INTRODUCTION:

Almost 70% of new chemical entities discovered in recent years have shown poor water solubility leading to low bioavailability in systemic circulation^[1]. These drugs get eliminated from the gastrointestinal tract before getting absorbed which greatly hinders the drug’s clinical translations. Therefore, it is necessary to safely increase the drug’s saturation solubility in the body^[1]. Drug delivery through nanocrystal approach has evolved few years ago for poorly soluble drugs which involve formation of nano sized particles. Electron-dense and nanometer in size, nano crystalline formulation basically comprise of drug, water, stabilizer and other excipients like buffer, salts and sugars etc. can be added if required. Drug concentration of 400 nanometer- mg/ml or less is usually preferred for nano crystal preparation.

Advantages of Nano crystals:

1 Increase in dissolution velocity of low soluble drug compounds^{[2, 3]}

2 Increase instauration solubility^{[2, 4]}

3 Higher bioavailability compared to micro particles^[2]

4 Small size and safe composition of nano crystals make the formulation easy to be injected parenteral promising 100% bioavailability^{[2, 3]}

THEORY:

Low dissolution is a characteristic feature of poorly soluble compounds. The principle of nano sizing rests primarily on the Noyes-Whitney and Prandtl equations, which describes an increase in the surface area by reducing the particle size, increasing its dissolution rate ^[4,5].

Drug NCs possess an outstanding feature of increasing adhesiveness to biological mucosa including gastrointestinal, which leads to an improved absorption of poorly soluble drugs^[4,5]_.The gastrointestinal mucoadhesion of drugs enables the release of drug exactly at the absorption sites, which leads to a high concentration gradient and sustained retention time^[6]_.Dissolution behavior of the drugs is also affected by the crystalline state. Transformation into crystalline structure or amorphous particle depends on the method of NC preparation^[7]. Amorphous drugs have high solubility, leading to faster dissolution rate due to high inner energy. Sometimes the amorphous drug may revert to a lower energy state, usually to crystalline state during storage^[7]. Unfortunately, prediction of such transformations is not easy, hence NC at crystalline state are preferred mainly.

FORMULATION CONSIDERATION:

Stabilizer:

Stabilizer is added to the drug particles primarily to avoid cluster formation of NC, called as Ostwald’s ripening^[3,5]. Nature and quantity of stabilizer has prominent effect on the physical constancy and in vivo behavior of NC formulation^[4,8].Excipients suitable for the use as polymeric stabilizers include cellulosic, such as hydroxypropyl cellulose and hydroxypropyl methylcellulose, povidone, and pluronics^[3,9]_.The surfactant stabilizers can be polysorbate, sodium lauryl sulfate and docusate sodium^{[10, 11]}

Organic Solvent:

Used for nano suspensions, solvents like as methanol, chloroform, isopropanol, propylene carbonate, benzyl alcohol etc. are preferred^[3].

Co-Surfactants and additives:

Co surfactants influence the phase behavior hence their effect should be studied properly. Solubilizers such as Dimethylacetamide, Cremophor EL etc. can be used as co-surfactants^[3]_.Additives like buffers, cryopreservatives can be use^[12]

PREPARATION OF DRUG NANOCRYSTAL:

Various preparation methods have been studied for drug NC and can be divided in two basic methods^[1113]:

Bottom up process:

In this process the drug is dissolved completely in a selected solvent which is then added to a non-solvent, which causes precipitation of the drug ^[3,14]_.Controlling the structure of the particles becomes an essential step. Controlling the influential factors and adding stabilizers such as surfactants can help in the same^[11]_.This technique may also be called as classical precipitation process. Various methods include:

a. Solvent Evaporation Method:

Solvent evaporation method was developed by Tachibani et al.^[15]. In this process, drug and matrix were dissolved in the common solvent followed by the solvent evaporation under vacuum^[16,17]. This method can result in achieving high dissolution rate but physical stability is matter of concern^[12]. During handling and storage, risk of uncontrolled crystallization can be seen in amorphous drug^[16]. Hence crystalline products are usually preferred.

b. Hydrosol Technique:

This is a traditional precipitation technique designed by List and Sucker^[15,18]. This technique involves dissolving of drug in an organic solvent followed by its mixing with an anti-solvent^[19]. To assure fast nucleation mixing of the solutions should be performed rapidly. Stabilizing of NC is done by dissolving it in the aqueous medium ^[20]. The solvent removal can be done by evaporation or lyophilization resulting in NC drug product.

c. Supercritical fluid technologies:

Formulations by this technology is more or less based on GAS and RESS ^[21]

Gas anti-solvent recrystallization (GAS): In 1991 Krukonis et al. obtained drug NC using GAS^[15]. A saturated solution of the drug in an organic solvent with supercritical fluids made which decreases the solubility of the drug in the solvent and subsequently causes precipitation of the drug^[11].

Rapid expansion of Supercritical Fluid technology (RESS): In 1999 Pace et al. prepared drug NC by RESS ^[15].The drug is dissolved in the supercritical fluid followed by decrease in the pressure which causes the drug to precipitate from the fluid ^[11].

The super critical fluid in GAS process is used as an anti-solvent, whereas in the RESS it’s used as a solvent ^{[11, 15].}

d. Freeze drying Method:

A newer technology called CCDF was established in recent years where the drug NC was obtained by dissolving the drug in the mixture of organic solvent and solution containing the matrix material. This procedure is carried out at high temperature resulting drug and matrix to crystallize ^[15].

e. Spray-freezing into liquid:

This method involves rapidly freezing the mixture of drug, polymer, organic solvent and water ^[22]. Modification in the process parameters such as the concentration of solvents used, temperature at which process was carried out etc. can yield crystalline NC ^[17].

Top down process:

Two basic disintegration technologies for preparation of drug NCs include ^[4]:

Pearl milling:

This basic technology was established by G. Liversidge et al ^[4,13]. The macro suspension of drug is filled into milling chamber^[23]. This chamber is filled with milling pearls made from glass, zircon oxide or special polymers ^[3]. The drug is ground to NC with the help of the pearls which are moved by the stirrer ^{[4, 24].} First products on the market launched in 2002 and 2003 were Rapamune and Emend respectively^[4].

High pressure homogenization:

Various process parameters can influence the drug NC formulation and hence their optimization becomes essential ^[23]. Optimization of the applied pressure during homogenization, number of cycles to be performed to obtain crystals of required size range and appropriate temperature for carrying out the homogenization process is required^[24,
25].

a. Microfluidisation:

Principle involves passing of drug suspension with a high velocity into the homogenization chamber. The drug suspension changes its direction of flow leading to particle collision in the Z type chamber^[26]. In the Y type, the second type of chamber, the suspension stream is divided into two which collides in the front^[4]. This method has been pursued by Research Triangle Pharmaceuticals (RTP).

b. Disso cubes:

The drug suspension, in this method is passed through a small orifice which causes decrease in the static pressure leading to boiling of water causing formation of gas bubbles^[3,4]. Upon reaching the typical pressure, the suspension leaves the gap and the gas bubbles collapse causing reduction in the particle siz^{[4, 26].} Large number of cycles may be required to achieve required size range ^{[4, 10, 24].}

c. Nano pure:

It is a homogenized suspension in water-free media. In this method, cavitation is the determining factor^[3]. Hence to initiate cavitation, static pressure should be maintained ^[4]. The drug suspension in the non- aqueous media were homogenized at 0⁰C or even below the freezing point and hence are called "deep-freeze" homogenization^{[10, 27].}

d. Nano edge:

Nano edge involves combination of precipitation and homogenization techniques which results in particle size of nano range^[4,27]. The precipitated suspension is homogenized causing decrease in particle size. This is carried in water using solvents like methanol, ethanol etc.^[3]. Evaporation is done to provide a solvent-free starting material followed by high-pressure homogenization for producing an effective nano suspension^[10,
26].

EVALUATION:

Commonly used characterization techniques for determining nano crystals stability are:

Particle size:

Techniques like photon correlation spectroscopy, laser diffraction and coulter counter are commonly used to measure the particle size and size distribution ^{[12, 13].}

PCS/DLS:

The mean particle size and size distribution are measured as Polydispersity Index (PDI)^[3]. A PDI value of 0.1 - 0.25 indicates a narrow size distribution while a broader distribution is indicated by a PDI greater than 0.5 ^{[4, 5].}

LD:

Detection range lies between 20 nm - 2000 μm ^[28]. Laser Diffraction parameters are LD50, LD90 and LD99 which indicates 50, 90 or 99% of the particles are below the given size, respectively^{[4, 5].}

CC:

Measures the absolute number of particles per volume unit.

SEM, TEM and AFM are widely used for assessment of particle morphology ^{[13, 29].}

Sedimentation/creaming:

Evaluation of sedimentation is done by visual observation over a period. This is measured by volume of the settled particle layer to the total suspension volume^[7]. Higher sedimentation volume indicates a steady suspension. Easily-dispersed suspension indicates loose flocs^[7].

Particle surface charge:

Evaluation of electrophoretic kinesis of particles suspended in the medium is measured through Laser Doppler ^[7]. According to the texts, a zeta potential of at least -30 mV for electrostatic and -20 mV for sterically stabilized systems is preferred to obtain a physically stable nano crystal^{[3, 13].}

Crystalline state:

Usually evaluated by XRD or DSC ^[13]. XRD distinguishes amorphous and crystalline nanoparticles ^[3]. Crystalline particles have a sharp melting peak which is absent in amorphous materials ^[30]. Hence melting point can be utilized to differentiate different polymorphs ^[7].

Chemical stability:

HPLC is the common characterization technique used to provide precise analysis on the degradation impurities. HPLC is coupled with MS to identify the impurities. FTIR and NMR can also be used for evaluation ^[7].

Evaluation of primary structure of large biomolecules can be done using size exclusion chromatography and electrophoresis.

APPLICATION:

Oral route drug delivery

Oral route is the most preferred and common route of drug delivery^[3].Large surface area of nano crystals increases the saturation solubility which eventually increases the dissolution rate, enhancing drug absorption ^[31].Rapamune® was the first US FDA approved oral nano crystals launched in the year 2000 by Wyeth Pharmaceuticals (Madison, NJ) ^[13].Muller et al. have refined oral delivery of thermo stable drugs utilizing melted PEG (melting point at 60 °C) which allows fixing of nano crystals in a solid PEG matrix. Nano crystals dispersed in melted PEG were milled to powder and directly compressed into the tablet or filled in capsules shell ^[9].

Table 1: Drug delivery explored through oral route.

Drug	Technique	Mean Particle size	Use
Itraconazole	Precipitation	267 nm	Antifungal
Cyclosporine	HPH	962 nm	Autoimmune disease
Fenofibrate	HPH	356 nm	Lipid lowering agent

Intravenous drug delivery:

Drug administration via intravenous route provides numerous benefits such as immediate action, reduced dosing and 100% bioavailability ^[9,13]. Nano crystals can be considered as the ideal candidates for intravenous delivery because their developmental processes do not employ excess use of harmful excipients ^[3,13].Thus, size range of <100 nm is preferred. Nano crystals of drugs such as ascularine, oridonin, itraconazoleand curcumin have been successfully developed thereby providing benefits like increase in Cmax, and AUC^[9].

Table 2: Drug Delivery explored through intravenous route.

Drug	Technique	Mean Particle size	Use
Atovaquone	HPH	279 nm	Improved activity against toxoplasma encephalitis
Oridonin	HPH	103.3 nm	Anticancer
Ascularine	HPH	133 nm	Anticancer

Pulmonary drug delivery:

Lungs are highly perfused organs with a fully expanded surface area^[6]. Pulmonary route is a viable option for delivery of therapeutics^[13]. Lack of hepatic portal drainage system acts as an advantage in rapidly transporting the drug into the systemic circulation with high efficiency. A nebulizer is generally required to administer powdered nano crystals which can incorporate nano crystals into small inhalable droplets (1–5 μm)^[9,32]. A literature study showed that aerosolized nanocrystals enhanced drug bioavailability when compared to inhalable dispersions containing micro particles^{[13, 25].}

Table 3: Drug delivery explored through pulmonary route.

Drug	Technique	Mean Particle Size	Use
Itraconazole	Precipitation	Less than 1 μm	Antifungal
Carvedilol	Solvent precipitation	190 nm	Anti hypertensive
Budesonide	-	Less than 1 μm	Anti-asthmatic

Ocular drug delivery

This is a challenging drug delivery route owing to critical pharmacokinetic environment and physiological barriers that hinder the drug delivery into eye^[13]. Ocular therapy is delivered through a topical formulation in the form of solution or suspension^[3]. Conventional formulations undergo rapid clearance from application site due to rapid eye movements and lacrimation ^{[31, 32].} Short retention time induces a need of repeated dosing ^[9]. NC technology played an innovative role by improving dispensability issues of poorly soluble drugs like budesonide etc. ^[18]. Ali et al. used combination technology based upon microfluidic nano precipitation and wet milling to create nano crystals of hydrocortisone and ocular bioavailability was evaluated in albino rabbits. Results demonstrated an extended duration of action and significantly improved AUC of developed nano crystals in comparison to free drug. Pharmacodynamics studies revealed that it also efficiently lowered the intraocular pressure up to 12 h in comparison to conventional suspension^[33].

Table 4: Drug delivery explored through ocular route.

Drug	Technique	Mean Particle Size	Use
Hydrocortisone	Precipitation	300 nm	Steroid
Mycophenolatemofetil	HPH	440 nm	Immunosuppressant
Forskolin	Wet milling	164 nm	Antiglaucoma agent

Dermal drug delivery:

Permeation of drugs across stratum corneum is a major concern in dermal delivery ^[6]. Formation of an occlusive layer increasing penetration and permeation of drugs can be done through NC ^[3]. Dispersed nano crystals are retained topically for sufficient period, slowly releasing the active constituents ^{[18, 32].} Suspended nano crystals of lipophilic compounds showed an increased permeation through a synthetic membrane ^[9].

Table 5: Drug delivery explored through dermal route.

Drug	Technique	Mean Particle Size	Use
Lutein	HPH	429 nm	Antioxidant
Hesperetin	HPH	300 nm	Antioxidant and Antiallergic
Tretinoin	Precipitation	324 nm	Anti-acne agent

CHALLENGES IN DRUG NANOCRYSTAL:

While multiple NC products have been approved clinically in the past, major challenges prevent their widespread acceptance^[24]. Relationship between pharmacological properties of drug NC and nanoparticle structure still need to be fully recognized. Proper valid methods for evaluation, characterization and reference standards for quality and safety of NC products needs to be described^[11].

Simultaneous monitoring of intermediate NC and evaluatory tests of final products should be made mandatory as they may affect ADME and can cause toxicity of drug NC. Evaluation of risk and benefit of NC will be essential to appreciate the clinical potential of this novel paradigm of therapy ^[18]. Lack of nontoxicity and cytotoxicity data, regulatory accepted status of the excipients, and large scale production lines are few reasons due to which many formulations are under investigation and only few made it to the market ^{[11, 24].} Moreover, only certain drugs are suitable for this technique as the use is restricted to BCS class II drugs ^[10,
18].

FUTURE AND SCOPE:

Drug NC represents a valid process to overcome problems related solubility^[4].Any poorly soluble drug can be transformed into nano crystals resulting in enhanced saturation solubility, dissolution rate, and enhanced adhesiveness to surface^[10].Diverse methods of preparation give options to produce different therapeutics with different routes of administration^[3,27].This will not only help to improve the efficacy of noxious drugs but will facilitate the alteration of suitable drug candidates that are considered failures because of lack of solubility^[10].With rapid advancement in science and technology, drug NC can potentially be vital for clinical applications of highly potent hydrophobic drugs ^[24].

CONCLUSION:

Drug NCs have become most successful in recent years since its discovery. They have potential advantages and reduced side effects^[24]. It is a unique approach to solve solubility and bioavailability related issues^[13,14,29].Major advantage of drug NC is that they can be delivered through almost all routes of administration ^3]. Nano crystal technology has huge potential to deliver pre-existing or newly developed poorly water-soluble drugs into effective dosage form with high commercial applicability and acceptability^[5,24].

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Received on 20.10.2017 Modified on 18.11.2017

Research J. Pharm. and Tech 2018; 11(4): 1685-1690.

DOI: 10.5958/0974-360X.2018.00314.1