Improved intestinal lymphatic drug targeting via phospholipid complex-loaded nanolipospheres of rosuvastatin calcium

Abstract

The present work describes the systematic development and characterization of nanolipospheres (NLPs) loaded with phospholipid complex of rosuvastatin for enhanced oral drug absorption trough lymphatic pathways. The construction of Job's plot revealed 3 : 1 as the apt stoichiometric ratio for formation of complex between the drug and phospholipid. The complex was characterized by FT-IR, DSC, PXRD, H¹-NMR, SEM and molecular docking studies. Evaluation of the complex for aqueous solubility and dissolution studies revealed 4.4- and 3.8-fold improvement in the solubilized fraction and release rate of the drug as compared to the physical mixture and pure drug. Further, the complex-loaded NLPs were prepared by phase inversion method and systematically optimized using Box–Behnken design, selecting the amounts of Compritol 888 (X₁) and Solutol HS15 (X₂) as the product parameters, and stirring speed (X₃) as the process parameter. The prepared formulations were evaluated for particle size, zeta potential, encapsulation efficiency and in vitro drug release. Ex vivo permeation and in situ intestinal perfusion studies revealed 4.2 to 6.5-fold improvement in the permeability and absorption of the drug from NLPs vis-à-vis the complex, physical mixture and pure drug. In vivo pharmacokinetic studies corroborated significant improvement in C_max (4.7-fold) and AUC (5.3-fold) by the complex-loaded NLPs in comparison to the physical mixture and pure drug. The lymphatic distribution studies construed 4.8-fold augmentation in the uptake of drug from NLPs over the erstwhile formulations. Pharmacodynamic studies also revealed superior efficacy of the NLPs in reducing the serum cholesterol, LDL, triglycerides, and increasing the HDL over the pure drug. Overall, the studies indicated enhanced biopharmaceutical performance of the NLPs loaded with phospholipid complex of rosuvastatin calcium.

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1. Introduction

Rosuvastatin calcium, (primarily referred to as rosuvastatin) is an antihyperlipidemic agent, which competitively inhibits the hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase and decreases the biosynthesis of cholesterol.¹ It is primarily indicated for treatment of dyslipidemia, hypercholesterolemia and hypertriglyceridemia like conditions.² Clinically, it is effective in the doses of 5 to 40 mg for reducing the elevated levels of cholesterol and low density lipids in the blood. Unlike other statins, it is considered to be highly potent and well-tolerated in humans up to the dose 80 mg for the management of hyperlipidemia.^3,4 However, being a BCS class II drug, rosuvastatin primarily exhibits poor oral bioavailability (<10%) owing to low aqueous solubility (0.01 mg mL⁻¹), limited intestinal permeability (log P of 1.92) and high hepatic first-pass effect.^2,5 A wide diversity of drug delivery strategies have been employed for enhancing the oral bioavailability of rosuvastatin, but with limited fruition as these address only the problem of poor solubility alone.^6–8

Lipid-based drug delivery systems, in this regard, offer immense promise in oral bioavailability enhancement by multiple mechanisms like improvement in aqueous solubility, dissolution rate, permeability and gastrointestinal absorption of drugs through intestinal lymphatic pathways.^9,10 Instances of these systems include lipidic dispersions, lipospheres, lipidic microparticles, solid lipid nanoparticles, nanostructured lipid carriers and self-nanoemulsifying systems.¹¹ However, these systems possess several drug delivery challenges like low solubility of the moderately lipophilic and high dose drugs in the lipidic excipients, poor drug loading, lower formulation stability, chances of gastric irritation, potential toxicity owing to higher concentration of surfactants, etc.^10,12

Of late, the lipid–drug complexes have been considered to be preferred modes of drug delivery over the aforesaid lipidic formulations for enhancing the oral bioavailability of drugs owing to their stellar merits like ease of formation, high drug loading capacity, long-term stability, and above all, ease of scalability to industrial milieu.¹³ These systems particularly uses biocompatible physiological lipids (e.g., phospholipids), which forms supramolecular complexes with drugs through weak hydrogen bonding and van der Waal's interaction forces.¹⁴ The complexation of drug molecules with phospholipids tend to enhance lipophilicity of the drugs, leading eventually to higher encapsulation efficiency, intestinal permeability and enhanced drug absorption through intestinal lymphatic pathways.¹⁵ Several literature reports have demonstrated the utility of phospholipid–drug complexes for enhancing the oral bioavailability of drugs like, diminazenediaceturate,¹⁴ decitabine,¹⁶ methotrexate,¹⁷ curcumin,¹⁸ ellagic acid,¹⁹ probucol,²⁰ rifampicin²¹ and tamoxifen.²² These literature reports vouch the immense utility of lipid–drug complexes as one of the versatile platform technologies for enhancing the oral bioavailability of drugs. However, no attempts in literature are traceable to explore the benefits of phospholipid complexes encapsulated in nanocolloidal carriers for improving the oral bioavailability of drugs through lymphatic pathways.

Attempts, therefore, have been made to explore the potential of dual formulation strategy involving use of rosuvastatin–phospholipid complex encapsulated in nanolipospheres (i.e., NLPs) for targeted delivery via intestinal lymphatic pathways for augmenting the biopharmaceutical performance and therapeutic efficacy of the drug.

2. Materials and methods

2.1 Materials

Rosuvastatin calcium was obtained ex gratis from M/s Ranbaxy Laboratories Limited, Gurgaon, India. Compritol 888 ATO was received as gift sample from M/s Gattefosse, Cedex, France. Phospholipon 90G (i.e., 90% soya phosphatidylcholine) was gifted by M/s Phospholipon GmbH, Germany, while Lutrol F188 and Solutol HS15 were gifted by M/s BASF, Mumbai, India. Solvents like dichloromethane, methanol and acetonitrile were purchased from M/s Thermo Fisher Scientific, Pennsylvania, USA, while dibasic potassium phosphate, hydrochloric acid, trehalose and Triton X-100 were purchased from the M/s Himedia Ltd., Mumbai, India. Triple-distilled and deionized water, obtained from Mili-Q distillation assembly (M/s Milipore, Mumbai, India), was employed throughout the experimental studies. All other chemicals, solvents and reagents used during the studies were of analytical reagent grade, and were used as obtained.

2.2 Preparation of rosuvastatin–phospholipid complex

The stoichiometric ratio for complexation of drug with Phospholipon 90G (i.e., PL) was determined by Job's plot method.²³ Briefly, equimolar solutions of each of drug and PL were prepared individually in dichloromethane : methanol (8 : 2, % v/v) mixture. From these stock solutions, various dilutions of drug and PL were prepared in the molar ratios ranging between 1 : 9 and 9 : 1, along with identical dilutions for the pure drug alone in the aforesaid solvent mixture. These two sets of mixtures were subjected for absorbance measurement at λ_max of the drug (i.e., 243 nm) using UV-Visible spectrophotometer (Win-UV 3000⁺, M/s Labindia, Mumbai, India). The net differences in absorbance (ΔA) between serial dilutions of the drug solution from the drug–PL mixture were determined. The Job's plot was drawn between ΔA and mole fraction of drug in the mixture for identifying the apt combination of drug and PL for formation of complex. Finally, the drug to PL ratio identified from Job's plot was refluxed at 60 °C for 30 min and evaporated under reduced pressure in a rotary evaporator to obtain the complex as solid residue. The product was passed through 30 mesh sieve, flushed with nitrogen gas and stored in a desiccator until use.

2.3 Preparation of rosuvastatin–PL physical mixture

The physical mixture of rosuvastatin with PL was prepared by admixing drug and PL in the ratio identified from the Job's plot followed by triturating both of them in a mortar–pestle for 10 min. Subsequently, the mixture was passed through 30 mesh sieve, flushed with nitrogen gas and stored in a desiccator till use.

2.4 Characterization of rosuvastatin–PL complex

2.4.1 Equilibrium solubility studies. The equilibrium solubility studies of the pure drug, physical mixture and complex was carried out in water and n-octanol by shake flask method.²⁴ Accurately weighed quantities of the pure drug (i.e., 100 mg), physical mixture and complex (equivalent to 100 mg of rosuvastatin) were taken separately in 100 mL conical flasks. Subsequently, 50 mL distilled water was added to each and stirred for 15 min to obtain uniform dispersion. The suspension was then transferred into a 250 mL separating funnel containing 50 mL n-octanol. The separating funnel was allowed to stand for 30 min with periodic shaking in every 5 min. Both the phases were separated and collected in different containers. Each fraction was subjected for extraction of the drug using methanol as the extracting solvent.²⁵ The amount of drug solubilized in aqueous and organic phase was estimated by previously developed and validated HPLC method of the drug.²⁶

2.4.2 Fourier transformed infrared (FT-IR) spectroscopy. The FT-IR spectroscopy was used for evaluating the formation of complex between drug and PL. The spectra of pure drug, PL, physical mixture and complex were recorded in KBr discs over the range of 4000–400 cm⁻¹ using an FT-IR spectrophotometer (M/s Perkin Elmer, Massachusetts, USA) and analyzed for any shift in characteristic peak(s) of the drug.

2.4.3 Differential scanning calorimetry (DSC). DSC thermograms were recorded for pure drug, PL, physical mixture and complex by placing the samples in hermetic pans at heating rate of 10 °C min⁻¹, up to 300 °C under nitrogen atmosphere using TA-DSC calorimeter (M/s TA Instruments, Delaware, USA).

2.4.4 Powder X-ray diffraction (PXRD). The diffraction patterns of pure drug, PL, physical mixture and complex were recorded in X'Pert³; powder X-ray diffractometer (M/s Panalytical, Almelo, The Netherlands). The samples were subjected to Ni-filtered, Cu kV radiation, at a voltage of 40 kV and 30 mA to observe the X-ray diffraction patterns.

2.4.5 Nuclear magnetic resonance (NMR) spectroscopy. The pure drug, PL, physical mixture and complex were dissolved in deuterated dimethylsulfoxide and ¹H-NMR spectra was recorded in Bruker Avance 400 MHz NMR spectrometer (M/s Bruker Daltonik GmbH, Bremen, Germany). The chemical shift values were recorded for the samples in parts per million (ppm).

2.4.6 Field emission scanning electron microscopy (FESEM). The FESEM studies were performed to visualize the surface morphology of pure drug and complex. Samples were mounted on aluminum stubs using double sided adhesive tape and sputter coated with a thin layer of gold at 10 Torr vacuum before examination. The specimens were scanned using an electron beam at acceleration potential of 1.2 kV and images were collected in secondary electron mode using the FESEM instrument (M/s Hitachi, Tokyo, Japan).

2.4.7 Molecular modeling and simulation studies. The computer-aided molecular modeling and simulation studies were performed for conformational analysis of the interaction between the drug and PL during complexation process. The chemical structure of rosuvastatin was drawn using Chem Draw Ultra 11.0 software and energy minimized to generate the 3D-model using ChemBio 3D Ultra-11.0 software (M/s Cambridgesoft, Cambridge, USA). Docking was performed between drug and phosphatidylcholine transfer protein (i.e., closest analogue of PL in the human body) using Schrodinger software version Glide 5.5 (M/s Schrodinger LLC, New York, USA). The protein data bank file for phosphatidylcholine transfer protein was acquired from the RSC protein data bank (PDB 1LN1) for docking studies. A grid box of 120 Å × 120 Å × 120 Å was created around the drug molecule to cover the entire molecule with fatty acid side chain of protein. One molecule of protein occupied the grid box with free 3D-movement of rosuvastatin to evaluate the interaction between them. Lamarckian genetic algorithm was then used to find the different docked conformations of rosuvastatin protein. Detailed analysis of the drug–protein interactions were carried out with the help of PyMol software (M/s Schrodinger LLC, New York, USA). The final coordinates of the drug and protein were saved in protein data bank file format for analyzing the changes in energy related parameters for the purpose.²⁷

2.5 Preparation of rosuvastatin–PL complex loaded NLPs

The drug–PL complex loaded NLPs were prepared by hot-melt emulsification and homogenization method with slight modifications.²⁸ Briefly, Compritol 888 (i.e., 200–600 mg) was heated at 80 °C and the complex (equivalent to 100 mg of rosuvastatin calcium) was dispersed in it with gentle mixing under magnetic stirring condition at 100 rpm. The aqueous solution of Lutrol F188 (0.5% w/v) and Solutol HS15 (i.e., 50–100 mg) was prepared in triple distilled water (approx. 10 mL) and added to the lipidic phase under continuous stirring condition with the help of mechanical stirrer (M/s Remi, Mumbai, India). The resultant mixture was subjected to homogenization for 30 min using laboratory scale probe homogenizer Ultra Turax (M/s IKA, Mumbai, India) in an ice bath. Further, solvent-diffusion was carried out by adding excess amount of distilled water (10 mL), followed by continuous stirring for additional 30 min at room temperature. Finally, the prepared dispersion containing NLPs was lyophilized using 5% trehalose as the cryoprotectant. The obtained dry powder was filled in glass vial, flushed with nitrogen gas and stored in a desiccator till use.

2.6 Optimization of NLPs employing experimental design

Systematic optimization of the NLPs was performed employing Box–Behnken Design (BBD), by selecting the amount of Compritol 888 (X₁), Solutol HS15 (X₂) and stirring speed (X₃) as the factors at three different levels, i.e., low (−1), intermediate (0) and high (+1) levels. Design Expert software ver. 10.1 (M/s Stat-Ease Inc., Minneapolis, USA) was employed for experimental design-guided formulation development and data analysis. ESI Table 1† illustrates the design matrix highlighting detailed composition of the prepared formulations, along with factors levels in coded units as well as in actual values. A total of 17 formulations were prepared as per the design including the quintuplicate studies at the center point (0, 0, 0) formulation. All the prepared formulations were evaluated for various quality attributes, viz. particle size, zeta potential, encapsulation efficiency and time required for 90% drug release (T_90%). Further, modelization of the experimental data, search for optimum formulation and validation studies were also performed, as already described in Section 1.1 of ESI† data text.

2.7 Characterization of rosuvastatin–PL complex loaded NLPs

2.7.1 Particle size and zeta potential. Aliquots of 1 mL of the prepared NLPs from each formulation were diluted 100-fold with distilled water and subjected for particle size distribution and zeta potential measurement by dynamic light scattering technique using Zetasizer ZS 90 instrument (M/s Malvern Instruments, Worcestershire, UK). Each sample was analyzed in triplicate and the mean values were noted.

2.7.2 Encapsulation efficiency. Encapsulation efficiency of the prepared NLPs from each formulation was determined as per the previously reported method with slight modification.^15,29 Briefly, the NLPs dispersion was centrifuged at 10 000 rpm (5590 × g), the supernatant was discarded and the sediment (pellet) was harvested. The pellet was subjected for digestion in 0.1% w/v solution of Triton-X100, followed by ultrasonication at 9 mW frequency for 15 min to allow complete removal of drug from the particles. The drug was extracted in methanol/chloroform mixture (96 : 4, % v/v) followed by vortex mixing and filtered through 0.45 μm membrane filter to remove the undissolved particles. The amount of entrapped drug was quantified by HPLC using the previously developed and validated method.²⁶ The encapsulation efficiency was calculated using eqn (1) as follows:where, W₁ represents the amount of drug encapsulated in the NLPs, and W₂ represents the total amount of drug added to the formulation.

2.7.3 In vitro drug release. The in vitro drug release studies for pure drug, physical mixture, plain complex and NLPs were carried out by dialysis bag method using 250 mL of 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) as the dissolution media for 12 h at 100 rpm and 37 ± 0.5 °C. The dialysis membrane with molecular weight cut-off, i.e., 12 kDa (M/s Himedia Limited, Mumbai, India) was employed during the study. An accurately weighed amount of the pure drug, physical mixture, plain complex and NLPs (equivalent to 20 mg of the drug) were dispersed in 1 mL of the dissolution medium and placed in the dialysis bag. Aliquot of 3 mL samples were withdrawn at predetermined time intervals followed by replenishment with an equal volume of fresh dissolution medium to maintain the sink condition. The samples were subjected to HPLC for quantifying the concentration of drug released at each time point. The raw data obtained from dissolution studies were analyzed using in-house ZOREL software with in-built provision for applying the correction factor for volume and drug losses during sampling using eqn (2),^30,31 as follows:where, C_i = corrected absorbance; A_i = absorbance of ith reading; V_s = sample volume; V_t = total volume of dissolution medium.

2.7.4 Transmission electron microscopy (TEM). An aliquot of 1 mL of the NLP dispersion was diluted 100-folds with distilled water and placed on copper grids for negative staining with 1% phosphotungstic acid solution (in phosphate buffer saline) for 30 s, followed by visualization under electron microscope, JEM-2100F (M/s Jeol, Tokyo, Japan).

2.8 Animal studies

All the studies involving animal experiments were carried out in accordance with the experimental protocols approved by the Institutional Animal Ethics Committee of the Panjab University, Chandigarh, India (reference number: PU/IAEC/S/14/110). Unisex Wistar rats (approx. 300 g) housed in polypropylene cages were kept under standard laboratory conditions at 25 ± 2 °C and 55 ± 5% RH with free access to standard diet and water ad libitum. The proper care and maintenance of the animals were undertaken following the guidelines of Committee for Prevention, Control and Supervision of Experimental Animals, Govt. of India.

2.8.1 Ex vivo permeation studies. The ex vivo permeation studies were carried out as per the previously reported method.^32,33 Rats were sacrificed by cervical dislocation, and the entire length of the small intestine was dissected out and washed with ice cold Kreb's Ringer Buffer (KRB) solution. Medial jejunum segment (i.e., middle 8–10 cm of jejunum section) was then carefully removed and washed thoroughly with KRB solution. This segment was then cut and ligated with thread to one end of a glass rod and carefully everted. One gram weight was tied at the end of everted gut segment to make an empty gut sac, and to prevent peristaltic contractions. The gut sac was filled with KRB solution and then placed inside the bath containing 50 mL of KRB solution. The bath was maintained at 37 ± 0.5 °C by an outer water jacket, followed by continuous supply of atmospheric air at 10–15 bubbles per minute. Accurately weighed amount of pure drug, physical mixture, complex and optimized NLPs (equivalent to 20 mg dose of rosuvastatin) were poured in the bath medium outside the gut sac. An aliquot (1 mL) sample was withdrawn from the gut sac at periodic time intervals (i.e., 5, 10, 15, 30 and 45 minutes), followed by replacement with fresh KRB solution and analyzed by HPLC to determine the amount of drug permeated and percent drug permeated in 45 minutes (Perm_{45 min}). Also the flux and permeation rate constant were calculated from the plots constructed between the cumulative percent of drug permeated versus time for each of the treatment formulations.

2.8.2 In situ intestinal perfusion studies. In situ intestinal perfusion studies were carried out after slight modification of the protocol, already described in various literature reports.^32–34 The animals were anesthetized using intraperitoneal injection of thiopental sodium in the dose of 50 mg kg⁻¹. The abdomen was opened up with a midline incision, and the entire small intestine was taken out carefully. The proximal part of the jejunum 2–5 cm below the ligament of Treitz was cannulated followed by second incision at 10–15 cm below the first incision. The intestinal segment was perfused with KRB solution maintained at 37 ± 1 °C until the perfusate became clear. Further, the intestine was perfused with treatment formulations viz. pure drug, physical mixture, complex and optimized NLPs (equivalent to 20 mg of rosuvastatin) at a perfusion rate of 0.2–0.3 mL min⁻¹. At specified time intervals, i.e., 5, 15, 30 and 45 minutes, the perfusates were collected and analyzed using HPLC after suitable dilutions. Various permeability parameters like, effective permeability (P_eff) and wall permeability (P_wall), and absorption parameters like, fraction absorbed (F_a) and absorption number (A_n) were calculated using the standard formulae given in literature reports.^35,36

2.8.3 In vivo lymphatic uptake studies. In vivo lymphatic uptake studies were carried out in rats under overnight fasting condition. The animals were divided into four groups with three animals each. Various treatment formulations viz. pure drug, physical mixture, complex and NLPs, each containing 20 mg kg⁻¹ of rosuvastatin calcium, were administered to the animals through peroral route. At predetermined time intervals of 0, 1, 3, 6 and 12 h, the rats were anaesthetized by 50 mg kg⁻¹ dose of intraperitoneal injection of thiopental sodium, followed by cannulation of the mesenteric lymph duct with the help of polyethylene tubing (15 cm length, i.d. 0.58 mm, o.d. 0.965 mm).³⁹ The lymph (approx. 0.2 mL) was collected in eppendorf tubes at the specified time periods and the drug was extracted by adding suitable volume of acetonitrile, followed by centrifugation at 10 000 rpm for 15 min. The supernatant organic fraction was collected and the organic phase was evaporated. The remaining dry residue was subsequently reconstituted in mobile phase and analyzed through previously developed and validated HPLC method of the drug in lymph. The concentration of drug uptaken in lymph was estimated from the previously constructed calibration plot of the drug in lymph and the obtained data was plotted with respect to time.⁴⁰

2.8.4 In vivo pharmacokinetic studies. A single dose and parallel design in vivo pharmacokinetic study was conducted in rats. The animals were divided into four groups (I–IV) with six animals in each group. The animals were administered with various treatment formulations viz. pure drug, physical mixture, complex and optimized NLPs, each containing rosuvastatin equivalent to 20 mg kg⁻¹, respectively. The formulations were administered through peroral route to the animals with the help of oral feeding cannula. Blood samples (approx. 0.2 mL) were periodically withdrawn from the retro-orbital plexus of the animals at specified time intervals like 0, 0.5, 1, 2, 3, 4, 6, 12, 16, 20 and 24 h in micro-centrifuge tubes containing heparin (10 μL) as the anticoagulant. Plasma was harvested by centrifugation at 10 000 rpm (5590 × g) for 10 min, followed by extraction of drug using acetonitrile. Further, the samples were subjected to centrifugation at 10 000 rpm (5590 × g) for 10 min for separation of the plasma proteins. The supernatant fraction was collected and the organic phase was completely evaporated to dryness using water bath maintained at 80 °C. The stable behaviour of drug at high temperatures is already documented in the literature reports thus corroborating the absence of any change during forced degradation studies.^37,38 Further, after evaporation of the organic phase, the remaining dry residue of the drug was reconstituted with mobile phase and filtered through 0.22 μm membrane filter (M/s mdi Technologies, Mumbai, India) to obtain the clear solution. The filtrate obtained was filled in the vials and subjected to HPLC analysis as per the previously developed and validated bioanalytical method of the drug in rat plasma.²⁶

Computer-based pharmacokinetic modeling and data analysis on plasma concentration–time data were carried out using Win-Nonlin software ver. 5.0 (M/s Pharsight Inc., California, USA). One-compartment open body model (1-CBM) with zero lag-time for extravascular administration was employed by selecting Model 3 in the software for Wagner–Nelson method. Various pharmacokinetic parameters like maximum observed plasma concentration during the study period (C_max) and the corresponding time (t_max), area under the curve (AUC_0–t), elimination half-life (t_1/2) and absorption rate constant (K_a) were computed and their statistical validity was ratified on the basis of minimization of various model fitness parameters like Akaike Information criterion (AIC), Schwartz criterion (SC), sum of squares of residuals (SSR) and maximization of Pearsonian correlation (R).

2.8.5 In vivo pharmacodynamic studies. In vivo pharmacodynamic studies were carried out in rats induced with dyslipidemia by administering high fat diet and free access to water ad libitum up to 4 week.⁴¹ After attaining the experimental dyslipidemia like condition with total cholesterol level >350 mg dL⁻¹, the animals were divided into five groups (I–V), each containing six rats. The animals in Group I were act as control, while the animals in Group II–V were orally administered with pure drug, physical mixture, complex and optimized NLPs. Blood samples were collected in heparin-coated glass vials from the animals under light ether anesthesia by puncturing the retro-orbital plexus on periodic time intervals, i.e., 7th, 14th and 21st day, respectively. The plasma was harvested by centrifugation at 10 000 rpm (5099 × g) for 10 minutes and stored under frozen conditions until further use. The samples were analyzed for estimating the levels of total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglycerides (TG) using ENZOPAK enzymatic diagnostic kit (M/s Reckon Diagnostics, Gujarat, India).

2.8.6 Statistical data analysis. All the experimental data from the in vitro, ex vivo and in vivo studies were expressed as mean ± standard deviation (SD). Statistical analysis was performed by Garph Pad Prism® software ver 6.01 (M/s GraphPad Inc., CA, USA) using two-way ANOVA followed by Tukey–Kramer multiple comparison test at 5% level of significance.

3. Results and discussion

3.1 Preparation of rosuvastatin–phospholipid complex

The drug–PL mixtures were prepared in various molar ratios (1 : 1 to 1 : 9, and vice versa) and subjected to UV absorbance measurement. Fig. 1 illustrates the Job's plot between the net difference in the absorbance versus molar ratios of drug–PL mixture. The maximum was observed at the stoichiometric ratio of 3 : 1 (drug : PL), confirming the formation of stable complex of drug with PL. This indicated the existence of plausible interaction between three molecules of drug with one molecule of phospholipid through weak hydrogen bonding between hydroxyl groups of rosuvastatin with hydrogen atom on the methyl group attach to quaternary amine moiety of PL. Thus, ratio 3 : 1 was selected as the optimum combination of drug and PL for forming complex and subjected for characterization studies.

Fig. 1 Job's plot depicting the net difference in absorbance of drug–PL90G mixture at different molar ratios.

3.2 Characterization of rosuvastatin–phospholipid complex

3.2.1 Equilibrium solubility studies. Table 1 enlists the equilibrium solubility data of pure drug, physical mixture and complex in water and n-octanol at 25 °C. The results indicated significant increase in solubility of the drug from complex in both water and n-octanol vis-à-vis the physical mixture and pure drug. Nearly 1.54- and 4.37-fold augmentation in the solubility of drug was observed in aqueous and organic phase from the complex over the physical mixture and pure drug, respectively. The results revealed that rosuvastatin being a sparingly soluble drug with low lipophilicity (i.e., log P of 1.92), the complex formation with phospholipid facilitated significant improvement in hydrophilic and lipophilic characteristics.^42,43 This indicated significant improvement in the solubility of the drug from the prepared complex compared to its free counterpart.

Table 1 Solubility data of pure drug, physical mixture and complex, in water and n-octanol at 25^a °C

Sample type	Solubility in water (μg mL^−1)	Solubility in n-octanol (μg mL^−1)
a All the data represented as mean ± S.D. (n = 3).
Pure drug	18.28 ± 1.25	7.59 ± 0.41
Physical mixture	21.61 ± 0.56	16.14 ± 0.68
Drug–PL complex	28.79 ± 0.39	33.21 ± 1.07

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3.2.2 Fourier transform infrared (FTIR) spectroscopy. The FTIR spectra of pure drug, PL90G, their physical mixture and complex are shown in Fig. 2. Comparative spectral analysis revealed mild shift in the absorption peaks of the drug in complex as compared to the physical mixture and pure drug. The drug–PL complex showed shift in the position of O–H peak of the drug from 3333.53 cm⁻¹, and quaternary nitrogen peak of the PL from 3405.98 cm⁻¹ to 3357.21 cm⁻¹. Moreover, the O–H peak of the drug was quite broad appearing as a single peak, while the quaternary nitrogen peak of PL was relatively suppressed, thus confirming the merger of two aforesaid functional peaks. This can be attributed to the formation of weak intermolecular hydrogen bonding interactions between the two functional groups, leading eventually to the shift in peak area and ratifying the formation of drug–PL complex. However, other signature peaks of the drug remained intact construing no significant change in the complex.

Fig. 2 FT-IR spectra of rosuvastatin, PL90G, physical mixture and complex.

3.2.3 Differential scanning calorimetry (DSC). Fig. 3 illustrates the overlaid DSC thermograms of pure drug, PL, physical mixture and complex. The pure drug showed a broad endothermic peak at 145.14 °C, indicating its semi-crystalline nature, while PL showed absence of any melting event ostensibly owing to its amorphous nature. On the other hand, endothermic peak of the drug was not present in the complex, thus confirmed vanishing of semi-crystalline nature of the drug owing to the formation of hydrogen bond and van der Waal's interaction forces with PL.^18,22

Fig. 3 DSC thermograms of rosuvastatin, PL90G and complex.

3.2.4 Powder X-ray diffraction (PXRD). The PXRD spectra of pure drug, PL, physical mixture and complex are shown in Fig. 4. The pure drug and PL indicates their semi-crystalline and amorphous nature, respectively. The physical mixture showed no significant change in the PXRD pattern. However, the complex showed disappearance of characteristic sharp peaks of the drug owing to transformation from semi-crystalline to amorphous state ostensibly by formation of hydrogen bonding with PL to form complex.^20,21 Moreover, the results obtained from the PXRD studies were found to be in accordance with the FT-IR and DSC studies.

Fig. 4 PXRD spectra of rosuvastatin, PL90G, physical mixture and complex.

3.2.5 Nuclear magnetic resonance (NMR) spectroscopy. The ¹H-NMR spectra of pure drug, PL, physical mixture and complex are portrayed in Fig. 5. The ¹H-NMR spectrum of drug–PL complex when compared with the spectra of pure drug revealed mild alteration in the chemical shift values of 3H from δ 4.33 to δ 4.19, and 6H from δ 4.41 to δ 4.32, respectively. This indicated downfield nature of chemical shift values, and confirmed the formation of drug–PL complex plausibly by hydrogen bonding interactions between the free hydroxyl groups of the drug with quaternary amine moiety of the PL. Moreover, the results obtained from the NMR studies were in consonance with the interpretation discerned from FT-IR and DSC studies.

Fig. 5 ¹H-NMR spectra of rosuvastatin, PL90G, physical mixture and complex.

3.2.6 Field emission scanning electron microscopy (FESEM). Fig. 6 depicts the surface morphology of the pure drug and its complex with PL. The surface morphology of pure drug showed appearance of rod-shaped crystals, while the complex showed complete absence of crystalline nature of the drug. This can be attributed to complete masking of the crystallinity of the drug owing to complexation with PL, which causes polymorphic changes in the crystal habit of the drug leading to its transformation into amorphous state. Further, the observations obtained from FESEM imaging were found to be inconsonance with the DSC and PXRD results for the purpose.^20,22

Fig. 6 SEM photomicrographs of the rosuvastatin and complex.

3.2.7 Molecular docking and simulation. The molecular docking and simulation studies revealed plausible site(s) of interaction between the drug and PL analogue (i.e., phosphatidylcholine transfer protein). Mechanistically, the studies revealed formation of weak intermolecular hydrogen bonding interactions between the drug and protein. Fig. 7 illustrates the docking patterns of the drug, where the yellow color dotted lines represent the hydrogen bonding between the free hydroxyl groups of the drug with terminal quaternary amine group of protein. This can be attributed to the mild reduction in the activation energy of the drug upon complexation with protein by forming H-bonds, leading eventually to minimization of the total free energy for attaining thermodynamically stable confirmation. Change in the free energy was particularly observed for the free hydroxyl groups present on the same plane on the drug exhibiting docking energy of −2.11 kcal mol⁻¹ and −1.58 kcal mol⁻¹, respectively, as compared to other hydroxyl groups exhibiting docking energy of −1.81 kcal mol⁻¹. Overall, the molecular docking studies confirmed that the drug is able to form complex with phosphatidylcholine transfer protein, plausibly owing to its structural similarity with phospholipid in its physiochemical properties.⁴⁴

Fig. 7 Molecular docking images depicting binding of rosuvastatin with phosphatidylcholine transfer protein.

3.2.8 In vitro drug release. Fig. 8 illustrates the in vitro drug release profiles of pure drug, physical mixture and the complex in 0.1 N HCl and pH 6.8 phosphate buffer solution. In both media, the pure drug and physical mixture showed almost analogous profile with a total of 24.4% to 28.7% and 26.8% to 29.5% drug release in 2 h, respectively. On the other hand, the complex showed faster and nearly complete drug release (>90%) within 2 h of the dissolution studies, with insignificant difference (p > 0.05) in the drug release profile between both the media. This revealed pH-independent drug release profile of the prepared formulation, which is considered to be highly beneficial for rapid absorption of the drug through GI tract. Further, comparative evaluation of the drug release profiles revealed highly significant differences in the dissolution of pure drug and complex, as is evident from the higher values of dissimilarity factor (f₁) of 45.8 and lower value for similarity factor (f₂) of 28.9 between them. The studies indicated improved dissolution performance of the drug upon complexation with PL molecule with nearly 3.8-fold improvement in the drug release rate vis-à-vis the pure drug. This can be explained owing to facilitation of drug solubilization by amphiphilic nature of PL in the prepared complex. Besides, conjugation of drug molecules with PL tends to form micellar structures, where polar head groups of the PL plays pivotal role in minimizing the surface interfacial tension, leading eventually to faster dissolution rate and enhanced dissolution performance as compared to the free drug.^45,46

Fig. 8 In vitro drug release profile of pure drug, physical mixture and complex in 0.1 N HCl (A), and pH 7.4 PBS (B); data points expressed in mean ± S.D. (n = 3).

3.3 Characterization of rosuvastatin–PL complex loaded NLPs

3.3.1 Particle size and zeta potential. The mean particle size distribution of all the prepared NLPs was found to be ranging between 32.4 and 112.5 nm, with polydispersity index 0.121 to 0.176, indicated the nanostructured nature of the developed formulations. All the factors selected during optimization studies exhibited significant influence on particle size distribution. The smaller particle size was observed at lower levels of lipid (i.e., Compritol 888) and stirring speed, and intermediate levels of surfactant (i.e., Solutol HS15). Increase in the concentration of lipid tends to produced larger particles owing to increase in surface area acquired by the lipidic phase to form the dispersion.⁴⁷ However, Solutol HS15 helped in reducing the particle size owing to its surfactant like properties, which helps in minimizing the surface interfacial tension and stabilizes the dispersion.⁴⁸

Likewise, zeta potential evaluation revealed values ranging between 11.2 and 28.6 mV for the NLPs prepared as per the experimental design. The positive values of zeta potential indicated thermodynamically stable nature of the prepared formulations, which helps in minimizing the particle growth and aggregation during storage.⁴⁹ This can be attributed to the net charge provided by Lutrol F188 and Solutol HS15, leading eventually to generation of stable surface charge on the system.^50,51

3.3.2 Encapsulation efficiency. The prepared NLPs showed values of encapsulation efficiency ranging between 64% and 89%. Higher encapsulation was observed at higher levels of Compritol 888 and Solutol HS15. Owing to the highly lipophilic characteristics of the Compritol 888, it is known to facilitate higher encapsulation of the drugs when used at higher levels, while Solutol HS15 tend to augment encapsulation efficiency by its solubilization property.⁵² Stirring speed also showed significant influence on the encapsulation efficiency, as it provides uniform congealing of the lipidic phase to produce the uniform dispersion.⁵³ Higher stirring speed showed lower values of encapsulation efficiency ostensibly owing to disruption of particles under high shearing forces.

3.3.3 In vitro drug release. The in vitro drug release profiles of the NLPs (F1–F13) performed in 0.1 N HCl and pH 6.8 PBS revealed quite analogous drug release profile (data not shown). All the formulations exhibited faster drug release characteristics with more than 60% release between 3 to 7 h and almost complete drug release within 12 h. This can be ascribed to the presence of varying levels of lipid and surfactant, which tend to influence the release profile of the drug from the prepared formulations. Faster drug release was observed for formulations (i.e., F2, F3, F7, F9) containing lower levels of lipid, while moderately slower drug release was observed for formulations (i.e., F4, F6, F10, F11, F12), ostensibly owing to the intermediate levels of lipid retarding the drug release rate. Comparatively more sustained drug release profile was observed at higher levels of lipid (i.e., F1, F5, F8, F13). The evaluation of drug release kinetic revealed highly predominant influence of Fickian-diffusion mechanism, as is clearly evident from the valued of “n” ranging between 0.23 and 0.41 studied. As Compritol 888 tend to form a spherical matrix structure, it facilitate slower release liberation of the drug from the system though diffusion phenomena.⁵⁴ Thus, the concentration of Compritol 888 showed higher influence on the values of “n” owing to its direct influence on the drug release rate by diffusion mechanism.^55,56

3.4 Optimization data analysis and validation studies

The generation of second-order quadratic polynomial model and data analysis was performed by multiple-linear regression analysis (MLRA). ESI data Table 2† enlist the coefficients of the polynomial equations obtained for each of the response variables. Further, the response surface analysis was conducted for analyzing the factor–response relationship. The detail interpretation on response surface analysis has been included in Section 1.2 of ESI† data text. The search for optimum formulation was carried out by “trading-off” various response variables to attain the desired objectives by minimization of the particle size (i.e., imperative for ease of permeation and absorption of the drugs), and maximization of encapsulation efficiency and T_90% (i.e., necessary for maximizing the absorption of drugs), and zeta potential (i.e., imperative for formulation stability). Based on the aforesaid objectives, the selection criterion was embarked upon for selecting the optimized formulation with particle size <100 nm, zeta potential >20 mV, encapsulation efficiency >80% and T_90% < 6 h. The optimized formulation was identified by numerical optimization with desirability function close to unity. Fig. 9 portrays the optimized formulation of NLP demarcated in the design space overlay plot, which contained Compritol 888 (422 mg), Solutol HS15 (89 mg) and stirring speed (3000 rpm), which corresponds to the particle size of 48.9 nm, zeta potential of 23.2 mV, encapsulation efficiency of 82.05% and T_90% of 4.6 h, respectively. ESI data Fig. 2† portrays the particle size distribution and TEM image of the optimized NLPs, which confirmed the appearance of particles in the nanosized range.

Fig. 9 Overlay plot depicting the design space region and optimized NLP formulation (flagged).

Validation of the DoE methodology revealed close proximity among the predicted and observed values of the check-point formulations. The linear correlation plots demonstrated high values of “r” ranging between 0.997 and 0.999, thus ratifying excellent goodness of fit of the observed and predicted data (p < 0.001 in each case). The percent prediction error (or percent bias) for the response variables varied between −2.24% and 3.51% with overall mean ± SD as −0.89% ± 1.05. Besides, the corresponding residual plots also showed nearly uniform distribution of the data and absence of any assignable variation around the zero-axis (data not shown), indicating the high degree of prognostic ability of the employed experimental methodology.

3.5 Ex vivo permeation studies

Fig. 10 portrays the ex vivo permeation profiles of various test formulations, which revealed that pure drug and physical mixture showed P_app values of 34% and 41% in 6 h, while the complex and NLPs revealed P_app greater than 85% within 3 h of the study period. This revealed enhanced permeation characteristics of the drug from the prepared NLPs and complex vis-à-vis the physical mixture and pure drug. Nearly 3.15-fold improvement in the permeability of the drug was observed from NLPs and complex as compared to the physical mixture and pure drug. The values of permeation flux of the drug also showed significant augmentation from the NLPs (3.06 μg cm⁻² h⁻¹) and complex (2.45 μg cm⁻² h⁻¹) over the physical mixture (1.02 μg cm⁻² h⁻¹) and pure drug (0.81 μg cm⁻² h⁻¹). The above results corroborated enhanced permeability characteristics of the developed formulations. This can be ascribed to the lipophilic nature of the prepared formulation coupled with nanosized structure, which facilitate faster permeation of the drug through intestinal barrier.⁵⁷ The formation of unique nanoscale supramolecular assemblies after oral administration of the prepared formulations plays a pivotal role for faster permeation of the drugs through GI environment. Besides, the presence of PL also helps in augmenting permeability, where polar head groups are responsible for drug solubilization, while non-polar groups helps permeation by loosening the epithelial tight junctional barrier.²⁵

Fig. 10 Ex vivo permeation profile of pure drug, physical mixture and complex during everted gut-sac technique; data points expressed in mean ± S.D. (n = 3).

3.6 In situ intestinal perfusion studies

Table 2 illustrates various permeability and absorption parameters for the pure drug, physical mixture, complex and NLPs indicating a remarkable increase in the values of P_eff, P_wall, F and A_n, respectively. Further, the comparative evaluation of net increase in permeability and absorption parameters by the developed formulation was calculated with respect to the pure drug (Fig. 11). The NLPs showed 1023% and 1456% increase in P_eff and P_wall, while complex showed nearly 696% and 848% increase in the P_eff and P_wall as compared to the pure drug. However, the physical mixture showed only 198% and 297% increase in the values, respectively. This confirmed significant improvement in the permeability of the drug from NLPs and complex ostensibly owing to presence of PL and its potential effect on drug solubilization capacity by avoiding precipitation of the drug in the GI environment.⁵⁸

Table 2 Values of permeability and absorption parameters of various treatment groups during intestinal perfusion studies

Treatment groups	Permeability parameters		Absorption parameters
	Peff^a	Pwall^a	Fa	An
a Peff and Pwall values expressed as ×10^−4 cm s^−1. All the data represented as mean ± S.D. (n = 3); NLPs: nanolipospheres.
Pure drug	2.78 ± 1.02	3.42 ± 0.79	0.021 ± 0.44	0.018 ± 1.33
Physical mixture	2.98 ± 3.41	3.97 ± 1.15	0.034 ± 0.89	0.027 ± 2.56
Drug–PL complex	7.96 ± 1.76	9.48 ± 2.34	0.065 ± 0.76	0.083 ± 3.87
NLPs	11.23 ± 2.78	15.56 ± 3.62	0.109 ± 1.58	0.136 ± 1.49

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Fig. 11 In situ intestinal perfusion study data depicting the percent increase in permeability and absorption parameters vis-à-vis the pure drug; values expressed in mean ± S.D. (n = 3).

Like permeability parameters, the dimensionless absorption parameters (i.e., A_n and F) also revealed drastic improvement in their values from the complex and NLPs vis-à-vis the pure drug. Fig. 11 also illustrates the percent increase in absorption parameters with respect to the pure drug. In this regard, the NLPs showed 427% and 628% improvement in A_n and F, while the complex showed 215% and 346% increase, respectively. On the contrary, the physical mixture revealed only 67% and 48% rise in the absorption parameters as compared to the pure drug, respectively. This can be explained by the faster permeation of the drug through intestinal paracellular and transcellular pathways.⁵⁹ Besides, the transportation of formulation through intestinal lymphatic pathways also helps in increasing the drug absorption parameters.⁶⁰

3.7 In vivo lymphatic uptake studies

Fig. 12 illustrates the lymphatic uptake of drug after oral administration of various test formulations with respect to time. Maximal uptake (211.4 ± 2.78 μg mL⁻¹) of the drug was observed from NLPs in 3 h time period, while the plain complex showed uptake up to 124.5 ± 4.12 μg mL⁻¹, respectively. On the contrary, the physical mixture and pure drug showed maximal uptake up to 52.1 ± 3.59 μg mL⁻¹ and 47.0 ± 1.89 μg mL⁻¹ in 3 h. The results indicated nearly 2.1- and 4.9-fold improvement in the uptake of drug from complex and NLPs, while the physical mixture showed only 0.3-fold improvement vis-à-vis the pure drug. This construed significant improvement in the uptake of drug in lymph from the NLPs and plain complex vis-à-vis the physical mixture and pure drug (p < 0.05), ostensibly owing to the absorption and transportation of the drug through intestinal lymphatic pathways. Faster the rate of drug absorption through lymph indicates superior drug absorption characteristics.³⁹ Multiple factors tend to influence the lymphatic absorption of drugs including lipid chain length, partition coefficient, and above all, formation of chylomicron end products, which possess higher affinity for transportation of the drugs through lymph.^11,61

Fig. 12 Bar chart depicting in vivo lymphatic uptake of rosuvastatin in rats treated with pure drug, physical mixture, complex and NLPs at different time intervals. Values expressed in mean ± S.D. (n = 6).

3.8 In vivo pharmacokinetic studies

Fig. 13 depicts the mean plasma concentration versus time profile observed after peroral administration of NLPs, complex, physical mixture and pure drug. Highly statistically significant difference in plasma concentration was observed for NLPs and plain complex over the physical mixture and pure drug at all the time-points (p < 0.0001). The pharmacokinetic data analysis explored using compartmental models indicated best model fitting with one-compartment open body model, as is evident from highly significant values of the statistical model parameters like, R, AIC, SBC and SSR.

Fig. 13 Graph depicting plasma concentration of rosuvastatin at various time-points from the pure drug, physical mixture, complex and NLPs formulation; values expressed in mean ± S.D. (n = 6).

As per the chosen model, the pharmacokinetic parameters showed superiority in the oral bioavailability owing to increase in the extent and rate of drug absorption (i.e., AUC, C_max, t_max, K_a and t_0.5) for the NLPs and complex vis-à-vis the physical mixture and pure drug (Table 3). Nearly 3.73- and 4.35-fold augmentation in the values of C_max and AUC was observed by NLPs, while the plain complex showed 2.66- and 2.01-fold enhancement as compared to the pure drug, respectively. However, the physical mixture showed only 1.04- and 2.70-fold increase in C_max and AUC vis-à-vis the pure drug, respectively. On the other hand, the NLPs showed 59% reduction in t_max as compared to the pure drug, while the plain complex and physical mixture revealed 30% and 13.5% reduction in the t_max vis-à-vis the pure drug, respectively. This confirmed faster onset of action owing to significant improvement in the rate of drug absorption from the prepared formulations.

Table 3 Pharmacokinetic data of rosuvastatin from various treatment groups in rats^a

Parameters (units)	Pure drug	Physical mixture	Complex	NLPs
a All the data represented as mean ± S.D. (n = 6).
Tmax (h)	1.94 ± 0.89	1.73 ± 1.28	1.41 ± 0.95	0.83 ± 1.55
Cmax (μg mL^−1)	183.95 ± 4.56	243.96 ± 2.71	674.17 ± 1.24	869.38 ± 2.12
AUC0–24 h (h μg mL^−1)	671.56 ± 5.64	1378.34 ± 4.11	2018.59 ± 3.79	3593.58 ± 2.48
Ka (h^−1)	1.19 ± 0.42	1.22 ± 0.96	1.34 ± 0.78	1.42 ± 0.26
t0.5 (h)	0.48 ± 0.84	0.51 ± 1.01	0.57 ± 1.22	0.64 ± 0.54

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Apart from these parameters, maximal change observed in K_a indisputably vouch the enhanced oral drug absorption characteristics of NLPs revealing nearly 2.9-fold increase followed by plain complex with 2.18-fold over the physical mixture and pure drug. On the contrary, remarkably significant reduction the in values of t_0.5 of the drug (i.e., 34.2- and 18.6-fold) also vouched superior drug absorption characteristics from NLPs and plain complex vis-à-vis the physical mixture and pure drug.

Overall, the pharmacokinetic studies corroborated supremacy of NLPs in augmenting the oral absorption of rosuvastatin over the plain complex. The enhanced absorption potential of the developed formulations can be attributed to the myriad mechanistic pathways. These primarily include the nanostructured nature of the NLPs facilitating drug absorption through intestinal transcellular and paracellular pathways.⁵⁷ Besides, the blend of lipids, solubilizers and absorption enhancers also helps in enhancing the drug absorption into the systemic circulation plausibly through micellar solubilization, followed by digestion in the GI fluid to produce the chylomicrons.¹⁰ This can be ascribed to the manifold enhancement in the permeability and absorptivity parameters of the drug, as is evident from ex vivo and in situ perfusion studies. Also, the role of lymphatic uptake of the drug through lipidic formulations is considered to be pivotal in augmenting the absorption of a drug into the systemic circulation.^61–63

3.9 In vivo pharmacodynamic studies

In vivo pharmacodynamic studies following administration of high fat diet exhibited significant elevation in the serum lipid levels in all the animals exhibiting hyperlipidemia-like condition. Post-treatment with various test formulations viz. NLPs and complex revealed remarkable alteration in the levels of serum lipids (i.e., TC, LDL, HDL and TG) vis-à-vis the physical mixture and pure drug. ESI data Table 3† illustrates the plasma lipid level data in control and treatment formulations at different time intervals, i.e., 7, 14 and 21 days. All the treatment formulations exhibited pharmacodynamic effects in altering the serum lipid levels from 7th day onwards up to 14th and 21st days, with statistically significant differences among all the treatment groups (p < 0.001). Fig. 14 portrays percent alteration in serum levels of various lipids after oral administration of various treatment formulations.

Fig. 14 Bar chart depicting percent alteration in serum lipid levels after 21 days of treatment with pure drug, physical mixture, complex and NLPs formulation in hyperlipidemic rats. Values expressed in mean ± S.D. (n = 6).

Among all the treatment formulations, NLPs revealed superior alteration in the levels of pharmacodynamic parameters as compared to the plain complex, physical mixture and pure drug. Nearly 65%, 60%, 44% reduction in TC, LDL and TG was observed by NLPs with respect to the control group (p < 0.001). Likewise, the complex also showed significant results for reduction in the levels of TC (52%), LDL (45%) and TG (35%) within 21 days of study period over the control group (p < 0.001). However, the physical showed 46%, 20% and 31% reduction in the values of TC, LDL and TG, while pure drug indicated 41%, 18% and 22% reduction vis-à-vis the control group, respectively (p < 0.05).

The observations also demonstrated that the complex loaded NLPs were found to be superior over the physical mixture and pure drug in reducing more than 50% of TC, LDL and TG within 7 days of treatment period. Also, the complex loaded NLPs showed statistically significant difference for reduction in the levels of TC, LDL and TG during all the studied time points (p < 0.001) over the physical mixture and pure drug, respectively. On the contrary, the above observations were further supported by significant elevations in the levels of HDL. Significant improvement in serum HDL level was observed by the NLPs and complex (i.e., 201% and 118%) post 21 days of treatment period (p < 0.0001), while the physical mixture and pure drug showed only 90% to 82% improvement (p < 0.001), respectively. The rise in HDL levels construed amelioration in the treatment efficacy by the developed formulation, where HDL is known as “good cholesterol” beneficial for house-keeping functions.^32,64 This construed high degree of supremacy of the NLPs in normalizing the elevated levels of TC, LDL and TG, which are considered to be the key markers in aggravating the dyslipidemia and hyperlipidemia like conditions.

Overall, the results corroborated improved biopharmaceutical performance of the prepared complex over the physical mixture and pure drug in the management of experimental hyperlipidemia. This can be ascribed to the attainment of superior plasma concentration of the drug, leading eventually to drastic reduction in the elevated levels of serum lipid when administered in NLPs.⁶⁵

4. Conclusions

The present studies demonstrated the successful development of NLPs loaded with phospholipid complex of rosuvastatin calcium for enhancing its oral bioavailability. In this regard, the implementation of dual formulation strategies not only improved the physicochemical attributes of the rosuvastatin, but also showed substantial enhancement in the oral bioavailability and the consequent pharmacodynamic performance. Use of experimental designs helped in judicious selection of the apt blend of lipidic excipients, stabilizers and process parameters for formulation of the optimized NLPs. Extensive characterization of the developed formulations through a gamut of in vitro, ex vivo, in situ and in vivo studies corroborated considerable enhancement in the biopharmaceutical performance of the drug. In a nutshell, the studies ratify formulation of drug–phospholipid complex loaded nanocolloidal carriers as the novel formulation strategy with immense potential for addressing the biopharmaceutical challenges encountered with rosuvastatin, and other similar BCS class II and class IV drugs too.

Declaration of interest

Authors declare no conflict(s) interest.

Acknowledgements

The financial grants received from the University Grant Commission (UGC), New Delhi, India, to Mr Sarwar Beg under the RFMS scheme-F. No. 5-94/2007(BSR) is deeply acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra24278a

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