sub-acute toxicity study

Accepted Manuscript Title: Mucopenetrating nanoparticles for enhancement of oral bioavailability of furosemide: in vitro and in vivo evaluation/sub-acute toxicity study Authors: Salma El-Sayed Radwan, Magda Samir Sokar, Doaa Ali Abdelmonsif, Amal Hassan El-Kamel PII: DOI: Reference:

S0378-5173(17)30383-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.04.072 IJP 16638

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

2-3-2017 25-4-2017 28-4-2017

Please cite this article as: Radwan, Salma El-Sayed, Sokar, Magda Samir, Abdelmonsif, Doaa Ali, El-Kamel, Amal Hassan, Mucopenetrating nanoparticles for enhancement of oral bioavailability of furosemide: in vitro and in vivo evaluation/sub-acute toxicity study.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.04.072 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mucopenetrating nanoparticles for enhancement of oral bioavailability of furosemide: in vitro and in vivo evaluation/ sub-acute toxicity study

Salma El-Sayed Radwan1, Magda Samir Sokar1, Doaa Ali Abdelmonsif2, Amal Hassan ElKamel*1 1

Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt 2

Department of Medical Biochemistry, Faculty of Medicine, Alexandria University, Egypt

*Correspondence: Professor Amal H El-Kamel Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, 1 Khartoum Square, Azarita, Alexandria 21521, Egypt Tel +20 100 508 0510 Email [email protected]

Furosemide mucopenetrating nanoparticles

Abstract The aim of this study was to formulate and evaluate chitosan (CS)/alginate (ALG) nanoparticles (NPs) loaded with furosemide (FSM) in an attempt to enhance its release, permeability and bioavailability. Non-everted gut sac method was used to evaluate the ex vivo permeation of FSM from its suspension and the selected CS/ALG NPs formulation. The pharmacokinetic parameters of FSM subsequent to oral administration of the selected formulation were assessed in rats. In vivo subacute toxicity study of the prepared blank and FSM loaded formulations was evaluated in rats. The selected optimized formulation (F3) showed optimum particle size (PS), polydispersity index (PDI), zeta potential (ZP) and acceptable percentage entrapment efficiency (%EE) of 253.8 nm ± 4.6, 0.25±0.03, -35 mV±1 and 96%±1, respectively. The release profile of FSM from the selected formulation was characterized by initial burst effect in 0.1 N HCl. Scanning electron microscope (SEM) demonstrated a smooth surface and spherical shape for the lyophilized optimized NPs. Selected CS/ALG NPs (F3) presented a significant enhancement (p≤0.01) in permeation parameters of FSM as well as in Tmax, Cmax, AUC0-24 and AUC0-∞. Subacute toxicity study results revealed that the selected formulation was safe and nontoxic. The histopathological inspection of the stomach and small intestine tissues of the loaded NPs (F3) and blank groups reflected no obvious signs of cellular toxicity or inflammatory reaction. CS/ALG NPs loaded with FSM enhanced both drug release and mucus-penetrating ability leading to an overall increase in FSM bioavailability. In addition, the in vivo subacute toxicity study results indicated the safety of the prepared NPs for oral drug delivery.

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Furosemide mucopenetrating nanoparticles

Keywords: nanoparticles, furosemide, mucopenetration, pharmacokinetics, in vivo subacute toxicity

1.Introduction

Challenges associated with oral delivery are a result of the number of barriers that must be overcome in the gastrointestinal tract (GIT) before a therapeutic agent is absorbed and enters the blood stream. The first barrier is the harsh pH environment of the stomach and small intestine and enzymes secreted for digestion (Pridgen et al., 2014). In addition, poor GIT permeability significantly affects oral bioavailability of many drugs (Desai et al., 2012). Nanotechnology presents novel promising methods aiming at preventing/treating many diseases by enhancing the therapeutic efficiency of oral drugs and minimizing their toxicity to organs or cells. Recently, the mucoadhesive properties of some materials have been employed as a strategy to improve the residence time of nanocarriers in the GIT by improving the association to mucus and minimizing direct transit and fecal elimination (Plapied et al., 2011).

The limitations of mucoadhesive nanocarriers encouraged

researchers to develop mucopenetrating nanocarriers that can penetrate across the mucus layer and release drug in the surrounding area of the epithelium cells (Lai et al., 2009). Crater and Carrier (Crater and Carrier, 2010) reported a 20–30 times less diffusion across the mucus barrier for cationic particles in comparison with anionic ones. Polyelectrolyte complexation is a technique based on the ionotropic gelation principle and is used in the formulation of nano-drug delivery systems without the necessity of harsh chemicals or raised temperature (Patil et al., 2010). Polyelectrolyte complexes (PECs) are composed of polymeric combinations in which stout electrostatic attractions take place 3

Furosemide mucopenetrating nanoparticles

spontaneously without the assistance of reaction initiators, catalysts or crosslinkers (Shovsky et al., 2012). Polyelectrolyte complexes are composed of complexes associated among polymer-polymer, polymer-drug and polymer-drug-polymer (Lankalapalli et al., 2009). Chitosan is a linear copolymer composed of repeating units of 2-amino-2-deoxy-d-glucan with glycosidic linkages, where the amine groups contribute to CS distinct properties; such as its increased charge density and its ability to form salts. Chitosan is regarded as safe, of minimum toxicity and biodegradable, therefore, it has been broadly used in drug delivery (Rinaudo, 2006). Alginate is also considered a safe and biodegradable polymer of natural origin (Laurienzo, 2010). Alginate is a sodium salt of alginic acid, a linear polymer composed of 1,4-linked βD-Mannuronic acid (M) and α-D-Gluronic acid (G) residues in changeable amounts and arrangements. Sodium alginate shows good water solubility and ability to form a reticulated structure which can be cross-linked with divalent or polyvalent cations, like calcium and zinc, resulting in a meshwork of poor solubility (Patil et al., 2010). Since ALG is a biopolymer with chelation and gelation ability it has been used in vast biomedical applications (Sirisha and Campus, 2015) and controlled drug release (Lan and Starly, 2011). CS/ALG PEC formation takes place as a result of the interaction of the CS (amino groups carrying positive charge) and sodium ALG (carboxylate groups carrying negative charge). This PEC overcomes the limitations encountered in pure CS and ALG, where the solubility of CS at low pH decreases by the presence of ALG and the solubility of ALG at neutral pH decreases by the presence of CS. This property of CS/ALG PEC combined with its

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Furosemide mucopenetrating nanoparticles

biocompatibility, mucoadhesivity, and pH sensitivity make it of great interest for controlling release of active agents, specifically for oral delivery (Mateescu et al., 2015). Alginate's colloidal abilities in conjunction with the biological attributes of CS can create a multipurpose material and be used in food industry and medicine (Kulig et al., 2017). It has been also reported that CS/ALG NPs has enhanced intestinal mucopenetration properties (Bagre et al., 2013). Jayachandran Venkatesan et al. (Venkatesan et al., 2017) stated various ALG-CS based composites with silver and drugs utilized for wound dressing, sensor, bone tissue engineering, antimicrobial, anticancer, and dental applications. Alginate-chitosan acyl derivative microcapsule formed by means of independent layers of polyelectrolyte complex hydrogel films is a patent product mainly used for embedding microcapsules carrier cell transplantation, cell culture, proteins, nucleic acids and other biologically active substances ( Ma Xiaojun et al., 2014). Furosemide, an oral loop diuretic, used in treating hypertension and oedema (Nielsen et al., 2015). It undergos site-specific absorption in the stomach and upper small intestine, this rather narrow absorption window of FSM leads to erratic and highly variable absorption and low bioavailability of FSM administered via the oral route (20–60%) (Iannuccelli et al., 2000). Furosemide is of both poor aqueous solubility in the physiological pH range (5–20 µg/mL) and poor intestinal permeability hence, according to the Biopharmaceutics Classification System (BCS) it‟s categorized as a class four drug. It‟s a weak acid possesing two pKa values of 9.9 and 3.5. There are two approaches to improve the oral bioavailability of FSM; either by increasing its solubility in the stomach or its intestinal permeation characteristics (Sahu and Das, 2014a).

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Trials to increase solubility of FSM using nanosuspensions were investigated (Sahu and Das, 2014a),(Sadr and Nabipour, 2013). Gaikwad et al. (Gaikwad et al., 2010) prepared FSM loaded Eudragit RS100 NPs by nanoprecipitation method, while Zhi et al. (Zhi et al., 2005) studied the adsorption of FSM from water in oil nanoemulsion system. However, in both cases some of the used additives may have been toxic (Youm and Youan, 2013). On the other hand, chitosan NPs were used to augment the permeability of FSM by Sadighi et al. (Sadighi et al., 2012). The present study aimed at formulation and characterization of a safe oral polymeric drug delivery system that improves solubility, permeability and hence the bioavailability of poorly soluble, poorly permeable FSM. 2. Material and methods 2.1. Materials Furosemide; was kindly provided as a gift from the European Egyptian Pharmaceuticals (Alexandria, Egypt). Chitosan; low molecular weight, viscosity of 1% w/v in 1% acetic acid is 200 cp, Naproxen and orthophosphoric acid were purchased from Sigma Aldrich Corporation (St Louis, MO, USA). Sodium Alginate; low viscosity (0.02 Pa.s) for a 1% solution at 20 C provided as a gift from Pharonia Pharmaceutical Company (Alexandria, Egypt). Calcium chloride was purchased from the United Egyptian Company for chemicals (Cairo, Egypt). Ethanol, Acetonitrile; HPLC grade were purchased from Fisher Scientific (UK). All other chemicals and organic solvents were of analytical grade. Assay kits used in the subacute toxicity study were purchased from Spectrum and Diamond Diagnostics (Hannover, Germany). Animals

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Furosemide mucopenetrating nanoparticles

Experimental animals used were male Wistar rats, weighing 200-250 g. They were provided by the animal facility of the Faculty of Medicine, Alexandria University. Rats were housed in ambient temperature. Experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (Faculty of Pharmacy, Alexandria University). 2.2. UV spectrophotometric assay of Furosemide Three FSM standard solutions (1mg%) namely: a) FSM methanolic solution, b) FSM methanolic solution diluted with 0.1 N HCl and c) FSM methanolic solution diluted with phosphate

buffer

pH

5.8

were

scanned

spectrophotometrically

(Helios

alpha,

ThermoSpectro, UK) over a range of 200-400 nm to determine the wavelength of maximum absorption (λmax). The calibration curves were constructed over a concentration range of 0.6-1.6 mg%, for standard solutions (a & b) and 0.4-1.4 mg% for (c). The assay procedure was validated in terms of linearity, interday and intraday precisions and accuracy (Shabir, 2004). 2.3. High performance liquid chromatography assay of furosemide The drug was assayed by HPLC (Agilent technologies 1260 infinity, quaternary pumpG1311C, DAD-G1315D, automatic inj.-G1329B, Santa Carla, CA, USA) method as reported by Youm and Youan (Youm and Youan, 2013) with slight modifications. A mobile phase 45:55 ACN: distilled water of pH 2.5 adjusted by orthophosphoric acid was used. The mobile phase was freshly prepared, filtered through 0.45µm Millipore filter and degassed under vacuum before use, then introduced onto the column (Reversed-phase C18 column, Kinetex™ 250 x 4.6 mm, particle size=5 μm, pore size= 100 Å, equipped with a security guard ULTRA cartridges UHPLC C18 (4.6 mm) (Phenomenex Co., USA)) at a

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Furosemide mucopenetrating nanoparticles

flow rate of 1mL/min. The injection volume was 10 µL and peaks were detected at 230 nm. Elution was carried out at room temperature. All solutions were protected from light and used within 24 h. Calibration curves of FSM in ringer‟s solution in a concentration range of 1–50 μg/mL (for ex vivo study) and in plasma in a range of 0.2-10 μg/mL (for in vivo study) were plotted. The linearity, intraday and interday precision and accuracy data were assessed (Shabir, 2004). 2.4. Preparation of CS/ALG NPs A two stage process has been adopted, where CS was added drop wise into pre-gel CaALG solution (Sæther et al., 2008). Formulation and process variables were optimized to prepare NPs with suitable PS and PDI along with maximum %EE. In the preliminary studies, two methods of stirring were tried for preparation of the NPs; homogenization (Ultra Turrax, IKA Labortechnik, T25 with S 25N-8G dispensing element, Germany) and magnetic stirring (RH-basic, IKA, Germany) using two different kinds of surfactants, namely; Pluronic F127 and Tween 80 in different concentrations (0%-2.5 g% w/v). The optimum NP preparation procedures were as follows: The pH of 10 mL ALG solution (300 mg/100mL) was modified to pH 5.1 by the addition of 0.5 M HCl. A calculated amount of CS was dissolved in 1% acetic acid solution overnight followed by sonication (ultrasonic bath sonicator USR3, Julabo, Germany) for 10 min. The pH of CS solution was adjusted to 5.4 using 2.5 M NaOH solution. Two mL CaCl2 solution (332 mg/100mL) were added drop wise, at a rate of 1mL/min to 10 mL ALG solution, while stirring by a magnetic stirrer at 480 rpm for 30 min. Four mL CS solution of 80, 160, 240 mg/100mL were then

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added drop-wise to the calcium ALG pre-gel and stirring was continued for an additional 1h. The formed NPs were centrifuged using high speed cooling centrifuge (Sigma 30K, Germany) at 14,000 rpm at 4 C for 30 min. The supernatant was removed and the precipitate was washed and reconstituted in 15 mL filtered distilled water and sonicated for 10 min. he N suspension was frozen for 24 h at -30 C, then dried in a laboratory freeze dryer (Cryodo-50; Telstar, SA, Terrassa, Spain). For drug loading, one mL of FSM in ethanol containing various amounts of drug (5, 10, 20, 40 mg), was incorporated into the ALG solution and sonicated for 1 min before adding the CaCl2 solution. 2.5. Solubility study of FSM Saturation solubility of FSM was determined in water, water containing 1% Tween 80, 0.1N HCl and in phosphate buffer pH 5.8. An excess quantity of FSM was added to 3 mL of the medium in a well-closed glass vial. Samples were kept in a shaking water bath (SW20 C, Julabo, Germany) at 100 rpm and 37 C for 24 h then left for equilibrium for another 24 h. Samples were centrifuged at 9000 rpm for 10 minutes and the supernatant was separated to analyze the drug content after Millipore filtration (0.45 µm). Drug concentration was measured spectrophotometrically at the predetermined λmax using the corresponding blank. 2.6. Physicochemical characterization of nanoparticles 2.6.1. Particle size, poly-dispersity index and zeta potential The PS, PDI and ZP of the different FSM loaded CS/ALG NPs were determined using dynamic light scattering technique (Malvern Zeta Sizer, Malvern Instruments, Malvern,

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UK). The measurements were done at 25 C at a scattering angle of 173 . Samples were diluted with filtered water (1:3) before measurement. Measurements were performed in triplicate. 2.6.2. Entrapment efficiency (%EE) CS/ALG NPs fresh suspension was centrifuged at 14,000 rpm for 30 min at 4 C. Then the precipitate was separated and washed with methanol and centrifuged at 3000 rpm for 5 min at 4 C. Following lyophilization of NPs precipitate, a weight theoretically equivalent to 5 mg drug was digested using drops of acetic acid for 20 minutes. An aliquot of 10 mL of methanol was then added and stirred on a magnetic stirrer at 240 rpm, then centrifuged at 3000 rpm for 10 min. The resultant supernatant was filtered using 0.45µm Millipore Teflon filter and assayed for drug content spectrophotometrically after suitable dilution at 277 nm using methanol as blank. Experiment was done in triplicates. Percentage EE and yield were calculated using the following equations:

2.6.3. Scanning Electron Microscope (SEM) SEM (JFC-1100 E, JEOL, Japan) was used to examine the surface morphology of the selected FSM loaded CS/ALG NPs. One drop of the NP suspension was poured onto a cover glass to evaporate the aqueous medium. Samples were then coated by Au-Pd spattering in a SPI-MODULE sputter coater for 5 min under vacuum. Scans were performed at an acceleration voltage of 20 kV.

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Furosemide mucopenetrating nanoparticles

2.6.4. In vitro release study The in vitro release studies were carried out using USP dissolution apparatus II (USP dissolution test apparatus II, SR8 PLUS, Hanson, USA) under sink conditions. The dissolution medium was 0.1 N HCl (450 mL) for 2 h, then the pH of the medium was adjusted to 5.8 by the addition of 125 mL 0.2M Na3PO4.12H2O as reported by Sokar et al. (Sokar et al., 2013). he dissolution medium was continuously stirred at 50 rpm at 37 C

0.5 C in amber glass

cups. Furosemide loaded NPs (equivalent to 10 mg drug) were added as lyophilized powder to the stirred dissolution medium. Dissolution profiles of the two selected CS/ALG NPs (F3 and F5) were compared with that of the pure FSM (10 mg). At different time intervals, 5 mL samples were withdrawn and filtered using Millipore filter (0.22 µm) and compensated by the same volume of the fresh corresponding medium. Samples in triplicates were then measured using UV spectrophotometer at predetermined λmax in 0.1 N HCl and phosphate buffer pH 5.8, respectively. The release profiles of the prepared NPs and that of pure FSM were compared using model dependent methods (Costa and Sousa Lobo, 2001). All calculations were done using the add in excel programme; DDsolver (Zhang et al., 2010b). 2.6.5. Fourier transform infrared spectroscopy (FT-IR) Samples of ALG, CS, FSM and selected FSM loaded CS/ALG NPs were mixed separately with dry crystalline KBr in a ratio 1:100 and compressed into discs. The scanning range was 4000-500 cm−1 at room temperature. FT-IR spectra were obtained using FT-IR spectrometer (PerkinElmer spectrum RXFT-IR, USA). 2.6.6. Differential scanning calorimetry (DSC)

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Furosemide mucopenetrating nanoparticles

Thermal analysis of FSM, ALG, CS, selected FSM loaded CS/ALG NPs, and their physical mixture (containing an equivalent amount of the drug) were performed using DSC (PerkinElmer Inc, Shelton, CT, USA). Samples of 5 mg were placed in aluminum pans and sealed, then heated at a speed of 10°C/minute from 35 to 350°C under nitrogen atmosphere (60 mL/minute). An empty pan was used as a reference and the instrument was calibrated with indium. 2. Stability studies The selected FSM NP formulation was tested for its phyiscal stability.

suspension of the

selected freshly prepared CS/ LG N s was kept in the refrigerator at 4 C for one week and examined for its mean PS, PDI and ZP before lyophilization. Lyophilized formulations were enclosed in polyethylene petridishes under long term storage conditions (25 C and 60

H) and protected from light for 6 months in stability

cabinet. At specified time intervals, NPs were reconstituted and examined for the PS and PDI. 2.7. Ex vivo permeation of furosemide from the selected CS/ALG NPs Ex vivo permeation of FSM (5mg) from its suspension and the selected NP formulation (F3) containing equivalent amount of drug was evaluated using non-everted gut sac method (Ruan et al., 2006). Rats were sacrificed by spinal dislocation and the small intestine was separated by cutting the upper end of the duodenum and the lower end of the ileum. inger‟s solution was used to clean the small intestine using a syringe with a blunt end. The intestinal tract was then cut into 7 ± 0.5 cm long sacs with a diameter of 0.5± 0.01 cm. Each sac was filled with 1.5 mL of either FSM pure drug suspension or the selected FSM NPs suspension (equivalent to 5 mg of FSM) using a blunt needle while both ends of the

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intestine were knotted. Non-everted intestinal sacs were put in a glass beaker containing 100 mL of inger‟s solution in order to obtain sink conditions. he entire system was kept at 37°C in a shaking water bath at 100 rpm and aerated with 5% CO2 and 95% O2 (10–15 bubbles/minute) using a laboratory aerator (Laboratory aerator, Linn company, Germany). An aliquot of 10 µL of filtered samples (0.22 µm Teflon syringe filter) was injected onto the HPLC system. The concentrations of FSM in the tested samples were determined using the constructed calibration curve. Apparent permeability (Papp), lag time (LT) and diffusion coefficient (D) were calculated (Martin et al., 1993). 2.8. In vivo study 2.8.1. Preparation of rat plasma samples Two hundred and fifty microliters of acetonitrile and 50 µL naproxen (1µg/mL) as an internal standard were added to 200 µL plasma samples. The mixture was then vortexmixed for 10 seconds and centrifuged at 7000 rpm for 20 min at 4°C. The supernatant was filtered through 0.22 µm Teflon syringe filter. An aliquot of 10 µL of the clear supernatant solution was injected onto the HPLC system. Blank plasma samples were first injected and tested for interference to ensure the selectivity of the method. The concentrations of FSM in the tested samples were determined from calibration curves prepared and injected on the same day and constructed using the same rats‟ plasma. 2.8.2. Study design of in vivo evaluation of the selected CS/ALG NPs Twelve white Male Wistar rats were randomly assembled into two groups (test and control) of equal size (n = 6). The rats were fasted overnight but supplied with water ad libitum previous to the experiment and until the end of the study (after 24 h). Each group received single doses equivalent to 40 mg/Kg of either lyophilized selected FSM loaded CS/ALG

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Furosemide mucopenetrating nanoparticles

NPs (F3) or pure FSM as aqueous suspension by oral gavage. Blood samples (0.5 mL) were withdrawn into EDTA tubes by optic puncture at the following time intervals: 0 (predose), 0.5, 1, 2, 3, 5, 7, 8 and 24 h following oral administration. The plasma was then separated from the collected blood samples by centrifugation at 7000 rpm for 20 min and then deepfrozen at -70 C. Pharmacokinetic analysis of the data was done by means of PKSolver, an add-in program for Microsoft excel for pharmacokinetics and pharmacodynamics data analysis (Zhang et al., 2010a). The areas under the plasma concentration-time profile (AUC0-t and AUC0-inf) were calculated. The maximum plasma concentration (Cmax) and the time to reach the Cmax (Tmax) were also calculated. 2.9. In vivo subacute toxicity study Male Wistar rats (8 weeks old, total of 24 animals) were kept in standard metal cages at 21ºC ± 1ºC and 65% relative humidity with a 10 h light/14 h dark cycle. They were given standard chow and water ad libitum for the duration of the study and they were given 2 weeks to adapt to their environment before the experiment. The rats were randomly selected and assigned to the following three test groups (eight animals per group): negative controls (received 2 mL normal saline), groups two and three administered 24.5 mg blank CS/ALG NPs (F3) and 24.5 mg FSM loaded CS/ALG NPs suspended in 2 ml water, respectively, via oral gavage route daily for duration of 14 days. A weight of 24.5 mg of FSM CS/ALG suspension in water is equivalent to 40 mg drug/kg (Hammarlund and Paalzow, 1982). 2.9.1. Animal observations

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Furosemide mucopenetrating nanoparticles

Sub-acute toxicity was assessed by measurement of mortality and survival time and also by observing clinical picture of intoxication and behavioral reactions. Animals on study were observed for any adverse reaction, like condition of eye, nose and motor activity during the first 24 h of treatment and then daily till the end of the study. Body weight of each rat was determined at the beginning of the experiment and once weekly. The amounts of daily food (g/d) and water (mL/d) consumption were also determined (Gelperina et al., 2002). The animals were sacrificed by decapitation 24 h after the last dose after day 15 of the experiment. Then they were necropsied, so control and treated animals' organs (liver, kidneys, spleen, brain, etc.) were subjected to gross examination to monitor any significant change in texture and shape. 2.9.2. Biochemical tests On the day of sacrifice, blood samples were taken from each animal and recovered serum was stored at -20 C for determination of liver functions ( L ,

S and

L ) and kidney

functions (urea and creatinine). Serum ALT and AST activity was measured by spectrophotometric method using Spectrum assay kit (Reitman and Frankel, 1957). Serum ALP activity was measured by spectrophotometric method using Spectrum assay kit (Belfield and Goldberg, 1971). Serum level of urea was measured by enzymatic colorimetric method using Diamond Diagnostics assay kit (Tabacco et al., 1979). Serum level of creatinine was measured by buffered kinetic Jaffe reaction using Spectrum assay kit (Bowers and Wong, 1980). 2.9.3. Histopathological study After animals' sacrifice, tissues of stomach and upper intestine were placed in 10% formalin then examined histopathologically. The samples tissues were embedded in

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Furosemide mucopenetrating nanoparticles

paraffin blocks, then cut and positioned on glass slides. After histological staining the slides were studied and photos were taken using light microscope (ZEISS, primo star, Germany) and histopathological inspection was performed (Dhana Lekshmi et al., 2010). 2.10. Statistical analysis Data were analyzed using SPSS software package (version 16, SPSS Inc, 2007, USA). ANOVA test was applied when required. For abnormally quantitative variables, data were described using range (minimum and maximum). Data of subacute toxicity study were analyzed using Kruskal Wallis test. Significance of the obtained results was judged at the 5% level (Chan, 2003).

3. Results and discussion 3.1. UV spectrophotometric assay of furosemide Scanning of 1 mg % FSM methanolic solution, phosphate buffer pH 5.8 and 0.1N HCl showed λmax at 276, 277 and 274 nm, respectively. Furosemide showed a linear relationship in MeOH, 0.1N HCl and phosphate buffer pH 5.8 over a concentration range 0.6-1.6 mg% for MeOH and 0.1N HCl and 0.4-1.4 mg% for phosphate buffer pH 5.8 with a value of coefficient of determination (R2) 0.9992, 0.9995 and 0.9994, respectively. Intraday and interday precision and accuracy; the CV% values were not more than 4.9 and % Recovery ranged from 97.4-103.3%. 3.2. Preparation of CS/ALG NPs A two stage process has been adopted, where CS solution was added drop wise into pre-gel Ca-ALG solution (Sæther et al., 2008). The PEC stability of CS and ALG was affected by the surrounding ambience like ionic strength and medium pH. The used pH values allowed

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Furosemide mucopenetrating nanoparticles

the amino group of CS to be protonated carrying a positive charge and carboxylic acid group of ALG to be ionized carrying a negative charge. Charges that didn‟t involve in the PEC formation intermingled with the aqueous medium and were held accountable for both the PEC being stable and charged. It has been reported by Patil et al. (Patil et al., 2010) that CS quickly bound on the exterior of the ALG with restricted diffusion into the inner core. Therefore, in an attempt to improve the stability of CS/ALG PEC, CaCl2 was added to ALG to form pre-gel before addition of chitosan. The pre-gel has an immense effect on the capability of ALG to bind to CS (Patil et al., 2010).

During optimization process, homogenization (13 Krpm) resulted in PS and PDI values of 390 nm ± 14.36 and 0.346 ± 0.013, respectively, using Pluronic F127 as a surface active agent, which agrees with the findings of Arora et al. (Arora et al., 2011). However, homogenizer results were not reproducible as the opalescence, which indicated the formation of NPs was missed sometimes because of the surface-active agent‟s froth. It has been reported that polyelectrolyte complexation mainly depends on the interaction between two oppositely charged polymers at a certain pH that ensures their ionization (Siyawamwaya et al., 2015) rather than applying severe shear stress. Consequently, in this study magnetic stirrer was used for mild stirring at 480 rpm to prepare CS/ALG NPs as reported by (Liu and Zhao, 2013). Unlike the results of the current study, Das et al. (Das et al., 2010) reported the formation of 100 nm ± 20 curcumin loaded CS/ALG NPs using Pluronic F127 (0.1% w/v) with mild stirring. In the current study, Pluronic F127 (0.2-2.5%) didn‟t provide any good results

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Furosemide mucopenetrating nanoparticles

using magnetic stirring at 480 rpm even at a concentration as high as 2.5%; with PS of 860 nm ± 53 and PDI of 0.53 ± 0.024. Therefore, it was replaced with Tween 80 for further trials. Tween 80 (1%) as surface active agent showed more promising outcomes for PS and PDI, 255 nm± 10.3 and 0.395 ± 0.011, respectively. However, only 9% drug was entrapped in this formulation, this could be attributed to the presence of 1% Tween 80 in aqueous medium which increased solubility of FSM 12 folds higher than that in water (0.9mg/mL± 0.004 in 1% Tween 80 compared with 0.075mg/mL± 0.0021 in water). The hydrophobic drug (FSM) preferred to dissolve in water containing surface active agent rather than to be entrapped in the hydrophilic polymeric NPs. From the aforementioned results, surface active agents showed negative effect on the efficiency of FSM loading in CS/ALG NPs and hence they were removed from the formulation throughout the subsequent studies. Similarly, Li et al. (Li et al., 2008) reported the formation of CS/ALG NPs without the aid of surface active agents. After eliminating surface active agents from the formula, 2 mL of 332 mg/100mL CaCl2 were dropped to 10 mL aqueous solution of 300 mg/100 mL ALG then left to mix using magnetic stirrer (480 rpm) for half an hour. After that, 4 mL of 80 mg/100 mL CS solution were added drop wise and left to stir for an additional hour then sonicated for 10 min (CS/ALG mass ratio of 1:9). CS/ALG NPs formation took place at ambient temperature; the method of preparation was characterized by being rather easy, fast and reproducible. From the previous preliminary studies, the optimized blank NPs chosen for further studies had PS and PDI values of 230.9 nm ± 9.1 and 0.264 ± 0.045, respectively. 3.2.1. Effect of drug loading on PS, PDI and %EE

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Furosemide mucopenetrating nanoparticles

Entrapment of the hydrophobic FSM into the hydrophilic CS/ALG NPs was of great challenge. Beads or NPs formed by using each polymer alone are often “leaky” to entrapped drugs because of charge repulsion. However, when CS and ALG polymers are mixed, ALG carbonyl groups carrying a negative charge and CS amine groups carrying a positive charge ionically interact forming the CS/ALG PEC. As a consequence to the PEC formation, ALG NPs porosity is reduced and in turn the entrapped drug leakage is also decreased (Sezer AD, 1999). In an attempt to solve the poor aqueous solubility of FSM it was dissolved in 1mL ethanol and sonicated for 30 seconds prior to its incorporation into the ALG solution. The effect of incorporation of various concentrations of FSM 5 (F1), 10 (F2), 20 (F3) and 40 (F4) mg on the characteristics of the CS/ALG NPs was investigated at CS to ALG mass ratio 1 to 9. When CS was added to the Ca-ALG pre-gel that contained FSM, NPs were formed instantaneously entrapping the homogenously suspended drug in the medium resulting in a suspension of CS/ALG NPs with entrapped FSM. Furosemide showed in-situ nanoprecipitation in the ALG solution after being dissolved in ethanol, hence, facilitated the loading in the CS/ALG NPs. Figure 1 illustrated the effect of addition of different amount of FSM on the PS and PDI of CS/ALG NPs. Loading the blank NPs with 5, 10 and 20 mg FSM showed no significant (p>0.05) increase in both PS and PDI. On the contrary, loading of 40 mg FSM had a significant increase (p≤ 0.05) in both PS and PDI. One possible underlying mechanism suggested by Goycoolea et al. (Goycoolea et al., 2009) to explain the variability in results among the blank and the loaded NPs was that the ionic interaction of drug with CS took place at the expense of

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Furosemide mucopenetrating nanoparticles

CS/ALG interactions. Also, it was possible that ALG interacted with FSM, which might affect the mechanism of interaction of CS and ALG. Concerning %EE; as the initial drug concentration increased, % EE also increased with no further increase in FSM entrapment upon increasing drug concentration from 10 to 20 mg then, started to decrease at 40 mg. Multiple comparison post hoc Duncan tests revealed the following order for drug entrapment in NPs: F1 (5mg) F4 (40 mg). These findings were in agreement with what was reported by Das et al. (Das et al., 2010) who stated that as the concentration of curcumin was increased, an improvement was observed in the %EE of curcumin till reaching a certain concentration then, the %EE started to decline after this point with further addition of the drug. The yield of the NPs augmented as the quantity of drug (FSM) loaded increased. The % yield data was found to significantly fit in the following order: blank < F1(5 mg) < F2 (10 mg) < F3 (20 mg). An explanation suggested for these results was that the interaction between the CS, ALG and FSM increased as the amount of FSM loaded increased (Goycoolea et al., 2009). These findings were similar to that reported by Rahaiee et al. (Rahaiee et al., 2015) who reported an increase in % yield in loaded NPs when compared to blank NPs. To sum up, FSM (20 mg) dissolved in 1 mL ethanol prior to its addition to ALG solution was the optimum drug concentration to be chosen for further investigations. The addition of 20 mg drug (F3) had no significant effect (p>0.05) on the PS and PDI. In addition, F3 showed %EE and %yield values of 96 % ±1 and 64.2 % ±4, respectively. 3.2.2. Effect of addition of various concentrations of CS

20

Furosemide mucopenetrating nanoparticles

The effect of addition of various concentrations of CS during formulation of CS/ALG NPs was evaluated. Four mL of different CS concentrations 80, 160, 240 mg/100 mL were added drop wise to the Ca-ALG pre-gel containing FSM (20 mg). The increase in CS concentration rendered the ALG:CS weight ratio to change from 9:1 at 80 mg/100 mL CS to 4.5:1 and 3:1 at CS concentrations of 160 and 240 mg/100 mL, respectively. Table 1 illustrates the effect of various CS/ALG weight ratio on PS, PDI, %EE and ZP of the prepared CS/ALG NPs. Post Hoc multiple comparison Duncan test for the effect of various CS concentrations showed insignificant difference (p>0.05) between the values of PS, PDI for NPs containing ALG:CS 9:1 (F3) and that containing 4.5:1 ALG:CS (F5). Whereas, PS and PDI of NPs containing 3:1 ALG:CS (F6) had a significantly higher (p≤0.01) values than that of F3 and F5. The increase in PS of NPs as CS concentration increased (AlG:CS weight ratio decreased) may be attributed to the repulsion between the positive charges provided by CS molecules in the NPs as previously reported by Mukhopadhyay et al. (Mukhopadhyay et al., 2015). Bhunchu et al. (Bhunchu et al., 2015) reported a similar finding of increase in both PS and PDI as CS:ALG weight ratio increased. However, an increase in the mass ratio above a certain point produced aggregated particles and an opaque suspension was formed. This could be explained by the development of a CS film covering the NPs, leading to a rapid increase in the PS and PDI (Bhunchu et al., 2015). According to Zhang and Kosaraju (Zhang and Kosaraju, 2007), the PS increased when the association between CS and ALG was relatively small. This could be due to the presence of amino groups in excess amounts, when the CS concentration was elevated, which competitively inhibited the crosslinking reactions between the polymers. Bagre et al. (Bagre et al., 2013) also witnessed an increase

21

Furosemide mucopenetrating nanoparticles

in PS as the CS weight ratio increased relative to tripolyphosphate, they credited this increase to increased collision of the CS polymer with tripolyphosphate ions. Concerning the entrapment efficiency, in the current study %EE decreased with high significance (p≤0.01) when ALG:CS weight ratio decreased from 9:1 (F3) to both 4.5:1 (F5) and 3:1 (F6). Duncan tests for % EE showed that there was no significant difference between F5 and F6, while F3 showed a significantly higher (p≤ 0.05) % EE when compared to F5 or F6. Entrapment efficiency was reported to vary with the concentration of polymers (Patil et al., 2012). Rahaiee et al. (Rahaiee et al., 2015) reported that for CS/ALG NPs of crocin, the %EE began to increase as the concentration of CS was increased till reaching a certain concentration after that, when CS was increased %EE decreased. They explained this observation by a decline in the capacity of NPs to entrap crocin as the increase in CS concentration decreased the vacancy for crocin. Zeta potential (ZP) less than -30mV or more than +30mV could be indicative of the stability of NP system (Arora et al., 2011). Post Hoc multiple comparison Duncan test for the effect of various CS concentrations showed insignificant difference (p> 0.05) between the values of ZP for weight ratio 9:1 ALG:CS (F3) and 4.5:1ALG:CS (F5). On the other hand, ZP of 3:1 ALG:CS (F6) significantly (p≤0.05) exhibited less negative charge than F3 and F5. Zeta potential of CS/ALG NPs suspension is a result of the total –ve charge due to FSM and ALG. By increasing concentration of CS, value of negative ZP decreased due to increased neutralization of the –ve charge by increasing the concentration of positively charged CS. This was in agreement with Bhuchu et al. (Bhunchu et al., 2015) who reported that ZP of CS/ALG is negative because ALG is the core material. At higher levels of CS

22

Furosemide mucopenetrating nanoparticles

concentration more positive charges resulted which neutralized the negative charges on the NP surface. Zeta potential for F3 and F5 remained less than -30 mV at the final pH (5.25) of CS/ALG NP suspension. The values of ZP indicated that the CS/ALG NPs suspension remained physically stable without aggregation (Arora et al., 2011). Lyophilizing of NPs is one of the critical steps in the formulation of NPs for oral drug delivery as it has been advised by various studies in an attempt to enhance the stability and shelf life of the NPs in addition to making their use of greater ease. Usually, the process of lyophilizing may cause the NPs to aggregate resulting in an enlargement of the NPs PS after their reconstitution in aqueous medium. Hence, more than one technique can be used to guarantee the full reconstitution of the NPs. Among these techniques are vortex mixing, sonication and even shaking manually (Rahaiee et al., 2015). In this study manual shaking and sonication for 15 min was used to reconstitute the lyophilized powder of both F3 and F5 CS/ALG NPs. Following the reconstitution of the lyophilized CS/ALG NPs in the filtered water, the PS and PDI were practically similar (p>0.05) to their original solution demonstrating full reconstitution of the CS/ALG NPs and the inexistence of any aggregates for both F3 (258±41nm and 0.47±0.057 for PS and PDI, respectively) and F5 (312±10 nm and 0.431±0.02 for PS and PDI, respectively). In conclusion, the ALG:CS ratio 9:1 (F3) and 4.5:1 (F5) showed PS and PDI values of 253.8 nm±4.6, 0.25±0.03 and, 290 nm±30, 0.228±0.028, respectively. Their ZP values indicated the presence of a stabilized NPs suspension. Whereas, the ratio between their components was acceptable and could maintain Ca-ALG in the pre-gel form and adequate CS concentration was present in order for the CS/ALG NPs to be formed (Li et al., 2008).

23

Furosemide mucopenetrating nanoparticles

Therefore, they were selected for further studies. Both F3 and F5 were lyophilized for the up-coming experimental purposes. 3.3. In vitro release study Concerning the release study, two formulas were selected, namely: F3 and F5. These formulations were compared with FSM pure powder. Figure 2 shows the in vitro release profiles of FSM from FSM powder and from F3 and F5 in 0.1 N HCl for 2 h then phosphate buffer pH 5.8 up to 8 h. It was worth mentioning Furosemide stability was related to pH of the media proving unstable in acidic media and very stable in basic media (Santos et al., 2011). On the other hand, Bundgaard et al. (Bundgaard et al., 1988) reported that FSM was stable in acidic conditions (pH 1-2) when protected from light. The in vitro release profiles of FSM from F3 and F5 NPs formulations were characterized by initial burst effect in 0.1 N HCl in the first 2 h. The burst release indicated that a certain amount of FSM initially associated with the surface of the NPs by weak interaction forces between the drug and polymers used (Mukhopadhyay et al., 2015). The release profiles of F3 and F5 were superimposed in 0.1 N HCl while in phosphate buffer, pH 5.8, F3 release profile was higher than that of F5 and both showed higher release profiles than that of FSM powder. This difference in release profiles between F3 and F5 in phosphate buffer could be attributed to the doubled CS concentration in F5 which may hinder the release of FSM from the prepared CS/ALG NPs due to the poor solubility of CS in alkaline medium. Similar results were reported by Mukhopadhyay et al. (Mukhopadhyay et al., 2015). Percent FSM released from F3 and F5 was found to be almost 64% ± 3 and 66% ± 4, respectively, in the first 2 h compared to 44 % ± 0.2 for pure FSM. These results indicated

24

Furosemide mucopenetrating nanoparticles

a highly significant increase (p≤0.01) in release of FSM from both F3 and F5 in 0.1N HCl at 2h compared with FSM powder. While, at 2h %FSM released showed no significant difference between F3 and F5 (p>0.05). The release enhancement in 0.1N HCl may be due to the increased surface area produced by the significant decrease in PS of the prepared NPs (Sahu and Das, 2014a). On the contrary, at pH 5.8 drug release from F3 and F5 illustrated a sudden increase followed by a plateau pattern (Figure 2) due to the improved association between the relatively alkaline solution and CS which carries a positive charge (Li et al., 2008). This interaction eased the movement of the dissolution medium as it penetrated the ALG core reaching the FSM. ALG swells in alkaline medium and becomes in its ionic form which might have enhanced the release of FSM (Mukhopadhyay et al., 2015). Since the CS/ALG PEC decreases the solubility of CS in acidic medium by the presence of ALG and on the contrary, ALG solubility is decreased in alkaline medium due to the presence of CS (Sharpe et al., 2014). The value of diffusional exponent „n‟ was 0.8 for pure FSM suggesting that the drug release mechanism was Non-Fickian transport (anomalous transport). While for both F3 and F5, the „n‟ values were 0.125 and 0.095, respectively, indicating Fickian diffusion. From the aforementioned findings, F3 and F5 showed an insignificant difference when it came to PS and PDI and release efficiency. However, F3 had a significantly higher %EE of FSM. In addition, from an economic and industrial point of view, amount of polymer used in F3 is less than that used in F5. On the bases of these findings, F3 was selected for additional characterization. 3.4. SEM

25

Furosemide mucopenetrating nanoparticles

Figure 3 shows the SEM micrograph of CS/ALG NPs (F3). The micrograph demonstrated that the dried CS/ALG NPs (F3) had a spherical shape and its surface was smooth. 3.5. FTIR study FTIR was performed to study the possible interaction between FSM and the other polymeric components of the NPs in the solid form. FTIR spectrum of pure FSM (Figure 4a) displayed the characteristic peaks at 3755 cm-1 (O-H stretch), 3286.42 cm-1 (N-H stretch), 3122.9 cm-1 (C-H stretch), 1566.08 cm-1(C=O stretch), 1672.76 cm-1 (N-H bending), 1324.5 cm-1 (S=O asymmetric stretch) (Sahu and Das, 2014b) and (C-Cl stretch) vibration (Das and Senapati, 2008) at 584 cm-1, respectively. The FTIR spectrum of ALG (Figure 4b) showed the main peaks at 1619.9 and 1419 cm−1, corresponding to the asymmetric and symmetric stretching of carboxylate salt groups. Moreover, a band at 1093.67 cm−1(C-O-C stretching) could be credited to ALG saccharide function group which was in agreement with the findings of Sahu and Das (Das et al., 2010). Mukhopadhyay et al. results for CS were similar to the found results; the characteristic peak of CS (Figure 4d) at 3443 cm−1(O-H stretch and N-H stretch, overlap), 2878 cm−1(C-H stretch), 1650.97 cm−1 (NH-CO (I) stretch) and 1599.43 cm−1(N-H bend) and 1083 cm−1(C-O stretch) were detected (Mukhopadhyay et al., 2015). The FTIR spectrum of FSM-loaded CS/ALG NPs (F3) spectrum (Figure 4c) demonstrated that ALG asymmetrical stretching of -COO− groups were shifted to 1673 cm−1 and the symmetrical stretching of -COO− groups are shifted to 1411.23cm−1. Furthermore, CS showed an absorption band at 1599 cm−1 which has shifted to 1500.55 cm−1 subsequent to its interaction with ALG. The stretching vibration of -OH and -NH2 at 3443 cm−1 shifted to

26

Furosemide mucopenetrating nanoparticles

3548 cm−1. FT-IR study designated that the CS amino groups interacted with the carboxylic groups of ALG resulting in the PEC NPs (Li et al., 2008), (Mukhopadhyay et al., 2015). Almost identical absorption bands of FSM (Figure 4a) were obtained in FSM-loaded CS/ALG NPs (F3) spectrum, but with less magnitude as shown in (Figure 4c). The observed absorption FSM bands were similar to the reported values by Das and Senapati (Das and Senapati, 2008). The characteristic absorption band of FSM appeared in the FSM loaded CS/ALG NPs indicated that FSM was entrapped in the PEC CS/ALG NPs. 3.6. DSC study The DSC thermogram of FSM presented a distinctive exothermic melting point at 222.7 C, which is indicative of the crystalline nature of the drug (Figure 5). The peak corresponding to the melting point of FSM in the CS/ALG NPs (F3) and physical mixture was present but with less intensity indicating the absence of interaction between FSM and polymers at a molecular level (Youm et al., 2012). In addition, a decrease in enthalpy of FSM entrapped in F3 (11 J/g) was observed when compared to that of pure drug (123 J/g). This indicated a deterioration in the crystalline nature of FSM entrapped into F3. This could be a reason for enhanced FSM release from F3 compared with FSM powder (Sahu and Das, 2014a). 3. Stability

The freshly prepared CS/ALG optimized NPs (F3) showed no statistically significant difference in mean PS, PDI and ZP (p>0.05) after 7 days storage in the refrigerator at 4 C compared to the freshly prepared formulation. The relatively high ZP of N3 (-35 mv) resulted in the stability of the suspension as it prevented the formation of aggregates; keeping the suspension in its nanosize. One way analysis of variance tests for the effect of storage conditions (25 C and 60

H) on the lyophilized CS/ALG NPs (F3) for 0, 3 and 6

27

Furosemide mucopenetrating nanoparticles

months on PS and PDI values showed insignificant increase (p> 0.05) in PS and PDI between 0 months and 3 or, 6 months. 3.7. Ex vivo permeation of furosemide through excised non-everted rat intestine Furosemide calibration curve in

inger‟s solution showed a linear relationship over the

concentration range 1-50 µg/mL, with a value of determination coefficient (R2 = 0.9999). Values of CV % of the intraday and interday data of different concentrations of FSM standards (1-50 µg/mL) in

inger‟s solution were not more than 1.24

, and

recovery

ranged from 86.5-101 %. Ex vivo absorption models are used in the study of the drug transport mechanisms, permeability classification, and prediction of the in vivo drug absorption behavior in humans decreasing both effort and investigational expenses in comparison to in vivo studies. Non-everted intestinal sac models in rats was used to study drug absorption (Barthe et al., 1998). The study took place in

inger‟s buffer medium (pH 7.4) achieving sink

conditions. Assuming that the non-everted intestinal sacs have a cylindrical shape, the inner diameter and surface area per sac were 0.5 ± 0.01 cm and 10 ± 0.02 cm2, respectively. Results (Figure 6) showed that for FSM suspension (dose 5 mg) only 14% ± 3.7 of FSM permeated after 2 hours. On the contrary, the CS/ALG formulation (F3) (dose equivalent to 5 mg FSM) exhibited 50% ± 1.3 permeation of FSM after 2 hours. The results revealed an increase in the values of Papp and, D for FSM loaded CS/ALG NPs (F3) (3.39 x 10-2± 0.01cm/min and, 3.5 x 10-3 ± 0.001 cm2/min, respectively) compared to that of FSM suspension (7.9 x10-3±0.002cm/min and, 7.26 x 10-4 ± 0.000 cm2/min, respectively). This may be due to the incorporation of the drug within the polymeric NPs which enhanced its intestinal permeability. In addition, LT of F3 (0.384±0.1 min) was less 28

Furosemide mucopenetrating nanoparticles

than that of FSM from its aqueous suspension (1.86 ± 0.38 min). In other words, the selected CS/ALG NPs (F3) enhanced the permeation of FSM through the intestinal barrier. Such highly significant enhancement in permeation (p≤0.01) could be attributed to the nanometric size of the polymeric formulation. One of the advantages of using NPs is the decrease in PS which gives a greater chance for drug full penetration during its stay in the intestine which is projected to be 3–4 h. Accordingly, an improvement in the drug permeability and in turn the time of drug delivery could be achieved (Goycoolea et al., 2009). Arora et al. (Arora et al., 2011); and Mukhopadhyaya et al. (Mukhopadhyay et al., 2015); similarly reported that the CS/ALG NPs PEC could improve bioavailability for hydrophobic drugs by the enhancement of the drug absorption through the intestinal tract. Siyawamwaya et al. (Siyawamwaya et al., 2015) explained the enhanced intestinal permeation of CS/ALG NPs as follows; initially, the NPs bioadhere to the intestinal cells accumulating CS in the epithelium which, consequently, allows the opening of the TJs during the drug delivery. This theory has been based upon previous observations of CS based NPs capability to reversibly decline the transepithelial resistance in epithelial cell cultures. Next, possibly a portion of the NPs might pass through the epithelial cells intracellularly delivering the associated drug. The presence of ALG increased the bioadhesive property of the NPs owing to its great affinity for Ca2+(Goycoolea et al., 2009). Similarly, Li et al. (Li et al., 2008) and Bagre et al. (Bagre et al., 2013) credited the increased paracellular uptake of drugs from CS/ALG NPs to the mucoadhesive nature of NPs. Cationic charge and mucoadhesivity of CS assist in paracellular transport across the

29

Furosemide mucopenetrating nanoparticles

intestinal epithelium owing to strong interaction between natural cationic polymer, CS and anionic epithelium lining. Chitosan/alginate NPs bioadhere and penetrate the mucus layer of intestinal epithelium; then the NPs penetrated disintegrate owing to their pH sensitivity releasing the FSM that passes through the TJs paracellularly (Siyawamwaya et al., 2015). Regression analysis showed a high correlation between the ex vivo % of FSM permeated through the non-everted intestinal sac and the % released in vitro over 120 min. Both cubic and quadratic models have the best fit for the ex vivo/in vitro correlation with a coefficient of determination (R2) 0.953 under the current experimental conditions according to the following equations, respectively: ((-

)

(Eq.3)

)

(Eq.4)

3.8. In vivo pharmacokinetic study For FSM calibration curve in rat plasma, linearity of FSM/naproxen peak-area ratio was confirmed (R² = 0.999) over the range of concentration 0.2-10 μg/mL. For interday and intraday precision data the values of CV % were less than 7.87 %, and % FSM recovery ranged from 87.3-107.6 %. The usual dosage of FSM in humans is 40–120 mg/day whereas for severe cases of edema doses as high as 600 mg/day may be required (Granero et al., 2010). The selected dose of 40 mg FSM/kg was a representative of the average oral dose of FSM in humans. The essential pharmacokinetic parameters of FSM resulting from the oral administrations of F3 and pure FSM (dose 40 mg/Kg) were calculated. The results shown in Figure 7 confirmed the superior bioavailability of FSM administered in the form of CS/ALG NPs

30

Furosemide mucopenetrating nanoparticles

(F3) when compared with pure FSM. Furosemide plasma profiles frequently display secondary or multiple peaks subsequent to either oral or I.V administration. This characteristic phenomena has been accredited to enterohepatic circulation of the drug (Granero et al., 2010). Maximum concentration (Cmax) achieved after administration of FSM powder was 0.73 g/mL (1st peak) compared to 1.54 g/ml for its CS/ALG NPs (F3). Time to reach maximum concentration (Tmax) for the nanoformulation was 5 h. On the other hand, Tmax of pure FSM was 1h (1st peak) and 5h (2nd peak). One-way ANOVA revealed statistical significant differences (p≤0.05) between the values of Cmax and, AUC0-∞. While the Tmax and AUC0-24 showed a high statistical significance difference (p≤0.01) between CS/ LG N s (5 1.5 h and 18 8 µg.h/mL) and FSM powder (1±2.25 h and 5±1 µg.h/mL). This was in agreement with the in vitro and ex vivo permeation data which showed higher drug release and permeation, respectively, from the selected CS/ALG NPs (F3). The enhanced oral bioavailability of FSM could be a consequence of the enhanced dissolution of FSM in 0.1N HCl and the improved FSM permeation from the small intestine which resulted in improving oral absorption of FSM from both the stomach, upper intestinal region. From the aforementioned findings, it can be deduced that the FSM CS/ALG NPs (F3) resulted in significant enhancement in bioavailability of FSM in rats since, both Cmax and, AUC0-24 increased by 2 folds while, AUC0-∞ increased by 3.6 folds. Similarly, Sahu and Das (Sahu and Das, 2014a) reported

that the AUC0-24 and, Cmax values of FSM

nanosuspension were approximately 2 fold greater than that of pure FSM preparation. They

31

Furosemide mucopenetrating nanoparticles

explained the improved oral bioavailability of FSM by the better dissolution of FSM in 0.1N HCl. 3.9. In vivo subacute toxicity study Due to the widespread use of NPs in the biomedical field, many concerns come up due to the increased entry of NPs to tissues and organs of the human body which might be a cause of toxicity. As NPs reach the systemic circulation, mostly they would be submitted to first pass metabolism and they might distribute through the vasculature to other organs (Yildirimer et al., 2011). The results of sub-acute toxicity study showed no mortality or adverse reaction in the condition of the eye, nose and motor activity till the end of the study. After animals were sacrificed and necropsied, no significant change in texture and shape of the animals' organs (liver, kidneys, spleen, brain, etc.) was noticed. Besides, assessment of animals' nutrition revealed gradual increase in body weight of rats of control, blank and FSM loaded formulation- treated groups. The amounts of daily food (g/d) and water (ml/d) consumption were also determined, showing no significant variations (p>0.05) between the three study groups. The feed consumption of the different study groups followed a similar pattern indicating the normal metabolism of the animals. These data indicated that feed intake and utilization of proteins and other nutrients were not affected by NPs intake (Lekshmi et al., 2011) precluding the potential toxicity of the CS/ALG NPs on the GIT at the administered dose of 40 mg drug/Kg for 14 days. On average, animals consumed 294 g of food and 496.3 mL of water daily. These values were within the physiological reference values for rats (George J, 2000). These findings were consistent with the results documented by

32

Furosemide mucopenetrating nanoparticles

Sonaje et al. (Sonaje et al., 2009) who reported non-appearance of toxicity in mice treated with 100 mg/kg blank CS NPs with a PS of around 220 nm. Liver functions (ALT, AST and ALP) and kidney functions (urea and creatinine) presented no significant difference (p>0.05) between the three groups as shown in Table 2. Mukhopadhyay et al. (Mukhopadhyay et al., 2015) performed an acute (1 day) in vivo study for blank CS/ALG NPs. Their results revealed that up to 150 mg/Kg blank NPs no mortalities were observed. They also found that ALT, AST and, creatinine levels showed a significant increase in values yet fall within the normal ranges indicating the safety of CS/ALG blank NPs at a dose as high as 150 mg/Kg which was in agreement with our findings. Investigation of whether NPs are tissue compatible or not is of great importance since, if they are not they might be a cause of an inflammatory reaction. Cells morphology could be indicative about the inflammatory state following the dosing of polymeric NPs. Consequently, from the toxicological point of view, it‟s vital to study the interaction between tissues and NPs (Mittal et al., 2007). Histopathological inspection of control rat fundus showed classical structure of the four major layers; mucosa, submucosa, muscularis externa and, serosa (Figure 8 A-C). The examination of the tested groups revealed an image similar to that of control image with no signs of cellular toxicity, degeneration abrasions, ulcers or excess inflammatory cells (Figure 8 D-F). he histopathological examination of control rat‟s small intestine revealed the classical structure with the same classical four layers. The inspection of the tested groups reflected the control image with no obvious signs of cellular toxicity or inflammatory reaction.

33

Furosemide mucopenetrating nanoparticles

Histopathological picture of the intestine is usually noticeable for the spreading of lymphocytes (inflammatory cells), which are part of body's defense mechanism. All three groups in this current study displayed no alteration in both the number and spreading of lymphocytes indicating that NPs did not induce harm or any inflammatory reaction (Figure 9 A-C). An evaluation of healthy tissues of untreated group with that of treated groups showed no signs of any inflammation (Figure 9 D-I) (Mittal et al., 2007). In addition, the epithelial membrane treated with the CS/ALG NPs were devoid of gross injury proving that the permeation-enhancing effect of CS/ALG PEC is of no harm to the mucosa (Qi et al., 2011).Thus, the sub-acute toxicity findings evidently showed that the FSM loaded CS/ALG NPs were nontoxic. 4. Conclusion In conclusion, preparation of chitosan/alginate nanoparticles loaded with furosemide was successfully achieved using polyelectrolyte complexation technique. The selected formula (F3) managed to improve the intestinal permeability of furosemide ex vivo and significantly improved its pharmacokinetic parameters in vivo while proving to be non-toxic

Acknowledgement The authors would like to thankfully acknowledge Professor Hoda M. Khalifa, Department of Histology, Faculty of Medicine, Alexandria University for her help in histological interpretation. Disclosure 34

Furosemide mucopenetrating nanoparticles

The authors report no conflicts of interest in this work

References

Arora, S., Gupta, S., Narang, R.K., Budhiraja, R.D., 2011. Amoxicillin loaded chitosanalginate polyelectrolyte complex nanoparticles as mucopenetrating delivery system for H. pylori. Sci. Pharm. 79, 673–694. Bagre, A.P., Jain, K., Jain, N.K., 2013. Alginate coated chitosan core shell nanoparticles for oral delivery of enoxaparin : In vitro and in vivo assessment. Int. J. Pharm. 456, 31– 40. Barthe, L., Bessouet, M., Woodley, J.., Houin, G., 1998. The improved everted gut sac: a simple method to study intestinal P-glycoprotein. Int. J. Pharm. 173, 255–258. 35

Furosemide mucopenetrating nanoparticles

Belfield, A., Goldberg, D.M., 1971. Revised assay for serum phenyl phosphatase activity using 4-amino-antipyrine. Enzyme 12, 561–73. Bhunchu, S., Rojsitthisak, P., Rojsitthisak, P., 2015. Effects of preparation parameters on the characteristics of chitosan-alginate nanoparticles containing curcumin diethyl disuccinate. J. Drug Deliv. Sci. Technol. 28, 64–72. Bowers, L.D., Wong, E.T., 1980. Kinetic serum creatinine assays. II. A critical evaluation and review. Clin. Chem. 26, 555–61. Bundgaard, H., Nørgaard, T., Nielsen, N.M., 1988. Photodegradation and hydrolysis of furosemide and furosemide esters in aqueous solutions. Int. J. Pharm. 42, 217–224. Chan, Y.H., 2003. Biostatistics 102: quantitative data--parametric & non-parametric tests. Singapore Med. J. 44, 391–6. Costa, P., Sousa Lobo, J.M., 2001. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 13, 123–133. Crater, J.S., Carrier, R.L., 2010. Barrier properties of gastrointestinal mucus to nanoparticle transport. Macromol. Biosci. 10, 1473–83. Das, M.K., Senapati, P.C., 2008. Furosemide-loaded alginate microspheres prepared by ionic cross-linking technique: morphology and release characteristics. Indian J. Pharm. Sci. 70, 77–84. Das, R.K., Kasoju, N., Bora, U., 2010. Encapsulation of curcumin in alginate-chitosanpluronic composite nanoparticles for delivery to cancer cells. Nanomedicine Nanotechnology, Biol. Med. 6, 153–160. Desai, P.P., Date, A.A., Patravale, V.B., 2012. Overcoming poor oral bioavailability using nanoparticle formulations - opportunities and limitations. Drug Discov. Today. Technol. 9, e71–e174. Dhana Lekshmi, U.M., Poovi, G., Kishore, N., Reddy, P.N., 2010. In vitro characterization and invivo toxicity study of repaglinide loaded poly (methyl methacrylate) nanoparticles. Int. J. Pharm. 396, 194–203. Gaikwad, A., Tamizhrasi, S., Sorti, A., Gavali, P., Mehare, G., 2010. Formulation and in vitro characterization of polymethacrylic acid nanoparticle containing frusemide. Int. J. PharmTech Res. 2, 300–304. Gelperina, S.E., Khalansky, A.S., Skidan, I.N., Smirnova, Z.S., Bobruskin, A.I., Severin, S.E., Turowski, B., Zanella, F.E., Kreuter, J., 2002. Toxicological studies of doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles in healthy rats and rats with intracranial glioblastoma. Toxicol. Lett. 126, 131–41. George J, K., 2000. The Laboratory Rat (Handbook of Experimental Animals). Routes of administration. Academic Press, London. Goycoolea, F.M., Lollo, G., Remuñán-López, C., Quaglia, F., Alonso, M.J., 2009. Chitosan-alginate blended nanoparticles as carriers for the transmucosal delivery of macromolecules. Biomacromolecules 10, 1736–1743. Granero, G.E., Longhi, M.R., Mora, M.J., Junginger, H.E., Midha, K.K., Shah, V.P., Stavchansky, S., Dressman, J.B., Barends, D.M., 2010. Biowaiver monographs for immediate release solid oral dosage forms: furosemide. J. Pharm. Sci. 99, 2544–56. Hammarlund, M.M., Paalzow, L.K., 1982. Dose-dependent pharmacokinetics of furosemide in the rat. Biopharm. Drug Dispos. 3, 345–59. Iannuccelli, V., Coppi, G., Leo, E., Fontana, F., Bernabei, M.T., 2000. PVP Solid Dispersions for the Controlled Release of Furosemide from a Floating Multiple-Unit 36

Furosemide mucopenetrating nanoparticles

System. Drug Dev. Ind. Pharm. 26, 595–603. Lai, S.K., Wang, Y.-Y., Hanes, J., 2009. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Deliv. Rev. 61, 158–71. Lan, S.-F., Starly, B., 2011. Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities. Toxicol. Appl. Pharmacol. 256, 62–72. Lankalapalli, S., Kolapalli, V.R.M., Lankalapalli S, K.V., 2009. Polyelectrolyte Complexes: A Review of their Applicability in Drug Delivery Technology. Indian J. Pharm. Sci. 71, 481–487. Laurienzo, P., 2010. Marine polysaccharides in pharmaceutical applications: an overview. Mar. Drugs 8, 2435–65. Lekshmi, U.M.D., Kishore, N., Reddy, P.N., 2011. Sub Acute Toxicity Assessment of Glipizide Engineered Polymeric Nanoparticles. J. Biomed. Nanotechnol. 7, 578–589. Li, P., Dai, Y.-N., Zhang, J., Wang, A., Wei, Q., 2008. Chitosan-alginate nanoparticles as a novel drug delivery system for nifedipine. Int. J. Biomed. Sci. 4, 221–8. Liu, P., Zhao, X., 2013. Facile preparation of well-defined near-monodisperse chitosan / sodium alginate polyelectrolyte complex nanoparticles ( CS / SAL NPs ) via ionotropic gelification : suitable technique for drug delivery systems. Biotechnol. J. 8, 847–854. Ma Xiaojun , Zheng Guochen , Yu Weiting , Xie Hongguo , Liu cave Xie Hongguo , Liu cave, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Alginatechitosan acyl derivative microcapsule, its preparation and application. Patent No. CN102475691 B, 16 April 2014. Martin, A., Bustamante, P., Chun, A.H.., 1993. Physical pharmacy (Diffusion and Dissolution), 4th ed. Lea & Febiger, Philadelphia. Mateescu, M.A., Ispas-Szabo, P., Assaad, E., 2015. Chitosan-based polyelectrolyte complexes as pharmaceutical excipients, in: Controlled Drug Delivery. Woodhead Publishing, US, pp. 127–156. Mittal, G., Sahana, D.K., Bhardwaj, V., Kumar, M.N.V.R., 2007. Estradiol loaded PLGA nanoparticles for oral administration : Effect of polymer molecular weight and copolymer composition on release behavior in vitro and in vivo. J. Control. Release 119, 77–85. Mukhopadhyay, P., Chakraborty, S., Bhattacharya, S., Mishra, R., Kundu, P.P., 2015. pHsensitive chitosan/alginate core-shell nanoparticles for efficient and safe oral insulin delivery. Int. J. Biol. Macromol. 72, 640–648. Nielsen, L.H., Rades, T., Müllertz, A., 2015. Stabilisation of amorphous furosemide increases the oral drug bioavailability in rats. Int. J. Pharm. 490, 334–340. Patil, J., Kamalapur, M., Marapur, S., Kadam, D., 2010. Ionotropic gelation and polyelectrolyte complexation : the novel techniques to design hydrogel perticulate sustained , modulated drug delivery system: a review. Dig. J. Nanomater. biostructures 5, 241–248. Patil, P., Chavanke, D., Wagh, M., 2012. A review on ionotropic gelation method: novel approach for controlled gastroretentive gelispheres. Int. J. Pharm. Pharm. Sci. 4, 27– 32. Plapied, L., Duhem, N., des Rieux, A., Préat, V., 2011. Fate of polymeric nanocarriers for oral drug delivery. Curr. Opin. Colloid Interface Sci. 16, 228–237. 37

Furosemide mucopenetrating nanoparticles

Pridgen, E.M., Alexis, F., Farokhzad, O.C., 2014. Polymeric nanoparticle technologies for oral drug delivery. Clin. Gastroenterol. Hepatol. 12, 1605–10. Qi, X., Wang, L., Zhu, J., Hu, Z., Zhang, J., 2011. Self-double-emulsifying drug delivery system ( SDEDDS ): A new way for oral delivery of drugs with high solubility and low permeability. Int. J. Pharm. 409, 245–251. Rahaiee, S., Shojaosadati, S.A., Hashemi, M., Moini, S., Razavi, S.H., 2015. Improvement of crocin stability by biodegradeble nanoparticles of. Int. J. Biol. Macromol. 79, 423– 432. Reitman, S., Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56– 63. Rinaudo, M., 2006. Chitin and chitosan: Properties and applications. Prog. Polym. Sci. 31 (2006) 603–632 Ruan, L.-P., Chen, S., Yu, B.-Y., Zhu, D.-N., Cordell, G.A., Qiu, S.X., 2006. Prediction of human absorption of natural compounds by the non-everted rat intestinal sac model. Eur. J. Med. Chem. 41, 605–10. Sadighi, A., Ostad, S.N., Rezayat, S.M., Foroutan, M., Faramarzi, M.A., Dorkoosh, F.A., 2012. Mathematical modelling of the transport of hydroxypropyl-beta-cyclodextrin inclusion complexes of ranitidine hydrochloride and furosemide loaded chitosan nanoparticles across a Caco-2 cell monolayer. Int. J. Pharm. 422, 479–488. Sadr, M.H., Nabipour, H., 2013. Preparation and identification of furosemide nanoparticles. J. basic Appl. Sci. Res. 3, 666–670. Sæther, H.V., Holme, H.K., Maurstad, G., Smidsrød, O., Stokke, B.T., 2008. Polyelectrolyte complex formation using alginate and chitosan. Carbohydr. Polym. 74, 813–821. Sahu, B.P., Das, M.K., 2014a. Formulation, optimization, and in vitro/in vivo evaluation of furosemide nanosuspension for enhancement of its oral bioavailability. J. Nanoparticle Res. 16, 1–16. Sahu, B.P., Das, M.K., 2014b. Nanoprecipitation with sonication for enhancement of oral bioavailability of furosemide. Acta Pol. Pharm. - Drug Res. 71, 129–137. Santos, C.A. dos, Mazzola, P.G., Polacwiecz, B., Knirsch, M.C., Cholewa, O., Penna, T.C.V., 2011. Stability of furosemide and aminophylline in parenteral solutions. Brazilian J. Pharm. Sci. 47, 89–96. Sezer AD, A.J., 1999. Release characteristics of chitosan treated alginate beads: II. Sustained release of a low molecular drug from chitosan treated alginate beads. J. Microencapsul. 6, 687–96. Shabir, G.A., 2004. A practical approach to validation of HPLC methods under current good manufacturing practices. J. Valid. Technol. 29–37. Sharpe, L.A., Daily, A.M., Horava, S.D., Peppas, N.A., 2014. Therapeutic applications of hydrogels in oral drug delivery. Expert Opin. Drug Deliv. 11, 901–15. Shovsky, ., Varga, I., Makuška, ., Claesson, .M., 2012. dsorption and Solution Properties of Bottle-Brush Polyelectrolyte Complexes: Effect of Molecular Weight and Stoichiometry. Langmuir 28, 6618–6631. Sirisha, V.L., Campus, K., 2015. Polysaccharide nanoparticles: preparation and their potential application as drug delivery systems. Int. J. Res. applied, Nat. Soc. Sci. 3, 69–94. 38

Furosemide mucopenetrating nanoparticles

Siyawamwaya, M., Choonara, Y.E., Bijukumar, D., Kumar, P., Du Toit, L.C., Pillay, V., 2015. A Review: Overview of novel polyelectrolyte complexes as prospective drug bioavailability enhancers. Int. J. Polym. Mater. Polym. Biomater. 64, 955–968. Sokar, M.S., Hanafy, A.S., El-Kamel, A.H., El-Gamal, S.S., 2013. Pulsatile core-in-cup valsartan tablet formulations: In vitro evaluation. Asian J. Pharm. Sci. 8, 234–243. Sonaje, K., Lin, Y.-H., Juang, J.-H., Wey, S.-P., Chen, C.-T., Sung, H.-W., 2009. In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery. Biomaterials 30, 2329–39. Tabacco, A., Meiattini, F., Moda, E., Tarli, P., 1979. Simplified enzymic/colorimetric serum urea nitrogen determination. Clin. Chem. 25, 336–7. Venkatesan, J., Lee, J.-Y., Kang, D.S., Anil, S., Kim, S.-K., Shim, M.S., Kim, D.G., 2017. Antimicrobial and anticancer activities of porous chitosan-alginate biosynthesized silver nanoparticles. Int. J. Biol. Macromol. 98, 515–525. Yildirimer, L., Thanh, N.T.K., Loizidou, M., Seifalian, A.M., 2011. Toxicology and clinical potential of nanoparticles. Nano Today 6, 585–607. Youm, I., Murowchick, J.B., Youan, B.C., 2012. Entrapment and release kinetics of furosemide from pegylated nanocarriers. Colloids Surfaces B Biointerfaces 94, 133– 142. Youm, I., Youan, B.-B.C., 2013. Validated reverse-phase highperformance liquid chromatography for quantification of furosemide in tablets and nanoparticles. J Anal Methods Chem 2013, 1–9. Zhang, L., Kosaraju, S.L., 2007. Biopolymeric Delivery System for Controlled Release of Polyphenolic Antioxidants. Eur. Polym. J. 43, 2956–2966. Zhang, Y., Huo, M., Zhou, J., Xie, S., 2010a. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306–14. Zhang, Y., Huo, M., Zhou, J., Zou, ., Li, W., Yao, C., Xie, S., 2010b. DDSolver : n Add-In Program for Modeling and Comparison of Drug Dissolution Profiles. AAPS J. 12, 263–270. Zhi, J., Wang, Y., Luo, G., 2005. Adsorption of diuretic furosemide onto chitosan nanoparticles prepared with a water-in-oil nanoemulsion system. React. Funct. Polym. 65, 249–257.

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List of Figures Figure 1: Effect of addition of different amounts of FSM on the mean particle size (PS) and polydispersity index (PDI) of the prepared CS/ALG NPs, n=3. .............................................. Figure 2: In vitro release profile of FSM from different CS/ LG N s (F3 and F5) compared to that of FSM powder at 100 rpm and 37 C in 0.1 N HCl for 2h then phosphate buffer pH 5.8 for 8h, n=3..........................................................................................................

Figure 3: SEM micrographs of FSM loaded CS/ALG NPs (F3) .............................................

Figure 4: FTIR spectrum of (a) free FSM, (b)ALG, (c) FSM loaded CS/ALG NPs (F3) and, (d) CS. ...............................................................................................................................

Figure 5: DSC thermograms of ALG, CS, FSM, their physical mixture and FSM loaded CS/ALG NPs (F3). ...................................................................................................................

Figure 6: Amount of drug permeated from pure FSM suspension compared to that permeated from CS/ALG NPs (F3) per unit area of non-everted rat intestine in 2 h at 37 C.

Figure 7: Average plasma drug concentration versus time profile after oral administration of CS/ALG NPs (F3) and pure FSM to rats in a dose corresponding to 40 mg/kg. .................

Figure 8: Photomicrographs of a cut section of stomach fundus showing mucosa (m), submucosa (sm) thrown into large gastric rougae (R). Multiple short gastric pits (p) are seen together with overcrowded fundic glands (g) occupying the whole mucosa. Oxyntic cells (↑) are well seen with other linig cells. H&E stain ( , B & C X 100) (D, E, & F X 400). A &, d : control, b &, e : blank, c &, f: formula..............................................................

Figure 9: Photomicrographs of a cut section of rats‟ small intestine showing mucosa (m), submucosa (sm), muscularis externa (M) and serosa (s). Multiple villi (v) and intestinal crypts (c) are seen. Note the goblet cells (↑) lining the villi and intestinal glands. H&E stain (a, b & c X100) (d, e, f, g, h & i X400). a, d &g: control, b, e & h: blank, c, f & i: formula. .. 40

Furosemide mucopenetrating nanoparticles

Table 1: Effect of various CS/ALG weight ratio on the particle size (PS), polydispersity index (PDI), zeta potenial (ZP) and percent entrapment efficiency (%EE) of the prepared CS/ALG NPs, (n=3). Formula code F3

CS concentration (mg%) 80

Wight ratio ALG:CS 9:1

PS (nm) ±SD

F5

160

4.5:1

290 ±30

F6

240

3:1

907±49

PDI ±SD

ZP(mV) ±SD

% EE ±SD

-35±1

96±1

0.33±0.062

-32.2±0

92.8±0.1

0.670 ±0.02

-28.5±1

93.2±0.5

253.8±4.6 0.312±0.066

41

Furosemide mucopenetrating nanoparticles

Table 2: Comparison between the biochemical parameters of the three experimental groups of rats according to liver & kidney functions.

Control

Blank

Formulation

(n = 8)

(n = 8)

(n = 8)

ALT(U/L)

29.13 ± 1.36

28.50 ± 1.20

29.38 ± 1.51

AST (U/L)

30.13 ± 5.03

31.38 ± 7.48

36.63 ± 8.55

ALP (IU/L)

147.75 ± 10.43

153.38 ± 23.69

145.75 ± 28.37

UREA (mg/dl)

30.13 ± 6.40

29.09 ± 4.45

29.40 ± 7.41

Creatinine (mg/dl)

0.81 ± 0.16

0.79 ± 0.21

0.76 ± 0.23

Parameters

42

*Graphical Abstract (for review)

In vitro solubility in 0.1N HCl Furosemide loaded chitosan/alginate nanoparticles

In vivo oral pharmacokinetics In vivo sub-acute liver and kidney function

Ex vivo intestinal permeation Sub-acute histopathological image of stomach and small intestine

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