Preparation and in vitro Evaluation of Darifenacin HBr as Nanoparticles Prepared as Nanosuspension

Rafid A. Rasool Alobaidy^1* and Nawal A. Rajab^2
*Correspondence: Rafid A. Rasool Alobaidy, Iraqi Ministry of Health, Baghdad Health Department, Al-Rusafa, Al-Baladyat Sector, Iraq, Email:

Keywords

Darfenacin hydrobromid • Nano suspension • Nano particle • Surfactant • Poly dispersity • Sustained release • Ultrasonication • Zeta potential

Introduction

Reduced drug particle size is considered the most promising strategy to improve drug bioavailability and solubility, leading to a new area of nanotechnology [1]. Different methods outlined in the conventional approach can be overcome by utilizing nanotechnology Nanoparticles have been explored for drug delivery to enhance the bioavailability, sustained release, and intracellular penetrability as Nano medicine continues to grow and improve [2].

Nanoparticles are colloidal particles with size varying from 10 to 1000 nm. The benefits of nanotechnology including the ability to deliver effective medicine (Nano medicine), which is expected to have a significant impact on the pharmaceutical and biotechnology sectors. Separation technologies, histology research, clinical diagnostic tests, and medication delivery systems are just a few of the areas in which they might be used [3]. Darifenacin hydro bromide is an anticholinergic medication that is frequently used in individuals with overactive bladder who do not respond to conventional therapy. Darifenacin Hydro-bromide (DH) is an (S)-2-{1-[2- (2, 3-dihydrobenzofuran-5-yl) ethyl]-3-pyrrolidinyl}-2, 2-diphenylacetamide hydro bromide, which is supplied as a white crystalline solid. It is slightly water soluble (6.03 mg/ml), and the pka equals to 9.2. Its melting point is 232℃-236℃ .The molecular formula of DH is C28H31BrN2O2, and the molecular weight is 507.472 g/mol. It is a powerful muscarinic receptor antagonist, available as a hydro bromide salt. The DH oral absorption is poor due to its low solubility and poor bioavailability (15%–19%), and oral bioavailability is limited by first pass metabolism [4,5]. The target of the current work was to study the formulation of Nano suspension of DH with polymers to improve the solubility of drug and to prepare long acting nanoparticles formulation as sustained release with high bioavailability.

Materials and Methods

Materials

Darifenacine HBr, Eudragit RS, Soluplus®, PVA, PVP, methanol, HPMC, glycerin.

Method of preparation of darifenacine HBr nanosuspension

A nano suspension is an easy and cost-effective approach to manufacturing a physically more stable product for low-soluble medicines. The production of nano suspension by two techniques: “Top-down method” and “Bottom-up technology”. DH nano suspension was generated by the solvent evaporation method or known as the anti-solvent precipitation method [6].

The generated organic drug solution (organic phase) was then introduced drop wise into a 30 ml aqueous stabilizer solution using a disposable syringe with the tip positioned directly into the aqueous medium.After that; stirring the mixture at an agitation rate of 1000 revolutions in each minute (rpm) on a magnetic stirrer for 30 min. to permit the evaporation of a volatile solvent. [7] The formulation was sonicated for 15 to 30 minutes ensuring that the formulation did not become hot. Further, the formulation was transferred to an amber-colored bottle and stored in the refrigerator [8].

Factors affecting the formulation of nano suspension

Method of preparation of darifenacine HBr nanosuspension: To reach the best formula different types of stabilizers at various concentrations were used in the preparation of DH nanosuspensions. The formulas (F1- F9) prepared by using dissimilar stabilizers and subjected to particle size analysis. The effect of using single stabilizer type was studied in F3 which contain soluplus and F8-F10 contains PVA, PVP K30, and HPMC at drug: stabilizer ratio 1:1.

Characterization of darfenacine HBr nanoparticles

Drug characterization: A The melting point of DH was measured by capillary tube technique, recognized by the United States Pharmacopeia (USP). A sufficient quantity of pharmaceutical powder was put into a capillary glass tube sealed from one side, then the capillary tube smoothly tapped on a hard surface to form a firm compact powder. The tube was then placed gently in the electrical melting point equipment, where the temperature was steadily increased and monitored the range value began from liquefaction the powder until when completely melted was recorded [9].

UV spectrum (Varian Cary 100): A specific amount of DH dissolved in phosphate buffer solution pH 6.8. The resulted solution Spectro- Photometrically scanned from 200-400 nm to obtained λ max of DH.

The UV scan revealed the wavelength of absorption peak (λ max), after which a calibration (standard) curve was prepared on the identified wavelength of higher absorption (λ max) [10].

P.S and PDI: Dynamic light scattering procedure was used to calculate the P.S and PDI of DH nanoparticles at 25℃ in a detection angle of 90o utilizing the ABT-9000 Nano Laser Particle Size Analyzer. All samples were examined in triplicate and without dilution (SD) [11].

Zeta potential evaluation of nano-suspension: Zeta-potential evaluated by the use of zetasizer (Zetasizer Nano ZS, Malvern instrument, Worcestershire, UK). In the dispersion media the zeta potential describes the extent of repulsion among neighboring and identically charged particles. The characteristics of surface charge were studied to assess the stability of the prepared Nano suspension. Minimum limit needed for electrostatic stabilization of Nano suspension is ± 30 mv [12]. The light scattering fluctuations caused by the Brownian motion of Nano suspension compositions were examined [13].

Dissolution pattern of DH as in vitro nanosuspension: Volume of nanosuspension equivalent to 15 mg DH was taken into dialysis membrane (M.w. cutoff 12,000-14,000 Hi-media), and set to paddle of USP dissolution apparatus-Type II applying rate of rotation reached to 100 rpm. Then solution of buffer phosphate (pH 6.8) was utilized as dissolution environment in a volume of 900 ml at 37°C ± 0.5°C. Volume of 5 ml was withdrawn on time scheduled basis of 30 min from starting, and then replaced by fresh media of dissolution, up to 24 hr. Samples were filtered using 0.22 micro filter syringe (0.22 Mm). Filtrate absorbance recorded by UV analysis versus blank (phosphate buffer). Depending on a calibration curve, percentage cumulative release was calculated at 284 nm[11,14].

Freeze drying of nanosuspension: Freeze drying used to convert the optimum formula to dry powder, later for further evaluation. Mannitol used as a cryoprotectant at 3% w/v. About 140 ml of optimized formula prepared and freeze dried to yield a dry powder for evaluation. Four flasks frozen in a deep freezer at -20°C for 24 hr. The frozen flasks were attached to the vacuum port of the device, then four flasks each containing 35 ml of nanosuspension, instrument operated till dry powder yielded. Sublimation of solvent from frozen samples took 48-72 hr [15,16].

Scanning Electron Microscopy (SEM): SEM images the surface of solidified sample. Information recorded by the signal such as surface topography, outer layer morphology, compositional chemistry, crystallinity of the particulate and electrical conductivity [17].

Fourier Transforms Infrared Spectroscopy (FTIR): The (FTIR) spectrum was documented for pure drug and optimized formulation utilizing KBr pellet method. The pellets were made with a KBr hydraulic press that was operated under hydraulic pressure of 150 kg/cm2. The measured spectra was above 3600-400 cm−1 at ambient temperature with a resolution of 4 cm−1, using FT-IR 2500 apparatus [18].

Differential Scanning Calorimetric (DSC): DSC experiments were occurred with a DSC apparatus model DSC-6. A little amount of pure DH and selected formula are inserted in an aluminum pan, as well as the test was performed under nitrogen atmosphere at a flow rate of 40 ml/min and scanning rate of 10°C/min in the range of 15°C-300°C [19].

Results and Discussion

The antisolvent precipitation technique was utilized to prepare ten formulas of nanosuspension (F1-F9), and then subjected to characterization, the following parameters were estimated.

Determination of DH melting point: The melting point of DH was 130°C-134°C. Indicating that the drug obtained is pure [20].

Analysis of particle size with poly dispersity index: The mean particle diameter and polydispersity index are shown in Tables 1 and 2. Poly dispersity index is a factor used to clarify the particle size distribution of nanoparticles achieved from a particle analyzer and afford an indication long term stability of Nano suspension, when poly dispersity index value below than 0.3 indicate narrow size distribution (homogenous), while if more than 0.3 considered a wide size distribution (heterogeneous) [21].

 

Types and concentration of stabilizer effect: Different parameters affect particle size and stability of Nano suspension but the type and concentration of stabilizer are critically important. The effect of stabilizer sorts and concentrations on Nano suspension formulation is demonstrated in F1-F9, The formulas stabilized by Soliplus have the lowest particle size than other formulas contained different stabilizer, this is due to the chemical nature of Soliplus which contain polyethylene glycol backbone as hydrophilic part and vinyl caprolactam/vinyl acetate side chain as a lipophilic part, the interfacial tension of the surface particles decreases due to adsorption of Soliplus onto drug particles, preventing aggregation of the freshly generated nanoparticles as a result of steric hindrance [22].

Furthermore, the adsorption affinity of non-ionic stabilizers on particle surfaces is also influenced by stabilizer concentration, increasing the concentration of stabilizer causes a drop in particle size to a certain limit, beyond which a particle size enlarge owing to an elevate in the thickness of the coating enclosing the nanoparticles and preventing diffusion between the solvent and antisolvent phases as in F3, F4 and F5 [23]. The formation of H-bond between DH particles and one of other stabilizer polymers like PVA, PVP, HPMC in formulas (F7,F8,F9) may lead to stabilization of DH Nano suspension, These polymers may efficiently adsorb onto drug particles, forming a stable barrier around the particle's surface, preventing it from growing [24].

Zeta potential: Zeta potential of darfenacin hydrobromid nanosuspension was found to be 28.03 in F4 as shown in Table 3 which ensures good stability of the NS on longer storage

 

Fourier Transforms Infrared Spectroscopy (FTIR): The spectra of FTIR of the pure drug, polymer, and physical mixture drug: polymer at same ratio (1:1) and selected formula was assay and obtained characteristic peaks as in Table 4.

 

The main characteristic peaks of FTIR spectrum of darfenacin hydrobromid that at wave numbers(in cm^-1) are: 3469 for N-H asymmetric stretching of amide (3500-3400), 2929 for C-H asymmetric stretching of aliphatic methyl and methylene group,1664 for C=O stretching of amide (1695-1630), 1581 for C=C stretching of aromatic ring (1610-1500), 1438 for C-H bending of aliphatic methyl and methylene groups (1450-1400), 1353 for C-N stretching of tertiary amine (1360-1310), 1243 for C-O stretching of furan (1300-1000), 1099, 1060, 1033 and 1002 for in-plane C-H bending of aromatic ring (1300-1000), 894,813 and 767 for out plan C-H bending of aromatic ring (900-675) and 705 for C=C bending of aromatic ring (700- 675).

According to the FTIR data, there is no chemical interaction and no differences between the peaks of the fingerprint region produced in the darfenacin hydrobromid spectrum and the spectra of the physical combination of darfenacin hydrobromid and polymers (Table 4).

Differential Scanning Calorimetric (DSC) study: The compatibility between drug and excipients can be determined by utilizing DSC, also to assess the crystalline condition of drug when transformed to nanoparticles (Figure 1).

Study the release of darfenacin hydrobromid nanosuspension in vitro: The USP dissolution test apparatus-II was used to conduct an in vitro dissolution investigation for 3 selected formulas with lowest particle size. The media in phosphate buffer solution (pH 6.8) and 37°C ± 0.5°C were shown, and the results showed sustained release may reach to 24 hr as in Table 5 below. The rise in DR dissolving rate may be explained using the Noyes Whitney equation, which says that by lowering the particle size, especially to the Nano scale limit, the surface area increases and increasing dissolution velocity. When compared F3, F4 and F5, the amphiphilic stabilizer (Solupus®) might increase the surface wet-ability of the weakly aqueous-soluble product, resulting in better drug release through the NS formulation. The surfactant inhibited the agglomeration of particles size by supply ionic or stearic barriers by way of which inter-particulate interactions in nano-suspensions are prevented (Figures 2-4).

 

Conclusion

This study confirms that the antisolvent technique is suitable for the preparation of darifenacin nanoparticles with sustained release efficiency. This formulation approach can be used to improve the therapeutic efficacy of poorly soluble drugs. The changes in nanoparticle size were affected by changes in polymer concentration. From the above, can conclude F4 has the best characteristic and suitable for formulation as nanosuspension for sustained release formula.

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