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Formulation of amlodipine nano lipid carrier: Formulation design, physicochemical and transdermal absorption investigation
Accepted Manuscript Formulation of amlodipine nano lipid carrier: Formulation design, physicochemical and transdermal absorption investigation Hema Kapoor, Mohd Aqil, Syed Sarim Imam, Yasmin Sultana, Asgar Ali PII:
S1773-2247(18)31031-1
DOI:
https://doi.org/10.1016/j.jddst.2018.11.004
Reference:
JDDST 826
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 8 September 2018 Revised Date:
31 October 2018
Accepted Date: 7 November 2018
Please cite this article as: H. Kapoor, M. Aqil, S.S. Imam, Y. Sultana, A. Ali, Formulation of amlodipine nano lipid carrier: Formulation design, physicochemical and transdermal absorption investigation, Journal of Drug Delivery Science and Technology (2018), doi: https://doi.org/10.1016/ j.jddst.2018.11.004. 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.
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Manuscript number:- JDDST_2018_980R2.
Formulation of amlodipine nano lipid carrier: formulation design, physicochemical and
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transdermal absorption investigation
Hema Kapoora, Mohd. Aqila*, Syed Sarim Imamb, Yasmin Sultanaa, Asgar Alia
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a. Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road New Delhi 110062, India.
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247121. Uttar Pradesh, India.
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b. Department of Pharmaceutics, Glocal School of Pharmacy, Glocal University, Saharanpur
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*Corresponding author: Dr. Mohd. Aqil Associate Professor Department of Pharmaceutics School of Pharmacuetical Education and Research Jamia Hamdard (Deemed University) M. B. Road, New Delhi 110062, India. Email: *[email protected]; †[email protected] Tel: +91-9811798725
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Abstract The present work was designed to formulate and statistically optimize transdermal amlodipine nanostructured lipid carriers (AMNLCs) using lipid blends. The formulation was optimized by
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using the independent variables Peceol (liquid lipid as X1), GMS (solid lipid as X2) and Tween80 concentration (surfactant as X3). Their effects were assessed on dependent variables particle size (Y1), transdermal flux (Y2), and entrapment efficiency (Y3). The optimized formulations was further evaluated for in vitro drug release, confocal laser scanning microscopy, physicochemical evaluation, and in-vivo absorption study. The optimized amlodipine nanostructured lipid carriers
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(AMNLCopt) showed low particle size (123.8 nm), enhanced transdermal flux (58.33µg/cm2/h), and higher entrapment efficiency (88.11%). Further, it showed prolonged drug release and
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followed higuchi release kinetics with R2 closer to unity. The rhodamine red (RR) loaded RRAMNLCopt revealed an enhanced permeation to the deeper layer of the skin after assessing through confocal laser scanning microscopy (CLSM). The in-vivo absorption study presented enhanced improvement in bioavailability of amlodipinein the wistar rats. From the study, it was concluded that the experimental design based AMNLCs showed the quadratic relationship between independent and dependent variables and also found to be a proficient carrier for
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transdermal delivery of amlodipine.
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Key words: Amlodipine, NLCs; Transdermal; In-vivo absorption; Histopathology
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Introduction The barrier nature of the stratum corneum (SC) is the major challenges that limit the entry of most of the drugs by the transdermal route (Lim et al., 2006). Many transdermal techniques have been tried to overcome the barrier of SC to achieve higher transdermal
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permeability. These techniques are designed to deliver the drug for a prolonged period of time (Imam et al., 2016), which is particularly encouraging to treat chronic disorders like hypertension (Qadry et al., 2016; Qumbar et al., 2016; Aqil et al., 2016). There are many antihypertensive drugs have shown low bioavailability due to poor solubility and it undergoes
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extensive first-pass metabolism which can be avoided by transdermal delivery. Many attempts have been taken to develop the transdermal delivery of antihypertensive drugs to circumvent the
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drawbacks of conventional drug delivery system (Kamran et al., 2016; Gungor and Ozsoy 2012; Selvam et al. 2010).
The application of lipid-based delivery systems offers suitable carrier for transdermal delivery due to their greater biocompatibility with the skin lipids (Chourasia et al., 2011). Among different lipid system, NLCs is the widely used carrier made up of the combination of solid lipid, liquid lipid and dispersed in an aqueous emulsifier solution. The addition of liquid
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lipid in NLCs gives the ordered crystalline state of solid lipid and enlarges drug storage space (Moghaddam et al., 2017; Pradhan et al. 2016). They are well suited for the systemic delivery across skin because they are composed of physiological and biodegradable lipids of low systemic toxicity and also low cytotoxicity (Jaiswal et al. 2016; Raj et al. 2015). The nano-size range
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gives a larger surface for the drug absorption across the skin and gives greater therapeutic efficacy (Muller & Keck, 2004). It acts by adhering to the skin, forms a thin film and gives an
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occlusive effect with a decrease in the transepidermal water loss (Jenning et al., 2000; Muller et al., 2007). The hydration of the SC could give broadening of the inter-corneocyte gaps and enhances drug permeation into deeper layer (Muller et al., 2007; Alam et al., 2016; Puglia & Bonina, 2012).
Amlodipine (AM), a third-generation dihydropyridine calcium channel antagonist, is
mostly choice of drug for the management of angina and hypertension (Sun et al., 2009). The poor aqueous solubility and low permeability through GIT gives low bioavailability (Chahbra et al., 2012), and hence limits the access of drug to their therapeutic targets like heart and cardiac smooth muscles. It is a weak base and mainly exists in its ionized form, has low permeability
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characteristics within the gastrointestinal pH range (Jang et al., 2012; Rausl et al., 2006). The oral bioavailability of amlodipine free base is low compared to amlodipine salt (Meredith., 2009; Saigal et al., 2008). The optimization AMNLCs was performed using Box–Behnken design using the independent variables (X1- Peceol, X2- glycerylmonostearate, X3- tween 80) at
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three level (low, medium and high) and their effect observed on dependent responses (Y1particle size; Y2- flux; Y3- encapsulation efficiency). The BBD design was used for the optimization because it requires fewer runs than a central composite design (Imam et al., 2017; Thapa et al., 2018; Jahangir et al., 2017). The present study was focused on the formulation of
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amlodipine loaded nano lipid carrier using the design expert software. Finally, the optimized nano lipid carrier formulation was analyzed for the morphology, in-vitro release, in-vivo
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absorption and skin interaction properties.
Material and Methods Materials
Amlodipine (AM) was received as a gift sample from Torrent Pharmaceuticals (India). HPLC grade methanol and water were purchased from Merck, Mumbai, India. Peceol (oil), Labrafil M
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2125, Glyceryl monostearate (GMS), Labrafil M 1944, received from “Gattefosse India Pvt. Ltd., (Mumbai, India)”and (Colorcon Asia Pvt Ltd, Mumbai, India). Tween-80, Carbopol 940, were purchased from S.D Fine chemicals, Mumbai, India) and all other chemicals and solvents used were of analytical grade.
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Methods Screening of liquid lipids
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The saturation solubility of amlodipine in different liquid lipids was determined by adding an excess amount of drug in oil (1mL) in an eppendorf tube. The eppendorf tube was subjected for the agitated until the drug gets completely dissolved, and the process was further continued until the saturation stage reaches. After that, the drug in the lipid sample was kept to withstand for 12 hours for stabilization. Finally, it was centrifuged at 3000rpm for 15 min and the supernatant was separated, dissolved in ethanol and the solubility was quantified by UV spectrophotometer at 356 nm.
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Screening of solid lipids To select the appropriate solid lipids, different solid lipids (stearic acid, Gleucire 44/14, Glyceryl monostearate (GMS)) were weighed (1 g), melted at 75 °C and agitated using a magnetic stirrer. The drug sample was added slowly to the liquefied lipid till saturation was attained and which
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was set aside for 12 h for stabilization. After that, a small portion of the supernatant from each vial was taken and ethanol was added to each tube. The sample was then analyzed by UV spectroscopy and concentration of each solution was determined.
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Experimental design
The Box-Behnken design was used to statistically optimize the formulation parameters keeping
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three factors at three levels for optimization study. The design used the response surface plot and second order polynomial models using Design Expert software (Version 9.0., Stat Ease Inc, Minneapolis, MN) to get the optimized formulation. The experimental design showed 17 experimental runs with 5 mid points to evaluate the effect of independent variables on dependent variables. The independent variables were solid lipid concentration (peceol as X1), liquid lipid concentration (GMS as X2) and surfactant concentration (tween 80 as X3) represented by the
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low, mid and upper limit (-1, 0, +1), respectively, while the dependent variables were particle size (Y1), transdermal flux (Y2) and % entrapment efficiency (Y3) as given in Table 1.
Formulation of amlodipine nano lipid carrier (AMNLCs)
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Amlodipine loaded NLCs were prepared by using melt emulsification ultrasound dispersion method with slight modification (Moghaddam et al., 2016; Rizwanullah et al., 2017). The lipid
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blend phase Peceol: GMS (3:7) and aqueous surfactant phase (Tween 80) were prepared separately in a beaker. The selected liquid lipid (Peceol) and solid lipid (GMS) was melted and calculated amount of AM was added to it. Both the phases were heated separately at 80°C on a temperature regulated water bath and the aqueous surfactant phase (tween 80) was added drop wise to the lipid blend phase to make a primary emulsion. Finally, the primary emulsion was probe sonicated at 50W to get small size (Elmowafy et al., 2015). The sample was sonicated in ice condition which helps to maintain the temperature of formulation at 4°C and a pause or a gap of 5 min. were taken in each cycles (Hielscher ultrasonicator, Germany). The prepared AMNLCs formulation was cooled to room temperature and stored for further use.
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Characterization Size and surface charge The particle size and surface charge of NLCs were determined through zetasizer (Zetasizer Nano ZS, Malvern, UK). The small amount (1mL) of the formulation was diluted in the distilled water
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and mixed thoroughly with vigorous shaking. The particle size, surface charge, and polydispersity index (PDI) were evaluated by adding the diluted dispersion into the disposable cuvette and analyzed using the zeta sizer. The polydispersity index (PDI) was determined as a measure of homogeneity of the particle. The ideal value of PDI must be between <0.1 - 0.3>
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indicate a homogeneous population and greater than that show high heterogeneity.
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Entrapment efficiency
The entrapment efficiency of the AMNLCs formulation was determined by ultracentrifugation method by calculating the amount of the untrapped drug. The sample (1mL) was diluted and kept in the upper compartment of the ultracentrifuge tube and run at 25,000 rpm for 15 min. The free drug moves to the lower chamber through the semipermeable membrane and the entrapped drug retained in the upper chamber. The sample collected from the lower chamber, diluted and
Surface morphology
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entrapment efficiency was determined using UV-spectrophotometer at 364nm.
The particle morphology was studied using TEM (MORGAGNI 268D Fei 155 Company,
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Netherlands) using the combination of bright field imaging at high magnification. The diluted sample was taken to the copper grid, left for 1 min to dry and a drop of phosphotungstic acid
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(PTA) was added (Hasnain et al., 2018). The excess of PTA was removed and analyzed at 6080KV at 1550 magnification. Further, SEM analysis was also performed to evaluate the surface morphology of the prepared NLC. The image was recorded on a scanning electron microscopy (ZEISS GEMINI 51530 FEG) and photomicrographs were obtained.
Differential scanning calorimetry (DSC) DSC was conducted to ascertain the compatibility of AM with the excipients used for the formulation using calorimeter (Pyris 6 DSC, Perkin Elmer). The study was done for the pure drug (AM) and lyophilized AMNLCopt formulation. The samples were kept in the hermetic pan
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and conducted at a heating rate of 10°C/min over the temperature range of 30–300°C using an inert nitrogen gas with keeping an empty aluminum pan as a reference.
Formulation of amlodipine nano lipid carrier based gel (AMNLCG)
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The epidermal retention of a transdermal formulation for a prolonged period of time would eventually help in delivering the drug optimally. The formulation AMNLCopt was insufficiently viscous and could be therefore quickly removed from the skin, therefore it was converted into gel formulation. The gel formulation (AMNLCGopt and control) was prepared by taking Carbopol
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940 (1%w/v) as gelling agent. Carbopol was mixed with distilled water to get the polymer dispersion and then kept aside in dark to allow for complete swelling. Finally, polyethylene
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glycol 400 (15%w/w) and chlorocresol (0.1%) were added slowly with stirring to get homogenous dispersion. The gel formulation was then neutralized using triethanolamine (TEA) and stirred slowly to get an optimized carbopol based gel (control gel). Same procedure was used to get gel formulation by adding the optimized AMNLCopt formulation into preformed gel with stirring (Thasleem et al., 2018), to get final AMNLCGopt for further characterization.
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Evaluation of AMNLCG
The prepared AMNLCGopt formulation was evaluated for different parameters like color, homogeneity, consistency and phase separation. The homogeneity was checked by visual evaluation after the gels have been kept in the container. The presence of any aggregates was
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observed for their appearance. The pH of the gel was determined by using digital pH meter
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(Mettler Tolledo, Japan) fitted with a glass microelectrode by allowing it to equilibrate for 1 min.
Viscocity and spreadability The viscosity of prepared AMNLCGopt was calculated using Rheometer (MCR101 Rheoplus of Anton Paar), connected to a thermostatically controlled circulating water bath. AMNLCGopt was evaluated for the spreadability by taking a specified amount of gel sample and kept within a circle of premarked 2 cm diameter on a glass plate. The second plate was kept over it and a weight was allowed to rest on the upper glass plate for 5 min (Bachhav et al., 2009). The increase in the diameter due to an application of weight was noted (n = 3). To determine the
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percent spread by area, calculate as follows: % Spread by area =A2 A1× 100, where, A1= 2 cm2, and A2= final area after spreading.
Texture analysis
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In order to characterize and evaluate the various textural parameters of the prepared gel their firmness, adhesiveness, force of adhesion, and gel strength were measured. The optimized formulation AMNLCGopt was placed into identical glass jars (height 40 mm, diameter: 55mm) to a fixed sample height (30mm). The entrapment of air bubble have been avoided into the
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sample and testing surface was kept as flat as possible to avoid early triggering of the test (Hagerstrom et al., 2003). The texture profile analysis was performed using a TA.XT2 plus
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texture analyzer (Stable Micro System, UK) in the compression mode. The parameters are the resultant force-time plots and several mechanical parameters of the gels were determined.
In-vitro drug release
The in-vitro release study was accomplished by using diffusion cell having receptor chamber of 20 mL capacity with the diffusion area of 1.5 cm2. The pretreated membrane with the pore size
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of 0.2µm was used for the study. Both samples (AMNLCopt and AMNLCGopt equivalent to 5mg AM) was filled into the donor compartment and the receptor compartment was filled with PBS: ethanol mixture (9:1) and the release study was performed at 37°C with continuous stirring. After predetermined time interval the sample (0.5mL) was withdrawn from the sampling port
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and replenished with the fresh media. The samples were filtered and diluted to analyzed by UV spectrophotometer. The collected release data from this study were fitted to kinetic models zero-
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order, first-order, Higuchi’s matrix (Higuchi 1963) and the Korsmeyer peppas model (Korsmeyer et al. 1983, Peppas 1985). The relevant correlation coefficients were taken into account for the selection of best model. The mechanism of drug release was determined by taking the data and fitted into the Korsmeyer peppas model and their release exponent (n) were calculated from the slope of the straight line (Uprit et al. 2013).
Skin permeation studies The excised abdominal rat skin was fixed between donor and receptor compartment of diffusion cell (area 1.5cm2) in such way that stratum corneum faced towards the donor compartment and
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dermis side faced towards the receptor compartment. Both AMNLCopt and AMNLCGopt formulations (equivalent to 5 mg of Amlodipine) were placed in a donor compartment and receptor compartment was filled with the PBS:ethanol (9:1; 10mL). The receptor compartment was stirred regularly on a magnetic stirrer at 100 rpm and the temperature was maintained at
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32oC±0.5oC during the whole study. The diffused samples (1mL) were withdrawn at predetermined intervals from receptor compartment replaced with the same medium. The collected samples were diluted further and drug content were analysed by HPLC. The cumulative
at steady state was calculated.
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Confocal laser scanning microscopy (CLSM) study
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amount of drug permeated was plotted as a function of time and the skin permeation rate (Flux)
The extent of the penetration of prepared NLC formulation through rat skin “confocal laser scanning microscopy (CLSM)” was performed. The fluorescent probe rhodamine B dye loaded AMNLCGopt and control gel used for this study. The study was performed for 8 hours similar to ex-vivo permeation study. After that, the treated skin was collected, washed with distilled water and the excess dye was removed. The skin was cut into the small size and fixed on the slide as
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stratum corneum facing upward. The depth of penetration was observed under a confocal microscope with an argon laser beam with excitation at 524nm and emission at 625nm. The skin sample was sliced in sections and visualized through the z-axis by CLSM (Dayan and Touitou,
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2000).
In-vivo absorption study
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The in-vivo absorption study was done with Wistar rats of either sex (200-250g) as the animal models. The animals were supplied by Central Animal House facility after the approval from Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi (application No-1130). All the rats were kept under standard laboratory conditions in 12-hour light/dark cycle at 25°C ± 2°C provided with pellet diet (Lipton, Kolkata, India) and water ad libitum. The study was performed with rats and dose administered equivalent dose of 2.5 mg/kg body weight of AM (Jang et al., 2013). The animals were fasted for 24 hours and divided into 2 groups having six animals in each group. Group A treated with AMNLCGopt (1.25 gm) and Group B was orally administered with a marketed tablet (Amdepine), respectively. AMNLCGopt was applied to the shaved
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abdominal rat skin and kept under observation. The blood samples were withdrawn from the tail vein at different time intervals (0, 1, 2, 4, 8, 12, 24h) and collected in eppendorf tubes containing disodium EDTA as an anticoagulant. The plasma samples were separated by centrifugation at 5000 rpm for 15min. and the separated samples were stored under -70°C for drug analysis by
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HPLC method. The different biopharmaceutical assessment such as Cmax (the maximum plasma concentration) and Tmax (time to reach maximum plasma concentration) was directly obtained from the plasma concentration versus time plot. The area under the curve AUC0-48 and AUC0-inf were calculated by trapezoid rule. HPLC system was (Shimadzu, Model LC-10 ATVP) Japan)
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equipped with a binary pump and UV detection system (SPD-10A). Chromatographic separation was accomplished by using C-18 (250mm×4.6mm i.d, 5µm particle size, Shiseido, Japan). The
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mobile phase used was acetonitrile: water (70:30) with the water phase containing 0.2% triethylamine (pH 4). The study was performed at the wavelength (364nm), flow rate (0.8 ml/min), with injection volume (20 µl) and total run time was 10 min.
Skin irritation and histopathology
Skin irritation was assessed for any irritation from developed formulation on Wistar rats using
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Draize score test (Draize et. al, 1944). The rats were divided into 2 groups, Group I was treated with AMNLCGopt, Group II received formalin (0.8%v/v) as a standard irritant (Imam et al., 2017). Both samples were applied to the rat skin and upon the removal, animals were examined for irritation by giving visual scoring for erythema and edema. An adjacent area of untreated skin
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was served as a control. After that, the animals were sacrificed and the skin samples from treated and untreated (control) area were excised and collected in buffered formalin saline (pH=7.4) and
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processed for histopathological studies. The specimens were cut into a small section and each section was dehydrated using ethanol, embedded in paraffin. The samples were fixed on the slide and stained with hematoxylin/eosin dye for clear visibility. These samples were observed in the light microscope (Motic digital microscope, DMB series) and compared with control skin sample (Sintov et al., 1999).
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Results and Discussion Solubility study From the solubility and miscibility studies, the different solid and liquid lipids were taken to evaluate the maximum solubility of AM. The solubility in liquid lipid was found in this order
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Peceol > iso propyl myristate > Labrafil M1944 > Labrafil M 2125 and in solid lipid GMS > stearic acid > Gleucire 44/14. The final selected component for the formulation was Peceol (liquid lipid), GMS (solid lipid) and Tween-80 (surfactant), respectively for the formulation of AMNLCs because they showed maximum solubility of the drug. The blend of liquid and solid
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lipid showed stable formulation at 3:7 ratio without any phase separation so finally used further
Experimental design optimization
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for the formulation.
The statistical design showed a total of 17 runs and characterized for different responses like size, flux and encapsulation efficiency as depicted in Table 1. The mathematical relationships between both independent and dependent variables were established, coefficients of the second order polynomial equations generated for size, flux, and encapsulation were found to be
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quadratic. The coefficients correlation of the polynomials fit well to the data, with the values of R2 ranging between 0.9650 and 0.9910. The polynomial equation represents negative sign for the variables gives the inverse relationship between the factor and the response whereas the positive value favors the optimization. The all three independent variables [liquid lipid (peceol), solid
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lipid (GMS) and surfactant (tween 80)] showed significant effect individually and combinely on
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all the three responses namely particle size, flux, and encapsulation.
Effect of independent variables (A) peceol (B) GMS (C) tween 80 on size The minimum and maximum particle size was found between 104 nm and 285 nm as shown in Table 1. The all independent variables shown their effects on particle size depicted in threedimensional Figure 1A-B. It is very evident from the model graph that the variation in concentration of total lipid affects the particle size. As the ratio of solid lipid and liquid lipid increases, the particle size increased. The increased size due to higher cross linking between lipid and drug takes place. As the lipid concentration increase there is more space available between solid lipid and liquid lipid to accommodate the drug. The increase in tween 80
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concentration decreases the particle size due to the reduction in interfacial tension between the two phases, leading to the formation of smaller size emulsion droplets (Liu et al. 2007). At high surfactant concentration, the particles were stabilized by forming a steric barrier on the surface and prevent the small particles to form coalescence (Rahman et al. 2010). In this case, A, B,
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AC, AB, BC, A2, B2, C2 were significant model terms. The “Predicted R-Squared” of 0.9089 was in reasonable agreement with the “Adj R-Squared’’ of 0.9795; i.e the difference was less than 0.2. “Adeq Precision” measures the signal to noise ratio.
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Effect of independent variables (A) peceol, (B) GMS (C) tween 80 on transflux
The minimum and maximum transdermal flux was found between 36.43 to 60.56 µg/cm2/h as
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shown in Table 1. The effects of independent variables on flux are presented by threedimensional graph (Figure 1C-D). It was observed that the transdermal flux has a direct relationship with the surfactant concentration and total lipids content. The proposed mechanism for improved permeation across skin from AMNLCopt may involve disruption of densely packed lipids structure that fills the extra-cellular spaces of the stratum corneum. The intercellular lipid barrier in the stratum corneum would be more permeable following treatment with NLCs. The
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drug permeation also enhances due to high thermodynamic activity gradient in the upper part of the stratum corneum by adsorption and fusion of drug loaded NLC onto the surface of the skin. The “Predicted R2” of 0.9364 was in reasonable agreement with the “Adj R2’’ of 0.9751. The adequate precision measures the signal to noise ratio and the model can be used to navigate the
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design space. In this case, all the individual and combined variables like A, B, C, AB, AC, BC,
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A2, B2, C2 showed significant model terms.
Effect of independent variables (A). peceol (B). GMS (C). tween 80 on encapsulation efficiency.
The minimum and maximum encapsulation efficiency was found between 67.4 to 92.35% as shown in Table 1. Figure 1(E-F) showed three-dimensional plot which shows the effect of different independent variables on EE (Y3). It has been observed that the entrapment efficiency of NLCs increased with an increase in the content of GMS. The increase in EE may be due to the blend of liquid lipids and solid lipids that lead to disturb the crystal order, which ultimately improved drug entrapment efficiency. The increase in the surfactant concentration gives the
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initial increase in EE, at high surfactant concentration in the external phase might increase the partition of the drug from internal to external phase. Due to increased partition, the drug solubility increased in the external aqueous phase so high concentration of drug can solubilize in it (Rahman et al. 2010). In this case A, B, C, AB, AC, BC A2, C2 were significant model terms.
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The “Predicted R2” of 0.9012 was in reasonable agreement with the “Adj R2’’ of 0.9439.
Point prediction
Finally, the optimized formulation (AMNLCopt) was formulated using the composition of liquid
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lipid (Peceol- 74.26 mL), solid lipid concentration (GMS- 210 mg), and surfactant concentrations (tween 80- 0.2mL). The experimental and predicted value of AMNLCopt for
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particle size (128.3 nm and 129.4nm), % encapsulation efficiency (88.11 and 87.34) and flux (57.33 and 58.24) and was found to be very close to each other. This result showed 98.23%, 99.51% and 100.08% validity of the predicted values of responses, confirming the selection of optimized formulation. The ANOVA model was assessed and revealed that the model terms for individual and interaction effects were statistically significant. Finally, on the basis of point prediction the optimized AMNLCopt was converted into gel using carbopol as gelling agent and
Characterization
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further evaluated for different parameters.
Particle size and surface charge
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The penetration of drug across skin requires smaller particle size which gives a larger surface area for absorption thereby drug absorption increases. The particle size of all the developed
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formulations was found in the range of 104 nm to 285 nm. The optimized AMNLCopt showed particle size of 118.3 nm (Figure 2A) with PDI of 0.202 indicated narrow size distribution. The zeta potential value of -29.3 mV was found to be revealing greater stability (Figure 2B). The high negative charge indicates the electrostatic repulsion between particles with the same electrical charge and will prevent the aggregation of the particle. Thus, the values obtained for the NLCs are found to be adequate to form a stable particle.
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Surface morphology The TEM and SEM image of lyophilized AMNLCopt showing almost spherical shape with uniform distribution without aggregation (Figure 3A-B). The size also found in agreement with
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the result obtained by photon correlation spectroscopy.
Thermal Behaviour
The DSC curve for AM showed sharp endothermic melting peak at 204.47°C indicating their crystalline nature. The lyophilized AMNLCopt curve did not showed any peak hence indicated
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that none of the formulation excipients had interference with the drug (Figure not shown). The
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drug is completely solubilized in the lipid used for the formulation.
In-vitro drug release
The comparative drug release profile of AMNLCopt (88.23%) and AMNLCGopt, (61.55%) were illustrated in Figure 4. The prepared AMNLCGopt showed biphasic release pattern with initial burst release followed by a steady release for 24 hrs. The reason for initial burst release due to diffusion of untrapped AM in initial hours and the reason of slow release attributed to the
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carbopol leading to the formation of a dense gel matrix structure. The drug has to undergo an additional barrier as a result of entrapment in gel matrix and giving sustained release over a long period of time. This type of release behavior is useful to achieve high concentration gradient required for successful transdermal drug delivery (Csoka et al., 2007). The drug release data
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were fitted to several kinetic models and the best fit model was found to be higuchi (0.981). The peppas model showed n value of 0.344 was obtained indicating that drug release was regulated
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through non fickian diffusion.
Characterization of AMNLCGopt The developed AMNLCGopt were evaluated for various parameters to check the quality of gel (Table 4). The viscosity, pH, spreadability and swelling index was found to be 240.5PaS (Figure 5A), 6.52, satisfactory spreadability (215.33) and 3.32, respectively. The various parameter of texture analysis like of were like firmness, toughness, consistency, cohesiveness and index of viscocity were evaluated. The result showed that the developed formulation depicted firmness
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(0.160±0.01), toughness (0.453±0.10), consistency (1.524±0.14), cohesiveness (-0.09±0.63), and index of viscocity (-1.071±0.12) (Figure 5B).
Confocal laser scanning microscopy
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The CLSM results showed that RR loaded AMNLCGopt was fairly and uniformly distributed deeply and uniformly throughout the skin layers to a greater extent throughout the subcutaneous, viable epidermis, and dermis with high fluorescence intensity (Figure 6A-B). The depth of penetration was found to be 45.23 µm in the skin shown by AMNLCGopt (Figure 6B), whereas
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the control formulation showed penetration of 12.76 µm depth only (Figure 6A). The prominently efficient delivery of AMNLCGopt suggested their enhanced penetration and
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consequent fusion with the membrane lipids in the depths of the skin.The deeper penetration of AMNLCGopt due to consequent fusion with membrane lipids of skin supported the hypothesis of many researchers (Godin and Touitou 2004; Jain et al. 2008).
In-vivo absorption study
The comparative plasma concentration profile of amlodipine following the application of
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optimized AMNLCGopt, and AMTab (Amdepine marketed tablet) in Wistar rats showed in Figure 7. The developed formulation AMNLCGopt showed Cmax and Tmax of 912.56 ng/ml and 4 hr, whereas AMTab showed Cmax of 1212.65 ng/ml and Tmax of 2 hr, respectively. The difference in the Cmax and Tmax of these two formulations was due to the barrier property of stratum
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corneum in case of transdermal gel formulation. The AUC0-24 and AUMC0-24 for AMNLCGopt formulation was 12686.3ng.h/ml, and 115388.7ng.h/ml, respectively. While after oral AMTab
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administration AUC0-24, and AUMC0-24 was found to be 10301.98 ng.h/ml, and 78103.6 ng.h/mL. The pharmacokinetic study revealed greater extent of amlodepine absorption from NLC based gel formulation (NLCGopt) than the marketed tablet (Amdepine marketed tablet). The significantly high AUC value observed with AMNLCGopt indicated increased bioavailability of the AM as compared to oral administration. The NLC based gel showed higher absorption may be due to the increased solubility of amlodipine, nano particle size and presence of efficient permeation enhancers (surfactant and lipid) in the formulation. Furthermore, increased in bioavailability of AMNLCGopt formulation (1.23 times) was acheived in comparison to oral formulation might be due to the avoidance of the hepatic first pass metabolism.
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Skin irritation The skin irritation study results revealed that the treated skin with AMNLCGopt showed no erythema and edema with the score (0.36± 0.03) and (0.82±0.11). The formalin treated skin showed higher irritation score for erythema (1.18±0.25) and edema (2.92±0.58). The erythema
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and edema score were added to calculate primary irritation index (PII) and the score was found to be for AMNLCGopt treated [PII 1.18] and formalin treated [PII=4.98]. The results confirm their non-irritant nature as the score was found less than two. The compounds producing scores of 2 or less are considered negative (Draize et al. (1944). No obvious erythema, edema or
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inflammation was observed on the skin after application of the formulation whereas formalin solution showed significant skin irritation. Hence, the developed transdermal formulations are
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free of skin irritation. Further, to study the internal damage to the skin the histopathology examination was performed. The result of the study revealed that AMNLCGopt treated site did not produce any internal damage in the skin (Figure 8A-B). There were no significant changes observed in rat skin specimens treated with formulation in comparison with the untreated SC suggest absence of any skin irritation. There was not any sign of infiltration of inflammatory cells or cyst and bubble formation as similar to control skin. The epidermal and dermal layers are
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well defined and the keratin layer was also well formed and was presenting just adjacent to the top most layer of the epidermis. The formalin treated skin showed marked damage to the internal structure of the skin. There was a clear sign of visible inflammatory cell and redness to the skin. The marked damage to skin and extraction of lipid has been seen in the formalin treated skin
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Conclusion
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confirms the score shown by visual scoring.
Amlodipine encapsulated NLC based transdermal formulation was successfully prepared by melt emulsification and ultrasonication method”. The optimized formulation AMNLCopt showed experimental and predicted value for particle size (128.3 nm and 129.4nm), % encapsulation efficiency (88.11 and 87.34) and flux (57.33 and 58.24µg/cm2/h). The in-vitro drug release profiles showed prolong release for AMNLCGopt. CLSM study established that the rhodamine B loaded formulation was efficiently deeply permeated into the skin. In vivo pharmacokinetic study revealed that the relative bioavailability of AMNLCGopt was significantly increased in comparison to the marketed formulation and pure drug suspension in Wistar rats.
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Animal rights The “European Community guidelines as accepted principles for the use of experimental
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animals”, were adhered to.
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Conflict of interest: None
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Figure 1: Three dimensional image showing effect of independent variables (A) peceol, (B) GMS (C) tween 80 on the dependent variables (Y1) particle size (Y2) flux, (Y3) EE Figure 2: AMNLCopt (A). Particle size image (B). Surface charge image Figure 3: Surface morphology of AMNLCGopt (A). Transmission electron microscopy (B).
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Scanning electron microscopy
Figure 4: Comparative in-vitro drug release profile of AMNLCopt and AMNLCGopt
Figure 5: Physicochemical evaluation of AMNLCGopt (A). Viscocity (B). Texture analysis
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Figure 6: Comparative CLSM image of transdermal treated skin (A). AMG (control) and (B). AMNLCGopt. Figure 7: Comparative in-vivo absorption image of (A). AMNLCGopt (B). AMTab
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Figure 8: Skin histopathology image (A). untreated vs (B). AMNLCGopt treated
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Table 1:- Optimization of AMNLCs formulation by Box Behnken statistical design
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Dependent variables Y1 Y2 Y3 (Size) (Flux) Entrapment (nm) (µg/cm2/h) efficiency(%) 118 56.46 85.35 206 51.53 81.23 212 52.12 82.07 224 58.88 70.87 246 42.75 68.07 285 54.55 67.43 104 50.01 83.13 211 51.97 82.66 212 60.56 85.96 235 51.44 82.72 279 46.27 85.68 222 36.43 85.15 211 51.05 86.62 277 58.16 87.55 185 48.85 75.94 221 50.22 81.64 189 56.48 84.38
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Independent variables X1 X2 X3 (Peceol) (GMS) (Tween(mg) 80) (mL) (mL) 105 210 0.2 105 155 0.35 105 155 0.35 40 155 0.2 40 100 0.35 105 100 0.2 105 210 0.5 105 155 0.35 170 155 0.5 105 155 0.35 105 100 0.5 40 155 0.5 170 155 0.2 170 100 0.35 40 210 0.35 105 155 0.35 170 210 0.35
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Table 2: ANOVA model of developed AMNLCopt formulation. R2
Adjusted R2
Predicted R2
Mean
SD
C.V%
0.9910
0.9795
0.9089
297.47
13.59
4.57
Flux
0.9844
0.9751
0.9364
51.62
0.95
1.85
EE%
0.9650
0.9439
0.9012
91.66
1.52
1.66
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Quadratic model Size
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Table 3: Point prediction assessed AMNLCopt with their observed and predicted value. Optimized concentration 74.26 210.0
Peceol (oil) GMS (solid lipid)
0.2
Size (nm) Flux (µg/cm²/h) Entrapment efficiency (%)
Predicted value 129.34 58.24
Experimental Value 128.3 57.33
87.34
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Tween-80 (surfactant)
Response
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Optimized formula
88.11
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Table 4: Evaluation parameter of AMNLCGopt for different physicochemical and mechanical properties. Mechanical properties Toughness Consistency Cohesiveness (g.sec.) (g)
Spreadability (gcm/s)
pH
Swelling Index
240.5
215.33
6.52
3.326 ± 0.050
0.160±0.01
0.453±0.10
1.524±0.14
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Viscosity (PaS)
Index of viscosity (g.sec.) -1.071±0.12
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Firmness (g)
Physicochemical property
-0.09±0.63
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Table 5: Pharmacokinetic absorption parameter of transdermal AMNLCGopt formulation and oral marketed formulation (Amdepine tablet).
AMNLCGopt
AMTab
Cmax (ng/mL)
912.56± 91.3
1212.65± 108.5
Tmax (h)
4 ± 0.7
2 ± 0.15
AUC0-t (ng.h/mL)
12686.3 ± 456.6
10301.98 ± 511.4
AUMC0-t (ng.h/mL)
115388.7 ± 1187.7
78103.6 ± 987.5
Elimination rate (h)
0.043315
Relative bioavailability (%)
1.23
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Absorption parameters
0.08823 -
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Highlights •
Amlodipine loaded nanostructured lipid carriers (NLCs) were formulate using lipid blends. The formulation was optimized using formulation design approach using the independent
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variables were Peceol (liquid lipid as X1), GMS (solid lipid as X2) and Tween-80 (surfactant as X3). •
The in vitro drug release, confocal laser scanning microscopy, physicochemical
•
AM-NLCopt
showed
particle
size
(123.8
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evaluation, and in-vivo transdermal absorption study were also performed. nm),
enhanced
transdermal
flux
(58.33µg/cm2/h), and higher entrapment efficiency (88.11%).
CLSM study revealed rhodamine loaded AMNLCopt showed an enhanced permeation to the deeper layers of the skin.
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The in-vivo transdermal absorption study presented enhanced improvement in bioavailability of amlodipine in the wistar rats.
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The formulation was also assessed for skin interaction study and found to be safe.
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S1773-2247(18)31031-1
DOI:
https://doi.org/10.1016/j.jddst.2018.11.004
Reference:
JDDST 826
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 8 September 2018 Revised Date:
31 October 2018
Accepted Date: 7 November 2018
Please cite this article as: H. Kapoor, M. Aqil, S.S. Imam, Y. Sultana, A. Ali, Formulation of amlodipine nano lipid carrier: Formulation design, physicochemical and transdermal absorption investigation, Journal of Drug Delivery Science and Technology (2018), doi: https://doi.org/10.1016/ j.jddst.2018.11.004. 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.
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Manuscript number:- JDDST_2018_980R2.
Formulation of amlodipine nano lipid carrier: formulation design, physicochemical and
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transdermal absorption investigation
Hema Kapoora, Mohd. Aqila*, Syed Sarim Imamb, Yasmin Sultanaa, Asgar Alia
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a. Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), M. B. Road New Delhi 110062, India.
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247121. Uttar Pradesh, India.
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b. Department of Pharmaceutics, Glocal School of Pharmacy, Glocal University, Saharanpur
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*Corresponding author: Dr. Mohd. Aqil Associate Professor Department of Pharmaceutics School of Pharmacuetical Education and Research Jamia Hamdard (Deemed University) M. B. Road, New Delhi 110062, India. Email: *[email protected]; †[email protected] Tel: +91-9811798725
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Abstract The present work was designed to formulate and statistically optimize transdermal amlodipine nanostructured lipid carriers (AMNLCs) using lipid blends. The formulation was optimized by
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using the independent variables Peceol (liquid lipid as X1), GMS (solid lipid as X2) and Tween80 concentration (surfactant as X3). Their effects were assessed on dependent variables particle size (Y1), transdermal flux (Y2), and entrapment efficiency (Y3). The optimized formulations was further evaluated for in vitro drug release, confocal laser scanning microscopy, physicochemical evaluation, and in-vivo absorption study. The optimized amlodipine nanostructured lipid carriers
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(AMNLCopt) showed low particle size (123.8 nm), enhanced transdermal flux (58.33µg/cm2/h), and higher entrapment efficiency (88.11%). Further, it showed prolonged drug release and
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followed higuchi release kinetics with R2 closer to unity. The rhodamine red (RR) loaded RRAMNLCopt revealed an enhanced permeation to the deeper layer of the skin after assessing through confocal laser scanning microscopy (CLSM). The in-vivo absorption study presented enhanced improvement in bioavailability of amlodipinein the wistar rats. From the study, it was concluded that the experimental design based AMNLCs showed the quadratic relationship between independent and dependent variables and also found to be a proficient carrier for
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transdermal delivery of amlodipine.
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Key words: Amlodipine, NLCs; Transdermal; In-vivo absorption; Histopathology
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Introduction The barrier nature of the stratum corneum (SC) is the major challenges that limit the entry of most of the drugs by the transdermal route (Lim et al., 2006). Many transdermal techniques have been tried to overcome the barrier of SC to achieve higher transdermal
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permeability. These techniques are designed to deliver the drug for a prolonged period of time (Imam et al., 2016), which is particularly encouraging to treat chronic disorders like hypertension (Qadry et al., 2016; Qumbar et al., 2016; Aqil et al., 2016). There are many antihypertensive drugs have shown low bioavailability due to poor solubility and it undergoes
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extensive first-pass metabolism which can be avoided by transdermal delivery. Many attempts have been taken to develop the transdermal delivery of antihypertensive drugs to circumvent the
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drawbacks of conventional drug delivery system (Kamran et al., 2016; Gungor and Ozsoy 2012; Selvam et al. 2010).
The application of lipid-based delivery systems offers suitable carrier for transdermal delivery due to their greater biocompatibility with the skin lipids (Chourasia et al., 2011). Among different lipid system, NLCs is the widely used carrier made up of the combination of solid lipid, liquid lipid and dispersed in an aqueous emulsifier solution. The addition of liquid
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lipid in NLCs gives the ordered crystalline state of solid lipid and enlarges drug storage space (Moghaddam et al., 2017; Pradhan et al. 2016). They are well suited for the systemic delivery across skin because they are composed of physiological and biodegradable lipids of low systemic toxicity and also low cytotoxicity (Jaiswal et al. 2016; Raj et al. 2015). The nano-size range
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gives a larger surface for the drug absorption across the skin and gives greater therapeutic efficacy (Muller & Keck, 2004). It acts by adhering to the skin, forms a thin film and gives an
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occlusive effect with a decrease in the transepidermal water loss (Jenning et al., 2000; Muller et al., 2007). The hydration of the SC could give broadening of the inter-corneocyte gaps and enhances drug permeation into deeper layer (Muller et al., 2007; Alam et al., 2016; Puglia & Bonina, 2012).
Amlodipine (AM), a third-generation dihydropyridine calcium channel antagonist, is
mostly choice of drug for the management of angina and hypertension (Sun et al., 2009). The poor aqueous solubility and low permeability through GIT gives low bioavailability (Chahbra et al., 2012), and hence limits the access of drug to their therapeutic targets like heart and cardiac smooth muscles. It is a weak base and mainly exists in its ionized form, has low permeability
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characteristics within the gastrointestinal pH range (Jang et al., 2012; Rausl et al., 2006). The oral bioavailability of amlodipine free base is low compared to amlodipine salt (Meredith., 2009; Saigal et al., 2008). The optimization AMNLCs was performed using Box–Behnken design using the independent variables (X1- Peceol, X2- glycerylmonostearate, X3- tween 80) at
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three level (low, medium and high) and their effect observed on dependent responses (Y1particle size; Y2- flux; Y3- encapsulation efficiency). The BBD design was used for the optimization because it requires fewer runs than a central composite design (Imam et al., 2017; Thapa et al., 2018; Jahangir et al., 2017). The present study was focused on the formulation of
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amlodipine loaded nano lipid carrier using the design expert software. Finally, the optimized nano lipid carrier formulation was analyzed for the morphology, in-vitro release, in-vivo
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absorption and skin interaction properties.
Material and Methods Materials
Amlodipine (AM) was received as a gift sample from Torrent Pharmaceuticals (India). HPLC grade methanol and water were purchased from Merck, Mumbai, India. Peceol (oil), Labrafil M
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2125, Glyceryl monostearate (GMS), Labrafil M 1944, received from “Gattefosse India Pvt. Ltd., (Mumbai, India)”and (Colorcon Asia Pvt Ltd, Mumbai, India). Tween-80, Carbopol 940, were purchased from S.D Fine chemicals, Mumbai, India) and all other chemicals and solvents used were of analytical grade.
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Methods Screening of liquid lipids
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The saturation solubility of amlodipine in different liquid lipids was determined by adding an excess amount of drug in oil (1mL) in an eppendorf tube. The eppendorf tube was subjected for the agitated until the drug gets completely dissolved, and the process was further continued until the saturation stage reaches. After that, the drug in the lipid sample was kept to withstand for 12 hours for stabilization. Finally, it was centrifuged at 3000rpm for 15 min and the supernatant was separated, dissolved in ethanol and the solubility was quantified by UV spectrophotometer at 356 nm.
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Screening of solid lipids To select the appropriate solid lipids, different solid lipids (stearic acid, Gleucire 44/14, Glyceryl monostearate (GMS)) were weighed (1 g), melted at 75 °C and agitated using a magnetic stirrer. The drug sample was added slowly to the liquefied lipid till saturation was attained and which
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was set aside for 12 h for stabilization. After that, a small portion of the supernatant from each vial was taken and ethanol was added to each tube. The sample was then analyzed by UV spectroscopy and concentration of each solution was determined.
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Experimental design
The Box-Behnken design was used to statistically optimize the formulation parameters keeping
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three factors at three levels for optimization study. The design used the response surface plot and second order polynomial models using Design Expert software (Version 9.0., Stat Ease Inc, Minneapolis, MN) to get the optimized formulation. The experimental design showed 17 experimental runs with 5 mid points to evaluate the effect of independent variables on dependent variables. The independent variables were solid lipid concentration (peceol as X1), liquid lipid concentration (GMS as X2) and surfactant concentration (tween 80 as X3) represented by the
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low, mid and upper limit (-1, 0, +1), respectively, while the dependent variables were particle size (Y1), transdermal flux (Y2) and % entrapment efficiency (Y3) as given in Table 1.
Formulation of amlodipine nano lipid carrier (AMNLCs)
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Amlodipine loaded NLCs were prepared by using melt emulsification ultrasound dispersion method with slight modification (Moghaddam et al., 2016; Rizwanullah et al., 2017). The lipid
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blend phase Peceol: GMS (3:7) and aqueous surfactant phase (Tween 80) were prepared separately in a beaker. The selected liquid lipid (Peceol) and solid lipid (GMS) was melted and calculated amount of AM was added to it. Both the phases were heated separately at 80°C on a temperature regulated water bath and the aqueous surfactant phase (tween 80) was added drop wise to the lipid blend phase to make a primary emulsion. Finally, the primary emulsion was probe sonicated at 50W to get small size (Elmowafy et al., 2015). The sample was sonicated in ice condition which helps to maintain the temperature of formulation at 4°C and a pause or a gap of 5 min. were taken in each cycles (Hielscher ultrasonicator, Germany). The prepared AMNLCs formulation was cooled to room temperature and stored for further use.
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Characterization Size and surface charge The particle size and surface charge of NLCs were determined through zetasizer (Zetasizer Nano ZS, Malvern, UK). The small amount (1mL) of the formulation was diluted in the distilled water
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and mixed thoroughly with vigorous shaking. The particle size, surface charge, and polydispersity index (PDI) were evaluated by adding the diluted dispersion into the disposable cuvette and analyzed using the zeta sizer. The polydispersity index (PDI) was determined as a measure of homogeneity of the particle. The ideal value of PDI must be between <0.1 - 0.3>
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indicate a homogeneous population and greater than that show high heterogeneity.
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Entrapment efficiency
The entrapment efficiency of the AMNLCs formulation was determined by ultracentrifugation method by calculating the amount of the untrapped drug. The sample (1mL) was diluted and kept in the upper compartment of the ultracentrifuge tube and run at 25,000 rpm for 15 min. The free drug moves to the lower chamber through the semipermeable membrane and the entrapped drug retained in the upper chamber. The sample collected from the lower chamber, diluted and
Surface morphology
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entrapment efficiency was determined using UV-spectrophotometer at 364nm.
The particle morphology was studied using TEM (MORGAGNI 268D Fei 155 Company,
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Netherlands) using the combination of bright field imaging at high magnification. The diluted sample was taken to the copper grid, left for 1 min to dry and a drop of phosphotungstic acid
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(PTA) was added (Hasnain et al., 2018). The excess of PTA was removed and analyzed at 6080KV at 1550 magnification. Further, SEM analysis was also performed to evaluate the surface morphology of the prepared NLC. The image was recorded on a scanning electron microscopy (ZEISS GEMINI 51530 FEG) and photomicrographs were obtained.
Differential scanning calorimetry (DSC) DSC was conducted to ascertain the compatibility of AM with the excipients used for the formulation using calorimeter (Pyris 6 DSC, Perkin Elmer). The study was done for the pure drug (AM) and lyophilized AMNLCopt formulation. The samples were kept in the hermetic pan
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and conducted at a heating rate of 10°C/min over the temperature range of 30–300°C using an inert nitrogen gas with keeping an empty aluminum pan as a reference.
Formulation of amlodipine nano lipid carrier based gel (AMNLCG)
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The epidermal retention of a transdermal formulation for a prolonged period of time would eventually help in delivering the drug optimally. The formulation AMNLCopt was insufficiently viscous and could be therefore quickly removed from the skin, therefore it was converted into gel formulation. The gel formulation (AMNLCGopt and control) was prepared by taking Carbopol
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940 (1%w/v) as gelling agent. Carbopol was mixed with distilled water to get the polymer dispersion and then kept aside in dark to allow for complete swelling. Finally, polyethylene
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glycol 400 (15%w/w) and chlorocresol (0.1%) were added slowly with stirring to get homogenous dispersion. The gel formulation was then neutralized using triethanolamine (TEA) and stirred slowly to get an optimized carbopol based gel (control gel). Same procedure was used to get gel formulation by adding the optimized AMNLCopt formulation into preformed gel with stirring (Thasleem et al., 2018), to get final AMNLCGopt for further characterization.
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Evaluation of AMNLCG
The prepared AMNLCGopt formulation was evaluated for different parameters like color, homogeneity, consistency and phase separation. The homogeneity was checked by visual evaluation after the gels have been kept in the container. The presence of any aggregates was
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observed for their appearance. The pH of the gel was determined by using digital pH meter
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(Mettler Tolledo, Japan) fitted with a glass microelectrode by allowing it to equilibrate for 1 min.
Viscocity and spreadability The viscosity of prepared AMNLCGopt was calculated using Rheometer (MCR101 Rheoplus of Anton Paar), connected to a thermostatically controlled circulating water bath. AMNLCGopt was evaluated for the spreadability by taking a specified amount of gel sample and kept within a circle of premarked 2 cm diameter on a glass plate. The second plate was kept over it and a weight was allowed to rest on the upper glass plate for 5 min (Bachhav et al., 2009). The increase in the diameter due to an application of weight was noted (n = 3). To determine the
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percent spread by area, calculate as follows: % Spread by area =A2 A1× 100, where, A1= 2 cm2, and A2= final area after spreading.
Texture analysis
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In order to characterize and evaluate the various textural parameters of the prepared gel their firmness, adhesiveness, force of adhesion, and gel strength were measured. The optimized formulation AMNLCGopt was placed into identical glass jars (height 40 mm, diameter: 55mm) to a fixed sample height (30mm). The entrapment of air bubble have been avoided into the
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sample and testing surface was kept as flat as possible to avoid early triggering of the test (Hagerstrom et al., 2003). The texture profile analysis was performed using a TA.XT2 plus
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texture analyzer (Stable Micro System, UK) in the compression mode. The parameters are the resultant force-time plots and several mechanical parameters of the gels were determined.
In-vitro drug release
The in-vitro release study was accomplished by using diffusion cell having receptor chamber of 20 mL capacity with the diffusion area of 1.5 cm2. The pretreated membrane with the pore size
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of 0.2µm was used for the study. Both samples (AMNLCopt and AMNLCGopt equivalent to 5mg AM) was filled into the donor compartment and the receptor compartment was filled with PBS: ethanol mixture (9:1) and the release study was performed at 37°C with continuous stirring. After predetermined time interval the sample (0.5mL) was withdrawn from the sampling port
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and replenished with the fresh media. The samples were filtered and diluted to analyzed by UV spectrophotometer. The collected release data from this study were fitted to kinetic models zero-
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order, first-order, Higuchi’s matrix (Higuchi 1963) and the Korsmeyer peppas model (Korsmeyer et al. 1983, Peppas 1985). The relevant correlation coefficients were taken into account for the selection of best model. The mechanism of drug release was determined by taking the data and fitted into the Korsmeyer peppas model and their release exponent (n) were calculated from the slope of the straight line (Uprit et al. 2013).
Skin permeation studies The excised abdominal rat skin was fixed between donor and receptor compartment of diffusion cell (area 1.5cm2) in such way that stratum corneum faced towards the donor compartment and
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dermis side faced towards the receptor compartment. Both AMNLCopt and AMNLCGopt formulations (equivalent to 5 mg of Amlodipine) were placed in a donor compartment and receptor compartment was filled with the PBS:ethanol (9:1; 10mL). The receptor compartment was stirred regularly on a magnetic stirrer at 100 rpm and the temperature was maintained at
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32oC±0.5oC during the whole study. The diffused samples (1mL) were withdrawn at predetermined intervals from receptor compartment replaced with the same medium. The collected samples were diluted further and drug content were analysed by HPLC. The cumulative
at steady state was calculated.
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Confocal laser scanning microscopy (CLSM) study
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amount of drug permeated was plotted as a function of time and the skin permeation rate (Flux)
The extent of the penetration of prepared NLC formulation through rat skin “confocal laser scanning microscopy (CLSM)” was performed. The fluorescent probe rhodamine B dye loaded AMNLCGopt and control gel used for this study. The study was performed for 8 hours similar to ex-vivo permeation study. After that, the treated skin was collected, washed with distilled water and the excess dye was removed. The skin was cut into the small size and fixed on the slide as
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stratum corneum facing upward. The depth of penetration was observed under a confocal microscope with an argon laser beam with excitation at 524nm and emission at 625nm. The skin sample was sliced in sections and visualized through the z-axis by CLSM (Dayan and Touitou,
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2000).
In-vivo absorption study
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The in-vivo absorption study was done with Wistar rats of either sex (200-250g) as the animal models. The animals were supplied by Central Animal House facility after the approval from Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi (application No-1130). All the rats were kept under standard laboratory conditions in 12-hour light/dark cycle at 25°C ± 2°C provided with pellet diet (Lipton, Kolkata, India) and water ad libitum. The study was performed with rats and dose administered equivalent dose of 2.5 mg/kg body weight of AM (Jang et al., 2013). The animals were fasted for 24 hours and divided into 2 groups having six animals in each group. Group A treated with AMNLCGopt (1.25 gm) and Group B was orally administered with a marketed tablet (Amdepine), respectively. AMNLCGopt was applied to the shaved
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abdominal rat skin and kept under observation. The blood samples were withdrawn from the tail vein at different time intervals (0, 1, 2, 4, 8, 12, 24h) and collected in eppendorf tubes containing disodium EDTA as an anticoagulant. The plasma samples were separated by centrifugation at 5000 rpm for 15min. and the separated samples were stored under -70°C for drug analysis by
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HPLC method. The different biopharmaceutical assessment such as Cmax (the maximum plasma concentration) and Tmax (time to reach maximum plasma concentration) was directly obtained from the plasma concentration versus time plot. The area under the curve AUC0-48 and AUC0-inf were calculated by trapezoid rule. HPLC system was (Shimadzu, Model LC-10 ATVP) Japan)
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equipped with a binary pump and UV detection system (SPD-10A). Chromatographic separation was accomplished by using C-18 (250mm×4.6mm i.d, 5µm particle size, Shiseido, Japan). The
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mobile phase used was acetonitrile: water (70:30) with the water phase containing 0.2% triethylamine (pH 4). The study was performed at the wavelength (364nm), flow rate (0.8 ml/min), with injection volume (20 µl) and total run time was 10 min.
Skin irritation and histopathology
Skin irritation was assessed for any irritation from developed formulation on Wistar rats using
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Draize score test (Draize et. al, 1944). The rats were divided into 2 groups, Group I was treated with AMNLCGopt, Group II received formalin (0.8%v/v) as a standard irritant (Imam et al., 2017). Both samples were applied to the rat skin and upon the removal, animals were examined for irritation by giving visual scoring for erythema and edema. An adjacent area of untreated skin
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was served as a control. After that, the animals were sacrificed and the skin samples from treated and untreated (control) area were excised and collected in buffered formalin saline (pH=7.4) and
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processed for histopathological studies. The specimens were cut into a small section and each section was dehydrated using ethanol, embedded in paraffin. The samples were fixed on the slide and stained with hematoxylin/eosin dye for clear visibility. These samples were observed in the light microscope (Motic digital microscope, DMB series) and compared with control skin sample (Sintov et al., 1999).
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Results and Discussion Solubility study From the solubility and miscibility studies, the different solid and liquid lipids were taken to evaluate the maximum solubility of AM. The solubility in liquid lipid was found in this order
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Peceol > iso propyl myristate > Labrafil M1944 > Labrafil M 2125 and in solid lipid GMS > stearic acid > Gleucire 44/14. The final selected component for the formulation was Peceol (liquid lipid), GMS (solid lipid) and Tween-80 (surfactant), respectively for the formulation of AMNLCs because they showed maximum solubility of the drug. The blend of liquid and solid
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lipid showed stable formulation at 3:7 ratio without any phase separation so finally used further
Experimental design optimization
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for the formulation.
The statistical design showed a total of 17 runs and characterized for different responses like size, flux and encapsulation efficiency as depicted in Table 1. The mathematical relationships between both independent and dependent variables were established, coefficients of the second order polynomial equations generated for size, flux, and encapsulation were found to be
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quadratic. The coefficients correlation of the polynomials fit well to the data, with the values of R2 ranging between 0.9650 and 0.9910. The polynomial equation represents negative sign for the variables gives the inverse relationship between the factor and the response whereas the positive value favors the optimization. The all three independent variables [liquid lipid (peceol), solid
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lipid (GMS) and surfactant (tween 80)] showed significant effect individually and combinely on
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all the three responses namely particle size, flux, and encapsulation.
Effect of independent variables (A) peceol (B) GMS (C) tween 80 on size The minimum and maximum particle size was found between 104 nm and 285 nm as shown in Table 1. The all independent variables shown their effects on particle size depicted in threedimensional Figure 1A-B. It is very evident from the model graph that the variation in concentration of total lipid affects the particle size. As the ratio of solid lipid and liquid lipid increases, the particle size increased. The increased size due to higher cross linking between lipid and drug takes place. As the lipid concentration increase there is more space available between solid lipid and liquid lipid to accommodate the drug. The increase in tween 80
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concentration decreases the particle size due to the reduction in interfacial tension between the two phases, leading to the formation of smaller size emulsion droplets (Liu et al. 2007). At high surfactant concentration, the particles were stabilized by forming a steric barrier on the surface and prevent the small particles to form coalescence (Rahman et al. 2010). In this case, A, B,
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AC, AB, BC, A2, B2, C2 were significant model terms. The “Predicted R-Squared” of 0.9089 was in reasonable agreement with the “Adj R-Squared’’ of 0.9795; i.e the difference was less than 0.2. “Adeq Precision” measures the signal to noise ratio.
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Effect of independent variables (A) peceol, (B) GMS (C) tween 80 on transflux
The minimum and maximum transdermal flux was found between 36.43 to 60.56 µg/cm2/h as
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shown in Table 1. The effects of independent variables on flux are presented by threedimensional graph (Figure 1C-D). It was observed that the transdermal flux has a direct relationship with the surfactant concentration and total lipids content. The proposed mechanism for improved permeation across skin from AMNLCopt may involve disruption of densely packed lipids structure that fills the extra-cellular spaces of the stratum corneum. The intercellular lipid barrier in the stratum corneum would be more permeable following treatment with NLCs. The
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drug permeation also enhances due to high thermodynamic activity gradient in the upper part of the stratum corneum by adsorption and fusion of drug loaded NLC onto the surface of the skin. The “Predicted R2” of 0.9364 was in reasonable agreement with the “Adj R2’’ of 0.9751. The adequate precision measures the signal to noise ratio and the model can be used to navigate the
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design space. In this case, all the individual and combined variables like A, B, C, AB, AC, BC,
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A2, B2, C2 showed significant model terms.
Effect of independent variables (A). peceol (B). GMS (C). tween 80 on encapsulation efficiency.
The minimum and maximum encapsulation efficiency was found between 67.4 to 92.35% as shown in Table 1. Figure 1(E-F) showed three-dimensional plot which shows the effect of different independent variables on EE (Y3). It has been observed that the entrapment efficiency of NLCs increased with an increase in the content of GMS. The increase in EE may be due to the blend of liquid lipids and solid lipids that lead to disturb the crystal order, which ultimately improved drug entrapment efficiency. The increase in the surfactant concentration gives the
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initial increase in EE, at high surfactant concentration in the external phase might increase the partition of the drug from internal to external phase. Due to increased partition, the drug solubility increased in the external aqueous phase so high concentration of drug can solubilize in it (Rahman et al. 2010). In this case A, B, C, AB, AC, BC A2, C2 were significant model terms.
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The “Predicted R2” of 0.9012 was in reasonable agreement with the “Adj R2’’ of 0.9439.
Point prediction
Finally, the optimized formulation (AMNLCopt) was formulated using the composition of liquid
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lipid (Peceol- 74.26 mL), solid lipid concentration (GMS- 210 mg), and surfactant concentrations (tween 80- 0.2mL). The experimental and predicted value of AMNLCopt for
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particle size (128.3 nm and 129.4nm), % encapsulation efficiency (88.11 and 87.34) and flux (57.33 and 58.24) and was found to be very close to each other. This result showed 98.23%, 99.51% and 100.08% validity of the predicted values of responses, confirming the selection of optimized formulation. The ANOVA model was assessed and revealed that the model terms for individual and interaction effects were statistically significant. Finally, on the basis of point prediction the optimized AMNLCopt was converted into gel using carbopol as gelling agent and
Characterization
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further evaluated for different parameters.
Particle size and surface charge
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The penetration of drug across skin requires smaller particle size which gives a larger surface area for absorption thereby drug absorption increases. The particle size of all the developed
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formulations was found in the range of 104 nm to 285 nm. The optimized AMNLCopt showed particle size of 118.3 nm (Figure 2A) with PDI of 0.202 indicated narrow size distribution. The zeta potential value of -29.3 mV was found to be revealing greater stability (Figure 2B). The high negative charge indicates the electrostatic repulsion between particles with the same electrical charge and will prevent the aggregation of the particle. Thus, the values obtained for the NLCs are found to be adequate to form a stable particle.
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Surface morphology The TEM and SEM image of lyophilized AMNLCopt showing almost spherical shape with uniform distribution without aggregation (Figure 3A-B). The size also found in agreement with
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the result obtained by photon correlation spectroscopy.
Thermal Behaviour
The DSC curve for AM showed sharp endothermic melting peak at 204.47°C indicating their crystalline nature. The lyophilized AMNLCopt curve did not showed any peak hence indicated
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that none of the formulation excipients had interference with the drug (Figure not shown). The
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drug is completely solubilized in the lipid used for the formulation.
In-vitro drug release
The comparative drug release profile of AMNLCopt (88.23%) and AMNLCGopt, (61.55%) were illustrated in Figure 4. The prepared AMNLCGopt showed biphasic release pattern with initial burst release followed by a steady release for 24 hrs. The reason for initial burst release due to diffusion of untrapped AM in initial hours and the reason of slow release attributed to the
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carbopol leading to the formation of a dense gel matrix structure. The drug has to undergo an additional barrier as a result of entrapment in gel matrix and giving sustained release over a long period of time. This type of release behavior is useful to achieve high concentration gradient required for successful transdermal drug delivery (Csoka et al., 2007). The drug release data
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were fitted to several kinetic models and the best fit model was found to be higuchi (0.981). The peppas model showed n value of 0.344 was obtained indicating that drug release was regulated
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through non fickian diffusion.
Characterization of AMNLCGopt The developed AMNLCGopt were evaluated for various parameters to check the quality of gel (Table 4). The viscosity, pH, spreadability and swelling index was found to be 240.5PaS (Figure 5A), 6.52, satisfactory spreadability (215.33) and 3.32, respectively. The various parameter of texture analysis like of were like firmness, toughness, consistency, cohesiveness and index of viscocity were evaluated. The result showed that the developed formulation depicted firmness
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(0.160±0.01), toughness (0.453±0.10), consistency (1.524±0.14), cohesiveness (-0.09±0.63), and index of viscocity (-1.071±0.12) (Figure 5B).
Confocal laser scanning microscopy
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The CLSM results showed that RR loaded AMNLCGopt was fairly and uniformly distributed deeply and uniformly throughout the skin layers to a greater extent throughout the subcutaneous, viable epidermis, and dermis with high fluorescence intensity (Figure 6A-B). The depth of penetration was found to be 45.23 µm in the skin shown by AMNLCGopt (Figure 6B), whereas
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the control formulation showed penetration of 12.76 µm depth only (Figure 6A). The prominently efficient delivery of AMNLCGopt suggested their enhanced penetration and
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consequent fusion with the membrane lipids in the depths of the skin.The deeper penetration of AMNLCGopt due to consequent fusion with membrane lipids of skin supported the hypothesis of many researchers (Godin and Touitou 2004; Jain et al. 2008).
In-vivo absorption study
The comparative plasma concentration profile of amlodipine following the application of
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optimized AMNLCGopt, and AMTab (Amdepine marketed tablet) in Wistar rats showed in Figure 7. The developed formulation AMNLCGopt showed Cmax and Tmax of 912.56 ng/ml and 4 hr, whereas AMTab showed Cmax of 1212.65 ng/ml and Tmax of 2 hr, respectively. The difference in the Cmax and Tmax of these two formulations was due to the barrier property of stratum
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corneum in case of transdermal gel formulation. The AUC0-24 and AUMC0-24 for AMNLCGopt formulation was 12686.3ng.h/ml, and 115388.7ng.h/ml, respectively. While after oral AMTab
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administration AUC0-24, and AUMC0-24 was found to be 10301.98 ng.h/ml, and 78103.6 ng.h/mL. The pharmacokinetic study revealed greater extent of amlodepine absorption from NLC based gel formulation (NLCGopt) than the marketed tablet (Amdepine marketed tablet). The significantly high AUC value observed with AMNLCGopt indicated increased bioavailability of the AM as compared to oral administration. The NLC based gel showed higher absorption may be due to the increased solubility of amlodipine, nano particle size and presence of efficient permeation enhancers (surfactant and lipid) in the formulation. Furthermore, increased in bioavailability of AMNLCGopt formulation (1.23 times) was acheived in comparison to oral formulation might be due to the avoidance of the hepatic first pass metabolism.
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Skin irritation The skin irritation study results revealed that the treated skin with AMNLCGopt showed no erythema and edema with the score (0.36± 0.03) and (0.82±0.11). The formalin treated skin showed higher irritation score for erythema (1.18±0.25) and edema (2.92±0.58). The erythema
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and edema score were added to calculate primary irritation index (PII) and the score was found to be for AMNLCGopt treated [PII 1.18] and formalin treated [PII=4.98]. The results confirm their non-irritant nature as the score was found less than two. The compounds producing scores of 2 or less are considered negative (Draize et al. (1944). No obvious erythema, edema or
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inflammation was observed on the skin after application of the formulation whereas formalin solution showed significant skin irritation. Hence, the developed transdermal formulations are
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free of skin irritation. Further, to study the internal damage to the skin the histopathology examination was performed. The result of the study revealed that AMNLCGopt treated site did not produce any internal damage in the skin (Figure 8A-B). There were no significant changes observed in rat skin specimens treated with formulation in comparison with the untreated SC suggest absence of any skin irritation. There was not any sign of infiltration of inflammatory cells or cyst and bubble formation as similar to control skin. The epidermal and dermal layers are
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well defined and the keratin layer was also well formed and was presenting just adjacent to the top most layer of the epidermis. The formalin treated skin showed marked damage to the internal structure of the skin. There was a clear sign of visible inflammatory cell and redness to the skin. The marked damage to skin and extraction of lipid has been seen in the formalin treated skin
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Conclusion
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confirms the score shown by visual scoring.
Amlodipine encapsulated NLC based transdermal formulation was successfully prepared by melt emulsification and ultrasonication method”. The optimized formulation AMNLCopt showed experimental and predicted value for particle size (128.3 nm and 129.4nm), % encapsulation efficiency (88.11 and 87.34) and flux (57.33 and 58.24µg/cm2/h). The in-vitro drug release profiles showed prolong release for AMNLCGopt. CLSM study established that the rhodamine B loaded formulation was efficiently deeply permeated into the skin. In vivo pharmacokinetic study revealed that the relative bioavailability of AMNLCGopt was significantly increased in comparison to the marketed formulation and pure drug suspension in Wistar rats.
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Animal rights The “European Community guidelines as accepted principles for the use of experimental
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animals”, were adhered to.
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Conflict of interest: None
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Development of transethosomes formulation for dermal fisetin delivery: Box–Behnken design, optimization, in vitro skin penetration, vesicles–skin interaction and dermatokinetic studies,
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DOI:
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Figure 1: Three dimensional image showing effect of independent variables (A) peceol, (B) GMS (C) tween 80 on the dependent variables (Y1) particle size (Y2) flux, (Y3) EE Figure 2: AMNLCopt (A). Particle size image (B). Surface charge image Figure 3: Surface morphology of AMNLCGopt (A). Transmission electron microscopy (B).
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Scanning electron microscopy
Figure 4: Comparative in-vitro drug release profile of AMNLCopt and AMNLCGopt
Figure 5: Physicochemical evaluation of AMNLCGopt (A). Viscocity (B). Texture analysis
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Figure 6: Comparative CLSM image of transdermal treated skin (A). AMG (control) and (B). AMNLCGopt. Figure 7: Comparative in-vivo absorption image of (A). AMNLCGopt (B). AMTab
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Figure 8: Skin histopathology image (A). untreated vs (B). AMNLCGopt treated
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Table 1:- Optimization of AMNLCs formulation by Box Behnken statistical design
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Dependent variables Y1 Y2 Y3 (Size) (Flux) Entrapment (nm) (µg/cm2/h) efficiency(%) 118 56.46 85.35 206 51.53 81.23 212 52.12 82.07 224 58.88 70.87 246 42.75 68.07 285 54.55 67.43 104 50.01 83.13 211 51.97 82.66 212 60.56 85.96 235 51.44 82.72 279 46.27 85.68 222 36.43 85.15 211 51.05 86.62 277 58.16 87.55 185 48.85 75.94 221 50.22 81.64 189 56.48 84.38
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Independent variables X1 X2 X3 (Peceol) (GMS) (Tween(mg) 80) (mL) (mL) 105 210 0.2 105 155 0.35 105 155 0.35 40 155 0.2 40 100 0.35 105 100 0.2 105 210 0.5 105 155 0.35 170 155 0.5 105 155 0.35 105 100 0.5 40 155 0.5 170 155 0.2 170 100 0.35 40 210 0.35 105 155 0.35 170 210 0.35
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Table 2: ANOVA model of developed AMNLCopt formulation. R2
Adjusted R2
Predicted R2
Mean
SD
C.V%
0.9910
0.9795
0.9089
297.47
13.59
4.57
Flux
0.9844
0.9751
0.9364
51.62
0.95
1.85
EE%
0.9650
0.9439
0.9012
91.66
1.52
1.66
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Quadratic model Size
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Table 3: Point prediction assessed AMNLCopt with their observed and predicted value. Optimized concentration 74.26 210.0
Peceol (oil) GMS (solid lipid)
0.2
Size (nm) Flux (µg/cm²/h) Entrapment efficiency (%)
Predicted value 129.34 58.24
Experimental Value 128.3 57.33
87.34
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Tween-80 (surfactant)
Response
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Optimized formula
88.11
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Table 4: Evaluation parameter of AMNLCGopt for different physicochemical and mechanical properties. Mechanical properties Toughness Consistency Cohesiveness (g.sec.) (g)
Spreadability (gcm/s)
pH
Swelling Index
240.5
215.33
6.52
3.326 ± 0.050
0.160±0.01
0.453±0.10
1.524±0.14
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Viscosity (PaS)
Index of viscosity (g.sec.) -1.071±0.12
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Firmness (g)
Physicochemical property
-0.09±0.63
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Table 5: Pharmacokinetic absorption parameter of transdermal AMNLCGopt formulation and oral marketed formulation (Amdepine tablet).
AMNLCGopt
AMTab
Cmax (ng/mL)
912.56± 91.3
1212.65± 108.5
Tmax (h)
4 ± 0.7
2 ± 0.15
AUC0-t (ng.h/mL)
12686.3 ± 456.6
10301.98 ± 511.4
AUMC0-t (ng.h/mL)
115388.7 ± 1187.7
78103.6 ± 987.5
Elimination rate (h)
0.043315
Relative bioavailability (%)
1.23
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Absorption parameters
0.08823 -
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Highlights •
Amlodipine loaded nanostructured lipid carriers (NLCs) were formulate using lipid blends. The formulation was optimized using formulation design approach using the independent
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variables were Peceol (liquid lipid as X1), GMS (solid lipid as X2) and Tween-80 (surfactant as X3). •
The in vitro drug release, confocal laser scanning microscopy, physicochemical
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AM-NLCopt
showed
particle
size
(123.8
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evaluation, and in-vivo transdermal absorption study were also performed. nm),
enhanced
transdermal
flux
(58.33µg/cm2/h), and higher entrapment efficiency (88.11%).
CLSM study revealed rhodamine loaded AMNLCopt showed an enhanced permeation to the deeper layers of the skin.
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The in-vivo transdermal absorption study presented enhanced improvement in bioavailability of amlodipine in the wistar rats.
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The formulation was also assessed for skin interaction study and found to be safe.
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