Fabrication , Characterization , and In Vivo Evaluation of Famotidine Loaded Solid Lipid Nanoparticles for Boosting Oral Bioavailability

Famotidine as H 2 receptor has antagonistic effects on gastric secretion. Unfortunately, its hydrophobic nature contributes to its variable and poor oral bioavailability. In the current study efforts are being made to fabricate famotidine loaded solid lipid nanoparticles with narrow size distribution. Prepared nanoformulations were pharmaceutically evaluated to confirm the desired boosted oral bioavailability. Famotidine loaded nanoformulation (FFSe-4) showed particle size 111.9±1.3 nm, polydispersity index 0.464 ± 0.03, zeta potential −33.46 ± 2mV, entrapment efficiency 84 ± 2.7%, and drug loading capacity 2.709 ± 0.13%. Drugexcipients compatibility was confirmed by Fourier transformed infrared spectroscopy. Scanning electron microscopy confirmed spherical shaped, nanosized particles. Differential scanning calorimetry and powder X-ray diffractometry confirmed the change in crystalline nature. Prepared nanoformulation was more stable at refrigerated temperature. In vitro study showed that drug release time is proportional to drug pay load and followed zero order kinetics. Release exponent (n > 0.5) confirmed non-Fickian-diffusion mechanism for drug release. In vivo pharmacokinetic studies showed 2.06-fold increase in oral bioavailability of famotidine dispersed in solid lipid nanoparticles compared to commercial product.These results authenticate solid lipid nanoparticles as drug delivery system and propose prolonged release with improved oral bioavailability for famotidine.


Introduction
Approximately 40% of commercialized active pharmaceutical ingredients (APIs) are poorly water soluble, due to which sufficient amount of drug absorption from the gastrointestinal tract (GIT) is being a challenge for the researchers [1].Low solubility and permeability lead to oral bioavailability issues which ultimately affect the drug safety and efficacy [2].Previously, different colloidal carrier systems have been investigated to overcome this problem.But certain disadvantages were associated with them such as drug expulsion upon storage, limited stability, low drug loading, and polymers cytotoxicity [3].
This leads to the rise of fabricating solid lipid based nanodrug delivery system termed as solid lipid nanoparticle.
Solid lipid nanoparticles (SLNs) were developed in the end of the 20th century [4].It potentially gathers pluses of the old systems but avoids some of their major documented shortcomings [5].The use of SLNs is a striking improvement because the solid matrix of the lipids presents high flexibility in controlling the drug release and protects the encapsulated drugs from gastric degradation.SLNs are generally composed of biodegradable and biocompatible solid lipid as solid core, coated by nonhazardous surfactant/cosurfactant as the outer shell [6].Use of solid lipids increases drug absorption mainly through enhanced drug dissolution and solubilization in the intestinal-milieu, improved lymphatic-transport, enhanced gastrointestinal permeability, and decreased gastric-emptying rate [7,8].Particle size and PDI are key characteristics and are critical parameters in the stability and fabrication of SLNs [9].These characteristics mainly depend upon particles composition and different fabrication techniques.
Famotidine is widely used as competitive H 2 receptor antagonist (H 2 RA) and prokinetic drug [10].Molecular formula of famotidine is C 8 H 15 N 7 O 2 S 3 and IUPAC name is 3-[[2-(diaminomethylideneamino)-1,3-thiazol-4-yl]methylsulfanyl]-N  sulfamoylpropanimidamide (Figure 1).Its key pharmacodynamic effect is the inhibition of gastric acid secretion [11].It decreases stomach acid production up to 90% when given in oral dosage form (20 mg or 40 mg) and promotes duodenal ulcer curing [12].It is used in the treatment of heart-burn, ulcer, and inflammation of esophagus and high doses are used for the treatment of conditions like Zollinger-Ellison syndrome.It is commercially available in different dosage forms like capsules, tablets, and also chewable tablets for adults.Powder was also prepared for oral suspension, but after reconstitution its stability was limited to thirty days only and also had extremely bitter taste [12].Hence, researchers also tried numerous techniques to mask its bitter taste [13].Hydrophobic nature of famotidine reduces its water solubility and also exposure to gastric degradation contributes to its variable and poor oral bioavailability [14].
Famotidine belongs to Class-IV drugs of biopharmaceutical classification system (BCS-IV).Drugs of this class show poor aqueous solubility and low permeability [15].Due to which its oral formulations have not been successful due to low water solubility issues (1.1 mg⋅ml −1 ) and unfavorable pharmacokinetic parameters, including low oral bioavailability (43%) and a short plasma half-life (2.59 hrs) [16][17][18].Before selecting famotidine as drug model for loading into SLNs, the available limited literature for addressing the oral bioavailability issues has been studied.Patel, Dhaval J., et al. 2010 have reported FTD nanosuspension having minimum particle size of only 566 nm and also lacking stability study.Also, there has not been reported any in vivo pharmacokinetic study.
This research work was carried out to fabricate FTD loaded SLNs to enhance its aqueous solubility which in turn boost its oral bioavailability.SLNs were fabricated by solvent emulsification evaporation technique which is most suitable for of thermosensitive drugs as it avoids thermal stress [19].SLNs have adhesive properties that could increase the residence time in the administered area and hence enhance its oral bioavailability [20].The use of tween-80 as surfactant and PVP as cosurfactant may also improve oral bioavailability as they contribute to enhancing permeability as well as affinity between lipids and intestinal membrane [21,22].1) [19].Specified amount of stearic acid was dissolved in chloroform which was then emulsified with aqueous phase having surfactant (Tween-80) and cosurfactant (PVP) under magnetic stirring (1000 rpm) to form microemulsion.In this microemulsion, aqueous phase contains micron-size droplets of organic solvent containing stearic acid.Organic solvent is evaporated from this microemulsion via magnetic stirring.As the organic solvent evaporates, the lipid starts precipitating as SLNs in the aqueous phase is followed by centrifugation using ultra-centrifuge Cs 150 GXL (Gx-Series) for 10 minutes, at 30,000 rpm [23].-average particle size and PDI of these formulations were figured out by photon correlation spectroscopy using zeta-sizer Nano (ZS-90, Malvern Instruments, Malvern, UK) [24].

Preparation of FTD-SLNs.
Best conditions of UFSe-11 formulation, that is, stearic acid (1.0 g), tween-80 (1.6 ml), and PVP (0.4 g), were further used for fabricating FTD loaded SLNs (FTD-SLNs).Different formulations of FTD-SLNs were prepared on the basis of lipid drug ratio (Table 2).Specified quantity of FTD and stearic acid was dissolved in  chloroform.The rest of process followed was same as adopted for unloaded SLNs.Schematic diagram for preparation of FTD-SLN is shown (Figure 2).

Lyophilization.
SLNs are thermodynamically insecure systems; therefore, FTD-SLNs were lyophilized using freeze dryer (Heto Power Dry LL1500-Thermo Electron Corporation, USA).Glucose solution (10%) was added as cryoprotectant before drying.FTD-SLNs were kept overnight at −20 ∘ C and then shifted to freeze dryer to be lyophilized at −75 ∘ C for 48 hrs at increasing rate of 5 ∘ C/h [25].

Entrapment Efficiency (EE) and Drug Loading Capacity (DLC).
Freshly fabricated FTD-SLNs samples were centrifuged and supernatants were analyzed to quantify unentrapped drug using nanodrop spectrophotometer (Thermo scientific 2000c/2000 UV-VIS Spectrophotometer).Entrapment efficiency of FTD was calculated by EE% = Total amount of drug added − Unloaded Drug × 100 Total amount of drug added . ( Percent drug loading capacity of FTD was calculated by DLC% = Total amount of drug (SLNs) × 100 Amount of Drug + Amount of Excipients .
2.3.Characterization 2.3.1.Dynamic Light Scattering.Zeta-sizer analysis was carried out by using zeta-sizer ZS-90 (Malvern Instruments, England).-average particle size, PDI, and zeta potential were analyzed.All SLN formulations were diluted with deionized water in order to get proper scattering intensity, measured at 90 ∘ scattering angle and 25 ∘ C.

Drug-Excipients Interaction.
Fourier transform infrared spectroscopy (IR Prestige 21 Shimadzu, Japan) was used to study drug-excipients interaction with diffuse reflectance principle [26].Spectra of unprocessed FTD and processed FTD (FFSe-4) were scanned over a frequency range of 2000 to 400 cm −1 .For compatibility of formulation components, the peaks and patterns shaped by the unprocessed FTD were compared with processed FTD (FFSe-4).

Morphological Study.
Scanning electron microscopy (SEM) was used to study the morphological characteristics and texture of SLNs by JSM5910 (JEOL, Japan) [27].SEM micrographs were recorded at magnification of 60,000x and accelerating voltage of 20 kV [28].

Powder X-Ray Diffraction (P-XRD).
Powder X-ray diffraction analysis was performed to verify new solid state formation [29].P-XRD analysis was conducted for unprocessed FTD and processed FTD (FFSe-4) using an X-ray diffractometer JDX-3532 (JEOL Japan).Cu K radiation in scanning range of 2 = 5 To examine the physical stability of FTD-SLNs, stability study was carried out for FFSe-4 formulation [31].The freshly fabricated sample was divided into two parts.Each part was put in two plain sealed glass vials and stored at different temperatures (5 ± 2 ∘ C and 25 ± 3 ∘ C) for 3 months.Samples were taken on 1st, 15th, 30th, 60th, and 90th day of storage and subjected to particle size and PDI measurements.Data was analyzed statistically by two tailed -test.Probability < 0.05 was considered significant.

In Vitro Release of FTD from SLNs.
In vitro drug release study was conducted using dialysis bag method [32].Dialysis bags were soaked in deionized water for 12 hours before use.FTD-SLNs dispersion (1 ml) from each formulation was poured into the dialysis bag and placed in 250 ml phosphate buffer solution (pH 7.4) at 50 rpm.After definite time interval (1-12 hr), samples were taken and equal volume of phosphate buffer solution was replaced.Samples were analyzed by using UV spectrophotometer ( max 265 nm) against blank phosphate buffer solution (pH 7.4) [33].Data obtained from in vitro drug release study was fitted into different kinetic models to find out both drug release rate and mechanism that followed [34].

In Vivo Pharmacokinetic Studies
2.6.1.Oral Drug Administration.Before conducting in vivo study, approval was taken from departmental research ethics committee (vide letter number DREC/20160503-14).Healthy rabbits (2 ± 0.3 Kg) were kept fasted (12 hrs) before dosing but access to water was given.Two groups of animals were made, each having six rabbits.FFSe-4 formulation was orally administered to Group I while Ricer5 to Group II (10 mg⋅kg −1 ).At various time intervals (0 to 24 hrs), blood samples (0.5 ml) were collected and kept in tubes (heparinized).Plasma was separated by centrifugation and stored at −20 ∘ C till further analysis.
2.6.2.Quantification of Plasma Concentration.Prepared plasma samples were analyzed for drug quantification by HPLC technique.Acetonitrile : Methanol : (0.016 mol/l) Phosphoric Acid (10 : 10 : 80) were used as mobile phase (retention time: 3 min, flow rate: 1 ml/min).Reversed phase column (Supelco C 18 , 25 cm in length, 4.6 mm width, and 5 m particle size), generally used for hydrophobic drugs, and precolumn (Supelco C 18 ) were used at 37 ∘ C. Prior to HPLC analysis, plasma samples were mixed with acetonitrile and then placed at −20 ∘ C for 10 minutes followed by centrifugation to precipitate proteins.The supernatant (20 l) was then injected for the determination of FTD concentration using UV detector at  max 254 nm [33].Famotidine concentration was determined from the area of chromatographic peak using the calibration curve.
2.6.3.Data Analysis.Different pharmacokinetic parameters were determined for non-compartmental model.Area under curve (AUC 0→ ) was calculated from concentration-time curve by trapezoidal rule.From the individual plasma concentration-time curve, peak plasma concentration ( max ) and peak plasma concentration time ( max ) were calculated.Total area under the curve (AUC 0→24 ) was determined by is FTD concentration at 24th hour and   is apparent elimination rate constant.
Relative bioavailability (  ) after 24 hours for equal dose was determined by One-way analysis of variance and -test ( < 0.05) were used for statistical analysis of data.

Drug-Excipients Interaction.
Fourier transform infrared analysis is used specifically for assessing drug-excipients interaction in different formulations [35].The major peaks of C=C stretch at 1639 cm −1 , SO 2 stretch peak at 1147 cm −1 , C-H bend at 1284 cm −1 , C=S stretch at 1146, and N-H bend at 984 cm −1 were present in both unprocessed FTD and  processed FTD (FFSe-4).This clearly indicates no interaction between FTD and other excipients.The obtained spectra are shown (Figure 7).

Scanning Electron Microscopy (SEM).
Shape and surface morphology of FFSe-4 formulation was studied by SEM.SEM analysis showed solid and fairly spherical shaped particles with well-defined periphery.The particles size was also in nanometric range (Figure 8).

Powered X-Ray Diffraction (P-XRD).
Unprocessed famotidine (FTD) showed a series of sharp peaks indicating its crystalline nature.In processed FTD (FFSe-4), most of these peaks were suppressed but few disappeared, indicating conversion to amorphous form (Figure 9).3.6.Thermal Analysis.DSC thermograms of FTD (unprocessed), stearic acid (SA), physical mixture, and processed FTD (FFSe-4) were recorded separately.Sharp endothermic peak was observed for unprocessed FTD at 166.9 ∘ C, SA at 69 ∘ C, and physical mixture of FTD and SA at 166.5 ∘ C and 68.6 ∘ C, respectively.Processed FTD (FFSe-4) showed endothermic peak at 160 ∘ C (Figure 10).at refrigerated temperature was less than 5% but at room temperature it was almost 15.91%.At both temperatures, the particles growth was in acceptable range but PDI at room temperature exceeded the acceptable range (Figures 11 and  12).Statistically analyzed data from two tailed -test showed  value for particle size was 0.044 and PDI was 0.046.
3.9.In Vivo Pharmacokinetic Study.The plasma concentration-time curve of FFSe-4 formulation and marketed product is shown (Figure 14) and pharmacokinetic parameters are also listed (Table 5).FTD plasma concentrations were significantly higher in rabbits treated with FFSe-4 than for those treated with marketed product.Peak plasma concentration ( max ) for marketed product and FFSe-4 formulation was 0.498 ± 0.14 g⋅ml −1 and 1.03 ± 0.204 g⋅ml −1 , respectively.AUC 0→24 for marketed product was 43.96 g⋅hr⋅ml −1 whereas for FFSe-4 was 231.22 g⋅hr⋅ml −1 .FFSe-4 formulation showed 2.06fold increase in  max and 5.25-fold increase in AUC 0→24 compared to marketed product.These results showed that FTD absorption was improved significantly in SLNs formulation compared with conventional dosage form (marketed product).

Discussion. Solvent emulsification evaporation (SEE)
method has been used to fabricate FTD loaded SLNs.Optimized conditions for unloaded SLNs were stearic acid (1.0 g),  Tween-80 (1.6 ml), PVP (0.4 g), and magnetic stirring time (15 minutes) (Table 1).-average particle size was reduced by increasing surfactant concentration (Tween-80); its higher concentration also gave better stability to small lipid droplets which prevent them from coalescence [39].Addition of cosurfactant (PVP) further reduced -average particle size as SLNs fabricated with surfactant/cosurfactant mixture have lower -average particle size and better stability.PDI has been controlled and reduced by increasing magnetic stirring time as it has almost no effect on particle size reduction but only on PDI [40].The optimized unloaded SLNs formulation (UFSe-11) showed particle size 127.8 ± 2.3 nm.After drug (FTD) loading the particle size was reduced to 111.9 ± 1.3 nm (FFSe-4) having PDI 0.464 ± 0.03.After drug pay load, particle size reduced due to decreased free lipid content [41].Zeta potential of FFSe-4 formulation was −33.46 ± 2 mV, sufficient for electrostatic stability [42].
The PDI < 0.5 and zeta potential ± 30 revealed that the fabricated nanodispersion would be stable in nature [43].Both of these values for FTD-SLNs were within the range, exhibiting electrostatic stabilization having no aggregation which led to preventing Ostwald ripening and particle growth [42].The formulation (FFSe-4) gave entrapment efficiency and drug loading capacity 84 ± 2.7% and 2.709 ± 0.13%, respectively, with maximum encapsulation and higher drug loading efficiency.It has been reported that in polymer and lipid based nanoparticulate drug delivery systems, the binding energy of the drugs with the polymers and lipids plays a key role in successful encapsulation of drugs [44].In this case, it might be attributed to the high binding energy of the FTD with stearic acid, tween-80, and PVP which results in maximum entrapment efficacy and drug loading capacity.
However, EE% decreased from 96% to 59% as FTD pay load increased from 40 mg (FFSe-1) to 200 mg (FFSe-5).This sudden fall in EE% might be due to loading of FTD beyond saturation level of lipid [23].Lipophilic drugs can gain super-saturation in melted lipids; on cooling, saturation level reduces and excessive quantity of drug tends to partition in outer shell or external solvent [5].
FT-IR spectra of unprocessed FTD and processed FTD (FFSe-4 formulation) confirm the compatibility of FTD with the formulation components (Figure 7).Scanning electron microscopy further confirmed nanometric size particles of SLNs loaded with FTD.Micrograph of SEM (Figure 8) shows solid, identical, and fairly spherical shaped particles with a well-defined periphery.Most of the SLNs are present in dispersed form with homogeneous distribution which exhibit amorphous nature of the produced nanoparticles.P-XRD studies also confirmed the amorphous nature of the FTD loaded SLNs, as the disappearance and reduction in intensities of the peaks are indicative for amorphous nature of the particles (Figure 9) [43,45].DSC studies confirmed the amorphous nature of the FTD loaded SLNs, because for unprocessed FTD, sharp melting point peak appeared on 166.6 ∘ C while for FTD loaded SLNs formulation it was 160 ∘ C (Figure 10).This small diffused peak indicated reduced particle size of FTD, enlarged surface area, and closed contact between solid lipid (stearic acid) and drug (FTD) which could be considered for the change of FTD from crystalline to amorphous state [46,47].
In comparison with room temperature, refrigerated temperature was best for the stability of FFSe-4 formulation.Three-month study showed no significant change in size and PDI of the sample when stored at refrigerated temperature (Figure 11).However, at room temperature some growth was observed for the initial 30 days which is because of the amorphous nature of the FTD-SLNs followed by stabilization for rest of the period.Additionally, at room temperature, amorphous solids have increased free energy which results in decreased stability [48,49].
In vitro study showed that increased payload of FTD resulted in prolonged drug release time (Figure 13) [23].Release of FTD from SLNs followed zero order kinetics.However Korsmeyer-Peppas model showed that the release exponent () was greater than 0.5 which confirmed non-Fickian diffusion kinetics for all SLNs formulations [37,38].
The interesting results obtained from statistically analyzed data of in vivo pharmacokinetics confirmed boosted oral bioavailability with sustained release profile of FTD-SLNs (FFSe-4) compared to marketed product (Table 5).SLNs as drug delivery system open angles to formulate already available drugs (BCS-II and BCS-IV) in the market to boost their oral bioavailability and attain sustained release behavior.SLNs are not only responsible for improvement of oral absorption but can correspondingly be formulated for parenteral administration, which need additional studies [50].

Conclusion
This research work concluded that various processing parameters are the characteristic key factors to prepare appropriate lipid carriers for efficient loading of the selected drug.SLNs have been surfaced as novel drug carriers for famotidine with boosted oral bioavailability and strong sustained drug release performance.We have exposed that famotidine in form of SLNs is an encouraging nanomedicine with valueadded physical stability and prolonged release profile.Also, there was good affinity found between famotidine and stearic acid.In vitro and in vivo release study confirmed that SLNs system is very suitable to improve oral delivery of poor water soluble drug like famotidine with increased solubility and permeability which in turn enhanced bioavailability.In future perspectives, the produced FTD loaded SLNs could potentially be transformed into solid dosage form followed by in vitro and in vivo assessments.Thus, it is concluded that sustained release FTD-SLNs were successfully fabricated by simple and reproducible technique (solvent emulsification-evaporation method) which has potential to be scaled up for commercial production and no sophisticated instrument is required during fabrication.
∘ -80 ∘ was used with tube current 30 mA, operated voltage of 40 kV, step time 1.0 sec, step size 0.05 ∘

Table 3 :
Cumulative percent release of FTD.

Table 4 :
2 value of different kinetic models for FTD-SLNs formulation.