Modified mesoporous silica nanoparticles for enhancing oral bioavailability and antihypertensive activity of poorly water soluble valsartan

Accepted Manuscript Modified mesoporous silica nanoparticles for enhancing oral bioavailability and antihypertensive activity of poorly water soluble valsartan

Nikhil Biswas PII: DOI: Reference:

S0928-0987(16)30555-3 doi: 10.1016/j.ejps.2016.12.015 PHASCI 3832

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

4 August 2016 20 October 2016 15 December 2016

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ACCEPTED MANUSCRIPT Modified mesoporous silica nanoparticles for enhancing oral bioavailability and antihypertensive activity of poorly water soluble valsartan

Nikhil Biswas*

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Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India-700032

Corresponding Author:* Nikhil Biswas

Department of Pharmaceutical Technology Jadavpur University, Kolkata India-700032 Ph. No. +91 – 9476167459 (M) E-mail: [email protected]/ [email protected]

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ACCEPTED MANUSCRIPT Abstract The aim was to improve the oral bioavailability and antihypertensive activity of poorly soluble drug valsartan (VAL) by modifying the design and delivery of mesoporous silica nanoparticles (MSNs). The synthesized MSNs were functionalized with aminopropyl groups (AP-MSN) through postsynthesis and coated with pH sensitive polymer eudragit L100-55 (AP-MSN-L100-55) for pH dependant sustain release of anionic VAL. MSNs were

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characterized by Brauner-Emmett-Teller (BET) surface area analyzer, zeta sizer, Field Emission Scanning Electron Microscope (FESEM), Powder X-Ray Diffraction (PXRD) and

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Differential Scanning Calorimetry (DSC). Functionalized MSNs showed highest entrapment efficiency (59.77%) due to strong ionic interaction with VAL. In vitro dissolution of M-MSN

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[MSN-VAL and AP-MSN-VAL-L100-55 mixed equally] at physiological conditions demonstrated immediate release (MSN-VAL fraction) followed by sustained release (AP-

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MSN-VAL-L100-55 fraction) of 96% VAL in 960 min. The dramatic improvement in dissolution was attributed to the amorphization of crystalline VAL by MSNs as evidenced by

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DSC and PXRD studies. No noticeable cytotoxicity was observed for MSN, AP-MSN and AP-MSN-L100-55 in MTT assay. Pharmacokinetic study of M-MSN confirmed 1.82 fold increases in bioavailability compared to commercial Diovan tablet in fasted male rabbits.

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Blood pressure monitoring in rats showed that the morning dosing of Diovan tablet

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more than 840 minutes.

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efficiently controlled BP for just over 360 minutes whereas the effect of M-MSN lasted for

Key words: Poorly water soluble drug; Mesoporous silica nanoparticles; functionalization; bioavailability; in vitro in vivo correlation; antihypertensive activity.

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ACCEPTED MANUSCRIPT 1. Introduction Valsartan is a promising angiotensin II receptor blocker which is indicated for the first line treatment of hypertension (Markham and Goa, 1997). Valsartan (class II drug) is rapidly absorbed from GI tract after oral administration but suffers from the drawbacks of poor oral bioavailability of about 23% primarily due to its lack of solubility in the acid milieu of the GI tract. Valsartan is an acid in nature and therefore, has good solubility at pH˃5 (Flesch et al.,

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1997). Valsartan has other problems also leading to its poor oral bioavailability. Valsartan has an absorption window and is mainly absorbed from the upper parts of GIT where its solubility is low and shows fast pass metabolism. Various technologies were developed

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previously to address poor bioavailabilty issue of valsartan like  cyclodextrin complexs

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(Cappello et al., 2006), microcapsules (Li et al., 2010), solid dispersion (Yan et al., 2012) etc. However the potential of nanotechnology in improving bioavailability of valsartan has not

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been studied in detail till date.

Nanotechnology has benefitted a number of biomedical areas including drug delivery

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(Ramsden, 2005; Sahoo and Labhasetwar, 2003). Among nanoparticles different MSNs and their therapeutic applications have been studied extensively for the last few years. Some unique features of MSNs have made it an excellent candidate in the field of drug delivery.

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Mesoporous materials improve the solubility of the guest molecules by converting unstable

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crystalline to stable amorphous state without altering their lattice energy (Slowing et al., 2007; Wang, 2009). These materials creates larger surface for better adsorption of therapeutic cargo and protect the loaded drug from external attack by steric hindrance (Slowing, 2008; Rosenholma and Linde, 2008). Moreover, tunable pore size and surface functionalization has

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made it a promising carrier for controlled release of therapeutic cargo (Kapoor et al., 2009; Lia et al., 2006). Free MSN consisting of negatively charged silanol groups (Lvov et al.,

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1997; Wani et al., 2012; Zhang et al., 2012) allows prompt release of entrapped drug due to the weak ionic interaction with negatively charged drug like valsartan (Tosco et al., 2008). In the past the focus of MSNs has been mainly on the development of slow release formulations, and fewer reports have been published on the application of the MSNs involving the dissolution enhancement of poorly water-soluble drugs (Wang, 2009; Salonen et al., 2005). Till date no study was documented on MSN for oral delivery of valsartan. Moreover the antihypertensive effect of commercial oral valsartan formulations lasts only 4-6 h. So a true once a day formulation with higher bioavailability is urgently required. This goal may be achieved by delivering it in pulses at different time intervals using mesoporous support. In

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ACCEPTED MANUSCRIPT addition the treatment side effects may be minimized by matching the drug release profile with the circadian pattern of hypertension. In view of the aforementioned, the current work aims to maximize the therapeutic potential of oral valsartan by modifying the design and delivery of MSNs. To the best of our knowledge, most reported polymer-MSN hybrids have been prepared by three methodslayer-by-layer technique (Wang et al., 2005), graft-to (Carniato et al., 2010), and graft–from

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technique (Liu et al., 2011). Defiance of their novelty these strategies still possess some pitfalls such as tedious step synthesis, very low surface graftings efficiency etc. (Fleming et

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al., 2001). It is still challenging for the researchers to explore a facile and efficient method for producing a hybrid that can controllably deliver entrapped guest molecules. Herein we have

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synthesized novel bean shaped MSN and functionalized with aminopropyl groups (AP-MSN) through postsynthesis. After drug loading into the mesoporous support AP-MSN were coated

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with Eudragit L 100-55 an anionic methacrylic acid-ethyl acrylate copolymer having dissolution pH threshold of 5.5. Different MSN samples were characterized with BET surface

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area analyzer, zeta sizer, FESEM, PXRD, DSC. MSN-VAL and AP-MSN-VAL-L100-55 were encapsulated in 1:1 ratio (M-MSN) to obtain a typical dissolution pattern in gastrointestinal pH, immediate release followed by a lag time and then a sustained release.

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Oral bioavailability of M-MSN and commercial Diovan tablets were compared in fasted male

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albino rabbits. A Level A IVIVC (in vitro in vivo correlation) model was developed. Blood pressure lowering potential of oral M-MSN and Diovan tablet was estimated in hypertensive

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rats employing tail cuff method.

2. Materials and methods

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2.1. Material

Gift samples of valsartan (VAL) and losartan (internal standard) with the assay value of 99.8% and 99.7% respectively were received from Ranbaxy Laboratories Ltd., India. Pluronic® P123 was a gift sample from BASF (USA). Tetraethyl orthosilicate (TEOS) and 1, 3, 5 tri methyl benzene (TMB) and (3aminopropyl)triethosysilane (APES) were purchased from Sigma Aldrich, USA. Analytical grade chemicals like water, hydrochloric acid, ethanol, etc. were purchased from Merck Ltd., Mumbai, India.

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ACCEPTED MANUSCRIPT 2.2. Synthesis of MSN In a typical preparation 2 gm of Pluronic® P123 was dissolved in 52gm water and 20 ml hydrochloric acid (37%) with stirring at 35°C. Then 4.28 gm of TEOS was added into the solution with vigorous stirring at 35°C for 24 hr (Zhao, 1998).The mixture was aged at 100°C overnight without stirring. Trimethylbenzene (TMB) was added during synthesis in a 1:1 ratio with the surfactant. The solid product was filtered and washed repeatedly with water and

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ethanol. The product was dried in an oven at 90°C for 20 hr. The polymeric template was removed from MSN through thermal treatment. Isothermal annealing of the silica sample was done at 550°C for 6 h in a PID controlled Muffle furnace (Grieken et al., 2003).

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2.3. Functionalization of MSN

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A post synthesis procedure (Zhang et al., 2010) was adopted to functionalize the MSN with aminopropyl groups. 1 gm MSN was placed in a three naked flask and dehydrated at

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100°C for 6 h under nitrogen atmosphere. 5 ml (3aminopropyl)triethosysilane (APES) was dissolved in 100 ml ethanol and added to the flask. The system was refluxed at 77°C with overnight magnetic stirring under nitrogen atmosphere. The obtained product was filtered and

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washed with ethanol repeatedly. The product was dried in an oven at 90°C for 24 h. 2.4. VAL loading and Eudragit L 100-55 coating

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Two separate ethanolic solutions were prepared by dissolving 100 mg of each VAL in 10 ml ethanol. Then 50 mg of each MSNs and AP-MSNs were added to this solution under

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magnetic stirring for 4 hr. Thus VAL loaded MSNs and AP-MSNs were produced in separate beakers. MSN-VALs were filtered and dried in a hot air oven overnight at 60°C. 100 mg Eudragit L 100-55 was dissolved in 5 ml ethanol under stirring for 4 hr. Then this coating

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solution was added dropwise to the drug loaded AP-MSNs suspension (VAL: MSN: Eudragit L 100-55 = 2:1:2) under magnetic stirring at 40°C for 12 hr. Finally Eudragit L 100-55 coated

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AP-MSNs (AP-MSN-VAL- L 100-55) were filtered using cellulose acetate membrane (0.22 µm). Filtered materials were dried in a hot air oven overnight at 60°C. Dried materials were scrapped, collected and stored in desiccator.

2.5. Characterization 2.51.1. Nitrogen Sorption Isotherms at 77 K MSNs of different batches were characterized by nitrogen gas desorption/adsorption isotherm using Surface area analyzer (Micromeritics Gemini VII-2390t, USA). It was measured at -196°C. Samples were degassed at 150°C for 4 hr. Total surface was determined

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ACCEPTED MANUSCRIPT using Brauner-Emmett-Teller (BET) model in the relative pressure range between 0.05 and 0.3.The pore volume and pore size distribution were derived from the adsorption branches of the isotherms using Barrett-Joyner-Halenda (BJH) method. Total pore volume was estimated from the amount adsorbed at a relative pressure of 0.99. Micropore volume and micropore areas were measured from t-plot. t values were calculated as a function of relative pressure using Harkins and Jura equation.

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2.5.2. Average particle Size and zeta potential measurements The average particle size, polydispersity index (PDI), and zeta potential of different MSNs samples were accessed by dynamic light scattering technique (DLS) using a Malvern

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Zeta Sizer (Nano ZS 90). A small quantity of MSN sample was dispersed in a large volume

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of milli-Q water to obtain suitable light scattering intensity and vortex mixed for 5 min to prevent particle clogging. The sample was analyzed at 25°C for 1 min in triplicate

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2.5.3. Differential Scanning Calorimetry (DSC)

The DSC curves were recorded for the sample powder (Drug alone, MSN, AP-MSN, MSN-VAL, AP-MSN-VAL, AP-MSN-VAL-L100-55) using Pyris Diamond TG/DTA

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(PerkinElmer, Singapore) instrument in the temperature range of 20–200°C, under a dynamic atmosphere of nitrogen (150 ml/min) and at a heating rate of 10°C/min, using platinum

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crucibles and weighed samples of 20 mg.

2.5.4. Powder X-Ray Diffraction (PXRD)

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The x-ray powder diffraction pattern were recorded at room temperature in Philips Analytical Powder XRD using Ni-filtered, and CuKα radiation operated at voltage of 35 Kv and current of 25 mA. The scanning rate employed was 1° min−1 over 5° to 50° diffraction

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angle (2θ) range. Samples of pure VAL, MSN, AP-MSN, MSN-VAL, AP-MSN-VAL, APMSN-VAL-L100-55 were analyzed.

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2.5.5. FESEM

The shape and surface morphology of the samples were determined using a field emission scanning electron microscope (FESEM, Hitachi S-4800). The samples were goldplated prior to imaging. 2.6. Cytotoxicity Assay (MTT) MTT assay was performed to evaluate the cytotoxicity of test materials MSN, APMSN, AP-MSN-L100-55 using doxorubicin as standard. Approximately 1 × 105 cells/ml (CHO-K1) in their exponential growth phase were seeded in a flat-bottomed 96-well polystyrene coated plate and were incubated for 24 hrs at 37°C in a CO2 incubator. Series of dilution (0.13, 0.41, 1.23, 3.70, 11.11, 33.33, 100 μg/ml) of test materials and doxorubicin 6

ACCEPTED MANUSCRIPT (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15 μM) was added in the medium. After incubating 24 hours, 10 μl of MTT reagent was added to each well and was further incubated for 4 hours. Formazan crystals formed in each well were solubilised in 150 μl of DMSO, the plates were placed instantly under microplate reader (Spectramax M5) and readings were taken at 570 nm. All experiments were conducted in triplicate. Wells with complete medium, test materials and MTT reagent, without cells were used as blanks for cell viability determination.

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Untreated cells exposed to the medium were used as solvent control (Vijayakumar et al., 2012). 2.7. Drug Loading Efficiency

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VAL loading in MSN-VAL, AP-MSN-VAL and AP-MSN-VAL-L100-55 were

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determined by suspending 10 mg nanoparticles in 100 ml phosphate buffer (pH 6.8) under magnetic stirring for 12 hr at room temperature. After stirring, the solution was filtered

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through filtered through a cellulose acetate membrane (0.22 µm). The amount of VAL in the filtrate was analyzed using JASCO V550 UV/VIS spectrophotometer at 248 nm. The drug

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loading content and entrapment efficiency were calculated by the following equation (Yuan, 2011).

Drug Loading (%) = (Weight of drug in MSNs/Weight of drug loaded MSNs) x 100

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Entrapment Efficiency (%) = (Weight of drug in MSNs/Initial weight of Drug) x 100

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2.8. In vitro Release

The in vitro studies of pure VAL and different formulations (test and commercial) were carried out in a franz-diffusion cell of 50 ml capacity at 37±0.5°C. Dialysis membrane having pore size 2.4 nm, molecular cut off between12-14 kDa, was mounted to the receptor

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compartment of the cell. 2 ml (1% w/v) VAL formulation was placed in donor compartment. The receptor compartment was filled with dialysis medium (0.1 N HCl pH 1.2, phosphate

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buffer pH 4.5, pH 5.6, pH 6.8 and pH 7.4). At particular time intervals 1 ml of the sample was taken out from the receptor compartment and immediately replaced with an equal volume of dissolution medium to maintain the sink condition. The samples were filtered through a cellulose acetate membrane (0.22 µm) and analyzed by using JASCO V550 UV/VIS spectrophotometer at 248 nm. 2.9. Pharmacokinetic Study 2.9.1. Animals Eighteen healthy New Zealand albino male rabbits weighing between 1.2 and 1.4 kg were used for in vivo pharmacokinetic studies. The study protocol was reviewed and

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ACCEPTED MANUSCRIPT approved by the Institutional Ethics Committee, Jadavpur University, Kolkata. Rabbits were divided into three groups of six each and were fasted for 24 h before the study. The group I was administered intravenous bolus injection of VAL at the rate of 2 mg/kg body weight. Group II and Group III received oral commercial Diovan tablet (the reference product) and optimized formulation (the test product) equivalent to 20 mg VAL respectively. 2.9.2. Instrument and Chromatographic Conditions

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The HPLC analysis was carried out on Shimadzu HPLC system consisting of LC-20 AD Prominence solvent delivery module. The mobile phase consisting of a mixture of 45% 20 mM KH2PO4 buffer (adjusted to pH 2.8 with H3PO4) and 55% acetonitrile was eluted at a

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flow rate of 1.0 ml/min through Phenomenex C18 column. The eluent was monitored with a

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photodiode array (PDA) detector at a wavelength of 214 nm. 2.9.3. Sample processing

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Blood samples (0.5 ml) were collected carefully from the marginal ear vein of male albino rabbits into heparinized centrifuge tubes at specific time intervals. The plasma was derived from the blood samples by centrifugation at 5000 ×g for 10 min. 100 μL of blank

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plasma was transferred to Ependorff tube and was spiked with 0.2 ml of each known concentrations of VAL and losartan solution prepared in acetonitrile. The tube was vortex-

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mixed for 5 min and 0.5 ml acetonitrile was added. The samples were kept on mechanical shaker for 10 min and centrifuged at 5000 rpm for 10 min to completely separate plasma

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protein. The supernatant was collected and evaporated to dryness under a stream of hot nitrogen. The residue was reconstituted with 1 ml of mobile phase. The samples were filtered through a 0.2 μm membrane filter using syringe filter holder and 20 μl was injected into the

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column. The method was validated according to the US Food and Drug Administration (FDA) guideline for bioanalytical method validation, 2001 (CDER, 2001)).

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2.9.4. Pharmacokinetic and statistical analyses of data The pharmacokinetic parameters for VAL following iv of VAL solution and oral administration of the two treatments were determined from the plasma concentration time data by means of a model independent method (non-compartmental module) using Kinetica software 5.1 (Thermo Fischer Scientific). All the parameters were expressed as mean (± SD) values of six rabbits and were statistically analyzed using two-way ANOVA to assume the statistical significance at P < 0.05. 2.10. In vitro in vivo correlation (IVIVC) A Level A IVIVC correlation model was chosen for establishing dissolution specification because it represents a point to point relationship between in vitro dissolution and in vivo 8

ACCEPTED MANUSCRIPT input and considered as highest level of correlation (Yasir et al., 2010; Emami, 2006; VlugtWensink et al., 2007). The model was developed in two stages. Since VAL featured a linear pharmacokinetics in vivo, in the first step numerical deconvolution is performed to determine the fraction of VAL absorbed. In the second step the fraction of drug absorbed is compared with fraction of drug dissolved by treating the data graphically. The deconvolution method was attempted with intravenous bolus data as the reference treatment using Kinetica 5.1. correlation coefficient (r2), slope and intercept was calculated. 2.11. Pharmacodynamic study

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2.11.1. Induction of hypertension

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Linear regression analysis was performed to the in vitro in vivo correlation plot and

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Thirty six albino male rats weighing between 220-250 g were used for pharmacodynamic studies. Rats were acclimatized in animal house for 1 week and were fed a

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fixed standard diet. A 185 µmol/kg dose of L-NAME was injected intraperitoneally to twenty eight rats twice daily for 7 days to induce hypertension (Bahgata et al., 2008; Johnson and

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Freeman, 1992). The volume of the dose of L-NAME was adjusted to 1 ml/100 g of body weight. Remaining eight rats did not receive L-NAME and considered as normotensive. After 7 days 21 rats showed blood pressure (BP) of 150–160 mmHg. They were screened as

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hypertensive rats and were used in the experiment. The study protocol was reviewed and

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approved by the Institutional Ethics Committee, Jadavpur University, Kolkata, India. 2.11.2. Experimental design

A BIOPAC MP 36 (BIOPAC System Inc., USA) used for the measurement of blood pressure was composed of an animal heating controller (Tail heating B), small animal tail

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noninvasive blood pressure system (NIBP 200A) and MP 36 data integrating software. The experiment was conducted early in the morning by taking eighteen hypertensive and six

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normotensive (normal BP) male albino rats. Six normotensive rats were considered as group I and hypertensive rates were randomly divided into three groups of six each (group II, III, IV) (n = 6). The rats in group II were given 1 ml saline solution orally (control group). Group III and group IV were administered commercial Diovan tablet (20 mg) and optimized formulation respectively with the help of long oral tubing. During this time L-NAME injection was continued in the above mentioned dose and manner. Noninvasive blood pressure was measured at different time interval for 24 h beginning from the early morning with the help of tail cuff method utilizing BIOPAC MP 36.

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ACCEPTED MANUSCRIPT 3. Result and Discussion 3.1. VAL loading Entrapment efficiency of unmodified MSNs, aminopropyl functionalized MSNs (APMSN) and coated AP-MSNs were 43.25%, 59.77% and 54.16% respectively. The highest drug loading into the mesopores of AP-MSN was attributed to the strong ionic interaction between the positively charged amine group on the surface of AP-MSNs and negatively

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charged carboxyl group of VAL. Whereas negatively charged silanol group of unmodified MSNs weakly interacts with negatively charged carboxyl group and poorly binds with VAL. So the least amount of drug was loaded into MSNs. After coating of AP-MSN-VAL with

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anionic eudragit L100-55 the loading was reduced due to the competition between the anions

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and leaching of VAL in the coating solution. 3.2. BET Analysis

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BET surface area plot, N2 desorption adsorption isotherm and pore size distribution analysis was performed for all the MSN sample and the results were presented in Table 1. The nitrogen adsorption/desorption study showed that all the synthesized MSNs displayed

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type IV isotherm according to IUPAC indicating the mesoporous nature of the silica sample. H1-type Hysteresis loops with sharp adsorption and desorption branches were indicative of

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capillary condensation in the mesopore of MSNs. A high surface area (SBET), total pore volume VT and pore diameter of MSN and AP-MSNs samples ensured the effective loading

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of the drug molecule within these mesoporous structures (Yang et al., 2008). Functionalization with aminopropyl groups resulted in reduction in the values of SBET, VT and WBJH (pore diameter) of the MSN sample. The attachment of the functionalized aminopropyl

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group on the pore surface of MSNs may explain this phenomenon. Functionalization did not altered the shape of the H1-Hysteresis which indicated that the pore shape was unaffected by

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postsynthesis of MSN. The dramatic reduction in the values of SBET, VT and WBJH of MSNVAL and AP-MSN-VAL can be assigned to the shrinkage of the hexagonal structure and closing of the mesopores after successful loading of VAL (Fig. 1(A) & 1(B)). The higher pore width of the MSN and AP-MSN (13.58 and 11.03 nm) has ensured loading of VAL with smaller dimension. 3.3. Average particle size and zeta potential Average particle size, PDI and zeta potential of various MSN samples measured by Malvern Zeta Sizer were shown in Table 1. The results showed that the average size of MSN, AP-MSN, MSN-VAL, AP-MSN-VAL were 119.5, 128.5, 126.9 and 133.3 nm respectively. A dramatic increase in the average particle size of AP-MSN-VAL-L100-55 (179.2 nm) was 10

ACCEPTED MANUSCRIPT the indication of successful coating of eudragit L100-55 on the surface of AP-MSN by ionic interaction. All the MSN formulation batches were highly uniform in their size as shown by smaller values of PDI. The free MSN particles consisting of silanol group were negatively (ZP= -26.9 mV) charged (Lvov et al., 1997), while AP-MSN exhibited much higher positive charge (ZP= 42.3 mV) owing to large quantities of aminopropyl group which was attached to the surface of MSN through electrostatic interaction (Fig. 1(D)) (Yang et al., 2010). VAL

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loading resulted in increase in negative charge of MSN and decrease in positive charge of AP-MSN due to the presence of negatively charged carboxyl group. 3.4. DSC

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DSC analysis was performed to identify the crystalline/amorphous state of a material.

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When a drug is in crystalline form it may be quantified and characterized by sharp melting peaks using DSC. On the other hand if the drug is in noncrytalline/amorphous state, no peaks

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can be recorded. The DSC thermograms of pure VAL, MSN, AP-MSN, AP-MSN-VAL, APMSN-VAL-L100-55 were shown in Fig. 2(A). The DSC curve showed that a sharp endothermic peak appeared at about 105.68°C for VAL, corresponding to its melting point

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and indicating its crystalline nature. However, no melting peak of VAL was recorded in the DSC curves obtained from other VAL loaded samples. The absence of sharp melting peaks in

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these samples was indicating that the VAL present inside the mesopores was in amorphous

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state (Salonen et al., 2005; Heikkilä et al., 2007). 3.5. XRD

The powder X-ray diffractometry patterns of the pure VAL, MSN-VAL, AP-MSNVAL, AP-MSN-VAL-L100-55 were shown in Fig. 2(B). VAL showed intrinsic peaks at the

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diffraction angles, exhibiting a typical crystalline pattern. After encapsulation in MSN and AP-MSN samples no crystalline peaks were detected. The absence of distinctive peaks in

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these samples was indicating that the VAL present inside the mesopores of MSNs was in amorphous state (Kapoor et al., 2009; Salonen et al., 2005). The absence of VAL peaks in MSN-VAL may be due to the hydrogen bonding between the carboxyl group of VAL and silanol group of MSN which causes disordering of the crystals and transforms in to amorphous state. On the other hand amine group of AP-MSN-VAL interacts with carboxyl group of VAL leading to amorphization of entrapped VAL. 3.6. FESEM Surface morphology of the MSN and AP-MSN analyzed by FESEM were shown in the Fig. 3 (A) and (B) respectively. As the photomicrograph shows, the MSN and AP-MSN sample contains cylindrical shaped particles with typical aspect ratio (p= L/d) of 2. The 11

ACCEPTED MANUSCRIPT particles also shows tendency to form linear type chain by overlapping head-to-tail. In some portion, we also observed some irregular shaped aggregates of the particles as shown in the Fig. 3 (A). Mesoporous structure was clearly visible in Fig. 3(C). However, after coating with eudragit L100-55, individual particles agglomerated and give rise to bigger assembly (Fig. 3(D)). 3.7. Cytotoxicity Assay

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Different formulated nanomaterials like MSN, functionalized MSN (AP-MSN) and coated MSN (AP-MSN-L100-55) were incubated with CHO-K1 cells for 24 h at various concentrations (from 0.13- 100 μg/mL), and the cytotoxicity of the particles was examined

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using the MTT cell viability assay. Cells which are viable after 24 hours exposure to the

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sample were competent of metabolizing a dye (3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide) completely and the purple colored precipitate obtained was studied

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spectrophotometrically after dissolving in DMSO. After 24 hours of post treatment, CHO-K1 cells showed excellent viability even up to the concentration of 100 μg/ml of all the nanomaterial sample as shown in Fig. 3(E). However the functionalized MSN showed

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relatively higher toxicity than free MSNs. Samples exhibited concentration dependant toxicity, higher concentration showed greater toxic effect. Doxorubicin was used as a

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standard cytotoxic drug and its observed cytotoxicity (IC50= 0.265) standardized the assay. These results clearly demonstrate that MSN, functionalized MSN (AP-MSN) and coated

3.8. In vitro dissolution

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MSN (AP-MSN-L100-55) are completely safe for use as carrier for drug delivery.

The in vitro dissolution profiles of VAL from free drug powder, commercial product

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and different prepared formulations in distinct dissolution media were shown in Fig. 4. About 13% VAL dissolved in 0.1N HCl (pH 1.2) medium in 120 min. The dissolution of pure

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crystalline VAL displayed pronounced pH dependence due to its weak acidic nature and its dissolution extent was very low at low pH. Commercial tablet Diovan released only 44% VAL in 60 min. A remarkable increase in both the dissolution rate and extent of VAL was noticed with MSN-VAL as almost 50%, 80% and 90% of the drug dissolved in 10, 30 and 60 min respectively. The improvement in the extent of dissolution may be related to the mesopores of MSNs which is changing the insoluble crystalline state of VAL to a soluble amorphous state as evidenced by DSC and PXRD. The initial burst release of VAL from MSN-VAL may be due to the presence of the drug molecules in the external pores of MSNs, which allowed 50% VAL to be released within 10 min into the release medium and satisfied the need for pulse effect after administration. The negatively charged silanol group of 12

ACCEPTED MANUSCRIPT unmodified MSNs weakly interacts with negatively charged carboxyl group of VAL and could not prevent the burst release. The remaining VAL released in next 50 min due to the slow dissolution of the drug molecules from the pores inside the MSNs. The dissolution profile of VAL from AP-MSN-VAL exhibited a different type of release pattern. Almost 30% of the drug was released in the first 60 min, while the remaining amount followed a typical sustained release pattern and dissolved completely over a time

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period of about 600 min. This kind of release pattern may be explained by the strong ionic interaction between the positively charged amine group on the surface of AP-MSNs and negatively charged carboxyl group of VAL. Moreover the steric hindrance provided by the

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alkyl chain in AP-MSN further slowed down the diffusion of drug molecules (Zhang et al.,

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2010).

The dissolution profiles of eudragit L 100-55 coated AP-MSN (AP-MSN-VAL-L100-

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55) and M-MSNs (MSN-VAL: AP-MSN-VAL-L100-55 =1:1) were studied in various dissolution media to closely follow gastric pH condition. As eudragit L 100-55 soluble at pH >5.5, only 4-5% VAL was released from AP-MSN-VAL-L100-550 due to leaching in the

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dissolution media of pH 1.2 and pH 4.5, which was sufficient to provide a lag time or off release period of about 5 h in GIT. Our modified release M-MSNs containing 50% each of

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MSN-VAL and AP-MSN-VAL-L100-55 showed first pulse immediately followed by a lag

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time of 240 min and then a sustained release over a period of 960 min. 3.9. In vivo absorption studies

The plasma concentration – time curves of commercial Diovan tablets and the

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modified MSNs formulation (M-MSN) in male rabbits following oral administration of 20 mg doses were portrayed in Fig. 5. The mean pharmacokinetic parameters, derived by noncompartmental fitting of data, were summarized in Table 2. After oral administration of M-

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MSN, plasma VAL concentration increased rapidly reaching a maximum concentration within 80 min followed by a steady fall which was an indication of lag period, continued upto 330 min. Then plasma concentration increased slowly again reaching Cmax at 735 min. It is worth to note that the lag time for the in vivo plasma profile could be correlated with that observed in vitro (290 min) for the same treatment. Relative bioavailability of the group treated with Formulation M-MSNs was found 1.82 fold higher than the group treated with marketed formulation Diovan. The commercial formulation showed only 47.36% bioavailability. The significantly larger AUC0-1200 min and immediate plasma spike obtained from the M-MSNs indicated that MSNs facilitated the fast dissolution of the drug in the

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ACCEPTED MANUSCRIPT gastrointestinal fluid which resulted in enhanced oral bioavailability of the poorly soluble drug VAL (Meng et al., 2010; Amidon, 2010; Thommes, 2011). Again the results suggest that the uptake of intact gastrointestinal MSN particles occurred by the mechanisms involving M-cells in Peyer’s patches of the gastrointestinal lymphoid tissue (Clark et al., 2001: Florence and Hussain, 2001). Oral delivery of valsartan presents a challenge because of its poor oral absorption. The main hurdle faced by valsartan in the GI tract is its lack of

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solubility in the acid milieu and absorption window. Encapsulation of valsartan in Eudragit L 100-55 coated aminopropyl functionalized MSN particle promoted valsartan paracellular or transcellular transport across the intestinal mucosa. The Eudragit L 100-55 coat degraded at

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any pH ˃ 5.5 and valsartan was released from AP-MSN in a sustained fashion in the blood.

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The evidence of intact uptake of mesoporous silica nanoparticles (30–90 nm) was found in previous studies (Zhang et al., 2012). Compared to IV administration, longer half-lives of

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valsartan following oral administration has been attributed to flip-flop kinetics in which the rate of VAL oral absorption is slower than that of elimination. Terminal phase of VAL pharmacokinetic profile was shown to reflect slower total body drug clearance due to slower

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drug release/absorption from oral commercial and M-MSN formulations (Li et al., 2016).

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3.10. In vitro-In vivo correlation (IVIVC)

From the perspective of successful delivery of VAL for the treatment of hypertension

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it is very important to establish a relationship between the in vivo absorption and in vitro drug release for pulsatile-release M-MSN formulation. A level A in vitro in vivo correlation was explored using percent dissolved vs percent absorbed data at each time point for the entire

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study period (Fig. 6(A)). Non compartmental analysis was carried out to calculate apparent in vivo absorption profiles. From the pharmacokinetic parameters it is clear that absorption

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phase of the test product M-MSN was extended from 0 to 80 min and from 330 min to 735 min. VAL did not absorbed between the intermediate periods from 80 min to 330 min. Therefore, fractions dissolved in 80 min and then from 330 min to 735 min were plotted against the fraction absorbed in the same time point. A good (statistically significant) linear regression relationship was found between fraction dissolved and fraction absorbed (r2 =0.9960, P˂0.001) for the M-MSN test formulation and was best described by the equation FA = 0.959(FD) + 4.824 (Figure 3B). The slope of 0.959 was lower than 1, indicating that the in vivo absorption rate was under estimated. The phenomenon might be attributed to the hepatic first pass effect and small absorption window of VAL (Sirisuth and Eddington, 2002).

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ACCEPTED MANUSCRIPT 3.11. Pharmacodynamic study M-MSNs as oral VAL delivery system was assessed by measuring L-NAME induced blood pressure in hypertensive albino rats using non invasive BP measuring technique. The antihypertensive profiles of the normal as well as different groups of hypertensive rats receiving normal saline, commercial Diovan tablet, and formulations M-MSNs were shown in Fig. 6(B). On early morning administration commercial Diovan tablet effectively

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controlled BP for 360 min. Since VAL is weakly acidic in nature, its solubility is pHdependent. Therefore, conventional tablet formulations of this type of drugs results in decreasing solubility and poor absorption during movement from lower to higher pH region

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of gastrointestinal tract. The hypertensive rat group received prepared M-MSN formulation

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showed a controlled antihypertensive activity for over 840 min. Mesopores of M-MSNs improved the dissolution significantly by changing the insoluble crystalline state of VAL to a

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soluble amorphous state. Again pulse release of VAL improves its absorption by reducing the hepatic fast pass effect and provided immediate control over blood pressure. Sustained release fraction AP-MSN-VAL-L100-55 quickly uptaken by m cells of Peyer’s patch and

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through mesenteric lymph it directly reaches to systemic circulation which has contributed to extend the antihypertensive activity for over 840 min. From ABPM trials it is confirmed that

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BP in uncomplicated essential hypertension drops after 720 min of awakening and reaches to minimum during night time sleep. So if the time of administration (morning dosing) is

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matched with circadian rhythm of hypertension the prepared formulation may be sufficient for once a day dosing as evidenced from the pharmacodynamic study. 4. Conclusion

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The modified MSN (M-MSN) design proposed in the present work had succeeded in providing a dramatic improvement in oral bioavailability and round the clock

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antihypertensive activity for poorly water soluble drug VAL. After calcinations, prepared MSNs were functionalized with aminopropyl groups (AP-MSN) through postsynthesis, loaded with VAL and coated with pH sensitive polymer eudragit L100-55. Functionalized MSNs successfully entrapped maximum amount of VAL (59.77%). pH responsive coating of eudragit L100-55 onto AP-MSN prevented VAL release at any pH below 5.5. The in vitro release profile M-MSN consisting of equal amount of VAL loaded MSN and coated APMSN was characterized by immediate release of 47% VAL (first pulse) within 60 min followed by a off release period of 240 min and then sustained release of 96% for the next 600 min. The improvement in the extent of dissolution was attributed to the mesopores of MSNs which is changing the insoluble crystalline state of VAL to a soluble amorphous state. 15

ACCEPTED MANUSCRIPT Pharmacokinetics study confirmed 1.82 fold increases in bioavailability in fasted male albino rabbits comparing to the commercial Diovan tablet. Level A IVIVC model showed good linear regression relationship (R2=0.996) between dissolution and absorption fraction of the drug. More over non invasive blood pressure monitoring in albino rats showed that the MMSN formulation more efficiently controlled blood pressure than the commercial Diovan

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tablet for almost 840 min when administered in early morning.

Acknowledgements

We greatly acknowledge Council of Scientific and Industrial Research (CSIR), India

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for funding this research. We would also like to thank BASF, USA and Ranbaxy Laboratories

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Ltd., India, for their free gift samples.

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ACCEPTED MANUSCRIPT Figure caption: Fig.1. (A) & (B) BJH pore width distribution (C) zeta potential of different MSN samples. Fig.2. (A) DSC thermogram and (B) PXRD pattern of valsartan and different MSN samples. Fig.3. FESEM images of (A) unmodified MSN (B) functionalized MSN, (C) mesoporous structure of MSNs (D) eudragit L100-55 coated MSN. (E) MTT assay on cellular toxicity of CHO-K1 cells transfected with different MSN materials.

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Fig.4. In vitro release profiles of (A) pure valsartan, commercial Diovan tablet and MSNVAL (B) AP-MSN-VAL in dissolution media of pH 1.2, (C) AP-MSN-VAL L100-55 and MMSN in various dissolution media. (mean ± SD, n = 3)

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Fig.5. Plasma concentration–time profile of VAL after administration of intravenous bolus,

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oral Diovan tablet and M-MSN in rabbits (mean ± SD, n = 6).

Fig.6. (A) IVIVC model linear regression plots of percentage dissolved vs percentage

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absorbed for M-MSN formulation. (B) Changes in blood pressure after oral administration of control (saline solution), formulation M-MSN and commercial Diovan tablets in L-NAME

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induced hypertensive rats. [Mean ± SD, n=6]

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ACCEPTED MANUSCRIPT Table 1: Textural properties, average size distribution and zeta potential (ZP) of different MSNs

Sample

BET Surface area

total pore volume

pore width

Average

(SBET) (m2/g)

(VT) (cm3/g)

(WBJH) (nm)

Particle

PDI

ZP (mV)

Size (nm) 467.38

0.5563

13.58

119.52±4.2

0.12±0.02

-26.9±1.4

AP-MSN

413.46

0.4237

11.03

128.58±6.8

0.08±0.01

42.3±2.6

MSN-VAL

83.35

0.0962

6.165

126.92±3.3

0.17±0.04

-31.3±2.8

AP-MSN-VAL

68.92

0.0734

4.363

133.38±6.2

0.11±0.02

37.7±3.6

AP-MSN-

16.53

0.0382

3.227

179.23±8.5

0.23±0.03

31.2±2.5

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VAL-L100-55

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MSN

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Table 2: The Pharmacokinetic parameters of valsartan following IV administration of VAL solution, oral administration of commercial Diovan tablets and M-MSNs formulation in

Tmax (min) AUC(0-1200 min) t1/2 (min)

MRT (min)

5.6680 ± 0.42

2.6139 ± 0.12

2.8749± 0.26

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735

330.388 ± 14.5

1563.09 ± 22.42

2804.31 ± 24.18

151.22 ± 3.26

249.81 ± 4.11

244.73 ± 7.29

117.51 ± 2.65

441.04 ± 8.30

725.29 ± 10.24

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47.36%

84.96%

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Relative

M-MSN

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(µg/ml/min)

Diovan tablet

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Cmax (µg/ml)

Intravenous solution

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parameter

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rabbits. [mean ± SD, n = 6].

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Graphical abstract

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