Efficiency of self‐assembled etoricoxib containing polyelectrolyte complex stabilized cubic nanoparticles against human cancer cells

The aim of the present research was to formulate chitosan‐kheri gum polyelectrolyte complex (CKGPEC) stabilized etoricoxib containing cubic nanoparticles and evaluate against various human cancer cell lines.


| INTRODUCTION
The demand for new active moiety is increased continuously for the better therapeutic effect. Despite having good therapeutic effect, many active pharmaceutical ingredients suffer from poor aqueous solubility. 1 Poor aqueous solubility is a major obstacle for the translation of newly developed active moiety. Popular solubility enhancement method such as solid dispersion, prodrug and salt formation leads to dose escalation, high cost, and higher excipient percentage in the formulation. To overcome these limitations, the solvent-antisolvent method is widely employed for the generation of drug nanocrystals.
The development of nanoformulation for poorly water-soluble molecules is an extensively used strategy to improve the dissolution properties of the drug in the last decades. Solvent-antisolvent method has been touted to be a very promising bottom-up method for the preparation of nanoparticles, especially to improve the dissolution profile of less water-soluble drug candidates. 2 Attempts have been made to overcome drug-associated lower solubility. The application of nanotechnology in solubility improvement gains good attention due to rich expectations of novel outcomes with procedural modification. Method attracts worldwide researchers due to their simplicity and versatility. 3 Strategic improvement in nanosizing leads to improved dissolution rate hence bioavailability. Studies have shown that cyclooxygenase-2 (COX-2) is involved in tumor growth and advancement.
Selective COX-2 inhibitors block tumor development through many mechanisms, in particular through antiangiogenic and proapoptotic impacts. 4 In a study, Alhakamy et al discussed that an antifungal drug itraconazole shows anticancerous by same mechanism. 5 Wong et al showed that NSAIDS, specially COX-2 inhibitors (celecoxib, etoricoxib) have potential anticancerous activity against various cancer types. 6 Etoricoxib have pyrazole as a basic moiety and show various pharmacological activities in which anticancer activity is also involved. 7 Cyclooxygenase-2 (COX-2), an inducible prostaglandin G/H synthase, is overexpressed in several human cancers, including colon cancer and thus the potential ability of a selective COX-2 inhibitor, etoricoxib, is regarded in the rat model to prevent 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in the rat model. 8 In the present investigation, solubility of etoricoxib was enhanced by formulating nanoparticles and prepared formulations were evaluated for anticancerous activity against breast cancer cells.
Last few decades, researchers have shown concern regarding shape dependent therapeutic effect of nanoparticles. Shape and size modification of nanoparticles is an emerging technique to achieve prerequisite characteristics. The surface volume ratio and curvature dimension of nanoparticles is a major input to modify cellular behavior. Formation of low curvature rod and cubic nanoparticles enables them for better cellular internalization, biodistribution, and achieves unmet clinical needs.
Nanoparticles with unique morphology show better properties in vitro and in vivo due to increased surface area to volume ratio.
Higher surface area to volume ratio of nanoparticles enables them to dominate over the cellular barrier for drug targeting. For better therapeutic effect, it becomes essential for nanoparticles to cross cellassociated barriers. The division of cells in the tumor tissues also positively affects the uptake of nanoparticles within the cells. The internalization of nanoparticles also depends upon the shape of formulated particles. Elongated particles provide more binding sites to interact with tumor cells receptor, hence better therapeutic effect obtained than curve shape spherical nanoparticles. 9 Dasgupta et al were found that elongated particles showed a high propensity for cellular uptake than spherical particles for the same size range due to the high aspect ratio. 10 Exocytosis of elongated particles also found lower than spherical particles.
Despite chitosan is superior and approved biomaterial, it lacks water solubility. 11 Formation of polyelectrolyte complex between chitosan (Ch), a cationic polymer and kheri gum polysaccharide (KGP), an anionic polymer control solubility/dissolution of prepared nanoparticles in acidic (stomach), and basic (intestine) bioenvironment. Shah et al had prepared self-assembled nanoparticles by noncovalent, electrostatic interaction between oppositely charged molecules. They were also successfully utilized prepared nanoparticles for biomedical applications. 12 In the present investigation, the solvent-antisolvent method occupied with ultrasonication was applied for the formulation of cubic nanoparticles stabilized with Ch-KGP polyelectrolyte complex. Furthermore, prepared formulations were evaluated for anticancerous effect against human breast cancer cell lines (MCF-7), human colon cancer cell line (HT-29), and human melanoma cell line (SK-MEL-2). To the best of our knowledge, it was first attempt to fabricate chitosan-neem gum polyelectrolyte complex stabilized nanoparticles and studied against various human cancer cell lines.

| Materials
Chitosan (molecular weight: 190 000-310 000 Da) was procured from Merck Specialties Private Limited, Mumbai, India. Ethyl alcohol and acetone were supplied by S.D. Fine Chemicals, Mumbai, India. All the chemicals were used as supplied, without any purification. In experiments, HPLC grade water was used as a solvent.
Drug etoricoxib was obtained as a gift sample from Cipla Ltd,

Mumbai.
As described in our previous publication, crude KGP was collected and purified using a water-based extraction process. 13

| Preparation of PEC stabilized nanoparticles
For the preparation of PEC stabilized nanoparticles, the antisolvent method was used. In a recent study, nonstoichiometric ratios of anionic and cationic polymers were used for the fabrication of nanoparticles as shown in Table 1. HPLC grade water and 5% acetic acid was used as a solvent to prepare KGP and chitosan solution (20 mL each), respectively. In the solution of Ch, KGP was added dropwise and stirred at 50 rpm and 45 C for 30 minutes (PEC solution). Drug solution (10 μg/mL) was prepared by using acetone as a solvent and transferred dropwise into PEC solution by using a syringe (BD Emerald 5 mL). Moreover, the solution was stirred at 45 C for 30 minutes followed by cooling up to 35 C. Prepared formulations were stored in an airtight glass container.

| Factorial design
PEC stabilized nanoparticles were prepared using 3 2 factorial design.
In the present research, the concentration of Ch and KGP was selected as independent variables while particle size and entrapment efficiency of nanoparticles were selected as dependent variables (response factor). Three levels were selected for each independent variables as shown in Table 1 and the result was analyzed using NCSS 12 software (Trail version 12/06/2018) and shown in Table 2.

| Characterization of nanoparticles
Prepared polyelectrolyte complex stabilized nanoparticles were characterized for the following parameters: Particle size and zeta potential analysis Fabricated nanosuspensions were diluted to prepare 1% w/v suspension using water, the size and zeta potential was determined using zeta seizer (Malvern Instrument, version 6.32, Model No. ZEN3500, United Kingdom).

SEM analysis
Zeis EVO analyzer was used to study the morphology of nanoparticles.

Loading efficiency (%)
Formulation was mixed with 10 mL of 0.1 N HCl for 2 hours. The whole solution was centrifuged at 16 000 rpm in REMI centrifuge Preparation of egg membrane. To prepare the egg membrane egg of chicken (Gallus gallus) was taken. Further, egg yolk was removed by an orifice made at one end of the egg. A beaker was taken, filled with acidified water and egg was kept in it. The temperature was increased

Kinetics of drug release
The release pattern of the drug was also determined by using various kinetic models. In the present research model dependent methods (viz zero-order, first-order, higuchi model, Karsemeyer-Pepass model, Hixon-Crowel model, and Baker-Lonsdale model) and modelindependent methods (viz the similarity factor determination) were applied to characterize drug release pattern.
As discussed in our previous study Scale-Up and Post Approval Changes (SUPAC) guidelines provide a mathematical tool, that is, similarity factor S, for the comparison of dissolution profile of two formulations. Similarity factor S measures the closeness between the dissolution profiles of formulations. 14 In the present investigation, dissolution profiles of optimized formulation, K5 was compared when drug release was performed through two different biological barriers, that is, egg membrane and tomato membrane was characterized in terms of difference factor (f 1 ) and similarity factor (S). Difference factor (f 1 ) defines the percent difference in drug release between two curves at the same time where n is the number of time points, R t is the dissolution value of K5 when the egg membrane was used as a biological barrier at time t, and T t is dissolution value of K5 when the tomato membrane was used as a biological barrier at same time t. The similarity factor is log reciprocal square root transmission of the sum of square error (Equation 3).
Similarity factor S ð Þ = 50 log 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Similarity factor analysis was used to identify either release pattern of etoricoxib was the same or not when two different biological membranes viz tomato membrane and egg membrane were used as a barrier.

Particle size growth analysis
In the present study, nanoparticles were formulated which were dispersed in an aqueous medium. As they are in direct contact with the aqueous medium; so, it becomes necessary to evaluate the effect of the presence of solvent in crystal growth. Formulations were withdrawn at regular intervals viz 7, 14, 30, and 45 days and the effect of "Ostwald ripening" or crystal growth was analyzed using zeta analyzer.

Cytotoxicity screening
In vitro cytotoxicity study of optimized formulation K5 was carried out against human breast cancer cell lines ( The characterization parameters of KGP-Ch PEC stabilized nanoparticles were shown in Table 2.  Gibb's energy and prevent crystal growth. 15 The use of a stabilizer also retards the conversion of high energy forms (low crystal packing energy) of drug nanoprecipitates into low energy forms (high crystal energy forms).
Gibb's free energy (ΔG) for the cubic system can be expressed as Equation (4).
where γ is the surface energy of a solute-solvent system, Δμ is chemical potential, A cube is of cubic particle and V cube is the volume of cubic particle, and r is the width of the cubic particle.

| Factorial design
In the present investigation, 3 2 full factorial design was employed to evaluate the effect of concentration of cationic polymer (Ch) and concentration of anionic polymer (KGP) (independent variables) on particle size and entrapment efficiency (dependent variable). The reduced equation to measure the response (particle size and entrapment efficiency) having statistical significance for 3 2 factorial design can be shown as below Equation (6).
where Y is the response (dependent variable), b 0 arithmetic mean response of nine batches, and b 1 estimated coefficient for factor X 1 .
The coefficients corresponding to linear effects (b 1 and b 2 ), interaction (b 12 ) and the quadratic effects (b 11 and b 22 ) were determined from the results of the experiment. X 1 and X 2 are the concentration of Ch and KGP, respectively.
From the experimental data, Equation (6)

| Nucleation
Nucleation without the presence of foreign particles is known as homogeneous nucleation and can be expressed as Equation (9).
where B 0 is the rate of nucleation, A hom is pre-exponential factor, γ sl is interfacial tension at solid particles and solvent system, ϑ is molar volume, and T is the absolute temperature.
It can be concluded from above Equation (8), that the rate of nucleation increases with interfacial energy. High nucleation rate leads to precipitation of solute particles and in this case, supersaturation is utilized mainly for nucleation, not for the growth of particles.
To achieve a narrow size distribution of polyelectrolyte stabilized etoricoxib nanoparticles, researchers create a high degree of supersaturation, uniform distribution of antisolvent (by mixing) and controlled the growth of nanoparticles.
The formation of nanoparticles using antisolvent-solvent methods involves the formation of saturation zone, nuclei generation and particle growth in a successive manner. The initiation of particle formation depends upon the characteristics of the supersaturation zone in the solution. The size, shape and morphology of the nucleus depend upon supersaturation conditions. Mathematically supersaturation of API insolvent can be expressed using Equation (10) where C and C* are an actual concentration of API in solution and equilibrium solubility of API in solvent-antisolvent system, respectively.
The high degree of supersaturation leads to a higher nucleation rate due to lower Gibb's free energy. Mathematically nucleation rate (B o ) can be expressed as Equation (11).
where A hom depends upon the growing mechanism of solute particles, k is Boltzman constant, γ is interfacial tension, and T is the absolute temperature. Equation (10)  Uniform and a high degree of supersaturation lead to the formation of lower size range nanoparticles with narrow size distribution.
Higher nucleation rates lead to the formation of nanoparticles and in such cases, particle growth inhibited due to consumption by nucleation. Nucleation and particle growths are a simultaneous process and both target for supersaturation area. Controlled mixing causes coprecipitation of etoricoxib with polyelectrolyte, results in the formation of nanosuspension. The stabilizer also increases the nucleation rate and stabilizes the nuclei by reducing interfacial energy. This results in reduce particleparticle interaction and a decrease in the overall size of precipitated particles. The stabilizer also prevents secondary nucleation, which is an undesirable phenomenon to prevent agglomeration and Ostwald repining.

| Role of stabilizer
The presence of electric charge over particles due to PEC formation causes repulsion between stabilized particles and prevents aggregation.

| Shape and size of nanoparticles
SEM study shows that etoricoxib formed acicular crystals without stabilizer when precipitated using the solvent-antisolvent method ( Figure 3). Reverchon et al also discussed that in most of the cases acicular crystals are formed during API crystallization. 18 They also showed that generally amorphous particles are formed during solventantisolvent precipitation method because the process is spontaneous and API molecules have not enough time to go through organized precipitation.
The shape of nanoparticles also influences the circulation time and dynamics, cell internalization, and transport behavior. Surface curvature depends upon both the size and shape of particles. Curvature defines the contact between nanoparticles and cell membranes. In a study, Chen et al found that spherical gold particle was easily taken by HeLa cells as compared to rod-shaped gold particles of the same size. 2 The reason behind this opposite behavior is due to the difference in nanoparticle curvature. 19 Present research reports a new technique for the preparation of Damkohler number is the ratio of t mixing and t precipitation . When the value of D a is greater than 1, the process is controlled by mixing. In the present study, t precipitation t mixing , hence the value of D a is greater than1 and the process is mixing controlled. It was also found in the literature that D a > 1 results in the broader size distribution of nanoparticles but utilization of stabilizer results in narrow size distribution. The narrow size distribution of particle is also supported by PDI value as shown in Table 2

| Effect of ultrasonication
Ultrasound breaks any formed agglomerate. Ultrasound also controls the size, size distribution and crystal habit of formed nanoparticles.
Crystal faces are influenced by cavitations and abrasion caused by ultrasound.
In a study, Dalvi and Dave observed that sonication dependent precipitation leads to the formation of diamond-shaped particles. 22 Ultrasound increases the formation of particles by accelerating the diffusion process that further results in the reduction in induction time. Formation of cubic shaped particles may be due to the utilization of polyelectrolyte complex as a stabilizer or due to ultrasound waves or both. Ultrasound waves convert less ordered high-energy form to more ordered low energy form. Ultrasound also helps in adsorption and reordering of stabilizer molecules at the interface. It also decreases surface energy and interface and improved the stability of nanoparticles. Drug release data shows a peculiar drug release pattern of nanoparticles. All the formulation shows three phases of drug release viz initial immediate release followed by sustained release and finally the burst release. Time of 80% of drug release (T 80 ) from the formulation was summarized in Table 4.

| Dissolution and drug release
Minimum and maximum t 80% were found for K4 and K2, respectively, when the egg membrane was used as a biological membrane.
Minimum and maximum t 80% were found for K8 and K4, respectively, when the tomato membrane was used as a biological membrane.  Baker-Lonsdale's model of drug release can be expressed as Equation (12).

T A B L E 4
Estimation of T 80 of drug release where, the release rate constant, k, corresponds to the slope.  Table 7, drug release kinetics study of Ch-KGP stabilized nanoparticles through tomato membrane, in Table 8 drug release kinetics study of Ch-KGP stabilized nanoparticles through the egg membrane.

| Stability concern
Particle size growth analysis of prepared formulations was carried out.
In suspended conditions, the size of nanoparticles may be decreased due to the relative movement of solvent or particle-particle collision.
These mechanical processes are responsible for the formation of the tiny embryo and lead to the initiation of secondary nucleation. Secondary nucleation also depends upon stirring (impeller rotation) and concentration of solute. Mathematically it can be expressed as Equation (13): where W is impeller rotation speed, M T is the concentration of solid, ΔC is the difference between primary and secondary nucleation.
The temperature of the medium is also responsible for the alteration of nanoparticle size. Temperature changes the solubility of nanoparticles. Effect of temperature on the crystal growth can be expressed by using Arrhenius Equation (14) where G is the crystal growth rate, k G is constant depends upon temperature, and g is crystal growth order.
At higher temperatures, the Brownian movement increases due to higher kinetic energy that further leads to agglomeration. were shown in Table 9.

| In vitro cytotoxic study
As depicted in Figure    was considered as optimized formulations. All the prepared formulations were able to release drugs up to 420 minutes. It was concluded from the release data that formulations release the drug in three phases viz initial immediate release followed by sustained release and finally the burst release. It was interesting to note that the egg membrane and tomato membrane were not changed the drug release kinetics when utilized as a biological barrier. All the formulations