure homogenization. This article reviews various techniques being employed for production, characterization, merits and limitations of nanosuspensions and mechanisms that play a role in the physicochemical stability of nanosuspensions. The common strategies applied so far to overcome their stability and commercialization related aspects are also highlighted.

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NANOSUSPENSION TECHNOLOGY: A REVIEW

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Nanocrystals and nanosuspensions: an exploration from classic formulations to advanced drug delivery systems

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Nanocrystals and nanosuspensions have become realistic approaches to overcome the formulation challenges of poorly water-soluble drugs. They also represent a less-known but versatile platform for multiple therapeutic applications. They can be integrated into a broad spectrum of drug delivery systems including tablets, hydrogels, microneedles, microparticles, or even functionalized liposomes. The recent progresses, challenges, and opportunities in this field are gathered originally together with an informative case study concerning an itraconazole nanosuspension-in-hydrogel formulation. The translational aspects, historical and current clinical perspectives are also critically reviewed here to shed light on the incoming generation of nanocrystal formulations.

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Nanocrystals for Delivery of Therapeutic Agents

State of the art of nanocrystals technology for delivery of poorly soluble drugs.

Drug Nanocrystals

Avoid common mistakes on your manuscript.

Introduction

An increasing number of new drug candidates are characterized by an extremely low solubility, belonging to class II or class IV of the Biopharmaceutics Classification System (BCS) [ 1 , 2 ]. This dramatically hampers their drugability unless they enter into a tedious reformulation process. The significant progress of nanotechnology has also provided new opportunities for the task over classic solubilization strategies such as salt formation, co-crystal formation, or cyclodextrin complexation. Among the different applications in the field, the conceptualization of nanocrystals (i.e., a crystalline drug with a particle size below the micrometer scale) and/or nanosuspensions (i.e., crystalline, semi-crystalline or amorphous drug aqueous nanosuspensions) has become a feasible approach to overcome the formulation challenges of poorly water-soluble drugs [ 3 , 4 ].

Nanocrystals and nanosuspensions are crystalline or partly amorphous nano-sized structures of active compounds obtained through two main methods: one involves size reduction of larger crystals mainly by milling or high-pressure homogenization techniques (known as the top-down approach), whereas the other involves precipitation of dissolved molecules mainly by crystallization or spray-drying (referred to as the bottom-up approach) [ 5 , 6 ]. One of their significant and well-known advantages is their high specific surface area. According to the Noyes-Whitney equation, this favors a faster dissolution; and additionally an increased saturation concentration (Freundlich-Ostwald relation) [ 7 , 8 ]. This, together with a very high drug loading, and a nanosized particle distribution has promoted their implementation in formulation development [ 4 , 9 ]. Taking advantage of these assets, formulation scientists have been mainly focused on improving the oral bioavailability of poorly water-soluble drugs over the last two decades [ 6 , 9 , 10 , 11 ]. Literature inputs various reports on this application with particular emphasis on the formulation methodology and solid-state characterization of nanocrystals (see for instance the following review articles [ 6 , 12 , 13 ]). However, there exists nowadays a growing interest in exploring other opportunities. This not only entails other administration routes but also a search for alternative strategies to improve current treatments lacking efficacy. Nanocrystals or nanosuspensions represent in fact a less known but versatile platform for multiple applications. They possess a high particle engineering potential and can be linked to a broad spectrum of drug delivery systems (Fig.  1 ).

This review article aims to provide an up-to-date, comprehensive, and cross-disciplinary overview of some of the latest improvements in the field of nanocrystal technology for different applications. Particular attention is given for the first time to their integration in different drug delivery systems, ranging from classic nanosuspensions or tablet formulations to novel microneedles or targeted nanocrystals. In order to illustrate further the potential of these approaches, and the technicalities, we have included an informative and original case study dealing with the formulation and characterization of a nanosuspension-hydrogel of the poorly water-soluble drug itraconazole. To conclude, the historical and current perspectives in the clinic are addressed. The section contains a critical view with relevant information about the challenges and opportunities of these technology approaches. All the insights gathered will help researchers, formulators, and clinicians to comprehend better their current translational reality.

Schematic representation of formulations and drug delivery systems recently proposed for drug nanocrystals and nanosuspensions matched with their corresponding administration routes (see color code). The topical route includes ocular delivery. Nanosuspensions have been implemented in the past for pulmonary drug delivery. (A) Nanosuspensions, (B) tablets, (C) hydrogels, (D) microneedles, (E) microparticles, and (F) coated nanocrystals and liposomes. (Designed by Biorender)

Literature data collection and analysis

Literature review.

For the selection of publications presented in Sect.  3 , a MEDLINE Boolean literature search was mainly conducted using the keywords “nanocrystals” OR “nanomilling” AND “application” AND “formulation”. Papers and reviews with the mentioned contents and published over the last 5 years were included in the review process. Only the most relevant studies and majorly containing in vivo studies are highlighted in the text.

Clinical trials research in Sect.  5 included studies exclusively on drug nanocrystals and nanosuspensions. Data was extracted from the database ‘clinicaltrials.gov’, powered by the United States National Library of Medicine. In Sect.  5 , Fig.  4 F, the total number of publications per year was extracted from MEDLINE using the following Boolean search query: “nanocrystal” OR “nanosuspension” AND “drug” NOT “nanoparticle”.

Formulation and characterization of an itraconazole nanosuspension

Size reduction was performed using a homogenizer (Precellys 24 ® ) from Bertin Instruments (Montigny-le-Bretonneux, France). Itraconazole (Parchem, New Rochelle, NY, USA) was added to a 2-mL tube with the stabilizer poloxamer 407 (Sigma-Aldrich, Saint Louis, MO, USA) at a drug-to-stabilizer w/w ratio 1/0.1. Then, 1 g of 0.5-mm zirconium oxide beads (Next Advance, Troy, NY, USA) and 1 mL of Milli-Q water were added. The complete sequence consisted of 50 cycles of 70 s at 6,600 rpm. The obtained nanosuspension was also loaded in a preformed hydrogel containing 1.9 MDa hyaluronic acid (Bloomage Biotech, Jinan, China) at 2% w/v. Briefly, the aqueous nanosuspension was added to the gel and mixed gently until a homogeneous blend was obtained at the final drug concentration of 0.5 mg/mL.

Particle size distribution by specific surface density was determined using a Mastersizer 3000 (Malvern, Worcestershire, UK) laser diffraction equipment. Particle population distribution by intensity and mean particle size diameter expressed as mean Z-average was determined by dynamic light scattering (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK). The refractive index was set at 1.33 and the scattering angle was 173°. Particles’ morphology was visualized using a JSM-7001 F emission scanning electron microscope (SEM) (JEOL, Tokyo, Japan). Samples were previously sputter-coated with gold and all micrographs were acquired at a 15 kV voltage.

In vitro dissolution studies were performed in a HCl (Acros Organics, Geel, Belgium) 0.1 N solution to increase the solubility of itraconazole. Suspensions were incubated at a concentration of 10 µg/mL in an orbital shaker at 80 rpm at 37 °C. At allotted time points, each tube was centrifuged at 4,000 rpm at 37 °C for 5 min to recover supernatants. Samples were centrifuged at 10,000 rpm at 37 °C for 10 min and then filtered using 0.22 μm centrifuge tube filters prior to quantification. Drug quantification was performed using an Acquity™ Ultraperformance LC (UHPLC) coupled to a photodiode array (PDA) detector (Waters Corp, Milford, MA, USA). Samples were separated using an Acquity UPLC ® BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters, USA) equilibrated at 40 °C. A 2-min gradient elution at a constant flow rate of 0.5 mL/min was performed using a mobile phase starting at 50% of a 0.05% (v/v) formic acid (Reactolab SA, Servion, Switzerland) aqueous solution and 50% of 0.05% (v/v) formic acid acetonitrile (Sigma-Aldrich, Saint Louis, MO, USA) solution. Wavelength detection was set at 262 nm on the photodiode array (PDA) detector. Peak due to itraconazole eluted at 1.27 min. Lower limit of quantification value was set according to signal/noise ratio > 6 and linearity R2 > 0.990 was obtained for the calibration curves ranging from 0.05 to 8 µg/mL. Statistical comparison between different groups was performed using One-Way analysis ANOVA (Tukey’s multiple comparison test).

Formulations and drug delivery systems proposed for nanocrystals

Nanosuspensions and tablets.

Suspensions of nanocrystals (or nanosuspensions) represent the most common, simple, representative, and precursory formulation of this family (Fig.  1 A). 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 AUC 0−∞ 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 , 16 , 17 ] but also others such as the ocular [ 18 , 19 , 20 , 21 ], nasal [ 22 , 23 ], topical [ 24 , 25 ], intra-articular [ 26 ], intra-muscular delivery [ 27 ] and even the inhalation 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.  1 B) [ 29 , 30 , 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 AUC 0−∞ 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 [ 33 ]. 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 [ 34 , 35 ]. 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.

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.  1 C). 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 [ 36 , 37 , 38 ]. 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 [ 39 , 40 , 41 , 42 , 43 , 44 ]. 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 [ 45 ]. 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 [ 41 ]. 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 [ 46 ]. 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 [ 47 ]. 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.

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 [ 48 , 49 ]. 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 [ 50 ]. Microneedles, or microarray patches, are micron-sized spikes used to deliver active compounds transdermally (Fig.  1 D). They can pierce and bypass the stratum corneum by forming micron-sized pores in the skin, directly delivering the drug into the dermal tissue [ 51 ]. 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 [ 49 , 50 , 52 , 53 , 54 , 55 ].

Vora and colleagues were in the first ones to incorporate vitamin D3 into dissolvable microneedles [56]. First, they used a sonoprecipitation method to obtain a 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

[ 57 ]. 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 (human immunodeficiency virus)-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 C max 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 formulations can improve the transdermal delivery of active compounds at high doses. In addition, other routes of administration could benefit from microneedle systems containing nanocrystals to improve both drug penetration and sustained release. In fact, recent investigations in microneedle-mediated minimally invasive intra-ocular delivery have shown promising outcomes [ 58 ]. This technology is also 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.

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.  1 E) can provide an additional biodegradable solid shell that modulates and prolongs the release kinetics [ 59 , 60 , 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. The formulation procedure shown in Fig.  2 A was recently implemented for the encapsulation of the GLPG0555 in collaboration with Galapagos NV (Mechelen, Belgium) [ 64 ]. A representative cross section micrograph showing the internal structure of this type of particles is also shown in Fig.  2 B.

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 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.

(A) Schematic representation of a spray-dryer and the formulation of nanocrystals (NCs) loaded in polymeric microparticles (MPs). Briefly, the feed solution containing the polymer poly(lactic-co-glycolic acid) (PLGA) dissolved in an organic solvent together with nanocrystals in suspension is sprayed into a pre-heated chamber. Solvent evaporation from droplets leads to NC-MP formation and collection. (B) Scanning electron microscopy representative micrograph of a NC-MP cross section under investigation by our research group. Magnification is x10,000. For further details about the formulation approach and sample preparation visit [ 64 ]

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 [ 65 ]. Some studies have recently investigated the incorporation of nanocrystals into liposomes (Fig.  1 F). Drug nanocrystals can be incorporated or formed in situ within the lipid layers [ 66 , 67 , 68 ]. Li et al. developed in situ forming nanocrystals of ciprofloxacin in liposomes for oral delivery [ 69 ]. 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 [ 70 , 71 ]. Alternatively, a two-step method was proposed for a targeted liposomal delivery system model for hydrophobic antitumoral drugs in another study [ 72 ]. 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 [ 73 , 74 , 75 ]. Park et al. published in 2017 a study using albumin-coated nanocrystals of paclitaxel to improve the therapeutic outcomes for solid tumors [ 76 ]. 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 ® ). In a recent study, polydopamine coated paclitaxel-PEG nanocrystals were dispersed in electrospun nanofibers. This innovative nanocrystalline-in-nanofiber implant device showed improved anti-tumor efficacy in a murine cervicovaginal tumor model together with a prolonged vaginal residence, higher transmucus penetration and minimal mucosal irritation [ 77 ].

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 [ 78 ]. For instance, the well-known anticancer nanomedicine Doxil ® is reported to display stabilized recrystallized doxorubicin inside the liposomes [ 79 ]. All these upgrades might prompt the consolidation of safe and effective parenteral administrations of nanocrystals, and this can be also applied to others such is the case of the ocular delivery [ 80 ]. 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.

Case study: development of an itraconazole nanosuspension

The present paper includes an informative practical case. It is meant to exemplify the simplicity, versatility and technicalities of the approaches described in the previous section. In this context, we aimed to improve the therapeutic perspectives of the poorly-water soluble drug itraconazole by preparing a nanosuspension and a hydrogel. Itraconazole is a model drug for development of small molecule formulations because of its extremely low aqueous solublity (1–4 ng/mL) [ 81 ]. This weak base (pKa = 3.7) molecule with a logP of 5.6 belongs to a class of drugs known as azole antifungals. For that reason, the marketed oral solution Sporanox ® solubilizes it in a concentrated cyclodextrin solution at pH 2 [ 82 ]. In the following case study, we aimed to formulate a nanosuspension by a top-down approach to overcome the limited solubility of this drug.

Wet milling was performed based on previous experience to obtain a main particle size distribution in the nanosize range (for further information see Sect.  2.2 ). Laser diffraction results in Fig.  3 A indicated that nanosize reduction of itraconazole (Dv50: 0.52 μm) succeeded in the presence of stabilizers (i.e., poloxamer 407), in agreement with previous reports [ 6 , 76 ]. After milling, the specific surface area values increased by 100-fold in comparison with the initial drug (50,610 vs. 445 m 2 /Kg, respectively) as clearly depicted in Fig.  3 A. Practically, dynamic light scattering results showed that this itraconazole nanosuspension exhibited a heterogeneous population of particles with a mean hydrodynamic diameter size and polydispersity index of around 630 nm and 0.483, respectively (Fig.  3 B). Scanning electron microscopy confirmed the size reduction of the initial material shown in Fig.  3 C. We could observe that in the absence of a stabilizer and even though particles showed certain attrition, they clearly presented agglomeration (Fig.  3 D). On the contrary, the main population of the wet-milled poloxamer 407-stabilized itraconazole particles in Fig.  3 E were below 1 μm, in agreement with the other techniques. Finally, dissolution studies demonstrated that the itraconazole nanosuspension (Fig.  3 G, in green) dramatically increased the dissolution rate profile of the pure drug (Fig.  3 G, in red) from the beginning of the experiment. Interestingly, when the nanosuspension was loaded in a hyaluronic acid hydrogel (Fig.  3 F), we were able to tailor the drug release profile of the formulation. For instance, the burst release of the native nanosuspension was no longer observed (Fig.  3 G, in blue). Finally, itraconazole concentrations quantified in solution at 24 h for the two nanosuspension formulations were significantly higher in comparison with the initial compound (4.6/4.7 vs. 3.1 µg/mL, respectively).

The present case study illustrates how via wet milling, a simple and scalable manufacturing process [ 11 ], an itraconazole nanosuspension formulation with improved characteristics was successfully developed. In agreement with the content in Sect.  3 , we have demonstrated that the higher specific surface area of the nanosuspension translates into higher dissolution rates and solubility. This is again consistent with the the Noyes-Whitney and Ostwald–Freundlich Eqs. [ 7 , 8 ], and the results were also in line with the itraconazole solubility values reported for this setup [ 82 , 83 ]. It is essential to keep in mind that this ultra-fine particle size also enables the possibility of a direct parenteral injection [ 84 , 85 ]. As seen in Sect.  3.2 , it was also easy to embed this nanosuspension in a biodegradable and biocompatible hydrogel matrix (hyaluronic acid) in order to control their release profile. This reinforces the versatility and feasibility of the approach and stands for a realistic alternative in the drug delivery field for poorly soluble drugs in the pipeline of pharmaceutical industries.

Characterization of wet-milled itraconazole (ITRA). (A) Particle size distribution representative graphs by specific surface density obtained by laser diffraction. (B) Particle population distribution by intensity representative peak curve of ITRA nanosuspension (NS) measured by dynamic light scattering. Representative scanning electron micrographs of (C) initial ITRA, (D) wet-milled ITRA without stabilizer, (E) poloxamer 407 (P407)-stabilized ITRA NS and (F) P407-stabilized ITRA NS dispersed in a hyaluronic acid (HA) gel. (G) Dissolution profile of ITRA NS (green), HA Gel-ITRA NS (blue), and initial ITRA (+ P407) (red). Blue and red curves were fitted according to a pseudo first-order release kinetics. All results are represented by the mean ± sd ( n  = 3). ** p  < 0.01

Historical and present panorama

At the beginning of Sect.  3 , we commented on the advent of nanocrystal technology in the clinic as an efficient but also lucrative tool to address the problems of poorly-aqueous soluble drugs. Looking at the historical timeline in Fig.  4 A, pioneer ultramicrosized suspensions began to appear in the late 90s. Just after, in 2000, the FDA approved the first ‘official’ nanocrystal-tablet formulation Rapamune ® (sirolimus) [ 86 ]. This marked the ‘bloom’ of nanocrystal formulations for oral administration as seen in Table  1 . In the following five years, up to nine products (half of the available repertoire) were licensed under the Elan’s nanocrystal ® technology umbrella with different partners [ 32 , 86 ]. Among them, Emend ® (aprepitant), Triglide ® (fenofibrate), or the previously mentioned Rapamune ® reached the market as tablets or capsules during these years. Figure  4 B shows that the majority (40%) of formulations on the market and clinical trials are intended for the treatment of metabolic diseases (hypertension, lipid disorders, etc). This may partially explain why the oral route prevails here, as seen in Fig.  4 C. We had to wait until the end of the first decade of the 21st century to see the first long-acting nanocrystal injectable formulation, Invega ® (paliperidone palmitate). An interesting attribute of this formulation platform is that the different particle size and concentration allows for modulation of the controlled release profile from 1 (Sustenna) to 4 months (Trinza), approximately [ 87 ]. This could have paved the way for the market access of new nanosuspensions in the following years, but the expectations have surprisingly cooled down to date. One solely but recent success is the approval of Cabenuva ® , the first long-acting antiretroviral injectable nanoformulation composed of cabotegravir and rilpivirine. This once-monthly injected drug combo nanoformulation is showing promising results in maintaining HIV-1 suppression in patients [ 88 ].

To better appreciate the reasons for this abrupt halt we should have in mind some insights. In the first place, Elan’s nanocrystal ® technology is estimated to be protected by more than 100 patents worldwide [ 89 ]. This might not have been affordable for small/middle-sized companies or attractive enough for the big pharmaceutical holdings, with a large number of soluble drugs still in their pipeline. In the same period, the approval in 2002 of the ‘blockbuster’ Humira ® (adalimumab) marked the consolidation of biologics at the expense of small molecules [ 90 ]. The investment context did not improve with the economic crisis of 2008, precisely when one of the last nanocrystal formulations (i.e., Invega ® in 2009) was approved. However, more importantly, the emergence of the so-called amorphous solid dispersions, along with the advances in spray-drying and hot melting extrusion came out on top for the efficient tableting of poorly water-soluble drugs [ 91 ]. In recent years, the Covid-19 crisis diverted efforts towards the development of the tandem gene therapy and lipid nanoparticles. Concerning regulatory compliance, this should be in any case beneficial for nanomedicines in the future, and that should also include nanocrystals. Finally, whether we link it or not to the high preclinical and clinical failure rate of innovative drugs, especially non-soluble, we must inexorably recount the limitations of this kind of formulations. Operational, long-term, and physiological stability, including crystal growth and sedimentation, is one of the main hurdles hampering safety and handling, but also efficacy [ 92 ]. In this line, formulation scientists often show, as we did with the case study presented in Sect.  4 , faster dissolution rate profiles in artificial media or in phosphate-buffered saline solutions in the best-case scenario. This may not always be sufficient to reach therapeutic concentrations over time in vivo. The “Spring Parachute” dissolution model can also explain this event [ 93 ]. Here, drug peak concentrations in solution (see Fig.  3 G, in green) are progressively reduced over time as the stabilizer leaves the nanocrystal surface. Regarding manufacturing, top-down approaches mainly entailing wet milling and high-pressure homogenization are the most popular (Fig.  4 D). This is probably because they are easily scalable, show low batch-to-batch variation, and do not require organic solvents [ 92 ]. Nevertheless, there is still a risk to consider, specifically the contamination of the nanocrystals by metals, ceramic materials, or other milling machine debris. This needs to be carefully assessed especially in the case of parenteral administration [ 94 ].

In spite of the above-mentioned challenges, it still draws our attention that there is a lack of ongoing Phase III clinical trials (Fig.  4 E). We only found two studies corresponding to a nasal spray ivermectin nanosuspension for Covid-19 management (NCT04951362). Moreover, many of the studies in Phase II correspond to different anticancer indications of the Panzem ® (2-methoxyestradiol) nanocrystal colloid dispersion. While this is underwhelming, it is convenient to bear in mind that there are around twenty nanocrystal or nanosuspension formulations on the market in 2023 (Fig.  4 E; Table  1 ). This represents around the 25–30% of the total nanomedicine market [ 95 ]. It is not negligible, considering that the global nanotechnology market size reached USD 210 billion in 2022, and is expected to double over the next 10 years [ 96 ]. The historical timeline in Fig.  4 A suggests that these marketed nanocrystals were ahead of their time as well. Precisely when the so-called ‘golden era’ of nanocrystals was practically over around 2009, publications on the topic started to grow progressively as depicted in Fig.  4 F. The approval of the first liposomal nanomedicine Doxil ® (liposomal doxorubicin) in 1995 took around 30 years since the first liposomal structures were reported [ 97 ]. Yet, time is needed to confirm if this curve will continue to grow exponentially or is just a fleeting trend. This means that nanotechnologists are nowadays actively integrating nanocrystals in their novel drug delivery systems or giving them new roles as summarized in Sect.  3 ; but why now? Among other reasons, because this technology is relatively simple, versatile and because 90% of the current drug pipeline faces solubility issues [ 98 ]. Drug loadings can be also very high (close to 100%), resulting in more drug concentration in less volume. A large part of the success of the anticancer nanomedicine Abraxane ® , considered also as a nanosuspension by some scientists, relies on being just paclitaxel bound to albumin [ 32 ].

Still, we have to acknowledge that there is significant work ahead of us. For instance, formulators should explore innovative ways to control nanocrystal redispersibility after tableting. It is essential to understand better how nanocrystals permeate and penetrate subcutaneous tissues and biological barriers when applied topically. In that sense, the incorporation of the above-mentioned cabotegravir/rilpivirine nanocrystals (Cabenuva ® ) in microneedles might further ameliorate the therapeutic outcomes and patient compliance in HIV patients. In addition, nanocrystal cell internalization and drug dissolution profiles should be thoroughly evaluated in vitro/ex vivo using biological fluids. The field of parenteral injections offers ample opportunities for exploration and growth, and this not only includes the intramuscular route but also others such as the intra-vitreal or intra-articular routes. Attention should also be given to the particle size, shape (i.e., irregular and not spherical) and crystalline state of nanocrystals. All these can affect sedimentation, crystal growth, and interaction with cells, proteins and tissues, altering especially their physiological solubility and clearance/filtration towards other compartments. As highlighted in Sect.  3 , formulations at the cutting edge such as liposomes, in situ forming hydrogels, or long-acting polymeric microparticles loaded with nanocrystals are promising candidates for this application (Fig. 1CEF). In addition, most of these studies report the implementation of polymers, lipids, surfactants and other excipients already approved by regulatory agencies (e.g., poly (DL-lactide), hyaluronic acid, PEG and poloxamers), which indeed facilitates translational research. The increasing number of studies shown in Fig.  4 F may bridge the current technological gap from the bench to the clinic in the near future as well. This incoming generation of nanocrystal formulations will likely be integrated as a component in future drug delivery systems.

Nanocrystal formulations: a state-of-the-art estimation, based on an October 2023 search on Clinicaltrials.gov and MEDLINE databases. For further details on methodology, see Sect.  2.1 . (A) Historical timeline in the field of marketed nanocrystal and nanosuspension formulations. (B) Therapeutic indications (diseases) by percentage correspond to the formulations found in clinical trials and on the market. (C) Administration routes by percentage correspond to the formulations in clinical trials and approved. (D) Formulation approaches by percentage correspond to the approved formulations and in clinical trials. (E) Number of studies in clinical phase trials and formulations in the market in 2023. (F) Publications per year in the field of drug nanocrystal and nanosuspension formulations

Data availability

The data presented in this study are available on request from the corresponding author.

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Acknowledgements

The authors would like to thank Ms. Nathalie Boulens for her technical support in acquiring the SEM micrographs and Ms. Justine Langham for her technical support with the UHPLC. We would like also to thank JRS (J. Rettenmaier & Söhne) Pharma (Rosenberg, Germany) for the donation of itraconazole.

This work was supported by the University of Geneva.

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BR worked on the methodology and investigation and wrote various sections for the manuscript. OJ and EA oversaw this work and revised the final manuscript. CRN led the conceptualization, writing, overseeing and editing of the manuscript.

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Rossier, B., Jordan, O., Allémann, E. et al. Nanocrystals and nanosuspensions: an exploration from classic formulations to advanced drug delivery systems. Drug Deliv. and Transl. Res. (2024). https://doi.org/10.1007/s13346-024-01559-0

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Accepted : 21 February 2024

Published : 07 March 2024

DOI : https://doi.org/10.1007/s13346-024-01559-0

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