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Second generation lipid nanoparticles (NLC) as an oral drug carrier for delivery of lercanidipine hydrochloride
Colloids and Surfaces B: Biointerfaces 116 (2014) 81–87
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Second generation lipid nanoparticles (NLC) as an oral drug carrier for delivery of lercanidipine hydrochloride Nisharani S. Ranpise ∗ , Swati S. Korabu, Vinod N. Ghodake Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411 041, Maharashtra, India
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Article history: Received 22 July 2013 Received in revised form 31 October 2013 Accepted 9 December 2013 Available online 30 December 2013 Keywords: Nanostructured lipid carriers Lercanidipine hydrochloride Hypertension Solvent evaporation In vivo pharmacodynamic study
a b s t r a c t Lercanidipine hydrochloride is a calcium channel blocker used in the treatment of hypertension. It is a poor water soluble drug with absolute bioavailability of 10%. The aim of this study was to design lercanidipine hydrochloride-loaded nanostructured lipid carriers to investigate whether the bioavailability of the same can be improved by oral delivery. Lercanidipine hydrochloride nanostructured lipid carriers were prepared by the method of solvent evaporation at a high temperature and solidification by freeze drying. The nanostructured lipid carriers were evaluated for particle size analysis, zeta potential, entrapment efficiency, in vitro drug diffusion, ex vivo permeation studies and pharmacodynamic study. The resultant nanostructured lipid carriers had a mean size of 214.97 nm and a zeta potential of −31.6 ± 1.5 mV. More than 70% lercanidipine hydrochloride was entrapped in the NLCs. The SEM studies indicated the formation of type 2 nanostructured lipid carriers. The in vitro release studies demonstrated 19.36% release in acidic buffer pH 1.2 indicating that the drug entrapped in the nanostructured lipid carriers remains entrapped at acidic pH. The ex vivo studies indicated that the drug release was enhanced from 10% to 60.54% at blood pH in 24 h. The in vivo pharmacodynamic study showed that NLCs released lercanidipine hydrochloride in a controlled manner for a prolonged period of time as compared to plain drug. These results clearly indicate that nanostructured lipid carriers are a potential controlled release formulation for lercanidipine hydrochloride and may be a promising drug delivery system for the treatment of hypertension. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Oral route is the most preferred route for drug administration due to greater ease of administration, negligible pain, high patient compliance and no needle based injuries. However, newer formulations and dosage forms are always on the go in oral drug delivery systems due to low drug solubility, poor GI absorption, and metabolism related issues, continuous fluctuation drug plasma levels and variability due to food effects which may compromise the conventional dosage delivery system [1,2]. Nanomedicine has been experimented with greatly in the past decade to overcome bioavailability and solubility related problems, some of the formulations being nanoemulsions, nanosuspensions, polymeric nanoparticles. Lipid nanoparticles with solid particle matrix are derived from o/w emulsions by simply replacing liquid lipid (oil) by a solid lipid, i.e. being solid at body temperature. They are categorized as first
∗ Corresponding author at: Department of Pharmaceutics, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411 041, Maharashtra, India. Tel.: +91 9822671472. E-mail addresses: nisha [email protected], nisha [email protected] (N.S. Ranpise). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.012
generation nanoparticles: Solid lipid nanoparticles and Second generation nanoparticles: Nanostructured lipid carriers (NLCs) [3]. Need for second generation nanoparticles arised since particles from solid lipid matrix of SLNs form a relatively perfect crystal lattice. Thus, there is only a limited space to accommodate the drug which limits the loading capacity and causes drug expulsion. In NLCs, particles are prepared by using the blend of solid lipids and liquid lipids preferably in a ratio of 4:1 to 9:1. Due to the differences in the structures of the solid and liquid lipids, formation of a perfect crystal is distorted. Thus, the mixture accommodates the active in molecular form or in amorphous clusters [4]. The lipids employed in these are usually physiological lipids, i.e. biocompatible and biodegradable with low toxicity as opposed to the polymeric nanoparticles, the in vivo degradation of which may cause toxic effects [5,6]. NLCs also show high drug loading for both lipophilic and hydrophilic drugs [7,8], long shelf life and hassle free large scale production [2,9,10,11]. NLCs promote oral absorption of encapsulated drug via selective uptake through lymphatic route or payer’s patches [12,13]. Also, nanoparticles rarely undergo blood clearance via reticuloendothelial system, i.e. liver and spleen filtrations are avoided [9,14,15]. Lercanidipine hydrochloride (Fig. 1) is a calcium channel blocker of the dihydropyridine class. It is used alone or with an
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molten lipid required to solubilize the drug was noted visually within 24 h. The end point of the solubility study was the formation of clear solution of molten lipid. 2.3. Compatibility study of solid and liquid lipids
Fig. 1. Molecular structure of lercanidipine hydrochloride.
angiotensin-converting enzyme inhibitor, to treat hypertension, chronic stable angina pectoris, and prinzmetal’s variant angina. Lercanidipine hydrochloride is similar to other peripheral vasodilators. It inhibits the influx of extracellular calcium across the myocardial and vascular smooth muscle and interferes with the release of calcium from the sarcoplasmic reticulum. The decrease in intracellular calcium inhibits the contractile processes of the myocardial smooth muscle cells, causing dilation of the coronary and systemic arteries, and decreased systemic blood pressure. The absolute bioavailability of lercanidipine hydrochloride is about 10%, because of high first pass metabolism, thus making it a suitable agent for nanoparticle formulation. 2. Materials and methods 2.1.a. Materials Lercanidipine hydrochloride with a purity of 99.26% was kindly gifted by one of the leading pharmaceutical company in India. Labrafil M 2130, Compritol ATO888, Gelucire 44/14, Gelucire 50/13, Labrafil 2130M, Labrafac, Labrafil 2125M, Lauroglycol FCC, Capryol 90, Labrasol, Triton X100, Crempher EL, Solutol HS 15, and Transcutol P were obtained as gift samples from Gattefosse, Mumbai, India. Glyceryl monostearate (GMS), stearic acid, beeswax, carnauba wax, Span 80, Tween 80 and Lutrol® F68 were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Linseed oil was purchased from Pure Chem Labs, Pune; Olive oil was a product of Figaro, Madrid, Spain. Capmul MCM EP, Captex 300, Captex 500, MCT, Capmul MCM were gift samples from Abitech Corp., USA. Soya lecithin was supplied by Hi Media Ltd., Mumbai. All other reagents and chemicals obtained were of analytical grade. 2.1.b. Animals Male Sprague–Dawley rats (180–200 g) were used for this study. All the rats were fed standard rat chow and were maintained on a 12-h light/dark cycle. The experimental protocol was reviewed and approved by the STES’s Sinhgad college of Pharmacy Institutional Animal ethics committee (IAEC) constituted in accordance with the rules and guidelines of the Committee of the Purpose of Control and Supervision on Experimental Animals (CPCSEA), India. 2.2. Saturated solubility studies This study was done for determining the solubility of the drug in the components to be used in the formulation, namely, solvents, surfactants, solid lipids and liquid lipids. An array of solvents, surfactants, solid lipids and liquid lipids are subjected to this study to select the most compatible out of each category. Excess drug was added to a known volume of the solvent systems and mixed for 2 min and sonicated for 10 min to dissolve the drug. Incubator shaker was further used for 8–12 h to dissolve the drug. The contents were then centrifuged at 5000 rpm for 15 min. The aliquots of supernatant were diluted appropriately and analyzed using a UV spectrophotometer (Shimadzu 1800, Japan) at 239 nm. In contrast to this, for solid lipids, a known amount of drug was added to a measured quantity of lipid. The minimum amount of
A compatibility screening of selected solid lipids with liquid lipids in the ratios 9:1, 4:1, and 3:1 was performed. The lipid mixtures used were namely GMS:Linseed oil, GMS:Capmul, Labrafil:Linseed oil and Labrafil:Capmul; each mixture taken in the above mentioned three ratios. Mixtures were heated up to 5 ◦ C more than the melting point of the solid lipid and then checked after one hour, immediately after solidification and then after 24 h. Mixtures with only one single phase were selected for further study. 2.4. Preparation of NLC Lercanidipine hydrochloride loaded NLCs were prepared using two methods, i.e. ultrasonication and emulsion evaporation technique as described by Liu et al. and Jia et al. respectively [16,17]. In ultrasonication method, solid lipid, liquid lipid and drug were dissolved in organic solvent by heating up to 80 ◦ C. This mixture was dispersed in 1.5% Lutrol® F68 solution kept at the same temperature using a high speed stirrer at 8000 rpm. Further, this pre-emulsion was subjected to ultrasonication for three cycles and cooled for the NLCs to solidify. In emulsion evaporation method, drug, solid lipid, liquid lipid and lipophilic surfactant were co-dissolved in the organic solvent in a water bath at 75 ◦ C. The resultant organic solution was added drop wise in the aqueous phase containing 1.5% Lutrol® F68 and was subjected to mechanical agitation at 1000 rpm and cooled for obtaining NLCs. The formulations are depicted in Table 1. 2.5. Particle size analysis The mean particle size of the nanoparticles was measured by photon correlation spectroscopy using Nanophox Sympatec GmbH, Germany, at room temperature. Before measurement, batches were diluted with filtered double distilled water until the appropriate concentration of particles was achieved to avoid multi-scattering events. 2.6. Entrapment efficiency Entrapment efficiency was determined by minicolumn centrifugation method. Sephadex® G25 M solution (10%, w/v) was prepared in distilled water and kept aside for 24 h for swelling. To prepare minicolumns, Whatman filter pad was inserted in 1 ml syringe and swelled sephadex was added slowly to it. Respective formulation (100 l) was slowly added to prepared column and centrifuged at 2000 rpm for 10 min. Obtained eluted NLC samples were ruptured using sufficient volume of methanol and the absorbance of the same was measured by UV spectrophotometer [18]. The encapsulation percentage (%) was determined using the following W equation:%E.E. = W E × 100,where WE is the amount of lercanidipA ine hydrochloride in the NLCs and WA is the amount of lercanidipine hydrochloride in the system. 2.7. Freeze drying of NLC suspension The NLC suspensions obtained were subjected to freeze drying using a freeze dryer (Martin Christ, Germany) at a pressure lower than 0.5 milibar to obtain solid form of the NLCs. Prior to the drying process the NLC suspension was frozen in a freezer for 4 h. The frozen samples were subjected to the freeze drying process for
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Table 1 Formulation table. Formulation
Drug (mg)
Lutrol® F68
Tween 80
GMS (mg)
Labrafil (mg)
Linseed oil (mg)
Capmul (mg)
Water (ml)
F1E F2E F3E F4E F1U F2U F3U F4U
150 150 150 150 150 150 150 150
1.5% 1.5% 1.5% 1.5% 1.5% 1.5% 1.5% 1.5%
500 500 500 500 – – – –
200 200 – – 200 200 – –
– – 200 200 – – 200 200
50 – 50 – 50 – 50 –
– 50 – 50 – 50 – 50
50 50 50 50 50 50 50 50
‘E’ indicates formulations prepared using solvent evaporation method and ‘U’ indicates formulations prepared using ultrasonication method.
8–12 h. Mannitol (3%) was added as a lyoprotectant to avoid the lysis of the nanoparticles present in the suspension [16]. 2.8. Zeta potential measurement Charge on the drug loaded droplet surface was determined using Zetasizer 300 (Malvern Instruments, Malvern, UK). The potential was measured after dilution of samples with distilled water at room temperature. 2.9. Stability of the reconstituted solution The freeze dried NLC powder was reconstituted in 10 ml distilled water and analyzed by HPLC with suitable dilutions immediately, after 24 h and after 48 h.
using DSC (SIIO 6300 with auto sampler, Japan). Samples were accurately weighed onto aluminum pans and then hermetically sealed with aluminum lids. Thermograms were obtained at a scanning rate of 10 ◦ C per minute conducted over a temperature range of 50–350 ◦ C in the environment of liquid nitrogen (Flow rate 60 ml/min.). 2.13. X-ray diffraction (XRD) Drug and excipients of the formulation were subjected to Xray crystallographic studies. The powder X ray diffraction patterns were recorded using an X-ray diffractometer (D8 Advance, BRUKER, Germany) with 2.2 KW copper as an anode material and dermic Xray tube as a source. The samples were analyzed using lynux eye detector and filtered using Ni filter. The samples were analyzed in the 2 angle of 3–30◦ .
2.10. In vitro drug release from NLCs 2.14. Scanning electron microscopy (SEM) studies The method followed for in vitro drug release from NLCs and plain drug is as described by Chen et al. [18]. The drug released from carriers was measured using a Franz diffusion cell. A cellulose membrane (up to 12,000 Da) was mounted between the donor and the receptor compartments and a drug equivalent to 3 mg dose was placed on it. The receptor medium consisted of 7 ml of acidic buffer pH 1.2. The stirring rate and temperature was 300 rpm and 37 ◦ C respectively. At appropriate intervals, 2 ml aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug released was determined UV spectrophotometrically. 2.11. Ex vivo study Ex-vivo skin penetration studies of lercanidipine hydrochloride loaded NLCs were performed with rat stomach membrane as done by Liu et al. [17,19] using Franz diffusion cell. Two ex vivo studies were done using acidic buffer pH 1.2 and phosphate buffer pH 7.4 as the receptor fluid. The procedure followed for both the studies were the same. A small quantity of NLCs (equivalent to 3 mg drug) was placed on the stomach mucosal surface. Serial sampling was performed at specified time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 and 24 h) by removing the contents of the receptor compartment and replacing it with fresh medium. The samples were analyzed using HPLC (Shimadzu LC 2010 HT, Japan) and mean cumulative amount diffused at each sampling time point was calculated. At the end of 24 h, the amount of drug remaining on the skin, and the drug concentration in the skin was determined by extracting into a suitable solvent followed by spectrophotometric analysis using an HPLC. 2.12. Differential scanning calorimetry (DSC) studies The possibility of any interaction between drug and excipients was assessed by carrying out thermal analysis of the formulation
Shape and surface morphology of drug loaded NLC was observed by scanning electron microscope (Jeol 5400, Japan). The sample was coated by gold ion and the coating was performed in 5–6 min. Further, the sample was analyzed at 5000× and 350× magnifications. 2.15. In vivo pharmacodynamic study 2.15.a. General protocol Male Sprague–Dawley rats, initially weighing 180–200 g, were used for the experiment. Rats were divided into four groups: 1, Control; 2, Hypertensive control; 3, NLC formulation; 4, Plain drug. Before dietary manipulation, all rats were fed standard rat chow and were maintained on a 12-h light/dark cycle. In addition, rats were acclimated to the procedure of blood pressure measurement daily for 1 week. Hypertension was induced in rats by Fructoseinduced hypertension model, for that rats were fed with a diet containing 66% fructose, 12% fat, and 22% protein for 11 days, after that systolic blood pressure was measured to check hypertension in rats [20–22]. Next day control group and hypertensive control group of rats were given plain water, NLC formulation group animals were given reconstituted NLC formulation (equivalent to 3 mg/kg lercanidipine HCl), plain drug group animals were given plain lercanidipine HCl (3 mg/kg). The oral dosing was performed by intubation using an 18-gauge feeding needle (the volume to be fed was 0.5 ml in all cases). Blood pressure was measured at an interval of 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h. Statistical analysis of the collected data was performed using two way ANOVA test. 2.15.b. Blood pressure measurement Animals were allowed free access to diet and water and were kept in a quiet area before the blood pressure was measured using magnetic animal holder, IITC life sciences, USA. The tail-cuff method, without external preheating, was used to measure the systolic blood pressure [21]. The temperature was maintained at 30 ◦ C.
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Table 2 Particle size and entrapment efficiency of the samples formulated using the two methods. Formulation
Particle size (nm)
F1E F2E F3E F4E F1U F2U F3U F4U
200.9 91.19 211.47 422.76 1027.8 355.45 655.86 309.95
± ± ± ± ± ± ± ±
2.3 1.4 0.7 1.6 4.4 2.2 3.5 2.4
Particle size with drug (nm) 214.9 99.19 214.47 466.76 1107.8 371.45 685.86 336.95
± ± ± ± ± ± ± ±
0.5 1.2 0.3 2.1 5.5 2.7 3.6 2.9
Entrapment efficiency (%) 60.954 50.209 71.576 50.112 61.452 50.102 56.722 51.135
± ± ± ± ± ± ± ±
2.1 2.8 2.5 6.7 4.3 2.5 5.2 4.1
‘E’ indicates formulations prepared using solvent evaporation method and ‘U’ indicates formulations prepared using ultrasonication method.
3.3. Particle size analysis NLCs were prepared by solvent evaporation method and ultrasonication method. Labrafil 2130M and GMS were used as a solid particle matrix and linseed oil as the liquid lipid. The study shows that the method of preparation is a critical parameter governing the particle size. As seen in Table 2, formulations F1E, F2E and F3E prepared by solvent evaporation method were found to have the least particle size viz. formulation containing GMS:linseed oil, GMS:Capmul and Labrafil:linseed oil, 214.9 ± 0.5 nm, 99.19 ± 1.2 nm and 214.47 ± 0.3 nm respectively. It also can be seen that for this particular formulation, ultrasonication method gives higher particle sizes. 3.4. Entrapment efficiency
The systolic blood pressure of the rats was measured in the conscious state. The mean of three consecutive readings was used as the measurement of the systolic blood pressure of each rat.
3. Results and discussion 3.1. Saturated solubility studies In case of solid lipids, the least amount of lipid needed for complete solubilization of lercanidipine hydrochloride shows the highest solubility of the drug. Among solid lipids, Labrafil 2130M and glyceryl monostearate showed highest solubility of the drug. In liquid lipids, linseed oil showed the maximum solubility of the drug. Similarly, for surfactants, the drug is more soluble in Tween 80. It was also seen through the study that out of all the organic solvents, lercanidipine hydrochloride is the most soluble in methanol.
3.2. Compatibility study of solid and liquid lipids The mixtures were checked for phase separation immediately after solidification, after one hour and after 24 h. Mixtures with only one single phase were selected for further studies. GMS:linseed oil, GMS:Capmul, Labrafil:linseed oil and Labrafil:Capmul in the ratios of 9:1 and 4:1 showed no phase separation and hold their physical configuration throughout the 24 h. But the same mixtures in 3:1 ratio are seen in a semisolid state thus showing phase separation. Thus, the ratio of solid lipid: liquid lipid 4:1 was selected for further study since the bulk of the solid lipid would have increased on selection of the 9:1 ratio.
An important parameter with respect to NLCs as drug carriers is their capacity for drug loading. As summarized in Table 2, lercanidipine hydrochloride shows highest entrapment of 71.576 ± 2.5% in the mixture of labrafil:linseed oil (F3E) and 60.954 ± 2.1 in the mixture of GMS:linseed oil (F1E) as compared to the rest of the formulations. It was also observed that ultrasonication method gives low entrapment efficiency for lercanidipine hydrochloride and as seen above it gives NLCs with higher particle size. Thus, the NLCs prepared by this method were not studied further. 3.5. Zeta potential measurement Zeta potential measures the surface charge of particles. As the zeta potential () increases, the particle surface charge also increases. Zeta potential greatly influences particle stability in suspension through the electrostatic repulsion between particles. The repulsive interactions will be larger between the particles as the zeta potential increases or decreases, leading to the formation of more stable particles with a more uniform size distribution. The NLC suspension had a mean zeta potential of −31.6 ± 1.5 mV. High negative charges of -potential indicate that the electrostatic repulsion between particles with the same electrical charge will prevent the aggregation of the spheres and could stabilize particle suspensions. Thus, the values obtained for the NLCs are adequate to form a stable nanoparticle suspension [20]. 3.6. Stability of the reconstituted solution The F3E formulation containing Labrafil was chosen as the final formulation due to its higher entrapment efficiency and drug content. Stability of the reconstituted solution of the Labrafil
Fig. 2. Drug release graph of in vitro diffusion study.
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Fig. 3. Ex vivo diffusion study at pH 7.4 and at pH 1.2 for 24 h.
containing NLC was checked. The reconstituted solution showed a negligible change in concentration over a period of 48 h. The drug content at 0 h was 98.50 ± 0.8% and at 48 h was 97.40 ± 1.5%. This shows that the reconstituted solution is stable for 2 days. 3.7. In vitro release kinetics of the drug from NLCs The in vitro release study using the dialysis bag technique was done using a Franz diffusion apparatus. This study is done to understand the release mechanism of lercanidipine hydrochloride from the NLC system. The in vitro lercanidipine hydrochloride release from Labrafil NLC vs plain drug release is plotted against time as shown in Fig. 2. The total drug release from NLC in 12 h is 19.36%. This indicates that at gastric pH, the unencapsulated drug is released. The inclusion of drug into lipid nanoparticles significantly reduced the drug release at acidic pH 1.2. 3.8. Ex vivo diffusion study In the present investigation, lercanidipine hydrochloride loaded NLCs produced a significantly higher release at the blood pH 7.4
(60.53%) as compared to the release at gastric pH 1.2 (23.25%) as seen in Fig. 3. The rat stomach membrane has pore diameters of about 400 nm [21]. Thus, diffusion of the NLC seems possible, their particle size being 214.47 nm and high adhesion due to very high surface may explain the increase in skin permeation. Due to the lipid nature of SLN, the NLC penetrates the skin and remain localized for a longer period of time. At the end of 24 h, the remaining NLCs were scrapped and washed off from the stomach membrane using methanol. In a fixed volume of methanol, the NLCs were sonicated and ruptured. Dilutions were made accordingly and analyzed using HPLC (Shimadzu LC 2010 HT, Japan). The amount of drug remaining after 24 h was found to be 31.34%. 3.9. Differential scanning calorimetry (DSC) studies DSC has been used to interpret the melting and crystallization behavior of crystalline materials like lipid nanoparticles. DSC interpretation confirms the melting point of lercanidipine hydrochloride to be around 192–193 ◦ C and the solid lipid peak is seen at 63.9 ◦ C. The endothermic peak of final NLC formulation
Fig. 4. DSC curves for Labrafil 2130M, lercanidipine hydrochloride, physical mixture of lercanidipine hydrochloride and Labrafil 2130M and lercanidipine hydrochloride NLC.
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Fig. 5. X-ray diffractograms of lercanidipine hydrochloride vs. NLC.
seen at 50.7 ◦ C confirms the formation of nanoparticle as seen in Fig. 4, i.e. small particle size and higher surface area and that the solid lipid has entirely covered the drug during formulation.
3.10. X-ray diffraction studies The diffraction pattern of pure drug showed that it is highly crystalline in nature as indicated by its numerous distinctive peaks with major characteristic diffraction appearing at a diffraction angle of 2 at 13.760◦ , 17.377◦ , 19.108◦ , 19.945◦ , 20.493◦ , 21.457◦ and 43.972◦ . The XRD interpretation of final NLC formulation shows more of an amorphous nature as compared to the plain drug as seen in Fig. 5. Some sharp peaks are also observed which may be due to the presence of mannitol in the NLCs which is crystalline in nature.
Fig. 6. SEM image of NLC suspension.
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Fig. 7. Systolic blood pressures after treatment.
3.11. Scanning electron microscopy (SEM) studies for NLC suspension Fig. 6 shows the SEM image of lercanidipine hydrochloride NLC almost spherical and non adherent to each other on a scale of 5 m. SEM shows irregularly shaped particles thus showing the formation of type 2 NLC. 3.12. In vivo pharmacodynamic study The results of the in vivo pharmacodynamic study are shown in Fig. 7. Blood pressure was checked at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h. Fig. 7 shows the effect of treatment on systolic blood pressure levels of hypertensive rats. At 0 h, all the groups of rats except normal control group showed a significant increase in BP. Significant decrease in systolic blood pressure level was seen in NLC fed rats as compared to plain drug fed rats (P < 0.001). The lowest blood pressure attained by the plain drug group was 118.66 ± 2.51 mm Hg at 4 h whereas, the NLC formulation performed better resulting in a significant reduction in blood pressure up to 117.23 ± 1.61 mm Hg for 8 h and up to 130.13 ± 1.97 mm Hg for 24 h. After 24 h the blood pressure of NLC group still remained below the hypertensive BP whereas the plain drug groups came back to the hypertensive state. This clearly indicated that NLCs promote oral absorption of encapsulated drug via selective uptake through lymphatic route or payer’s patches [12,13]. The entire study puts forward superiority of NLC as compared to the plain drug and thus, validates the rationale behind the study. 4. Conclusion NLCs have evolved as second-generation lipid nanocarriers which retain the advantages of SLN but unlike SLN they exhibit good physical stability and higher drug loading. In view of this, NLCs of lercanidipine hydrochloride were successfully fabricated by employing solvent evaporation technique by using Labrafil 2130M and linseed oil as the lipid phases and Tween-80 and Lutrol® F68 as lipophilic and hydrophilic surfactant phases. The components used for the fabrication of the NLCs are biocompatible. The solvent evaporation technique successfully yielded nanostructured lipid carriers with particle size of 214.47 ± 0.3 nm. The pharmacodynamic study showed that NLCs released lercanidipine hydrochloride in a controlled manner for a prolonged period of time. The overall investigations present an alternate drug delivery system for increasing the lercanidipine hydrochloride bioavailability through the nanostructured lipid carriers (NLCs) methodology.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Second generation lipid nanoparticles (NLC) as an oral drug carrier for delivery of lercanidipine hydrochloride Nisharani S. Ranpise ∗ , Swati S. Korabu, Vinod N. Ghodake Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411 041, Maharashtra, India
a r t i c l e
i n f o
Article history: Received 22 July 2013 Received in revised form 31 October 2013 Accepted 9 December 2013 Available online 30 December 2013 Keywords: Nanostructured lipid carriers Lercanidipine hydrochloride Hypertension Solvent evaporation In vivo pharmacodynamic study
a b s t r a c t Lercanidipine hydrochloride is a calcium channel blocker used in the treatment of hypertension. It is a poor water soluble drug with absolute bioavailability of 10%. The aim of this study was to design lercanidipine hydrochloride-loaded nanostructured lipid carriers to investigate whether the bioavailability of the same can be improved by oral delivery. Lercanidipine hydrochloride nanostructured lipid carriers were prepared by the method of solvent evaporation at a high temperature and solidification by freeze drying. The nanostructured lipid carriers were evaluated for particle size analysis, zeta potential, entrapment efficiency, in vitro drug diffusion, ex vivo permeation studies and pharmacodynamic study. The resultant nanostructured lipid carriers had a mean size of 214.97 nm and a zeta potential of −31.6 ± 1.5 mV. More than 70% lercanidipine hydrochloride was entrapped in the NLCs. The SEM studies indicated the formation of type 2 nanostructured lipid carriers. The in vitro release studies demonstrated 19.36% release in acidic buffer pH 1.2 indicating that the drug entrapped in the nanostructured lipid carriers remains entrapped at acidic pH. The ex vivo studies indicated that the drug release was enhanced from 10% to 60.54% at blood pH in 24 h. The in vivo pharmacodynamic study showed that NLCs released lercanidipine hydrochloride in a controlled manner for a prolonged period of time as compared to plain drug. These results clearly indicate that nanostructured lipid carriers are a potential controlled release formulation for lercanidipine hydrochloride and may be a promising drug delivery system for the treatment of hypertension. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Oral route is the most preferred route for drug administration due to greater ease of administration, negligible pain, high patient compliance and no needle based injuries. However, newer formulations and dosage forms are always on the go in oral drug delivery systems due to low drug solubility, poor GI absorption, and metabolism related issues, continuous fluctuation drug plasma levels and variability due to food effects which may compromise the conventional dosage delivery system [1,2]. Nanomedicine has been experimented with greatly in the past decade to overcome bioavailability and solubility related problems, some of the formulations being nanoemulsions, nanosuspensions, polymeric nanoparticles. Lipid nanoparticles with solid particle matrix are derived from o/w emulsions by simply replacing liquid lipid (oil) by a solid lipid, i.e. being solid at body temperature. They are categorized as first
∗ Corresponding author at: Department of Pharmaceutics, Sinhgad College of Pharmacy, Vadgaon (Bk.), Pune 411 041, Maharashtra, India. Tel.: +91 9822671472. E-mail addresses: nisha [email protected], nisha [email protected] (N.S. Ranpise). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.12.012
generation nanoparticles: Solid lipid nanoparticles and Second generation nanoparticles: Nanostructured lipid carriers (NLCs) [3]. Need for second generation nanoparticles arised since particles from solid lipid matrix of SLNs form a relatively perfect crystal lattice. Thus, there is only a limited space to accommodate the drug which limits the loading capacity and causes drug expulsion. In NLCs, particles are prepared by using the blend of solid lipids and liquid lipids preferably in a ratio of 4:1 to 9:1. Due to the differences in the structures of the solid and liquid lipids, formation of a perfect crystal is distorted. Thus, the mixture accommodates the active in molecular form or in amorphous clusters [4]. The lipids employed in these are usually physiological lipids, i.e. biocompatible and biodegradable with low toxicity as opposed to the polymeric nanoparticles, the in vivo degradation of which may cause toxic effects [5,6]. NLCs also show high drug loading for both lipophilic and hydrophilic drugs [7,8], long shelf life and hassle free large scale production [2,9,10,11]. NLCs promote oral absorption of encapsulated drug via selective uptake through lymphatic route or payer’s patches [12,13]. Also, nanoparticles rarely undergo blood clearance via reticuloendothelial system, i.e. liver and spleen filtrations are avoided [9,14,15]. Lercanidipine hydrochloride (Fig. 1) is a calcium channel blocker of the dihydropyridine class. It is used alone or with an
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molten lipid required to solubilize the drug was noted visually within 24 h. The end point of the solubility study was the formation of clear solution of molten lipid. 2.3. Compatibility study of solid and liquid lipids
Fig. 1. Molecular structure of lercanidipine hydrochloride.
angiotensin-converting enzyme inhibitor, to treat hypertension, chronic stable angina pectoris, and prinzmetal’s variant angina. Lercanidipine hydrochloride is similar to other peripheral vasodilators. It inhibits the influx of extracellular calcium across the myocardial and vascular smooth muscle and interferes with the release of calcium from the sarcoplasmic reticulum. The decrease in intracellular calcium inhibits the contractile processes of the myocardial smooth muscle cells, causing dilation of the coronary and systemic arteries, and decreased systemic blood pressure. The absolute bioavailability of lercanidipine hydrochloride is about 10%, because of high first pass metabolism, thus making it a suitable agent for nanoparticle formulation. 2. Materials and methods 2.1.a. Materials Lercanidipine hydrochloride with a purity of 99.26% was kindly gifted by one of the leading pharmaceutical company in India. Labrafil M 2130, Compritol ATO888, Gelucire 44/14, Gelucire 50/13, Labrafil 2130M, Labrafac, Labrafil 2125M, Lauroglycol FCC, Capryol 90, Labrasol, Triton X100, Crempher EL, Solutol HS 15, and Transcutol P were obtained as gift samples from Gattefosse, Mumbai, India. Glyceryl monostearate (GMS), stearic acid, beeswax, carnauba wax, Span 80, Tween 80 and Lutrol® F68 were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Linseed oil was purchased from Pure Chem Labs, Pune; Olive oil was a product of Figaro, Madrid, Spain. Capmul MCM EP, Captex 300, Captex 500, MCT, Capmul MCM were gift samples from Abitech Corp., USA. Soya lecithin was supplied by Hi Media Ltd., Mumbai. All other reagents and chemicals obtained were of analytical grade. 2.1.b. Animals Male Sprague–Dawley rats (180–200 g) were used for this study. All the rats were fed standard rat chow and were maintained on a 12-h light/dark cycle. The experimental protocol was reviewed and approved by the STES’s Sinhgad college of Pharmacy Institutional Animal ethics committee (IAEC) constituted in accordance with the rules and guidelines of the Committee of the Purpose of Control and Supervision on Experimental Animals (CPCSEA), India. 2.2. Saturated solubility studies This study was done for determining the solubility of the drug in the components to be used in the formulation, namely, solvents, surfactants, solid lipids and liquid lipids. An array of solvents, surfactants, solid lipids and liquid lipids are subjected to this study to select the most compatible out of each category. Excess drug was added to a known volume of the solvent systems and mixed for 2 min and sonicated for 10 min to dissolve the drug. Incubator shaker was further used for 8–12 h to dissolve the drug. The contents were then centrifuged at 5000 rpm for 15 min. The aliquots of supernatant were diluted appropriately and analyzed using a UV spectrophotometer (Shimadzu 1800, Japan) at 239 nm. In contrast to this, for solid lipids, a known amount of drug was added to a measured quantity of lipid. The minimum amount of
A compatibility screening of selected solid lipids with liquid lipids in the ratios 9:1, 4:1, and 3:1 was performed. The lipid mixtures used were namely GMS:Linseed oil, GMS:Capmul, Labrafil:Linseed oil and Labrafil:Capmul; each mixture taken in the above mentioned three ratios. Mixtures were heated up to 5 ◦ C more than the melting point of the solid lipid and then checked after one hour, immediately after solidification and then after 24 h. Mixtures with only one single phase were selected for further study. 2.4. Preparation of NLC Lercanidipine hydrochloride loaded NLCs were prepared using two methods, i.e. ultrasonication and emulsion evaporation technique as described by Liu et al. and Jia et al. respectively [16,17]. In ultrasonication method, solid lipid, liquid lipid and drug were dissolved in organic solvent by heating up to 80 ◦ C. This mixture was dispersed in 1.5% Lutrol® F68 solution kept at the same temperature using a high speed stirrer at 8000 rpm. Further, this pre-emulsion was subjected to ultrasonication for three cycles and cooled for the NLCs to solidify. In emulsion evaporation method, drug, solid lipid, liquid lipid and lipophilic surfactant were co-dissolved in the organic solvent in a water bath at 75 ◦ C. The resultant organic solution was added drop wise in the aqueous phase containing 1.5% Lutrol® F68 and was subjected to mechanical agitation at 1000 rpm and cooled for obtaining NLCs. The formulations are depicted in Table 1. 2.5. Particle size analysis The mean particle size of the nanoparticles was measured by photon correlation spectroscopy using Nanophox Sympatec GmbH, Germany, at room temperature. Before measurement, batches were diluted with filtered double distilled water until the appropriate concentration of particles was achieved to avoid multi-scattering events. 2.6. Entrapment efficiency Entrapment efficiency was determined by minicolumn centrifugation method. Sephadex® G25 M solution (10%, w/v) was prepared in distilled water and kept aside for 24 h for swelling. To prepare minicolumns, Whatman filter pad was inserted in 1 ml syringe and swelled sephadex was added slowly to it. Respective formulation (100 l) was slowly added to prepared column and centrifuged at 2000 rpm for 10 min. Obtained eluted NLC samples were ruptured using sufficient volume of methanol and the absorbance of the same was measured by UV spectrophotometer [18]. The encapsulation percentage (%) was determined using the following W equation:%E.E. = W E × 100,where WE is the amount of lercanidipA ine hydrochloride in the NLCs and WA is the amount of lercanidipine hydrochloride in the system. 2.7. Freeze drying of NLC suspension The NLC suspensions obtained were subjected to freeze drying using a freeze dryer (Martin Christ, Germany) at a pressure lower than 0.5 milibar to obtain solid form of the NLCs. Prior to the drying process the NLC suspension was frozen in a freezer for 4 h. The frozen samples were subjected to the freeze drying process for
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Table 1 Formulation table. Formulation
Drug (mg)
Lutrol® F68
Tween 80
GMS (mg)
Labrafil (mg)
Linseed oil (mg)
Capmul (mg)
Water (ml)
F1E F2E F3E F4E F1U F2U F3U F4U
150 150 150 150 150 150 150 150
1.5% 1.5% 1.5% 1.5% 1.5% 1.5% 1.5% 1.5%
500 500 500 500 – – – –
200 200 – – 200 200 – –
– – 200 200 – – 200 200
50 – 50 – 50 – 50 –
– 50 – 50 – 50 – 50
50 50 50 50 50 50 50 50
‘E’ indicates formulations prepared using solvent evaporation method and ‘U’ indicates formulations prepared using ultrasonication method.
8–12 h. Mannitol (3%) was added as a lyoprotectant to avoid the lysis of the nanoparticles present in the suspension [16]. 2.8. Zeta potential measurement Charge on the drug loaded droplet surface was determined using Zetasizer 300 (Malvern Instruments, Malvern, UK). The potential was measured after dilution of samples with distilled water at room temperature. 2.9. Stability of the reconstituted solution The freeze dried NLC powder was reconstituted in 10 ml distilled water and analyzed by HPLC with suitable dilutions immediately, after 24 h and after 48 h.
using DSC (SIIO 6300 with auto sampler, Japan). Samples were accurately weighed onto aluminum pans and then hermetically sealed with aluminum lids. Thermograms were obtained at a scanning rate of 10 ◦ C per minute conducted over a temperature range of 50–350 ◦ C in the environment of liquid nitrogen (Flow rate 60 ml/min.). 2.13. X-ray diffraction (XRD) Drug and excipients of the formulation were subjected to Xray crystallographic studies. The powder X ray diffraction patterns were recorded using an X-ray diffractometer (D8 Advance, BRUKER, Germany) with 2.2 KW copper as an anode material and dermic Xray tube as a source. The samples were analyzed using lynux eye detector and filtered using Ni filter. The samples were analyzed in the 2 angle of 3–30◦ .
2.10. In vitro drug release from NLCs 2.14. Scanning electron microscopy (SEM) studies The method followed for in vitro drug release from NLCs and plain drug is as described by Chen et al. [18]. The drug released from carriers was measured using a Franz diffusion cell. A cellulose membrane (up to 12,000 Da) was mounted between the donor and the receptor compartments and a drug equivalent to 3 mg dose was placed on it. The receptor medium consisted of 7 ml of acidic buffer pH 1.2. The stirring rate and temperature was 300 rpm and 37 ◦ C respectively. At appropriate intervals, 2 ml aliquots of the receptor medium were withdrawn and immediately replaced with an equal volume of fresh buffer. The amount of drug released was determined UV spectrophotometrically. 2.11. Ex vivo study Ex-vivo skin penetration studies of lercanidipine hydrochloride loaded NLCs were performed with rat stomach membrane as done by Liu et al. [17,19] using Franz diffusion cell. Two ex vivo studies were done using acidic buffer pH 1.2 and phosphate buffer pH 7.4 as the receptor fluid. The procedure followed for both the studies were the same. A small quantity of NLCs (equivalent to 3 mg drug) was placed on the stomach mucosal surface. Serial sampling was performed at specified time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 and 24 h) by removing the contents of the receptor compartment and replacing it with fresh medium. The samples were analyzed using HPLC (Shimadzu LC 2010 HT, Japan) and mean cumulative amount diffused at each sampling time point was calculated. At the end of 24 h, the amount of drug remaining on the skin, and the drug concentration in the skin was determined by extracting into a suitable solvent followed by spectrophotometric analysis using an HPLC. 2.12. Differential scanning calorimetry (DSC) studies The possibility of any interaction between drug and excipients was assessed by carrying out thermal analysis of the formulation
Shape and surface morphology of drug loaded NLC was observed by scanning electron microscope (Jeol 5400, Japan). The sample was coated by gold ion and the coating was performed in 5–6 min. Further, the sample was analyzed at 5000× and 350× magnifications. 2.15. In vivo pharmacodynamic study 2.15.a. General protocol Male Sprague–Dawley rats, initially weighing 180–200 g, were used for the experiment. Rats were divided into four groups: 1, Control; 2, Hypertensive control; 3, NLC formulation; 4, Plain drug. Before dietary manipulation, all rats were fed standard rat chow and were maintained on a 12-h light/dark cycle. In addition, rats were acclimated to the procedure of blood pressure measurement daily for 1 week. Hypertension was induced in rats by Fructoseinduced hypertension model, for that rats were fed with a diet containing 66% fructose, 12% fat, and 22% protein for 11 days, after that systolic blood pressure was measured to check hypertension in rats [20–22]. Next day control group and hypertensive control group of rats were given plain water, NLC formulation group animals were given reconstituted NLC formulation (equivalent to 3 mg/kg lercanidipine HCl), plain drug group animals were given plain lercanidipine HCl (3 mg/kg). The oral dosing was performed by intubation using an 18-gauge feeding needle (the volume to be fed was 0.5 ml in all cases). Blood pressure was measured at an interval of 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h. Statistical analysis of the collected data was performed using two way ANOVA test. 2.15.b. Blood pressure measurement Animals were allowed free access to diet and water and were kept in a quiet area before the blood pressure was measured using magnetic animal holder, IITC life sciences, USA. The tail-cuff method, without external preheating, was used to measure the systolic blood pressure [21]. The temperature was maintained at 30 ◦ C.
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Table 2 Particle size and entrapment efficiency of the samples formulated using the two methods. Formulation
Particle size (nm)
F1E F2E F3E F4E F1U F2U F3U F4U
200.9 91.19 211.47 422.76 1027.8 355.45 655.86 309.95
± ± ± ± ± ± ± ±
2.3 1.4 0.7 1.6 4.4 2.2 3.5 2.4
Particle size with drug (nm) 214.9 99.19 214.47 466.76 1107.8 371.45 685.86 336.95
± ± ± ± ± ± ± ±
0.5 1.2 0.3 2.1 5.5 2.7 3.6 2.9
Entrapment efficiency (%) 60.954 50.209 71.576 50.112 61.452 50.102 56.722 51.135
± ± ± ± ± ± ± ±
2.1 2.8 2.5 6.7 4.3 2.5 5.2 4.1
‘E’ indicates formulations prepared using solvent evaporation method and ‘U’ indicates formulations prepared using ultrasonication method.
3.3. Particle size analysis NLCs were prepared by solvent evaporation method and ultrasonication method. Labrafil 2130M and GMS were used as a solid particle matrix and linseed oil as the liquid lipid. The study shows that the method of preparation is a critical parameter governing the particle size. As seen in Table 2, formulations F1E, F2E and F3E prepared by solvent evaporation method were found to have the least particle size viz. formulation containing GMS:linseed oil, GMS:Capmul and Labrafil:linseed oil, 214.9 ± 0.5 nm, 99.19 ± 1.2 nm and 214.47 ± 0.3 nm respectively. It also can be seen that for this particular formulation, ultrasonication method gives higher particle sizes. 3.4. Entrapment efficiency
The systolic blood pressure of the rats was measured in the conscious state. The mean of three consecutive readings was used as the measurement of the systolic blood pressure of each rat.
3. Results and discussion 3.1. Saturated solubility studies In case of solid lipids, the least amount of lipid needed for complete solubilization of lercanidipine hydrochloride shows the highest solubility of the drug. Among solid lipids, Labrafil 2130M and glyceryl monostearate showed highest solubility of the drug. In liquid lipids, linseed oil showed the maximum solubility of the drug. Similarly, for surfactants, the drug is more soluble in Tween 80. It was also seen through the study that out of all the organic solvents, lercanidipine hydrochloride is the most soluble in methanol.
3.2. Compatibility study of solid and liquid lipids The mixtures were checked for phase separation immediately after solidification, after one hour and after 24 h. Mixtures with only one single phase were selected for further studies. GMS:linseed oil, GMS:Capmul, Labrafil:linseed oil and Labrafil:Capmul in the ratios of 9:1 and 4:1 showed no phase separation and hold their physical configuration throughout the 24 h. But the same mixtures in 3:1 ratio are seen in a semisolid state thus showing phase separation. Thus, the ratio of solid lipid: liquid lipid 4:1 was selected for further study since the bulk of the solid lipid would have increased on selection of the 9:1 ratio.
An important parameter with respect to NLCs as drug carriers is their capacity for drug loading. As summarized in Table 2, lercanidipine hydrochloride shows highest entrapment of 71.576 ± 2.5% in the mixture of labrafil:linseed oil (F3E) and 60.954 ± 2.1 in the mixture of GMS:linseed oil (F1E) as compared to the rest of the formulations. It was also observed that ultrasonication method gives low entrapment efficiency for lercanidipine hydrochloride and as seen above it gives NLCs with higher particle size. Thus, the NLCs prepared by this method were not studied further. 3.5. Zeta potential measurement Zeta potential measures the surface charge of particles. As the zeta potential () increases, the particle surface charge also increases. Zeta potential greatly influences particle stability in suspension through the electrostatic repulsion between particles. The repulsive interactions will be larger between the particles as the zeta potential increases or decreases, leading to the formation of more stable particles with a more uniform size distribution. The NLC suspension had a mean zeta potential of −31.6 ± 1.5 mV. High negative charges of -potential indicate that the electrostatic repulsion between particles with the same electrical charge will prevent the aggregation of the spheres and could stabilize particle suspensions. Thus, the values obtained for the NLCs are adequate to form a stable nanoparticle suspension [20]. 3.6. Stability of the reconstituted solution The F3E formulation containing Labrafil was chosen as the final formulation due to its higher entrapment efficiency and drug content. Stability of the reconstituted solution of the Labrafil
Fig. 2. Drug release graph of in vitro diffusion study.
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Fig. 3. Ex vivo diffusion study at pH 7.4 and at pH 1.2 for 24 h.
containing NLC was checked. The reconstituted solution showed a negligible change in concentration over a period of 48 h. The drug content at 0 h was 98.50 ± 0.8% and at 48 h was 97.40 ± 1.5%. This shows that the reconstituted solution is stable for 2 days. 3.7. In vitro release kinetics of the drug from NLCs The in vitro release study using the dialysis bag technique was done using a Franz diffusion apparatus. This study is done to understand the release mechanism of lercanidipine hydrochloride from the NLC system. The in vitro lercanidipine hydrochloride release from Labrafil NLC vs plain drug release is plotted against time as shown in Fig. 2. The total drug release from NLC in 12 h is 19.36%. This indicates that at gastric pH, the unencapsulated drug is released. The inclusion of drug into lipid nanoparticles significantly reduced the drug release at acidic pH 1.2. 3.8. Ex vivo diffusion study In the present investigation, lercanidipine hydrochloride loaded NLCs produced a significantly higher release at the blood pH 7.4
(60.53%) as compared to the release at gastric pH 1.2 (23.25%) as seen in Fig. 3. The rat stomach membrane has pore diameters of about 400 nm [21]. Thus, diffusion of the NLC seems possible, their particle size being 214.47 nm and high adhesion due to very high surface may explain the increase in skin permeation. Due to the lipid nature of SLN, the NLC penetrates the skin and remain localized for a longer period of time. At the end of 24 h, the remaining NLCs were scrapped and washed off from the stomach membrane using methanol. In a fixed volume of methanol, the NLCs were sonicated and ruptured. Dilutions were made accordingly and analyzed using HPLC (Shimadzu LC 2010 HT, Japan). The amount of drug remaining after 24 h was found to be 31.34%. 3.9. Differential scanning calorimetry (DSC) studies DSC has been used to interpret the melting and crystallization behavior of crystalline materials like lipid nanoparticles. DSC interpretation confirms the melting point of lercanidipine hydrochloride to be around 192–193 ◦ C and the solid lipid peak is seen at 63.9 ◦ C. The endothermic peak of final NLC formulation
Fig. 4. DSC curves for Labrafil 2130M, lercanidipine hydrochloride, physical mixture of lercanidipine hydrochloride and Labrafil 2130M and lercanidipine hydrochloride NLC.
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Fig. 5. X-ray diffractograms of lercanidipine hydrochloride vs. NLC.
seen at 50.7 ◦ C confirms the formation of nanoparticle as seen in Fig. 4, i.e. small particle size and higher surface area and that the solid lipid has entirely covered the drug during formulation.
3.10. X-ray diffraction studies The diffraction pattern of pure drug showed that it is highly crystalline in nature as indicated by its numerous distinctive peaks with major characteristic diffraction appearing at a diffraction angle of 2 at 13.760◦ , 17.377◦ , 19.108◦ , 19.945◦ , 20.493◦ , 21.457◦ and 43.972◦ . The XRD interpretation of final NLC formulation shows more of an amorphous nature as compared to the plain drug as seen in Fig. 5. Some sharp peaks are also observed which may be due to the presence of mannitol in the NLCs which is crystalline in nature.
Fig. 6. SEM image of NLC suspension.
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Fig. 7. Systolic blood pressures after treatment.
3.11. Scanning electron microscopy (SEM) studies for NLC suspension Fig. 6 shows the SEM image of lercanidipine hydrochloride NLC almost spherical and non adherent to each other on a scale of 5 m. SEM shows irregularly shaped particles thus showing the formation of type 2 NLC. 3.12. In vivo pharmacodynamic study The results of the in vivo pharmacodynamic study are shown in Fig. 7. Blood pressure was checked at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h. Fig. 7 shows the effect of treatment on systolic blood pressure levels of hypertensive rats. At 0 h, all the groups of rats except normal control group showed a significant increase in BP. Significant decrease in systolic blood pressure level was seen in NLC fed rats as compared to plain drug fed rats (P < 0.001). The lowest blood pressure attained by the plain drug group was 118.66 ± 2.51 mm Hg at 4 h whereas, the NLC formulation performed better resulting in a significant reduction in blood pressure up to 117.23 ± 1.61 mm Hg for 8 h and up to 130.13 ± 1.97 mm Hg for 24 h. After 24 h the blood pressure of NLC group still remained below the hypertensive BP whereas the plain drug groups came back to the hypertensive state. This clearly indicated that NLCs promote oral absorption of encapsulated drug via selective uptake through lymphatic route or payer’s patches [12,13]. The entire study puts forward superiority of NLC as compared to the plain drug and thus, validates the rationale behind the study. 4. Conclusion NLCs have evolved as second-generation lipid nanocarriers which retain the advantages of SLN but unlike SLN they exhibit good physical stability and higher drug loading. In view of this, NLCs of lercanidipine hydrochloride were successfully fabricated by employing solvent evaporation technique by using Labrafil 2130M and linseed oil as the lipid phases and Tween-80 and Lutrol® F68 as lipophilic and hydrophilic surfactant phases. The components used for the fabrication of the NLCs are biocompatible. The solvent evaporation technique successfully yielded nanostructured lipid carriers with particle size of 214.47 ± 0.3 nm. The pharmacodynamic study showed that NLCs released lercanidipine hydrochloride in a controlled manner for a prolonged period of time. The overall investigations present an alternate drug delivery system for increasing the lercanidipine hydrochloride bioavailability through the nanostructured lipid carriers (NLCs) methodology.
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