DEVELOPMENT OF MESOPOROUS SILICA NANOPARTICLES OF RITONAVIR WITH ENHANCED BIOAVAILABILITY POTENTIAL: FORMULATION OPTIMIZATION, IN-VITRO AND IN-VIVO EVALUATION

DEVELOPMENT OF MESOPOROUS SILICA NANOPARTICLES OF RITONAVIR WITH ENHANCED BIOAVAILABILITY POTENTIAL: FORMULATION OPTIMIZATION, IN-VITRO AND IN-VIVO EVALUATION

Mohit Mahajan and Sadhana Rajput ^*

Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Kalabhavan Campus, Vadodara - 390001, Gujarat, India.

ABSTRACT: The objective of the study was to develop mesoporous silica nanoparticles for the poorly water soluble drug ritonavir (RTV) for enhancement of in-vitro dissolution and corresponding in-vivo bioavailability. A comparative assessment between 2D-hexagonal silica nano-structured MCM -41NPs and 3D cubic pore system MCM -48NPs on drug release rate was also investigated. RTV (BCS class II drug), was loaded in the synthesized MCM-41NPs and MCM-48NPs by the solvent evaporation technique. The obtained MCM-41NPs, MCM-48NPs and RTV loaded mesoporous nanoparticles were characterized by different analytical techniques like UV spectrophotometry, differential scanning calorimetry, thermogravimetric analysis, FTIR, N₂ adsorption-desorption technique, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and powder-XRD. The in-vitro release profile of RTV was studied in 900 mL 0.1N hydrochloric acid (HCl) medium using USP apparatus-II at 50 rpm. Further; In-vivo studies were performed in Wistar rats where drug loaded mesoporous nanoparticles were compared with pure RTV. In dissolution study MCM-48NPs showed better and fast release of RTV than the MCM-41NPs. In pharmacokinetics study, the maximum peak plasma concentrations of RTV, R- MCM-41NPs and R- MCM-48NPs reached 3.8 ± 0.85 µg/ml, 5.5 ± 0.72 µg/ml and 9.2 ± 0.77 µg/ml by 1 h. The AUC_0–t values of the R- MCM-41NPs and R- MCM-48NPs were found 1.34-fold and 1.94-fold higher respectively, as compared with pure RTV. The results demonstrated superiority of MCM-48NPs against MCM-41NPs in enhancing dissolution and improving the bioavailability of RTV.

Ritonavir, MCM-41NPs, MCM-48NPs, Drug loading, Dissolution, Bioavailability

INTRODUCTION: Human Immunodeficiency Virus (HIV) is the major cause of Acquired Immune Deficiency Syndrome (AIDS) which was the leading cause of mortality globally ¹.

Antiretroviral therapy has controlled the mortality rate significantly HAART recognizes as the most effective treatment and protease inhibitor play an important role in this.

But most of the anti-HIV drugs are administered orally and very few are delivered through parenteral or topical route. Most of these drugs show poor solubility when administered orally and thus affecting their bioavailability. Because of low and variable oral bioavailability antiretroviral drugs require a high dose to produce the desired effect ^{2, 3}.

Ritonavir (RTV), a HIV protease inhibitor comes under a BCS class II category drug, indicating that it has low solubility and high permeability. RTV is highly lipophilic as its log-D value is 4.3, at 25 ºC at pH 6.8. RTV is poorly water soluble and it shows an extremely slow dissolution rate 0.03mg/cm²-min in 0.1 N HCl and pH-dependent solubility which could exhibit limited absorption. This may be the reason for the variation in solubility and bioavailability ^{4, 5}.

RTV is reported to boost up the circulating concentration of other PI resulting in less dosing frequency though its higher dose could lead to higher toxicity effect in patients and because of their high dose and side effects ^{6, 7} there is a need for an innovative formulation approach to enhance the bioavailability.

It is widely accepted that formulation development helps BCS class II drugs to achieve much higher bioavailability by enhancing their solubility. As per literature, there are several techniques that have been utilized to increase the solubility, dissolution rate and bioavailability of RTV by using various approaches such as solid dispersion ^{8, 9}, solid self-micro emulsifying drug delivery ¹⁰, solid self nano emulsifying drug delivery system ¹¹, nanoparticles ^{12, 13}, inclusion complex ^{14, 15} etc. From last decade, mesoporous silica nanoparticles are considered to be the best possible and unique approach for poorly water-soluble drugs to increase dissolution as well as the bioavailability.

Literature survey shows that a number of poor soluble drugs such as atrovastatin ¹⁶, celecoxibe ^{17, 18}, itraconazole ¹⁹, carbamazepine ²⁰ and dasatinib ²¹ could be successfully loaded into various types of mesoporous silica nanoparticles for various applications like solubility and bioavailability enhancement, controlled drug/gene release and targeted delivery carriers. Mesoporous silica nanoparticles have several attractive features like high surface area, high adsorption capacity of drug, ability to convert the crystallinity of the drug to an amorphous state, reduction of the particle size to nanometre range, the stability of loaded drugs within pores and pores can be easily modified as per drug delivery kinetics, that makes it useful for drug delivery. MSNs surface has free hydroxyl groups which easily interact with the particular functional group of drug molecules. Due to this feature, MSNs have opened new possibilities in drug delivery system ^{22, 23}.

In the present study, different structural and pore size mesoporous silica nanoparticles have been used with the objective for improving the solubility and dissolution rate of RTV an antiretroviral drug, which ultimately affects its bioavailability. MCM -41NPs and MCM-48NPs (MSNs) belong to the M41S family of mesoporous material; MCM-41NPs is a 2D-hexagonal structure ²⁴, whereas MCM -48NPs having a 3D cubic structure ²⁵, both having high surface area and tunable pore size. Apart from this, no literature is available for RTV loaded in mesoporous silica nanoparticles till date.

The major aims of this study were: (i) synthesis and characterization of different mesoporous silica nanoparticles such as MCM-41NPs and MCM-48NPs (ii) loading of RTV in MSNs by solvent evaporation method (iii) preparation and evaluation of tablet formulation of R-MSNs as per IP (iv) performing in-vitro dissolution study of prepared formulation and (v) performing in-vivo pharma-cokinetic (PK) study of prepared formulation in Albino Wistar rats.

MATERIALS AND METHODS:

Materials: The active pharmaceutical agent RTV was received as gift sample from Hetero drug limited, Hyderabad, India. Polyoxyethylene 10 lauryl ether (PLE), Cetyltrimethylammonium bromide (CTAB; ≥ 98 %), tetraethyl ammonium hydroxide (TMAOH; ≥ 98 %), tetraethyl orthosillicate (TEOS; ≥ 99 %) and fumed silica were purchased from Sigma-Aldrich (USA). Hydrochloride acid (HCl) and methanol (HPLC and AR grade) were purchased from Rankem (India). All other solvents and material were of AR grade and were used without further purification. Deionized water was utilised in the synthesis of mesoporous silica nanoparticles.

Synthesis of Mesoporous Silica Nanoparticles (MSNs):

Synthesis of MCM-41NPs: MCM-41NPs was synthesized as per the procedure given in the literature ²⁶. Accurately weighed 4.42 g of CTAB was added in 36 g of deionized water and stirred for 15 min. Then 3.46 g TMAOH was added drop wise in the surfactant solution of CTAB with constant stirring. The mixture was stirred for 30 min. Then 3 g of fumed silica was added slowly and stirred continued for 1.5 h.

The obtained gel was treated by hydrothermal crystallization technique for 48 h at 110 °C in a reactor. The solid product was recovered by filtration, washed with deionized water and was kept at room temp overnight. Further removal of surfactant was carried out by calcination of the product in a muffle furnace at 550 °C for 6 h. The recovered final product was named as MCM-41NPs.

Synthesis of MCM-48NPs: The MCM-48NPs synthesis was carried out as per the procedure given in literature ²⁷. Firstly, 0.4 g CTAB was added in 30 mL of methanol-water (1:2) solution and stirred the solution for 15 min. Then 5.4 mL ammonium hydroxide (NH₄OH) and 1.7 mL ethyl acetate were added in the surfactant solution and stirred the solution for 10 min. afterward, 0.9 mL TEOS was added drop wise into the solution with continuous stirring.

Further; 150 mL water was added into the solution and kept the solution overnight under stirring at room temperature. The pH of the solution was around 11.5. Then the resulting solid sample was filtered and washed with methanol and finally dried at 60 ºC in an oven. Then the final product was calcined in a muffle furnace for removing of surfactant at 550 °C for 6 h. Recovered material was labelled as MCM-48NPs.

Loading of RTV in Mesoporous Silica Nanoparticles (R-MSNs): A solvent evaporation method was used to load RTV in MCM -41NPs and MCM-48NPs (MSNs). MCM-41NPs was added in the methanolic solution of RTV (10 mg/ml); at drug to carrier ratio was 1:1.5 (w/w). Afterwards, the mixture was stirred for 2 h at room temperature for achieving maximum drug loading in the MCM-41NPs. Finally, the methanol was evaporated at 50 °C on a Bucchi rotary evaporator until completely dry. The recovered solid was dried at room temperature, which was named as R- MCM -41NPs. The same solvent evaporation method was used for loading RTV in the MCM-48NPs and after drug loaded it was designated as R-MCM-48NPs.

Physical Mixture of RTV and Mesoporous Silica Nanoparticles: The physical mixtures (PM) were prepared by mixing of pure RTV with MCM-41NPs and RTV with MCM-48NPs in a mortar and pastel with gentle triturate which were designated as R-MCM-41NPs-PM and R- MCM-48NPs-PM respectively.

Characterization of MSNs and RTV Loaded MSNs (R-MSNs):

Scanning Electron Microscopy (SEM): The morphology of synthesized MSNs was determined by SEM operated at an acceleration voltage of 15 kV. The MSNs samples were affixed to an aluminium stubs with double side adhesive carbon tape and gold coated with ion sputter MC1000. The samples were examined using a Hitachi-SU 1510 scanning electron microscope.

Transmission Electron Microscopy (TEM): The porous structure and particle size of MSNs was confirmed by TEM Analysis. A TEM image of MSNs was taken with a TECHNAI-G2 Spirit-Biotwin, operated at 120 kV. The MSNs samples were dispersed in deionized water in an ultrasonic bath for ten minutes. Several drops were deposited on 200 mesh, copper grid coated with a holey carbon film. The electron micrographs images were recorded on electron negative films and digital PC system attached to the TEM system.

UV-VIS Spectroscopy and Thermogravimetric Analysis for Evaluation of Drug Loading Efficiency: UV-VIS spectroscopy (UV-1700, Shimadzu) was used for determining the loading efficiency of synthesized MSNs at 240 nm wavelength. A small amount of RTV loaded MSNs were dispersed and mixed in a certain volume of methanol respectively; so that the RTV gets easily solubilised into the preferred solvent and subsequent filtration of sample was carried out. The amount of drug was calculated with the help of the standard calibration curve. The loading efficiency was confirmed by using thermogravimetric analysis (TGA) on Shimadzu thermogravimeter TGA-50. Around 3-10 mg sample of RTV and R-MSNs were kept into the platinum pan respectively, then heated up to 500 °C at a scanning rate of 10 °C/min under a nitrogen gas flow of 50 mL/min. The thermo-grams were analyzed using the TA-60 software.

The % entrapment efficiency for RTV and % loading efficiency for MSNs were calculated by using a formula:

% Entrapment efficiency =Weight of RTV in nanoparticles / Weight of RTV initially added× 100

% Loading Efficiency =Weight of RTV in nanoparticles / Total Weight of sample × 100

FT-IR Analysis: FT-IR spectra of RTV, MCM -41NPs, MCM-48NPs, R-MCM-41NPs- PM, R- MCM-48NPs-PM R- MCM-41NPs and R- MCM-48NPs were recorded on a BRUKER ALPHA-T (GERMANY) FT-IR Spectrophotometer at room temperature. The samples were gently mixed with KBr powder in mortar and pestle. Then pallets were prepared under 5000 psi pressure. The IR spectras of samples were taken in the spectral region 4,000 to 700 cm⁻¹ using the resolution 1cm⁻¹.

Differential Scanning Calorimetry (DSC): To study the physical state of RTV, MCM-41NPs, MCM-48NPs, R-MCM-41NPs-PM, R-MCM-48NPs-PM R- MCM-41NPs and R- MCM-48NPs were examined by DSC on a Shimadzu DSC-60. Around 3-5 mg of samples were put into an aluminium pan, crimped it with an aluminium lid to provide an adequate seal and heated under nitrogen purging (flow rate 40 mL/min) from room temperature to 300 °C. The temperature rise rate was fixed at 10 °C per min. The data were analyzed using TA 60-WS software.

Powder X-ray Diffraction (PXRD): The crystalline arrangements and the nature of pure RTV, MCM-41NPs, MCM-48NPs, R-MCM-41NPs and in R- MCM-48NPs were studied using a powder X-ray diffractometer (EMPYREAN, PAN alytical) using CuKa radiation beam operating at 40 kV and 30 mA. The samples (both MSNs and R-MSNs) were scanned at a low angle from 1 to 10 degrees in continuous mode at scanning speed 0.02 2θ/5s and also RTV and R-MSNs scanned from 5 to 50 degrees in continuous mode.

N₂ Adsorption-Desorption Analysis: N₂ adsorption and desorption analysis is a most reliable method to study the porosity of mesoporous material and was carried out to get information regarding BET surface area, pore size (nm) and pore volume of plain MSNs and R-MSNs by using micromeritics ASAP 2010. Earlier to characterization, plain MSNs samples were degassed under vacuum at 200 °C for 5 h, while the R-MSNs samples were degassed at 40 °C for overnight in order to avoid sublimation of RTV. The BET specific surface area was calculated by application of the BET method to the isotherm. The pore volume and pore diameter of both plain MSNs and R-MSNs were calculated by application of Barrett-Joyner-Halenda (BJH) method to the isotherm.

Formulation of Tablet and Evaluation: R- MCM -41NPs and R- MCM -48NPs were formulated in tablets by direct compression method. Nano-particles equivalent to 100 mg RTV and different excipients like low- hydroxypropyl cellulose (L- HPC), microcrystalline cellulose, cross-povidone, lactose monohydrate (SUPERTAB 11SD) and magnesium stearate were blended and punched in single punch tablet machine having 12 mm diameter punches with flat faced beveled edges. Prepared tablets were characterized by various parameters such as weight variation, hardness, friability and disintegration time etc.

In-vitro Dissolution Study: In-vitro dissolution and release study was performed in dissolution apparatus (Veego dissolution test apparatus). Six dissolution units were studied for in-vitro dissolution of the RTV pure drug and R-MSNs. R- MCM-41NPs and R- MCM-48NPs equivalent to 100 mg tablets of RTV and pure drug RTV tablets were taken for the in-vitro dissolution study. In-vitro dissolution studies were conducted in the 0.1 N HCl media using USP dissolution apparatus II, 900 mL media volume at 37± 0.5 °C temperature and the rotation speed of 50 rpm.

At predetermined time intervals of 10, 20, 30, 45 and 60 min, five mL of dissolution sample was removed from the vessels with the help of cannula, replacing the same amount with fresh dissolution medium. Samples were filtered through 0.22 µm syringe filter and RTV content was determined by UV spectro-photometry (λ = 240 nm) method.

In-vivo Pharmacokinetic Study: The in-vivo studies were carried out for comparing the plasma profile of the pure RTV, R- MCM -41NPs and R- MCM-48NPs. In order to establish that the improved bioavailability was achieved with a preparation of R-MSNs as compared with the pure RTV. In this study, either sex of Albino Wistar rats (250-300 g) were used having oral dose administration. The research protocol for the animal studies was approved by the Institutional Animal Ethics Committee (IAEC, file No. 1404), The Maharaja Sayajirao University of Baroda, Vadodara, India. All the experiments were performed in triplicates and all the results are given in the mean ± standard deviation (SD).

The bioavailability of R- MCM-41NPs and R- MCM-48NPs was compared to the pure RTV. The pure RTV and R-MSNs equivalent to 10 mg/kg dose of RTV were dispersed in 2 mL of CMC solution (0.5 % w/v) and administrated orally to Wistar rat. The rats were anesthetized by using ether before blood withdrawing. Blood samples (0.3 mL) were collected through the retro-orbital vein into 60 μL EDTA (0.5% w/v) containing micro centrifuge tubes at 0, 0.5, 1, 1.5, 2, 4, 6, 8 and 12 h after administration. Collected blood samples were mixed with the anticoagulant by properly shaking and centrifuged at 5000 rpm for 10 min at 4 ºC using a high-speed centrifuge machine and then plasma samples were collected and stored at -20 °C.

A simple protein precipitation method was used for extraction of RTV from collected plasma samples. Acetonitrile was used as a protein precipitating solvent. 100 μL of drug contain plasma samples were piped into a micro centrifuge tubes and 400 μL of acetonitrile was added into it and mixed onto vortex for 2 min. Further, the samples were centrifuged at 10000 rpm at 4-5 °C for 15 min. The supernatants of the centrifuged samples were transferred into a sample loading vials and which were injected into the HPLC system. In the PK study, parameters like C_max, t_max, t_1/2 and Area Under Curve (AUC) were calculated from plasma concentration vs. time profile curve and results were showed as mean ± SD.

Storage Stability Studies: Storage stability study were performed by following the European Agency for the Evaluation of Medicinal Products guidelines for solid dosage forms which prescribes to keep the samples at 40 ºC ± 2 and 75% ± 5 of relative humidity (RH) for 6 months ²⁸. R-MSNs was kept into a glass vial respectively and then thermo stated at 40 ºC ± 2 and 75% ± 5 RH. The samples were withdrawn at established time 1, 3 and 6 month and changes in R-MSNs were observed by DSC and P-XRD.

RESULTS AND DISCUSSION:

SEM and TEM Study: The morphology, pore structure and particle size of MSNs were confirmed by SEM and TEM analysis respectively.

FIG. 1: SEM IMAGE OF (A) MCM-41NPs AND (B) MCM-48NPs

FIG. 2: TEM IMAGE OF (A) MCM-41NPs AND (B) MCM-48NPs

Fig. 1A and B showed the morphology of MCM-41NPs and MCM-48NPs were uniform in shape and having a smooth surface. Fig. 2A and B illustrated pore structure of MCM-41NPs and MCM-48NPs and it clearly showed regular 2D hexagonal honeycomb like structure arrangement and the cylindrical 3D cubic network formed by MCM-41NPs and MCM-48NPs respectively. Well structured MCM-41NPs and MCM-48NPs shows mean particles size 150 nm.

Analysis of Drug Loading Methods: After RTV loading in MSNs by solvent evaporation method, % loading efficiency and % entrapment efficiency were determined by UV spectrophotometry using above formulas (1 and 2). Alike, the thermal analysis was also carried out for the same samples for confirmation of % drug loading. TGA thermograms are shown in Fig. 3. Both analyses showed comparable results for % loading and % entrapment efficiency i.e. 88.5% and 38% for R- MCM-41NPs and 98.5% and 45% for R-MCM 48-NPs respectively.

FIG. 3: TGA THERMOGRAM OF (A) RTV, (B) R-MCM-41NPs AND (C) R-MCM-48NPs

Fourier Transforms-Infrared Spectroscopy (FT-IR): For functional group identification and confirming the compatibility between RTV and silica nanoparticles, FT-IR study was carried out. FT-IR spectra of pure RTV, MCM-41NPs, MCM-48NPs, R- MCM-41NPs-PM, R-MCM-48NPs-PM, MCM-41NPs and R-MCM-41NPs are shown in Fig. 4. The RTV spectrum shows peaks at 3327 cm^-1 relative to the N-H stretching of an amide group, 2962 cm^-1 relative to hydrogen-bonded acid within the molecule, 1706 cm^-1 relative to the ester group, 1660, 1611 and 1540 cm^-1 relative to –C=C– stretching aromatic carbons.

The FT-IR spectrum of MCM-41NPs and MCM-48NPs Fig. 4B and C gave a broad peak between 3350-3500 cm^-1 which proving the presence of isolated terminal silanol groups. The Si-O-Si and Si-OH stretching vibrations were shown at 1084 and 801 cm^-1 respectively. In R-MCM-41NPs-PM spectrum showed characteristic peaks of RTV and MCM-41NPs which proves compatibility between both drug and silica nanoparticles. Similar results were obtained for R-MCM-48NPs-PM. Fig. 4D and E.

On the other hand, in case of R- MCM-41NPs and R- MCM-48NPs spectrum Fig. 4F and G showed a remarkable decrease of the peak at 2962 cm^-1, 1706 cm^-1 and slight shifting of –C=C– stretching aromatic carbons with the disappearance of other peaks indicating that the complete uptake of the drug by MSNs. These changes suggested that the isolated terminal silanol group present in MSNs have some interactions with RTV functional groups.

FIG. 4: FT-IR (A) RTV, (B) MCM-41NPs, (C) MCM-48NPs, (D) R-MCM-41NPs-PM, (E) R- MCM-48NPs-PM, (F) R-MCM-41NPs AND (G) R- MCM-48NPs

Differential Scanning Calorimetry (DSC): The DSC thermogram of crystalline pure RTV, MCM-41NPs, R-MCM-41NPs-PM, R-MCM-48NPs-PM, R-MCM-41NPs and R-MCM-48NPs are shown in Fig. 5.

FIG. 5: DSC THERMOGRAM OF (A) RTV, (B) MCM-41NPs, (C) MCM-48NPs, (D) PHYSICAL MIXTURE OF RTV AND MCM-41NPs, (E) PHYSICAL MIXTURE OF RTV AND MCM-41NPs, (F) R- MCM -41NPs AND (G) R-MCM-48NPs

Crystalline RTV thermogram exhibited a sharp endothermic peak at 123 °C which corresponds to its fusion temperature point Fig. 5 A. MCM-41NPs and MCM-48NPs thermogram did not show any transition because the fusion point of silica is very high Fig. 5B and C. In both physical mixtures, the sharp endothermic peak of RTV was present indicating the compatibility between MSNs and pure RTV Fig. 5D and E. The R-MCM-41NPs and R-MCM-48NPs thermogram did not show any sharp endothermic peak of RTV, it suggested that no drug was present on the outer surface of nanoparticles Fig. 5F and G confirmed successful loading of RTV.

Powder XRD (PXRD): Low-angle powder XRD (LPXRD) patterns of MCM-41NPs and MCM-48NPs are shown in Fig. 6. LPXRD patterns of R- MCM-41NPs and R- MCM-48NPs are shown in Fig. 6A (b) and 6B (b) respectively, in that the intensity of the nanoparticles peak was slightly decreased that confirmed the drug load in MSNs and no structural changes in the structure of nanoparticles. The high angle powder XRD pattern of plain RTV is shown in Fig. 6C (a) showed several characteristic peaks at region 5-40° in the 2θ/5s region, which confirmed the crystalline nature of the drug.

FIG. 6: LOW ANGLE POWDER-XRD (A) (a) MCM-41NPs, (b) R-MCM-41NPs; (B) (a) MCM-48NPs, (b) R- MCM-48NPs. HIGH ANGLE POWDER XRD (C) (a) RTV, (b) R-MCM-41NPs, (c) R-MCM-48NPs

Whereas in Fig. 6C (b and c) showed that the drug characteristic peaks were completely disappeared in R-MCM-41NPs and R-MCM-48NPs respectively. That confirmed the RTV was completely loaded and no crystalline drug remains on the outer surface of MSNs respectively. This also shows that MSNs can stabilize the amorphous state due to confinement.

N₂ Adsorption Analysis: N₂-adsorption-desorption isotherms relative to MCM-41NPs, MCM-48NPs and R-MSNs are shown in Fig. 7. The N₂ adsorption-desorption isotherms gave data related to specific surface area, pore volume and pore size of nanoparticles.

FIG. 7: BET ISOTHERM OF (A) (a) MCM-41NPs, (b) R-MCM-41NPs (B) (a) MCM-48-NPs, (b) R-MCM-48NPs. PORE SIZE DISTRIBUTION OF (C) MCM-41NPs AND R- MCM-41NPs, (D) MCM-48-NPs AND R-MCM-48NPs

All the N₂ adsorption-desorption isotherms showed typical type IV isotherms and hysteresis loop (according to IUPAC) which confirmed that the nanoparticles have mesoporous property. After the drug loading, in R-MCM-41NPs, the type IV pattern and hysteresis loop of isotherm remain intact, reduction in surface area, pore volume and pore size as compared with MCM-41NPs Fig. 7A and C. The similar results were obtained for MCM-48NPs and R-MCM-48NPs Fig. 7B and D. However, MCM-48NPs having the higher surface area, cubic structure and smaller pore size compared to MCM-41NPs which could possibly have a significant impact on in-vitro and in-vivo profile of RTV. N₂- adsorption- desorption parameters of MCM-41NPs, MCM-48NPs and R-MCM-41NPs, MCM-48NPs are given in Table 1.

TABLE 1: EVALUATION PARAMETER OF R- MCM-41NPs AND R- MCM-48NPs BY N­­₂ ADSORPTION-DESORPTION

Evaluation of RTV Tablets: Evaluations of formulated RTV tablets were carried out by using several parameters like weight variation, hardness, friability, disintegration time and drug content (%). The results of all parameters are shown in Table 2. The Hardness for both prepared tablet formulations were in ranging from 6.8 to 7.8 kP, indicating that the hardness of tablet was good enough to withstand the external pressure. Tablets were prepared using standard excipients exhibited disintegration time of 1 ± 0.3 min; friability was less than 1% and % drug content values were obtained in the range of 98.12 - 101.56%.

TABLE 2: EVALUATION OF PREPARE R-MCM-41NPs AND R- MCM-48NPs TABLETS

In-vitro Dissolution Study: Drug release studies were performed to see the release pattern of pure RTV and drug loaded nanoparticles in 0.1 N HCl. The dissolution rate was significantly enhanced in the R-MCM-41NPs and R-MCM-48NPs as compared to pure RTV. The augment in dissolution rate in silica nanoparticles was observed due to the conversion of RTV into amorphous form after loading into mesoporous nanoparticles.

The RTV release profiles are illustrated in Fig. 8. R-MCM-48NP showed more than 95% drug release in dissolution media within 45 min, whereas pure RTV and R-MCM-41NP showed almost 39% and 72% drug release respectively in 0.1 N HCl. The reason for MCM-48NPs showing better dissolution profile than MCM-41NPs is might be due to the high surface area, small pore size and 3D-cubic pore structure of MCM-48NPs which adsorbed the drug molecule very efficiently.

FIG. 8: IN-VITRO DISSOLUTION STUDY IN 0.1 N HCl

The RTV molecules adsorbed in the high surface of the 3D interconnected MCM-48NPs gave faster dissolution and more rapid diffusion in the dissolution medium while, MCM-41NP has 2D independent long channels, appeared to prevent the drug molecules in the pore channels from diffusing into the dissolution medium resulting slow drug release.

In-vivo Study: RTV is a typical BCS II drug; whose absorption will be rate limited through the dissolution process. In the present study, the results of in-vitro dissolution studies were confirming the enhanced dissolution of RTV by R- MCM-48NPs and R-MCM-41NPs. To study the silica nano-particles effect, the in-vivo studies were performed in which the drug suspension was given orally to Wistar rat.

FIG. 9: PLASMA CONCENTRATION–TIME PROFILES OF RTV AFTER ORAL ADMINISTRATION AT A DOSE OF 10 mg/kg IN MALE RATS (n=3)

The results of plasma concentration-time profiles and the PK parameters of RTV are shown in Fig. 9 and Table 3, respectively. In Fig. 9, it is clearly shown that the absorption rate of R-MCM-48NPs was higher than R-MCM-41NP and pure RTV; that exhibited maximum plasma concentration was 9.21µg/mL by 1 h. Additionally, compared with the pure RTV the C_max and AUC_0–t values of the R-MCM-48NPs were increased 2.48-fold and 1.94-fold respectively.

TABLE 3: PHARMACOKINETIC PARAMETERS OF PURE DRUG AND PREPARED FORMULATION

However, regarding the R-MCM-41NPs, the C_max and the AUC_0–t values were increased 1.44-fold and 1.34-fold respectively. The PK profile clearly showed improvement in the bioavailability of R-MSNs as compared to pure drug. Maximum RTV concentration in plasma was achieved by the R-MCM - 48NPs formulation which was approximately 1.67 - fold more than the R-MCM-41NPs formulation.

Stability Study: Improvement in the dissolution rates were occurred due to drug adsorption on silica nanoparticles which having high surface area and very small pore size that converted drug in to amorphous form. Therefore, it is mandatory to conduct physical stability study for adsorbed drug. The effect of humidity and temperature was observed. The 40 ºC ± 2 and 75% ± 5 relative humidity for six months accelerated stability test samples were analyzed by DSC and PXRD. PXRD of samples that stored at 40 ºC ± 2 and 75% ± 5 relative humidity for six months showed in Fig. 10 C and D, absence of characteristic crystalline peaks of RTV for the R-MSNs samples. The result shows the mesoporous material upkeep the RTV in an amorphous state. These results were again confirmed by DSC as well; where RTV fusion point could not be detected in Fig. 10A and B. The results showed that the silica nanoparticles can hold the drug in amorphous form for a longer duration of time.

FIG. 10: DSC AND P-XRD OF STABILITY SAMPLES (A) AND (C) R-MCM-41NPs; (B) AND (D) R-MCM-48NPs RESPECTIVELY

CONCLUSION: In this study, the synthesized mesoporous silica nanoparticles MCM-41NPs and MCM-48NPs were suitable carriers for poorly soluble RTV drug. RTV was loaded in both the silica nanoparticles to examine the effect on solubility through the drug loading.

To achieve maximum drug loading, solvent evaporation method was preferred with an appropriate ratio of drug and carrier (1:1.5). In addition, characterization results like DSC, PXRD and N₂ adsorption-desorption confirmed that the RTV was successfully loaded into the mesoporous silica nanoparticles. In in-vitro drug dissolution study, both MCM-41NPs and MCM-48NPs showed several advantages as a carrier for drug delivery respectively. Both MCM-41NPs and MCM-48NPs could notably increase the dissolution rate of RTV as compared to the pure RTV, but R- MCM-48NPs showed better in the fast release.

The PK studies results affirmed the ability of the mesoporous silica nanoparticles, especially the MCM-48NPs enhancing the in-vitro dissolution rate and improve the bioavailability. The reason behind the above results is that MCM-48NPs having 3D cubic pores structure which offers easy drug diffusion from the interconnected pores into the dissolution media. From all the above facts revealed that MCM-48NPs contribute faster drug release as compared to MCM-41NPs with 2D hexagonal long channels. Thus, MCM-48NPs shows more propitious mesoporous carrier giving fast and maximum release compare with MCM-41NPs.

ACKNOWLEDGEMENT: Mr. Mohit Mahajan is highly thankful to the University Grants Commission, New Delhi, Government of India, for availing Senior Research Fellowship. The authors are thankful to the Hetero Pharmaceuticals Pvt., Ltd., Hyderabad, India for providing RTV gift sample.

CONFLICT OF INTEREST: There are no conflicts of interest.

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    How to cite this article:

    Mahajan M and Rajput S: Development of mesoporous silica nanoparticles of ritonavir with enhanced bioavailability potential: formulation optimization, in-vitro and in-vivo evaluation. Int J Pharm Sci & Res 2018; 9(10): 4127-37. doi: 10.13040/IJPSR.0975-8232.9(10).4127-37.

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