Nanosuspension For the Delivery of a Poorly Soluble Anti-Cancer Kinase Inhibitor

Poor solution of many anti -river drugs or drugs Candidates are serious obstacles to their development Their clinical use. To overcome this problem Strategy has been developed, such as the use of cyclodeslin. Use or co -mobile devices or co -mobile devices for ionized drugs Suitable active substance (often toxic, for example, Creo Fore EL) ^[1]. Instead, many nanosystems are being investigated. Colloidal dispersions of poorly water-soluble drugs, including liposomes, nanoparticles, polymeric micelles, dendrimers, etc.

Nanocarriers allow The drug to be specifically targeted to the tumor, thus reducing systemic side effects. Some nanoformulas have been clinically approved, e.g. Abraxane and Doxil ^[2]. Due to the presence of fenestrations in tumor endothelial cells, these nanocarriers can penetrate the interstitium, be captured by the tumor, and remain within the tumor tissue due to poor lymphatic drainage. This is “passive”. This targeting is based on the enhanced permeability retention (EPR) effect discovered by Matsumura and Maeda ^[3]. Various nanoscale strategies have recently emerged. Nanosuspensions (also called nanocrystals) are nanoscale crystals of compounds, with sizes less than 1 ?m ^[1]. Nanosuspensions have several advantages: (i) Nanosuspensions allow for the colloidal dispersion of poorly water-soluble drugs ^[4]. (ii) These particles, unlike matrix nanoparticles, have very high drug loadings (pure drug coated with surfactants or polymers as stabilizers) ^[5]. (iii) Nanosuspension techniques are easily scalable, offering a cost-effective and simple alternative ^[5]. (iv) Nanosuspensions ensure the physical and chemical stability of drugs ^[1].(v) Nanosuspensions can exhibit passive targeting (EPR effect) similar to colloidal drug carriers [[af]]ter intravenous injection ^[6]. (vi) Finally, Nanosuspensions are one of the most important strategies to Enhance the oral bioavailability of hydrophobic drugs but also for Their intravenous route ^[5]. More than 10,000 patent applications for kinase inhibitors have Been field since 2001 in the United States ^[7]. These significant investments were made because of the observation that kinases are closely linked To participate in the growth of cancer cells ^[7]. MTKi-327 (JNJ-26483327) is An anticancer multitargeted kinase inhibitor developed by Johnson And Johnson (Fig. 1) Kinase inhibitors have revolutionized the treatment of a certain group of diseases, such as chronic myeloid leukemia and gastrointestinal stromal tumors, which are caused by a single oncogenic kinase. However, most of cancers involve multiple kinases ^[7]. In this context, the development of multi-targetedvKinase inhibitor has emerged. MTKi-327 has been shown in vitro to be a potent inhibitor of various kinases including EGFR; (ii) Her-2, Her-4 and Ret (playing a role in proliferation and migration); (iii) VEGFR-3 and Yes (reducing lymphangiogenesis and angiogenesis); and (iv) Src, VEGFR and R[[af]] (limiting metastatic processes) ^[8]. In vivo, MTKi-327 (administered orally) has demonstrated antitumor activity against various subcutaneous tumors (e.g. A-431 and A-549), orthotopic tumors (e.g. DU-145), intracranial tumors (A-431) and bone tumors (MDA-MB-231). MTKi-327 inhibited tumor growth more effectively than lapatinib or erlutinib.[9] In a phase I study in patients with advanced solid tumors, MTKi-327 was well tolerated and showed a predictable pharmacokinetic profile^.[9] Therefore, the current challenge is to formulate this drug for intravenous and oral administration in a non-toxic formulation. Currently, there is no suitable formulation for parenteral administration. We hypothesized that nanosuspensions may be promising for the delivery of MTKi-327. Therefore, the objectives of this study were to (i) evaluate the parenteral and oral administration of MTKi-327 nanosuspension and (ii) compare this nanosuspension with other nanocarriers in terms of its anticancer efficacy and pharmacokinetics. Thus, different formulations of MTKi-327 were studied: (i)PEGylated PLGA-based nanoparticles. The polymer poly(lactide-co-glycolide) (PLGA) (approved by the FDA and EMA in parenteral products) was chosen for its biocompatibility and biodegradability properties. Poly(?-caprolactone-co-ethylene glycol) (PCL-PEG)PEG was added to take advantage of its repellent properties ^[10].The suitability of these nanoparticles has already been demonstrated.The encapsulation of poorly water-soluble drugs such as paclitaxel(PTX) or cycline dependent kinase inhibitor, JNJ-7706621[11,1}.

(ii) e-caprolactone (CL), trimethylene carbonate (TMC), and mmePEG750 (mmePEG750-p-(CL-co-TMC)) self-assembling diblock copolymers have been demonstrated to spontaneously form micelles when gently mixed with water. These copolymers are non-cytotoxic, non-hemolytic, and biocompatible. They make poorly soluble water drugs (like PTX) at least two orders of magnitude more soluble. The toxicity of paclitaxel was considerably decreased by these polymeric micelles, enabling the dosage to be increased for a greater therapeutic response ^[12,13]., (iii) an MTKi-327 nanosuspension; and (iv) a Captisol solution (pH = 3.5). Due to its acidity, this solution was not modified for repeated intravenous injections of MTKi-327, which was intended for oral delivery (pKa of MTKi327 = 3.9). MTKi-327 was given orally to mice, with a maximum tolerable dose of 100 mg/kg/day. Thus, the Captisol solution served as the control for this investigation. The concentration of MTKi-327 was ascertained and these four formulations were described. It was decided what the maximum tolerable doses were. Nude mice with A-431 tumours were used to test the in vivo anti-tumor efficacy. MTKi-327 nanosuspension was more thoroughly examined in comparison to the Captisol solution in order to illustrate the potential of the substance. The impact of the dosage, the mode of administration, and the quantity of doses given were all assessed. Studies on pharmacokinetics were also

       
           
       
Fig.1.chemical structure of the tyrosine kinase inhibitor pharmacophore

Abelson (Abl), Src, JNK, and numerous other intracellular protein tyrosine kinases are important players in signal transduction pathways and the formation of cancer. While they are abundantly expressed and active in malignant tumor cells, they are seldom active or expressed in normal cells ^[14].. As a result, numerous small molecule tyrosine kinase inhibitors (TKIs) have improved cancer treatment during the past three decades by entering clinical trials and receiving approval to treat both hematologic and non-hematologic cancers. The application of TKIs to the treatment of chronic myeloid leukemia (CML) has achieved the most advancements. Most of these compounds work on receptor tyrosine kinases, including vascular endothelial growth receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), and are ATP-competitive inhibitors rather than select   anti-tumor activity was performed in A-431-tumor bearing nude mice. Nanosuspension, nanosuspension MTKi-327 was studied more precisely compared to Captisol solution: the effects were evaluated dosage, route of administration, number of doses. Also, pharmacokinetic studies

Need Of Nanosuspension:

The medicine is kept in the necessary crystalline form with smaller particles thanks to nanosuspension technology, which increases the rate of dissolution and, consequently, the drug's bioavailability. Increases in surface area and, thus, dissolution speed are associated with higher rates of dissolution of micronized particles (particle size < 10>[15,16].

Properties Of Nanosuspension:

1. Nanosuspensions Have The Following Characteristics:
2. long-term physical stability
3. internal structure
4. adhesiveness
5. crystalline state and particle morphology
6. increased drug solubility and dissolution velocity
7. nano- suspension provides passive targeting.

1. Long-Term Physical Stability

Ostwald ripening, which causes crystal growth to generate microparticles, causes physical instability in dispersed systems. The differential in the dissolution velocity and saturation solubility of tiny and large particles is what causes Ostwald ripening. Since all of the particles in nanosuspensions have the same size, there is minimal variation in the drug particles' saturation solubility, which results in the complete absence of Ostwald ripening. ^[26].

2. Internal Structure

The drug particles undergo structural alterations as a result of the high energy input during the disintegration process. The drug's particles change from crystalline to amorphous states when subjected to high-pressure homogenization. The drug's hardness, the number of homogenization cycles, its chemical makeup, and the power density used by the homogenizer all af]fect the state change.

3. Adhesiveness

Ultra-fine particles have a higher adhesiveness than coarse powders. This little object's adhesivenessDrugs that are poorly soluble can be better delivered orally by using drug nanoparticle

d) crystalline state and particle morphology

Morphology of Particles and Crystalline States A drug's potential                                                                                                        polymorphism or morphological alterations upon nanosizing can be better understood by evaluating the crystalline state and particle morphology together. ^[12]. Due to high-pressure homogenization, nanosuspensions may experience a change in their crystalline structure, perhaps leading to an amorphous or other polymorphic form ^[13]. Differential scanning calorimetry can be used in conjunction with X-ray diffraction analysis ^[14] to ascertain the extent of the amorphous fraction and the changes in the solid state of the drug particles. ^[15] Scanning electron microscopy is the primary method for obtaining a true understanding of particle morphology.

e )  increased drug solubility and dissolution velocity

When the surface area of the drug particles increases from micrometers to nanometers, the medication dissolves more readily. The Noyes-Whitney equation states that when the surface area of particles grows from micron to nanoscale in size, the dissolving velocity increases. [1] dx/dt= D x A / h Cs X / V (1) where D stands for diffusion coefficient, A for particle surface area, dx/dt for dissolving velocity, V for dissolution medium volume, h for diffusion layer thickness, and X for concentration in surrounding liquid. ^[16]. When the surface area of the drug particles increases from micrometers to nanometers, the medication dissolves more readily. The Noyes-Whitney equation states that when the surface area of particles grows from micron to nanoscale in size, the dissolving velocity increases. ^[1]. dx/dt  = D x A / h Cs X / V (1) where D stands for diffusion coefficient, A for particle surface area, dx/dt for dissolving velocity, V for dissolution medium volume, h for diffusion layer thickness, and X for concentration in surrounding liquid.

f) nanosuspension provides passive taegating

Passive Targeting using Nano Suspension Due to their inability to reach the intended site of action, the majority of medications have not produced positive results. Serious side effects are frequently the result of a large portion of the administered medication being dispersed throughout healthy tissues or organs that are not a part of the pathological process. a practical strategy to address this important problem in the creation of targeted medicine delivery systems. Versatility: Nanosuspensions can be incorporated into a variety of dosage forms, including tablets, pellets, suppositories, and hydrogels, for a range of administration routes, thanks to their ease of post-production processing and flexibility in altering surface characteristics and particle size. Advantages Of Nanosuspension [19,20]

*It makes chemicals more soluble and bioavailable.
* Suitable for hydrophilic compounds

* Potential for higher drug loading

*Potential for dose reduction
* Increases the medications' chemical and physical stability 

*May be administered via any method
When administered subcutaneously or intramuscularly, there is less tissue irritation; tissue targeting and rapid dissolution are possible. through the intravenous route
* Increasing the particles' amorphous proportion, which could result in a change in their crystalline structure and increased solubility; * Potentially, large-scale product

Disadvantages Of Nanosuspension

• Physical stability, sedimentation & compaction cause problems

• It is bulky sufficient care must be taken during handling & transport.

• Improper dose.

• Uniform & accurate dose cannot be achieved. ^[20].

Application Of Nanosuspension

1.    Mucoadhesion of the nanoparticles

2.    targeted drug delivery

 3.    pulmonary drug delivery

 4.     oral drug administration

 5 .    parental drug delivery

 6 .    ocular drug delivery

 7.    topical drug  delivery

 8.    bioavailability enhancement

1.mucoadhesion of the nanoparticles

Nanoparticle Adherence to The Mucosa the Nanosuspension Adheres to The Mucosal Surface and Diffuses into The Liquid Medium Prior To Absorption If It Is Administered Orally [21]. For Instance, Buparvaquone Improves Absorption and Targeting of The Parasite Within The Gut When Used Against Cryptosporidium Parvum ^[21,22,23].

2.targeted drug delivery

Targeted medication administration Nanosuspensions can also be used for targeting since their surface properties and the stabilizer's behavior can be readily altered in vivo. The drug will be taken up and administered locally by the mononuclear phagocytic system ^[22]. Antifungal, antifungal, or antileishmanial drugs can be delivered to the cells if the infectious pathogen is still inside the macrophages

3.pulmonary drug delivery

Drug delivery in the lungs Nanosuspensions may be the most efficient way to provide medications with low solubility in pulmonary secretions. Aqueous nanosuspensions can be nebulized for lung delivery using mechanical or ultrasonic nebulizers ^[21]. Because of their tiny size, each aerosol droplet probably contains one or more drug particles, which makes the medication's distribution throughout the lungs more uniform. At the site of action, rapid diffusion and disintegration are made possible by the drug's nanoparticulate shape

4.Oral Drug Administration

 Oral medication administration Nanosized medications have greater oral absorption and, thus, greater bioavailability. The enhanced concentration gradient between the blood and the lumen of the gastrointestinal system and the drug nanoparticles' higher saturation solubility are the two elements that lead to better bioavailability. Direct use is possible for both liquid and dry dosage forms, including aqueous nanosuspensions and tablets or hard gelatine capsules with pellets ^[21]. Granulates can also be created by spray-drying nanosuspensions.

5.parentral drug delivery

Nanosuspensions are administered via a variety of different routes, such as intra-articular, intraperitoneal, and intravenous ^.The goal of parenteral administration of nanocrystals is to provide customized effects while reducing the toxicity issues related to non-aqueous formulations. A parenterally administered nanosuspension formulation shows lower toxicity than conventional drug delivery techniques ^[21]. A size range of 100 to 300 nm is appropriate for the enhanced permeability and retention effect (EPR effect), which is used to achieve drug concentration in solid tumors, which are thought to have a very dense vasculature ^[23]. In an attempt to overcome the limited effectiveness of conventional solubilization techniques, which involve the use of surfactants and cyclodextrins to promote bioavailability, the poorly soluble drug tarazepide has been converted into injectable nanosuspensions.

6.Occular Drug Delivery and Topical

Drug delivery through the eyes Ocular drug delivery is the suggested technique for treating conditions [[af]]fecting the eyes, including retinopathies, glaucoma, dry eye syndrome, infections, and inflammation ^[21]. Lachrymal fluid does not dissolve all drugs equally. Research has focused on using nanocarrier-based drug delivery systems (such liposomes and polymeric micelles) to improve the bioavailability of ocular medications because they can bypass many of the biological barriers of the eye. Because of the quick clinical development Compared to other forms of nanotherapeutics, such liposomes and dendrimers, and the commercialization of nanocrystals , the use of nanocrystals as an ocular formulation technique for medications that are poorly watersoluble has gained popularity recently. Using nanocrystals for drug delivery to the eye has several advantages, including enhanced ocular safety, increased formulation retention in cul-de-sac, improved corneal permeability across the corneal and conjunctival epithelium, improved ocular bioavailability, dual drug release profile in the eye, and improved tolerability.

7.Topical drug delivery

Topical medication administration Drug nanoparticles can be found in creams and waterless ointments. The increased saturation solubility of the drug in the topical dose form of nanocrystalline forms improves the diffusion of medication into the skin. Therefore, the finest example of suspensions with delayed settling rates is topical lotions

8.Bioavaibality enhancement

Improvement of Bioavailability By addressing the twin issues of weak solubility and limited permeability across the membrane, nanosuspensions address the issue of low bioavailability. A nanosuspension formulation was used to increase the bioavailability of oleanolic acid, a hepatoprotective drug that is poorly soluble. Higher bioavailability was demonstrated by the markedly improved therapeutic efficacy.

2.MATERIAL

2.1. MATERIALS

PLGA, PCL-b-PEG and PLGA-b-PEG polymers were synthesized by ring-opening polymerization and kindly provided by Prof. C.Jerome (CERM, ULg). Molecular weights were determined by size-exclusion chromatography (SEC) and NMR as previously described^[14,15]. The PEG-p(CL-co-TMC) diblock copolymer was synthesized by Johnson & Johnson Advanced Technologies for Regenerative Medicine (ATRM) (Somerville, NJ, USA). The polymer properties are shown in Table 1. MTKI-327 was provided by Johnson, Johnson, and Research Pharmaceutical.Development, Yanssen Pharmaceutical Division (Belgian, beer)^[9]. A-431 cells (human epithelium cancer) have been acquiredATCC (American Type Culture Collection, Va, Manassas,united states of america). Dubelco modified Eagle medium (DMEM), fetal bovine serum, trypsin-EDTA, penicillin-streptomycin mixture,L-glutamine were purchased from Invitrogen (Mellerbeke, Belgium). Ultrapure water was used during the experiments and otherchemicals were of analytical grade. NMRI and naked mousePurchased at Genest-ST-SLE, France in January

2.2. Composition

2.2.1. Nanoparticles

35 mg PLGA, 7.5 mg PLGA-PEG, 7.5 mg PCL-PEG and 5 mgMTKi-327 were dissolved in 600 ?l dichloromethane. 2 ml was added to the polymer solution.Sodium cholate (1%) was added and sonicated twice for 15 s (60 W). This emulsion was added to the glass syringe with 100 ml of sodium cholate (0.3%) while stirring with a magnetic stirrer2 hours to allow the dichloromethane to evaporate. Nanoparticles ultracentrifuged for 1 h at 4 °C at 22,000 g and washed three times with water. Finally, the pellet was suspended in 3 ml of ultra-purified water ^[24].

2.2.2. Polymeric micelles

500 mg of PEG-p(CL-co-TMC) 50:50 was added in excess. MTKi-327 (12.5 mg) was added to the formulation vial and mixed overnight. At 37°C. 4.5 ml of ultra purified water have been added to the polymer. Figure 2 shows the anti-tumor efficacy of various formulations of MTKi-327 on A-431-tumor-bearing mice. (A) Changes in the A431 tumor volume in naked mice following five days of intravenous injections of MTKi-327 nanosuspension (650 mg/kg/day) and five days of intravenous injections of polymeric micelles (6 mg/mg/day). The mean ± SEM is represented by each point. (B) Mean survival times and the Kaplan Meyer survival curve. p < 0>

3.Formulation Consideration:

Following agents are used in the preparation of nanosuspension

• Surfactant
• Cosurfactant
• Stabilizers
• organic solvent

• other additives

*Surfactant

Surfactants The SURFACE tension between two liquids, a liquid and a solid, or a gas and a liquid is decreased by surfactants ^[13]. Wetting agents, detergents, emulsifiers, foaming agents, dispersants, and emulsifiers are all possible uses for surfactants. In nanosuspension, surfactants such as Tweens and Spans are commonly used ^.[25]

*Co-surfactant

Cosurfactant Selecting the right co-surfactant is essential when making nanosuspensions with microemulsions. Examining how cosurfactants alter drug loading and the uptake of the inside section for a particular microemulsion composition is crucial because they have a big influence on section behavior ^[26] Transcutol, glycerol, ethanol, isopropanol bile salts, and dipotassium glycyrrhizinate are a few examples of cosurfactants.

*Stabilizers

A stabilizing agent The high surface energy of nanoparticles can lead to drug crystals clumping or aggregating in the absence of an appropriate stabilizer ^[27]. A stabilizer must completely moisten the drug particles and supply steric or ionic barriers to stop Ostwald's ripening and agglomeration of nanosuspensions in order to produce a physically stable formulation The physical stability and in vivo behavior of nanosuspensions are strongly influenced by the type and amount of stabilizer used .Lecithin, cellulosic, polysorbate, and poloxamer have all been used as stabilizers thus far .

*Organic solvent

Solvent That Is Organic The Pharmaceutical Industry's Acceptance Of Organic Solvents ^. Their Potential For Toxicity, And Their Ease Of Removal From The Formulation Must All Be Considered When Creating Nanosuspensions Using Emulsions Or Microemulsions As Templates. Water-Miscible Solvents That Are Less Hazardous And Pharmaceutically Acceptable, Like Ethanol And Isopropanol, As Well As Solvents That Are Only Partially Water-Miscible, Like Ethyl Acetate, Ethyl Formate, Butyl Lactate, Triacetin, Propylene Carbonate, And Benzyl Alcohol, Are Preferred In The Formulation .

*Other Additive

The Addition Of Buffers ^[28]. Salts, Polyols, Cosmogenic, And Cryoprotectants  To Nanosuspensions May Depend On The Drug's Properties Or The Mode Of Administration.Additives including buffers, salts, polyols, osmogent, and cryoprotectant may be included in nanosuspensions, depending on the needs of the drug moiety and the requirements of the route of administration

Nanosuspension:

The previously mentioned roller milling technique was used to create the MTKi-327 nanosuspension ^[1,4]. The MTKi-327 powder was dissolved in an aqueous solution that contained a number of ionic agents and stabilizers, including glucose (40 mg/ml), lipoid S75 (3 mg/ml), and pluronic F108 (25 mg/ml). The resulting pre-mixture was ground with 0.5 mm ZrO2 for 66 hours at room temperature. Using a syringe fitted with a fine bore needle microlance G23, the nanosuspension was extracted.

In vivo anti-tumoral activity

Anti-tumoral efficacy in vivo All experiments were authorized by the Université Catholique de Louvain's Ethical Committee for Animal Care (Permit number: UCL/MD/2008/025) and carried out in accordance with national rules. Every tumor implantation procedure was carried out under anesthesia, and every attempt was made to reduce patient pain. DMEM supplemented with 10% (v/v) fetal bovine serum, 100 IU/ml of penicillin G sodium, 100 lg/ml of streptomycin sulfate, and 1% L-glutamine was used to cultivate A-431 tumor cells (human epidermoid carcinoma). At 37 degrees Celsius, cells were kept in an incubator with 5% CO2 and 95% humidified air. Female naked athymic mice that were 4–5 weeks old and weighed about 25 g at the start of the trial. When a subcutaneous injection was administered, a solid tumor formed. Distribute 100 ll of a suspension of A-431 human cancer cells (1 107 cells per milliliter) onto the mice's right flank. When the tumor's volume (measured with an electronic caliper) reached 190 mm3, treatment began

Pharmacokinetic

The study's objective was to ascertain the pharmacokinetics (PK) of MTKi-327 administered orally and intravenously as a nanosuspension or in the Captisol formulation. Female athymic mice who were 4–5 weeks old were given the following treatments: Group 1 had five individuals receiving MTKi-327 nanosuspension (3 mg/kg) intravenously; Group 2 had five individuals receiving MTKi-327 nanosuspension (10 mg/kg), per os; Group 3 had five individuals receiving intravenous Captisol solution, MTKi-327 (at the MTD = 3 mg/kg); Group 4 had five individuals receiving Captisol solution, per os; MTKi-327 (10 mg/kg). At specified intervals (5, 15, 30 minutes, 1, 2, 6, 24 hours),: Orbital punctures were used to obtain blood samples. Each mouse had a maximum of three blood samples taken. Using EDTA as an anticoagulant, 300 ll of blood were drawn at each time point (producing slightly more than 100 ll of plasma) in Multivette 600K3E, No./REF 15.1671 tubes. [[Af]]ter two hours, the tubes were centrifuged at 1500g for ten minutes at room temperature. The plasma was then moved into an amber-colored container and kept at 20°C.

 A validated liquid chromatography–mass spectrometry bioanalytical technique (Johnson & Johnson) was used to determine the plasma concentrations of MTKi-327 ^[9]. To put it briefly, acetonitrile was used to de-proteinize plasma samples, and LC/MS/MS was used to examine the supernatant. A C18-ODS3 column (2 mm 50 mm, 3 lm particles) from Torrance, CA used as the HPLC column. It was kept at 60 °C and had a flow rate of 0.5 ml/min. Acetonitrile (B) and 5 mM ammonium acetate pH 3.75 (A) made up the mobile phase. The initial composition of the mobile phase was 87.5% A/12.5% B. Following sample insertion, the mobile phase was adjusted over the course of two minutes to 37.5% A/62.5% B, and it was maintained at that composition for one and a half minutes. An ion-trap mass spectrometer, the Finnigan LCQ Advantage (Thermo Electron Corp, San Jose, CA, USA), was connected to the HPLC and operated in both full MS/MS and positive ion electrospray modes. A quadratic regression weighed by reciprocal concentration (1/) was used to fit the standard curve, which had a range of 0.5 to 5000 ng/ml. For this test, 0.5 ng/ml was the limit of quantification (LOQ) ^[17]. The area under the plasma concentration-time curve from time zero and extrapolated to infinity (AUC0–1) following the dose and computed during the 24-hour dosing interval at steady state (AUC0–24h) were the PK parameters collected. maximum observed plasma concentrations (Cmax), apparent terminal half-life (t1/2), and duration to attain Cmax (Tmax)

Nanosuspension Characterisation

1. size
2. crystaline state
3. particle charge
4. stability

1.size

The polydispersity index (PI: particle size distribution) and particle size are the two most crucial properties of nanosuspensions. The following properties of nanosuspensions are significantly influenced by the size of the particles in them ^[29]

• Drug satueation solubility
• Physical stability
• Dissolution rate
• Bioavaibility

Based on Fick's first law of diffusion, the Noyes-Whitney equation (1) states that as particle size decreases, particle surface area increases, increasing drug solubility in aqueous solutions and accelerating the rate of dissolution .

dMdt

(1)where D is the average diffusion coefficient,  is the solid's surface area, and Cbulk dT/dt is the rate of dissolution. CBulk denotes the drug concentration in the bulk solution, CEq denotes the drug concentration in the drug's surrounding diusion layer, and "?" is the thickness of the diusion layer. Ostwald-Freundlich equation (2) also shows increased solubility with decreasing particle size .

Cr=C?exp(2yMrpRT)

(2)The solubilities of a particle of radius C(r),C(?)

2.Crystaline state

Drugs in their high-energy amorphous form are

Thermodynamically.  

Stable nanosuspensions are mostly dependent on particle charge. Drug nanoparticles are electrostatically repelled by an electric charge on their surface, which stops the particles from aggregating and precipitating. The electric double layer surrounding a charged particle is depicted in the diagram in Figure 3. A stern layer and a diusion layer of opposing ions make up the  double layer. The zeta potential is the name given to the electric potential near the shear plane Pure electrostatic stabilization is thought to require a minimum zeta potential of ±30 mV. A zeta potential of ±20 mV may be adequate to stop drug particles from aggregating and precipitating when electrostatic stabilization and steric stabilization are combined (by choosing the right polymers) . Adsorbed and hydrated polymer layers on the scattered polymer layers are what produce steric stabilization. Particle . When an electric field is applied, the charge of the particles is usually ascertained by measuring their electrophoretic mobility, which is subsequently transformed into zeta potential using the Helmholtz-Smoluchowski equation . An ultrasonic wave that causes the so-called electroacoustic phenomena can also be used to detect the zeta potential ^[31].

4.stability

Because there are more been unstable surface atoms and molecules when particle size is reduced, surface energy rises. This causes the colloidal suspension to become unstable. Stabilizers are therefore frequently required to prevent particle aggregation and lessen the likelihood of Ostwald ripening . Polysorbates, povidones, poloxamer, lecithin, polyoleate, and cellulose polymers are common stabilisers used to create nanosuspensions . Long-term stabilization of nanosuspensions has reported to benefit from a mixture of polymers and surfactants . Surfactants and polymeric materials function as ionic barriers and/or prevent particles from interacting closely with one another. By changing the zeta potential, surfactants can enhance particle stability and increase electrostatic repulsion . Another aspect to consider when evaluating the stability of nanosuspensions is particle precipitation. Stoke's law (3) states that the precipitation velocity decreases with decreasing particle size, decreasing solid phase density difference, and rising medium viscosity ^[32].

V=2r(?1-?2)g/((9n))                                                                                                (3)Where v stands for precipitation velocity, rfor particle size, ?

Method of nanosuspension

Although they are less complicated technically than liposomes and other popular colloidal drug carriers, nanosuspension preparations are said to be more cost-effective. It creates a more physically stable product, particularly for poorly soluble drugs (Fig. 1). Two competing methods for creating nanosuspensions are referred to as "Top-down process technology" and "Bottom-up process technology"  ^[33].The disintegration strategy used by the top-down method begins with big particles and advances to microparticles and nanoparticles

*High-pressurehomogenization

  a. Homogenization in aqueous media

  b. homogenization in non-aqueous media (Nanopure )

 c. Homogenization and precipitation combined (Nanoedge)

 d. Nanojet

a. Homogenization in aqueous media (Disso cube)

This technology was developed in 1999 by R.H.

Muller using a high-pressure homogenizer of the piston-gap type. High pressure with a 40 ml volume capacity and pressures ranging from 100 to 1500 bar and up to 2000 bar (for laboratory size) is the fundamental concept. Applying this pressure makes it simple to transform micron-sized particles into nano-sized ones. In order to use the jet mill to reduce the particle size to 25 microns ^[35]., which is what it originally was, we must take the sample from it. has must be a particle in the micron range. Additionally, we can use this equipment for both continuous and batch processes. In this case, the particles must first be put into a presuspension state

Principle:

This approach is based on the cavitation concept . The dispersion of the 3 cm diameter cylinder is abruptly pushed into a 25 m-wide aperture. Bernoulli's law states that the drift volume of liquid per crosssection in a closed system is constant. Below the boiling point of water at room temperature, it results in a decrease in static pressure and an increase in dynamic pressure due to the diameter decreasing from 3 cm to 25 m. After the suspension exits the gap (a process called cavitation) and the air pressure returns to normal, the water starts to boil at room temperature and produces imploding gas bubbles. When the particle cavitation forces are high enough, the drug nanoparticles are formed.

b. Homogenization In Non-Aqueous Media (Nanoedge)

Homogenized suspensions in water-based or water-free medium, such as PEG 400, PEG 1000, etc., make up nanopure ^[36]. Known as "deep-freeze" homogenization, the nonaqueous drug solutions were homogenized at 0°C or even lower than the freezing point. They can be used successfully for thermolabile chemicals in more tolerant situations because the results were similar to those of Disso Cubes. Gelatin or HPMC capsules can be filled with drug suspensions manufactured from drug nanocrystals suspended in liquid polyethylene glycol (PEG) or other oils.

c. combined precipitation and homogenization (Nanopure)

A miscible anti-solvent is combined with the organic solvent the drug is dissolved in to cause it to precipitate. The drug's poor solubility in the water-solvent mixture causes it to precipitate. Precipitation and high-shear processing have also been used together ^[37]. This is achieved through high-pressure homogenization and rapid precipitation. The nanoedge proprietary process from Baxter uses high shear and/or thermal energy to cause friable materials to precipitate, which breaks up materials. The blended solution unexpectedly becomes supersaturated and generates fine crystalline or amorphous particles when a medicine solution is rapidly added to an antisolvent. An amorphous material may also precipitate at high supersaturation when the amorphous state's solubility is surpassed. The basic ideas of homogenization and precipitation are the same as those of nanoedge. When these methods are combined, stability is improved more quickly and particle sizes are reduced. The precipitation method's main shortcomings, including crystal growth and long-term stability, can be addressed using nanoedge technology .

d.Nanojet

This method, sometimes referred to as opposing stream or nanotechnology, uses a chamber to split a stream of suspension into two or more parts that collide under extreme pressure. The process's high shear force causes a reduction in particle size . This idea underlies the operation of the Microfluidics M110L and M110S microfluidizers, which are used to prepare atovaquone nanosuspensions . This method's primary disadvantage is the large number of passes through the microfluidizer and the consequently high proportion of microparticles in the finished product ^[38].

*Media milling (Nanocrystal)

After being created by Liversidge et al. in 1992, this procedure was initially patented by the "Nanosystems" group [39]. The license to "Elan medication delivery" has finally been granted. In this case, the particle size is decreased by the high shear rate. Furthermore, the entire process is conducted at a regulated temperature . If not, a temperature will build up at high shear rates, causing part of the contents of the dosage form to degrade. This equipment is referred to as pearl mills or high-shear media miling .

This mill is composed of three major columns (Fig. 2):

• The recirculation chamber
• the milling chamber
• the milling shaft

       
           
       
Principle:

The high energy and shear pressures created by the drug's impaction with the milling media provide the energy input needed to convert the drug's microparticulate form into nanoparticles. The milling media is composed of glass, zirconium oxide, or polystyrene resins that are highly cross-linked. Either batch mode or recirculation mode can be used to execute the process. Dispersions with unimodal distribution patterns and mean diameters of 200 nm are produced in batch mode in 30 to 60 minutes. The media milling method can effectively handle drug crystals that are micronized or non-micronized. The quality of the dispersion varies comparatively little from batch to batch after the process and formulation are modified.

*Bottom- up process

it is a method for getting particles from the molecular to the nanoscale zone in order to reach nanosize. The term "bottom-up technique" describes the traditional precipitation ("hydrosol") techniques. Using the precipitation method, the drug is dissolved in an organic solvent, and the resultant solution is mixed with a miscible anti-solvent ^[40]. Because of its poor solubility in the water-solvent mixture, the medicine precipitates. The primary problem is that surfactant must be used to control crystal growth in order to stop the formation of microparticles during the precipitation process .

Advantages:

1. It may be used with easy-to-use, low-cost equipment

 2. It has greater saturation solubility ^[40].

Disadvantages:

1.  Drugs that are not adequately soluble in fluid and non-watery environments are no longer present in precipitation systems. At least one dissolvable that is miscible with a non-solvent should dissolve the medication in this system ^[40].
2.  Prevent Ostwald ripening, which is brought on by unique saturation solubilities close to particles of varying sizes, from causing crystal formation.

Precipitation Method

*Combined Method

Emulsion diffusion method

Making a pharmaceutical solution and then emulsifying it in a separate liquid that isn't the drug's solvent are the steps involved in this method. The drug precipitates as the solvent evaporates. Crystal formation and particle aggregation can be managed by using high-shear forces generated by a high-speed stirrer.

Micro-emulsion templates

The bulk of drugs that can be dissolved in this way are those that dissolve in volatile organic solvents or partially water miscible solvents ^[42]. In this procedure, the medication is dissolved in a suitable organic solvent and then emulsified in an aqueous phase with a suitable surfactant. In order to produce drug particles that precipitated in the aqueous phase, the organic solvent was then progressively evaporated under lower pressure. produced the required particle size for the drug's aqueous solution. Nanosuspensions can then be made by suitably diluting the produced suspension. Additionally, microemulsions can be used as templates to make nanosuspensions. Microemulsions are thermodynamically stable, isotopically transparent dispersions of two immiscible liquids, such water and oil, that are kept together by an interfacial coating of cosurfactant and surfactant. The medicine can either be put into the internal phase or carefully blended into the pre-formed microemulsion. The microemulsion is suitably diluted to create the drug nanosuspension. One advantage of using lipid emulsions as templates for the creation of nanosuspensions is that they are easy to manufacture and scale up. But using organic solvents has an effect. impact the environment, making the use of large amounts of stabilizer or surfactant necessary.

*Never Method

1. Supercritical fluid method

Many methods are used to produce nanoparticles, such as the precipitation with compressed antisolvent (PCA) process, the supercritical antisolvent process, and the rapid expansion of supercritical solution (RESS) process. The RESS approach involves expanding a drug solution via a nozzle into a supercritical fluid, which causes the drug to precipitate as tiny particles by losing some of its solvent power ^[43]. Young et al. used the RESS approach to manufacture cyclosporine nanoparticles with a diameter of 400–700 nm. Using the PCA process, the drug solution is atomized into the CO2 compressed chamber. When the solvent is eliminated, the solution becomes oversaturated, causing precipitation. During a supercritical antisolvent operation, a drug solution is injected into a supercritical fluid, causing the solvent to evaporate and the drug solution to become supersaturated

2. Dry co-griding method

The dry milling method is being used to create a lot of nanosuspensions. Dry-co-grinding eliminates the requirement for organic solvents and can be completed rapidly and economically. Co-grinding enhances the physicochemical properties and dissolution of weakly watersoluble medications by improving surface polarity and converting crystalline to amorphous drugs.

Evaluation Parameter

a) Saturation Solubility and Dissolution Velocity

b) Surface Hydrophilicity

c) Adhesion Properties

d) Particle Charge (Zeta Potential)

e)Mean Particle Size and Particle Size Distribution

 f)Crystal Morphology

g) Interaction With Body Proteins

a) saturation solubility and dissolution velocity

The ability of nanosuspension to accelerate both the dissolving velocity and saturation solubility gives it a significant edge over other methods. Compound-specific saturation solubility is a function of temperature and the characteristics of the dissolution media. Increases in saturation solubility can be explained using the Kelvin equation and the Ostwald-Freundlich equations. ^[19].

b) surface hydrophilicity p

One of the key factors influencing the in vivo organ distribution following intravenous administration is surface hydrophilicity/hydrophobicity. In addition to determining the contact with cells before phagocytosis, surface hydrophobicity is a significant parameter for the adsorption of plasma proteins, which is essential for organ distribution. The surface hydrophobicity must be assessed in the drug nanoparticles' original environment in order to prevent artifacts, which means in medium for aqueous dispersion. Hydrophobic interaction chromatography (HIC) is an appropriate method that was first used to assess the hydrophobicity of bacterial surfaces before being applied to the assessment of drug carriers in nanoparticulates. ^[44].

c ) Adhesion properties

Male Wistar rats can be utilized in in vivo bioadhesive studies. Typically, one oral dosage of a 1 ml water mixture comprising 10 mg of the drug-loaded nanoparticles (about 45 mg particles/kg body weight) is given to each animal. At one and three hours after administration, the animal is sacrificed by cervical dislocation. The stomach, small intestine, and cecum are extracted from the abdominal cavity, which is then opened lengthwise along the mesentery and cleaned with phosphate saline buffer (pH 7.4) Additionally, the cecum, small intestine, and stomach are divided into 2-cm-long segments and digested for 24 hours in an appropriate alkali. Two militeres of methanol were added to the digested samples, vortexed for one minute, and then centrifuged to extract the drug. Spectrophotometry is used to measure the percentage of adherent nanoparticles to the mucosa in an aliquot (1 ml) of the supernatants used for drug analysis. The drug's standard curve can also be created for computations.

4. Particle chaege (Zeta potensial)

Zeta potential gives certain information about the surface charge properties and further the long term physical stability of the nanosuspension. ^[45] Particle charge determines the stability of Nano suspension. For electrostatically stabilized Nano suspension a minimum zeta potential of ±30mV and for combined steric and electrostatic stabilization there should be a minimum of ± 20Mv. ^[44].

5. Mean particle size and particle size distribution

Two crucial characteristic characteristics that impact the saturation solubility, dissolution rate, physical stability, and even in-vivo behavior of nanosuspensions are the mean particle size and the span of particle size distribution (polydispersity index, PI). Laser diffraction (LD), coulter counter multiplier, and photon correlation spectroscopy (PCS) can all be used to determine the particle size distribution. The width of the particle size distribution (polydispersity index, or PI) can even be measured using PCS. For long-term stability, the PI, a crucial parameter that controls the physical stability of nano suspensions, should be as low as feasible. A size distribution that is somewhat tight is indicated by a PI value between 0.1 and 0.25, whereas one that is very broad is indicated by a PI value larger than 0.5.^[45].

f) Crystal morphology

It is possible to use methods such as X-ray diffraction analysis in conjunction with differential scanning calorimetric or differential thermal analysis to describe the polymorphic changes brought about by the influence of high-pressure homogenization in the drug's crystalline structure. Differential scanning calorimetry can be used in conjunction with X-ray diffraction analysis to ascertain the extent of the amorphous fraction and the change in the solid state of the drug particles. Scanning electron microscopy is the primary method for obtaining a true understanding of particle morphology. ^[45].

8. Interaction with body proteins

By incubating mucin and nanoparticles (1:4 weight ratio) in either an acidic or neutral media, the in vitro interaction between the two can be investigated. The incubation process is conducted at 37°C with stirring. Following centrifugation of the dispersions, 150 ?l of each supernatant is transferred to a test plate. After adding 150?l of the Micro BCA Protein Assay Reagent Kit to the plate and supernatants, it is incubated for two hours at 37°C. This method uses colorimeters to quantify the drug's ?max, which is the absorbance of mucin. The difference between the mucin's initial concentration and the concentration in the dispersion following incubation and centrifugation can be used to calculate the amount of mucin adsorbed to the nanoparticles. Mucin standard curves can be used as the foundation for the computations.^[44].

CONCLUSION:

Three nano-formulations—polymeric micelles, nanoparticles, and nanosuspension—included the multi-targeted kinase inhibitor MTKi-327. These intravenous injectable nanocarriers, which are smaller than 200 nm and tailored for the EPR effect, can deliver therapeutic dosages of MTKI-327. It was shown that the MTKi-327 nanosuspension given intravenously at a dose of 650 mg/kg produced the greatest regrowth delay in A-431 tumor-bearing nude mice. Because of

1. the greater MTKi-327 MTD,
2. the potential for intravenous injection of MTKi-327 for potential targeting by EPR effect,
3. its capacity to augment the supplied dose, and
4. its higher efficacy, nanosuspension may therefore be regarded as an effective anti-cancer MTKi-327 delivery system.

RESULT:

Physicochemical Characterization

Table 2 provides a summary of the drug concentration, size, and f potential results. In order to accommodate the EPR effect, all of the nanoformulations displayed sizes below 200 nm ^[46]. The polydispersity index (PDI) was below 0.2 for all of them, indicating a limited size distribution. As opposed to 10.6 mg/ml for the Captisol solution, the attainable drug concentration ranged from 109 mg/ml for the nanosuspension to 0.6 mg/ml for nanoparticles.

Table 2

Physico-chemical characterization of the 4 different formulations of MTKi-327 (n = 3).