Lipid nanocarrier-based transport of docetaxel across the blood brain barrier

Abstract

Successful treatment of brain cancer remains a formidable challenge in neuroscience research due to sub-therapeutic permeation of conventional chemotherapeutics across the blood–brain barrier (BBB). By optimizing various conditions and process parameters, we developed a phospholipid based nanosize carrier (NL) encapsulating docetaxel (DTX) and investigated its BBB crossing potential, both qualitatively and quantitatively, in vivo. The optimized NLs had a nanosize below 100 nm, smooth surface with intact lamellarity, 7.8% drug loading and a sustained drug release profile in vitro. Pharmacokinetic and biodistribution data showed an enhanced residence time of the drug in blood and efficient permeation of the drug from the DTX loaded NL through the BBB, as compared to free DTX. The technetium-99m labeled NL effectively crossed the BBB and accumulated in the brain tissue in a time dependant manner compared to technetium-99m labeled DTX. NL may provide a promising platform for an improved management of brain cancer.

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Introduction

Effective treatment of brain cancer remains a tough challenge due to the inefficiency of many current therapeutics to cross the blood brain barrier (BBB).¹ The BBB comprises of brain endothelial cells with tight junctions and it limits paracellular transit of substances between the blood and brain extracellular fluid, thus making the brain a formidable zone for most of the chemotherapeutics and diagnostic agents. To combat this, novel strategies are being investigated over the years to facilitate prognosis and treatment of brain cancer. Nanosize lipid-based vesicular drug delivery system is currently in the limelight as a promising drug carrier in various therapies, including brain cancer. The architectural uniqueness of such nanosize phospholipid-based carrier (NL) compensates for the blood brain barrier (BBB)-limiting characteristics of the conventional therapeutics.² The lipophilic feature coupled with nanosize facilitates NL to cross BBB to enter into the brain by endocytosis. Furthermore, due to their biocompatibility and ability of sustained drug release, the dose related side effects are significantly reduced.³

Docetaxel (DTX), an important member of taxanes, exhibits notable anticancer efficacy against various cancers.⁴ Though DTX structurally resemblances with paclitaxel (PTX), but it is almost twice more effective as inhibitor of microtubule depolymerization than PTX.⁵ However, its use in the treatment of glioma is highly limited due to its low BBB permeation capacity.⁶ Most of the available commercial formulations of DTX are unable to cross BBB efficiently, and are also accompanied with several side effects such as allergic reactions, nephro-toxicity, neuro-toxicity, low white blood cell count, cumulative fluid retention etc.^7,8 In recent years, numbers of alternative sub-micron novel delivery systems have been reported for delivery of DTX to brain cells.⁶ However, reports on the clear evidence of increased BBB permeation and sustainability of DTX within the brain by in vivo imaging technique are still scarce. As a result an optimized delivery system of DTX for clinical application in glioma therapy is yet to be well-established.

In order to fill up this gap, the study was intended to develop and characterize a phospholipid based 1,2-distearoyl-sn-glycero-3-phosphotidylethanolamine (DSPE) incorporated nanostructured delivery system of DTX for enhanced brain delivery of the drug. Seeing the abundant presence of DSPE in the white mater of brain, and other nervous tissues, where it constitutes almost 45% of all phospholipids, it is assumed that the presence of DSPE would further drive the formulation to the brain tissue. Here we have investigated both qualitatively and quantitatively the potential of our developed formulation to cross BBB in vivo. Various critical formulation and process parameters were optimized in order to keep the size of the final formulation within 100 nm along with the satisfactory physicochemical properties. We wanted to see whether the experimental formulation carrying DTX was able to permeate through BBB sufficiently and lead to sustained drug release in the brain as compared to free drug. Thus, the overall aim was to develop an optimized method of preparation of a biocompatible drug nanocarrier with a reasonable drug payload for efficient BBB permeation, sustained drug release in brain cells with an improved blood residence time and better efficacy for brain cancer treatment.

Materials and methods

Materials

DTX was obtained as a gift sample from Cipla laboratories, Goa, India. Soya-α-lecithin (SLE) was purchased from HiMedia Laboratories Pvt. Ltd (Mumbai, India). Cholesterol (CHL) was obtained from E Merck Ltd (Mumbai, India). Butylated hydroxyl toluene (BHT) was purchased from Qualigens Fine Chemicals (Mumbai, India). DSPE and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich Co. (Bangalore, India). All other chemicals used were of analytical grade.

Animals

For confocal microscopic investigations, and plasma and brain pharmacokinetic studies, Swiss albino mice of either sex (male : female ratio 1 : 1), weighing 20–25 g were used. For biodistribution and gamma scintigraphy imaging studies, male Sprague–Dawley rats weighing 250–300 g were used. The animals were purchased from Indian Institute of Chemical Biology, Kolkata, West Bengal. Animals were kept in polypropylene cages and housed in the university animal house at 25 ± 1 °C and 55% relative humidity environment with normal day and night cycle. Animals were fed standard diet and drinking water ad libitum. All the experimental procedures were reviewed and approved by the Animal Ethics Committee (AEC), Jadavpur University, Kolkata and the guidelines of AEC were followed throughout the study. The animals were acclimatized to the animal house environment at least for 2 weeks before beginning of the experiments.

Preformulation study

Fourier transform infrared spectroscopy (FTIR) was carried out as a preformulation study to depict any possible interaction between the drug and the excipients.

FTIR spectroscopy

The FTIR spectroscopy of pure drug, CHL, SLE, DSPE, a mixture of drug with CHL, SLE, DSPE, a mixture of CHL, SLE and DSPE, lyophilized formulation (with and without drug) was carried out by KBr pellet technique as described previously.⁹ The pellets were scanned over a wave number range of 4000 cm⁻¹ to 600 cm⁻¹ in an FTIR instrument (Magna-IR 750, Series II, Nicolet Instruments, Madison, Wiscosin, USA).

Procedure for the development of docetaxel-loaded nanosize lipid carriers (NLs)

NLs were prepared by the conventional thin film hydration method with some modifications of critical process parameters. Briefly, weighed amounts of DTX, SLE, CHL (Table 1) were taken in 250 ml round bottom flask and were dissolved in chloroform by vigorous shaking. Butylated hydroxyl toluene (BHT) [1% (w/v)] was added to the above mixture to prevent oxidation of lipid. The mixture was placed in a rotary vacuum evaporator [Rotavap superfit, PBU-6] fitted with an aspirator A3S (Eyela, Rikakikai Co. Ltd., Tokyo, Japan) and a circulating water bath (Spac N service, Kolkata, India) and was rotated at 130 rpm at 40 °C. The solvent was evaporated to form a thin film of lipids around the wall inside the round bottom flask. The flask was kept in a vacuum dessicator overnight for complete removal of residual organic solvent. It was then hydrated with phosphate buffer, pH 7.4, in a rotary vacuum evaporator fitted with a water bath and rotated at 150 rpm at 60 °C until the lipid film dispersed in the aqueous phase. The dispersion was sonicated in a bath type sonicator (Trans-o-sonic, Mumbai, India) for one hour to convert multilamellar vesicles (MLVs) to small unilamellar vesicles (ULVs). After sonication, preparation was kept at room temperature for two hours for vesicle formation followed by overnight storage at 4 °C. Then the preparation was centrifuged at 16 000 rpm for one hour. The product was collected, stored overnight at −20 °C for pre-cooling followed by lyophilization (laboratory lyophilizer; IIC Industrial Corporation, Kolkata, India) for 12 h. For the development of NLs with DSPE, weighed amount of DSPE was taken along with DTX, SLE, CHL, and BHT in a 250 ml round bottom flask and were dissolved in chloroform.⁹ All other procedures remained same as described previously.

Table 1 Composition, % yield, % drug loading and % drug loading efficiency of selected experimental formulations^b

Formulation code	SLE : CHL : DSPE ratio (w/w)	Drug : lipid ratio (w/w)	% yield	Practical % drug loading^a	% drug loading efficiency^a
a Data show mean ± SD (n = 3).b Abbreviations: DNL, docetaxel loaded NLs; DNL-PE, docetaxel loaded DSPE incorporated NLs.
DNL-1	75 : 50 : 0	1 : 8	51.5	6.7 ± 0.1	63.8 ± 1.2
DNL-2	125 : 50 : 0	1 : 8	67.3	8.4 ± 1.3	82.3 ± 2.1
DNL-PE	125 : 50 : 7	1 : 9	68.2	7.8.± 0.4	78.7 ± 0.8

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Development of fluorescent NLs

FITC was used as a marker to visualize the uptake of NLs by brain cells. Fluorescent NLs were prepared by the above described procedure, except 0.4% (w/v) FITC stock solution in chloroform and ethanol mixture at 3 : 1 ratio was prepared and 50 μl of this stock was dissolved in the organic phase (chloroform) during the first step of preparation.¹⁰ The rest of the procedure was same as mentioned above.

Characterization of docetaxel loaded NLs

Evaluation of drug loading. Weighed amount (5 mg) of NL was taken with a mixture of ethanol and water (7 : 3), vortexed and centrifuged at 12 000 rpm for 15 min. The absorbance of supernatant was measured at 229.9 nm using UV/VIS spectrophotometer (Beckman, Fullerton, CA, USA).⁹ The percentage drug loading and the drug loading efficiency were calculated using the following formula.

Drug loading (%) = (amount of DTX in NL/amount of NL obtained) × 100

Drug loading efficiency (%) = (practical drug loading/theoretical drug loading) × 100.

Determination of size distribution and zeta potential. Mean particle diameter (Z-average), polydispersity index (PDI) and zeta potential of the prepared formulations were determined by a dynamic light scattering (DLS) instrument (DLS-nano ZS, Zetasizer, Malvern Instrument Ltd, Malvern, UK). The data were interpreted by the instrument software (DTS software version 4.0).

Surface morphology study by field emission scanning electron microscopy (FESEM). The surface morphology of experimental NLs was analyzed by FESEM. Lyophilized samples were spread on a carbon tape over a stub, vacuum dried, platinum coated and were examined using FESEM (JSM 6100; JEOL, Tokyo, Japan).

Energy dispersive X-ray (EDX) analysis. The elemental composition of the experimental NLs was investigated by EDX technique which works as an integrated feature of the scanning electron microscope (JSM 6100; JEOL, Tokyo, Japan).

Cryo-transmission electron microscopy (Cryo-TEM). For Cryo-TEM analysis, 1 mg of lyophilized NLs was dispersed in 1 ml of Milli-Q water in a microcentrifuge tube. About 4 μl of sample suspension was applied to a clean grid, blotted away the excess with filter paper, and immediately vitrified into liquid ethane.¹¹ The grid was stored in liquid nitrogen until imaging using an electron microscope (Tecnai Polora, version 4.6 FEI Tecnai G2, Netherlands) operating at 300 kV equipped with an FEI Eagle 4k × 4k charge-coupled device (CCD) camera.

In vitro drug release study. Weighed amount (5 mg) of lyophilized formulation was reconstituted in 1 ml PBS and was taken into a dialysis bag (Himedia dialysis membrane-60, Mumbai, India).⁹ The two ends of the dialysis bag were tightly bound with cotton thread. The whole assembly was then put inside a beaker (100 ml capacity), containing 50 ml of phosphate-buffered saline (PBS) as drug release medium. The entire set up was then kept on a magnetic stirrer and stirred with a magnetic bead at 300 rpm at room temperature. At predetermined time intervals, 1 ml sample was withdrawn from the drug release medium and replenished with an equivalent volume of fresh medium. The samples were analyzed using a spectrophotometer at 229.9 nm against PBS as blank. The concentration was calculated from the calibration curve.

Drug release kinetics study. To predict the mechanism of drug release from the experimental NLs, data obtained from the in vitro drug release studies were plotted in various kinetic models such as zero order (cumulative amount of drug released versus time), first order (logarithmic value of cumulative amount of drug remained versus time), Higuchi model (cumulative amount of drug released versus square root of time), Korsmeyer–Peppas model (logarithmic value of cumulative amount of drug released versus logarithmic value of time), and Hixson–Crowell model (cube root of percentage drug remained versus time).⁹ The linearity of the plots was assessed from the calculated R² values.

In vivo investigations

Detection of the presence of fluorescent NLs in brain by confocal microscopy. FITC-labeled experimental NLs (FITC-DNL-PE/FITC-DNL-2) were injected through the tail vein in different groups of mice. The mice were sacrificed after 3 h and 5 h post injection. The brain tissue in each case was taken out. It was then fixed with 8% formalin solution, subsequently processed and embedded in paraffin block. Thin sections (5 μm) were cut and mounted on glass slides.⁹ The unstained samples were observed using a confocal laser scanning microscopy system (TCS SP2/AOBS, Leica, Mannheim, Germany).

Radiolabeling of DTX and DTX loaded NLs. Radiolabeling of DTX and DTX loaded NLs was done with ^99mTc according to the reported tin(II) chloride reduction method.^13,14 Briefly, the free drug (4 mg) was dissolved in 0.5 ml ethanol and the drug loaded NLs (equivalent to 4 mg of free drug) were suspended in 0.5 ml nitrogen purged water. To them, aqueous ^99mTc-pertechnetate (^99mTcO₄⁻) 40–100 MBq was added followed by addition of about 40 μl of aqueous SnCl₂·2H₂O (2 mg ml⁻¹) solution. The mixtures were incubated at room temperature for 10–15 min. The radiolabeled efficiencies were then determined by ascending thin layer chromatography using methyl ethyl ketone as mobile phase and silica gel coated aluminum strips (Merck, Darmstadt, Germany) as stationary phase. After developing the spots, the sheets were dried and cut into 5 strips (1 cm each) and quantitatively analyzed by counting in a well type gamma counter at 140 keV (Electronic Corporation of India, model LV4755, Hyderabad, India).

Gamma scintigraphy. Gamma scintigraphy imaging was carried out to give direct information regarding location of radiolabeled particles inside the body of the experimental rats. For gamma scintigraphy, male Sprague–Dawley rats (body weight 250–300 g) were used. The animals were divided in three groups. One group was administered ^99mTc-labeled free drug and the other two groups of rats were administered with ^99mTc-labeled DNL-PE and DNL-2 through tail vein (300 μl). The animals were anesthetized by intramuscular injection of ketamine hydrochloride (1 ml) followed by fixation on a board in the posterior position for imaging. Images were taken at two different time intervals (3 h and 5 h) using a planar gamma camera (GE Infinia Gamma Camera equipped with Xeleris Workstation, GE, Cleveland, OH, USA).

Biodistribution study. Biodistribution studies of the radiolabeled complexes were performed in Sprague–Dawley rats (body weight 250–300 g). The animals were anesthetized with ketamine (30–50 mg kg⁻¹ IM) and femoral vein was cannulated with polyethylene (PE-50) catheter tubes. All animals were well hydrated by i.p. administration of normal saline (0.9%, 2 ml) for 1 h. ^99mTc-labeled complex (^99mTc-labeled free drug/^99mTc-labeled DNL-PE/^99mTc-labeled DNL-2) at a volume 0.03 ml, (10–15 MBq kg⁻¹) was injected intravenously through the tail vein cannula.¹³ The rats were sacrificed at different time points (0.5 h, 2 h, 4 h, 8 h). The organs or tissues of interest were excised, washed with normal saline and transferred to preweighed counting vials. Blood sample was collected by heart puncture method. The radioactivity of the samples was measured using a well-type gamma scintillation counter along with an injection standard. The results were expressed as percent injected dose per g (% ID per g) of tissue or organ.

Pharmacokinetics study. In plasma and brain, the drug pharmacokinetic study was carried out on Swiss albino mice of either sex (male : female ratio 1 : 1) (body weight 20–25 g). The animals were divided into three groups each containing 6 animals. In group I, animals were administered DTX suspension intravenously (10 mg kg⁻¹ body weight). Animals of the second group received DNL-PE, injected intravenously with a quantity containing DTX equivalent to 10 mg kg⁻¹. Animals of the third group were untreated (control) animals. For plasma pharmacokinetic study, at a predetermined time interval of post i.v. dose, blood samples were withdrawn by heart puncture and stored in heparinized tubes. The blood samples were immediately centrifuged using cold centrifuge (HERMLE Labortechnik GmbH, Wehingen, Germany) at 5000 rpm for 10 min and the plasma was stored at −70 °C until further analysis.¹²

For studying drug pharmacokinetics in brain, the animals were sacrificed at 0.5, 1, 3, 6, 9 and 24 h post i.v. dose. Brains were removed, weighed, and homogenized in PBS, pH 7.4. The homogenates were stored at −70 °C until further analysis.

A LCMS/MS technique was used to determine the drug concentration in plasma and brain samples. In short, for LCMS/MS analysis, the plasma and brain samples were first mixed with about three volume of methyl-tert-butyl ether, vortexed and centrifuged to extract DTX. The supernatant in each case was collected. The organic solvent was evaporated to dryness at 40 °C under a steam of nitrogen. Before analysis, the dry samples were reconstituted with 100 μl of mobile phase. About 50 μl of internal standard solution (containing PTX 1 μg ml⁻¹) was added in each sample before analysis and 20 μl sample was injected into the LCMS/MS column (Agilent 6410, Triple Quad MS-MS, Agilent, USA). Different pharmacokinetic parameters i.e. area under the curve (AUC), area under the first moment curve (AUMC), total body clearance (Cl_t), volume of distribution (V_d), mean residence time (MRT) etc. were determined using non-compartmental PK Solver software (JiangSu Province, China, Version 2.0).

LCMS/MS conditions. The LCMS/MS Agilent C₁₈ column (75 × 4.6 nm; 3.5 μm) was used for quantification of DTX from the plasma and brain samples. The mobile phase for the chromatographic separation was composed of acetonitrile, HPLC water and formic acid (60 : 40 : 0.05 v/v/v, respectively) at a flow rate of 0.4 ml min⁻¹. The analyte was monitored using mass spectrometer equipped with a double quadruple and an electrospray ionization interface, operated in a positive mode (ESI+). The analysis was carried out at 40 °C with a sample injection volume of 20 μl.

Statistical analysis. All the experiments were conducted in triplicate and each value was expressed as mean ± standard deviation (SD). Statistical calculations of the various sets of data were performed using GraphPad Prism™ (version 5.0) software (California, USA). Statistical significance among three or more groups was analyzed by one-way analysis of variance (ANOVA) and Dunnett's multiple comparison tests.

Results

FTIR analysis

Drug–excipient interaction was investigated using FTIR spectroscopy to detect any interactions between the drug and the excipients. When the spectra of pure drug, each of the excipients (such as SLE, CHE, DSPE), physical mixture of the excipients and physical mixture of DTX with the excipients were compared, the characteristic peaks of the drug, that is, 3856 cm⁻¹ and 3807 cm⁻¹ (for amines), 709 cm⁻¹ (aromatic mono substituted benzene), 1247 cm⁻¹ and 1173 cm⁻¹ (for esters) etc. were found to be present in the mixture (Fig. 1). The data suggest that no chemical interaction exists between the drug and the selected excipients since all the characteristic peaks of the drug were detected. However, when spectra of the physical mixture of the excipients were compared with the spectrum of each of the excipients, it was observed that although the characteristic peaks such as 800 cm⁻¹ (aromatic phenyl ring substitution band), 1549 cm⁻¹ and 1640 cm⁻¹ (phenyl ring substitution overtone, finger print region) were present for CHL, there was shifting of peaks for alcohol from 3435 cm⁻¹ to 3381 cm⁻¹, which suggests that OH group of CHL had physical interaction. Likewise, shifting of peaks from 1234 cm⁻¹ to 1247 cm⁻¹ (carboxylic acid, C=O stretching) and from 1067 cm⁻¹ to 1058 cm⁻¹ (amine) showed the involvement of CHL and SLE in physical interactions. In case of DSPE, shifting of characteristic peaks were observed from 1210 cm⁻¹ (for acids) and 1000 cm⁻¹ (amines, C–N medium stretching vibration) which might be due to the formation of weak hydrogen bond between amino group of DSPE and OH group of CHL. Additionally, formation of weak H-bond between the amino group of SLE and OH-group of CHL might also take place. Further, some other physical interactions such as van der Waals force of attraction or dipole–dipole interaction etc. might also exist among SLE, CHL and DSPE molecules. These interactions could be responsible for the development of NL structure. When the lyophilized NLs (loaded with drug/without drug) were compared, no peak of drug was detected. This suggests that the drug was encapsulated completely and no free drug was available on the surface of the formulation.

Fig. 1 Fourier transform infrared spectroscopy (FTIR) spectra of (A) drug (docetaxel) (B) soya–α-lecithin (C) cholesterol (D) 1,2-distearoyl-sn-glycero-3-phosphotidylethanolamine (DSPE) (E) physical mixture of excipients (F) physical mixture of drug and excipients (G) lyophilized formulation without drug (H) lyophilized formulation loaded with drug.

Characterization of NLs

After optimization of the critical process and formulation parameters of all the prepared formulations by in vitro characterizations, two optimized formulation of NLs (DNL-1 and DNL-2) were selected and reported here. DNL-1 and DNL-2 showed the desired physicochemical characteristics among all the prepared formulations. Among the two formulations, based on surface morphology, average particle size data, homogenous distribution pattern, percentage drug loading etc., we further selected DNL-2 for the development of DSPE incorporated drug loaded NLs (DNL-PE).

Evaluation of percentage drug loading and drug loading efficiency

The percentage drug loading for DNL-1 was 6.7 ± 0.1% whereas for DNL-2, the value was 8.4 ± 1.3% with the drug loading efficiencies, 63.8 ± 1.2% and 82.3 ± 2.1%, respectively (Table 1). Further, DNL-PE formulation showed slightly lower percentage of drug loading than DNL-2. DNL-PE had 7.8 ± 0.4% drug loading with 78.8 ± 0.8% drug loading efficiency.

Particle size, size distribution and zeta potential of NLs

The average size of the experimental NLs was 74.6 ± 2.7 nm (DNL-2) and 82.1 ± 2.2 nm (DNL-PE) respectively (Fig. 2). DNL-PE showed that the average size of the particles became little higher than DNL-2 but was well below the Z-average value of DNL-1 (Fig. 2). PDI data suggests that the formulations had a narrow size distribution pattern (Table 2). The zeta potentials of DNL-2 and DNL-PE were −79.3 mV and −60.7 mV respectively (Table 2). DNL-PE showed slightly lower value of zeta potential than DNL-2, which may be due to the presence of DSPE containing cationic amino group. Higher values of zeta potential for the selected formulations signify their higher stability in a suspended state.

Fig. 2 Particle size distributions of selected docetaxel-loaded nanosize lipid carriers (A) DNL-1 (B) DNL-2; and (C) docetaxel-DSPE loaded nanosize lipid carriers (DNL-PE).

Table 2 Size distribution, PDI and zeta potential of various experimental formulations^b

Formulation code	Z-avg.^a (d nm)	PDI^a	Zeta potential (mV)
a Data show mean ± SD (n = 3).b Abbreviations: DNL, docetaxel loaded NLs; DNL-PE, docetaxel loaded DSPE incorporated NLs.
DNL-1	138.6 ± 1.3	0.21 ± 0.05	−71.7
DNL-2	74.6 ± 2.7	0.19 ± 0.81	−79.3
DNL-PE	82.1 ± 2.2	0.18 ± 0.32	−60.7

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Surface morphology study of NLs

We have given two FESEM images (Fig. 3) for each of the selected formulations, taken from different portions of the samples to justify our findings. FESEM photographs showed that all the formulations had smooth surface. DNL-1 showed a mixture of larger and smaller particles within nano-dimensions. However, the other two formulations had more homogenous size distribution patterns.

Fig. 3 FESEM photographs of experimental formulations, taken from different portions of samples (A and B for DNL-1, C and D for DNL-2, E and F for DNL-PE).

Elemental analysis of selected NLs

EDX analysis showed weight% and atomic% of different elements such as carbon (C), oxygen (O), and phosphorous (P) in experimental NLs (Fig. 4A and B, Table 3). The data showed proportional changes in values of weight% and atomic% of various elements in DNL-PE, suggesting the presence of DSPE in DNL-PE.

Fig. 4 Energy dispersive X-ray (EDX) of (A) docetaxel-loaded nanosize lipid carriers (DNL-2); and (B) docetaxel-DSPE loaded nanosize lipid carriers (DNL-PE), and (C) in vitro drug release profile of DNL-2 and DNL-PE. Data show mean ± SD (n = 3).

Table 3 Weight% and atomic% of elements in experimental NLs^a

Formulations	Weight%			Atomic%
	CK	OK	PK	CK	OK	PK
a Abbreviations: DNL, docetaxel loaded NLs; DNL-PE, docetaxel loaded DSPE incorporated NLs; CK, carbon counts; OK, oxygen counts; PK, phosphorous counts.
DNL-2	47.59	39.67	12.74	57.82	36.18	6.00
DNL-PE	40.96	41.92	17.11	51.80	39.80	8.39

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In vitro drug release and analysis of drug release kinetics

In vitro drug release study was carried out for DNL-2 and DNL-PE and data were compared (Fig. 4C). Results showed that cumulative 52.60% ± 0.03% and 58.39% ± 0.01% of DTX released from DNL-2 and DNL-PE respectively over a period of 24 h of drug release study. For DNL-PE, DTX release increased initially up to first 8 h of the study. Then from the 9th hour, the drug release was slightly slower. Further, the non-DSPE formulation (DNL-2) showed slightly slower DTX release than DNL-PE over the period of 24 h of DTX release. The drug release data was fitted in different kinetic equations to depict the drug release pattern and the corresponding R² values were calculated (Table 4). For DNL-2 and DNL-PE, Koresmeyer–Peppas kinetic model (R² = 0.978) indicated good linearity as compared to the other models. However, for DNL-PE, DTX release pattern primarily obeyed more Koresmeyer–Peppas model and to some extent Higuchi kinetic model (R² = 0.964) than the other models. This suggests that drug release from DNL-PE was governed by more than one process.

Table 4 In vitro drug release kinetics with R² values for selected formulations^a

Kinetic model	DNL-PE	DNL-2
a Abbreviations: DNL, docetaxel loaded NLs; DNL-PE, docetaxel loaded DSPE incorporated NLs.
Zero order kinetics	y = 2.520x + 7.536, R^2 = 0.866	y = 2.387x + 3.658, R^2 = 0.900
First order kinetics	y = −0.016x + 1.978, R^2 = 0.933	y = −0.014x + 1.994, R^2 = 0.928
Koresmeyer–Peppas	y = 0.942x + 0.634, R^2 = 0.978	y = 1.111x + 0.376, R^2 = 0.978
Higuchi	y = 14.91x − 10.65, R^2 = 0.964	y = 13.87x − 12.99, R^2 = 0.953
Hixson–Crowell kinetics	y = −0.051x + 4.547, R^2 = 0.913	y = −0.046x + 4.606, R^2 = 0.913

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Cryo-TEM study of experimental NLs

Cryo-TEM study was conducted for the selected NLs to provide details on internal structure, lamellarity etc. The Cryo-TEM images showed formation of unilamellar lipid vesicles along with very few bilamellar vesicles as depicted in Fig. 5. The formulation showed a mixture of smaller (30 nm) and little larger vesicles (50–60 nm). The NLs were spherical showing darker outer lipid layer enclosing the inner core and without any perforation or leakage on the outer membrane justifying good formation.

Fig. 5 Cryo-TEM images of DNL-PE (A) showing unilamellar vesicles and one bilamellar vesicle with intact lamellarity; size less than 100 nm, normal view (40 000×) and (B) magnified view of one larger size unilamellar vesicle (6 times magnified).

In vivo investigations

Detection of the presence of fluorescent NLs in brain by confocal microscopy. We have investigated the uptake of the fluorescent NLs (FITC-DNL-PE/FITC-DNL-2) by brain cells in vivo after 3 h and 5 h post i.v. injection. Confocal microscopic images showed that the NLs were well penetrated into the brain in experimental mice and distributed throughout the organ (Fig. 6). However, more internalization of NLs was observed for FITC-DNL-PE than FITC-DNL-2 at both the experimental time periods. The study thus gave us a preliminary idea that the experimental NLs could cross BBB and reach brain tissue.

Fig. 6 Confocal microscopic photographs of (I) brain tissue of experimental mice treated with fluorescein isothiocyanate labeled NLs (FITC-DNL-PE) at 3 h post i.v. injection; and (II) brain tissue of experimental mice treated with fluorescein isothiocyanate labeled NLs (FITC-DNL-PE) at 5 h post i.v. injection. (III) Brain tissue of experimental mice treated with fluorescein isothiocyanate labeled NLs (FITC-DNL-2) at 3 h post i.v. injection. (IV) Brain tissue of experimental mice treated with fluorescein isothiocyanate labeled NLs (FITC-DNL-2) at 5 h post i.v. injection.

Gamma scintigraphy. In order to visualize the brain uptake, gamma scintigraphy was performed in different groups of rats received ^99mTc labeled DNL-PE, ^99mTc labeled DNL-2 and ^99mTc labeled free drug. The images were taken at 3 h and 5 h post i.v. administration (Fig. 7). For animals treated with radiolabeled DNL-PE, radioactivity signal was observed in the vital organs and tissues (as mentioned in the Table 5), including brain, indicating that the lipid vesicles could successfully cross the BBB. Further, the signal was clearly visible in brain at 5 h (Fig. 7), which confirmed that the drug loaded NLs were reaching brain tissue in a time dependent manner. A relatively low radioactivity signal in brain was observed from the animals treated with ^99mTc labeled DNL-2 at 3 h. The signal further diminished at 5 h (data not shown) which signifies relatively lower brain retention property of non-DSPE formulation (DNL-2) than DSPE-loaded formulation (DNL-PE). On the other hand, the radioactivity signal (at 3 h) in the brain from the animals treated with free drug was too weak justifying the low BBB permeation capacity of the free drug (Fig. 7). No signal of free drug was detected in 5 h also (data not shown).

Fig. 7 Gamma scintigraphy images of (I) animals received ^99mTc labeled DNL-PE at 3 h post i.v. injection, A and B; (II) animals received ^99mTc labeled DNL-PE at 5 h post i.v. injection, C and D; (III) animals received ^99mTc-labeled DNL-2 at 3 h post i.v. injection, E and F; (IV) animals received ^99mTc labeled free drug at 3 h post i.v. injection, G and H. A, C, E, G, whole animal image; B, D, F, H, brain portion magnified.

Table 5 Biodistribution studies of ^99mTc labeled NLs containing DTX and ^99mTc labeled free DTX in rats at different time points^a

Formulations/free drug	Time	Heart	Blood	Liver	Lung	Spleen	Muscle	Intestine	Stomach	Kidney	Brain	Brain/blood
a Data are expressed in % mean of injected dose (ID) per gram of organ/tissue ± SD (n = 5).
DNL-PE	0.5 h	0.03 ± 0.01	2.10 ± 0.32	22.10 ± 3.22	4.56 ± 1.10	4.93 ± 0.19	0.01 ± 0.00	1.24 ± 0.31	1.21 ± 0.30	0.02 ± 0.00	0.83 ± 0.21	0.39
	2 h	0.14 ± 0.03	1.73 ± 0.45	35.17 ± 3.11	2.99 ± 0.21	2.76 ± 0.45	0.03 ± 0.00	2.46 ± 0.22	1.30 ± 0.32	0.09 ± 0.03	1.27 ± 0.32	0.73
	4 h	0.11 ± 0.02	1.51 ± 0.33	37.24 ± 4.21	2.89 ± 1.01	2.56 ± 0.43	0.05 ± 0.01	2.67 ± 0.77	2.56 ± 0.65	0.11 ± 0.03	1.79 ± 0.05	1.18
	8 h	0.10 ± 0.02	1.21 ± 0.32	38.65 ± 4.78	1.34 ± 0.5	1.89 ± 0.31	0.09 ± 0.01	4.65 ± 1.11	5.67 ± 2.11	0.16 ± 0.08	1.37 ± 0.55	1.13
DNL-2	0.5 h	0.02 ± 0.01	1.42 ± 0.07	26.23 ± 5.11	3.46 ± 0.08	3.35 ± 1.02	0.09 ± 0.05	2.01 ± 0.08	1.81 ± 0.20	0.06 ± 0.01	0.31 ± 0.04	0.21
	2 h	0.18 ± 0.11	0.95 ± 0.01	33.90 ± 2.44	2.08 ± 0.12	2.85 ± 0.04	0.14 ± 0.02	2.88 ± 0.69	2.39 ± 0.03	0.22 ± 1.13	0.56 ± 1.05	0.58
	4 h	0.13 ± 0.03	0.71 ± 0.09	38.21 ± 4.55	1.65 ± 1.33	2.01 ± 0.33	0.12 ± 0.22	3.26 ± 1.02	3.55 ± 0.07	0.18 ± 0.07	0.78 ± 0.06	1.09
	8 h	0.01 ± 0.02	0.25 ± 0.12	32.52 ± 2.11	0.81 ± 0.41	1.51 ± 0.02	0.04 ± 0.01	4.95 ± 0.09	7.01 ± 0.33	0.09 ± 0.11	0.13 ± 0.08	0.52
Free DTX	0.5 h	0.21 ± 0.01	0.61 ± 0.22	30.11 ± 3.12	1.22 ± 0.45	1.54 ± 0.67	0.11 ± 0.02	1.02 ± 0.33	4.20 ± 1.11	0.17 ± 0.05	0.09 ± 0.02	0.14
	2 h	0.12 ± 0.03	0.41 ± 0.21	41.21 ± 4.42	2.31 ± 1.11	2.22 ± 0.43	0.12 ± 0.08	2.56 ± 0.21	6.52 ± 1.21	0.21 ± 0.21	0.10 ± 0.06	0.24
	4 h	0.10 ± 0.00	0.32 ± 0.05	40.19 ± 4.21	2.67 ± 0.67	2.37 ± 0.78	0.11 ± 0.06	3.66 ± 1.11	8.54 ± 2.11	0.12 ± 0.00	0.09 ± 0.02	0.28
	8 h	0.08 ± 0.01	0.19 ± 0.02	30.32 ± 5.32	2.78 ± 0.78	2.35 ± 0.76	0.10 ± 0.00	6.54 ± 1.02	12.45 ± 2.34	0.12 ± 0.00	0.04 ± 0.00	0.21

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Biodistribution of radiolabeled NLs and free drug. Biodistribution studies following i.v. administration of ^99mTc labeled DNL-PE, ^99mTc labeled DNL-2 and ^99mTc labeled DTX (free drug) were performed in rats up to 6 h. Results expressed in % injected dose per gram (% ID per g) of tissue/organ have been given in Table 5. The brain/blood biodistribution ratio and brain concentration ratio of DNL-PE and free drug at all the experimental time points were calculated and compared. The residence time of DNL-PE in blood (1.51% ID per g at 4 h) was more than that of free drug (0.23% ID per g at 4 h) at all the time points indicating prolong circulation of DNL-PE in blood. Significant uptake of DNL-PE and free drug was found in liver and kidneys, the main excretory organs. The uptake in the intestine increased from 0.5 h (1.24% ID per organ) to 6 h (4.65% ID per organ) for both DNL-PE and free drug (1.02% ID per organ to 6.54% ID per organ). The brain accumulation for the DNL-PE was much higher than that of the free drug at all the time points studied. At 4 h, the brain accumulation for ^99mTc labeled DNL-PE was maximum (1.79% ID per organ), followed by DNL-2 (0.78% ID per organ), whereas it was very less (0.11% ID per organ) for the radiolabeled free drug. The brain/blood ratio of ^99mTc labeled DNL-PE and ^99mTc labeled DTX was found to be higher for DNL-PE than that of free drug at all the time points of the study. Data show that at 4 h, uptake of ^99mTc labeled DNL-PE in brain was about 9 times higher than that of ^99mTc labeled DNL-2 whereas the same was about 20 times higher than that of ^99mTc labeled free drug, indicating sufficient BBB permeation of the optimized formulation (DNL-PE) than DNL-2 and the free drug.

Plasma pharmacokinetics. A clear difference in important plasma pharmacokinetic parameters was observed between DNL-PE administration and free drug suspension treatment (Fig. 8). AUC_0–∞ values were 12 658.2 ± 1138.4 ng ml⁻¹ h⁻¹ and 6948.3 ± 121.2 ng ml⁻¹ h⁻¹ for DNL-PE and free drug suspension, respectively. Following i.v. administration, the MRT was 3.6 ± 0.3 h; with DTX suspension where as it was 5.7 ± 0.1 h with DNL-PE (Table 6). About 2.5 time enhancement in AUMC_0–∞ was also observed in DNL-PE treated animals as compared to the animals treated with DTX suspension. The data overall suggest improved pharmacokinetic parameters of DNL-PE as compared to the free drug suspension.

Fig. 8 (A) Plasma concentration–time profiles of DTX in mice after i.v. administration of DNL-PE and free drug suspension (10 mg kg⁻¹). Data show mean ± SD (n = 5). (B) Brain concentration–time profiles of DTX in mice after i.v. administration of DNL-PE and free drug suspension (10 mg kg⁻¹). Data show mean ± SD (n = 5).

Table 6 Plasma and brain pharmacokinetic parameters of DTX after intravenous bolus administration of free drug (DTX) suspension and drug loaded NLs (DNL-PE)^b

Pharmacokinetic parameters	Plasma^a		Brain^a
	DNL-PE	DTX	DNL-PE	DTX
a Data show mean ± SD (n = 5).b Abbreviations: AUC, area under the plasma concentration time curve; AUMC, area under the first moment curve; MRT, mean residence time; t1/2, plasma half life; Clt, total body clearance; Vd, apparent volume of distribution.
AUC0–∞ (ng h ml^−1)	12 658.2 ± 1138.4	6948.3 ± 121.2	10 739.3 ± 2331.6	2907.9 ± 116.3
AUMC0–∞ (ng h^2 m^−1)	74 421.4 ± 231.4	26 169.1 ± 3321.6	128 584.2 ± 2113.3	34 502.1 ± 332.6
MRT0–∞ (h)	5.7 ± 0.1	3.6 ± 0.3	11.7 ± 2.7	4.1 ± 0.3
Clt (L h^−1)	0.003 ± 0.011	0.005 ± 0.012	0.003 ± 0.022	0.15 ± 0.43
Vd (L)	0.02 ± 0.02	0.01 ± 0.51	0.04 ± 0.12	0.15 ± 0.18

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Brain pharmacokinetics. Brain pharmacokinetic data showed increased brain availability of DTX from DNL-PE than DTX suspension (Fig. 8). The AUC_0–∞ value of DTX from DNL-PE (10 739.3 ± 2331.6 ng h ml⁻¹) was reasonably higher than that from DTX suspension (2907.9 ± 116.3 ng h ml⁻¹). A significant difference in AUMC_0–∞ (128 584.2 ± 2113.3 ng h² ml⁻¹ for DNL-PE vs. 34 502.1 ± 332.6 ng h² ml⁻¹ for DTX suspension) and Cl_t (0.15 ± 0.43 l h⁻¹ for DNL-PE vs. 0.003 ± 0.022 l h⁻¹ for DTX suspension) values were also observed for DNL-PE and DTX suspension (Table 6). The MRT_0–t for DNL-PE (11.7 ± 2.7 h) increased almost three times as compared to the free drug suspension (4.1 ± 0.3 h), signifying higher residence of the formulation in brain tissue. After i.v. administration, the drug concentration for free DTX suspension decreased rapidly and at 12 h it was found almost non-detectable (the lowest limit of detection of the LCMS/MS used, <5.00 ng ml⁻¹), whereas the same was detectable even after 24 h in case of DNL-PE, suggesting sustained drug release property of the formulation in the brain tissue.

Discussion

In the present study, drug–excipients interaction, if any, was assessed using FTIR analysis. Data demonstrate that there was no chemical interaction between the drug and the selected excipients. However, minor shifting of some peaks might be due to the physical interactions by formation of weak physical bonds such as H-bonding, van der Waals force of attraction or dipole–dipole interaction etc. among the various functional groups of SLE, CHL and DSPE molecules. These physical interactions might have helped to form NLs structure. Further, in the lyophilized formulations (with and without drug), absence of any peak of the drug suggests that the drug was encapsulated completely.

Drug loading, surface morphology, average size (Z-average), PDI of the NLs varied with the changes in the quantity of their contents and in different process parameters, such as speed and duration of hydration, hydration temperature, duration of sonication, speed and duration of centrifugation, etc. We have first optimized the formulation parameters. We chose a fixed drug–polymer ratio of 1 : 9 (w/w) and CHL–SLE ratio of 1 : 2.5 (w/w) for the desired physicochemical properties. Initially, we found that with an increase in phospholipid concentration (at a fixed drug amount), percentage of drug loading increased. However, at a ratio of mixing by weight above 1 : 3 of CHL : SLE, the morphology of NLs was damaged. Clearly, CHL is responsible for stabilizing the NLs structure. When we decreased CHL and SLE ratio below 1 : 1 (w/w), the drug loading decreased significantly, though the structure was well formed as evidenced by FESEM. Similarly, above 1 : 9 drug–lipid ratio, an enhancement in the amount of drug did not increase drug loading. This suggests that the drug quantity and its loading efficiency are not proportional to each other. In the present study, duration of sonication was also optimized. Ultra-sonication for 1 h in bath type sonicator resulted in NLs of very small size (below 100 nm). However, sonication for more than 1 h produced larger NLs (as observed by FESEM) with a decreased drug loading (data not shown). Alternatively, less sonication time yielded less homogenous vesicles with variable sizes. During hour long sonication in a bath type sonicator, the larger size NLs might have broken to smaller size NLs. After sonication, a reasonable standing period of 2 h is important (as we have seen in our process) before refrigeration. This parameter is not often considered critical in many reports.^15,16 However, we have found that sonication followed by refrigeration without a sufficient period of standing led to a breakage of NLs structure. Variation in centrifugation speed and duration of centrifugation has been reported to vary the size and PDI of nanoformulations.^16,17 Optimization of speed of centrifugation and duration of centrifugation are important to form NL structures. In our work, we have optimized the centrifugation speed of 16 000 rpm for 1 h to get NLs. Thus, we chose a fixed ratio of 1 : 9 drug–lipid, 1 : 2.5 (w/w) ratio of CHL : SLE along with 1 h hydration at 130 rpm at 60 °C, 1 h sonication (bath type sonicator), 2 h standing period before freezing and 1 h centrifugation at 16 000 rpm as the optimized parameters for balancing the loading of drug and controlling the size of formulations below 100 nm. Again, based on size distribution, and the maximum drug loading obtained, DNL-2 was selected for further study.

A reasonable DTX loading of 7.8 ± 0.6% with 78.8 ± 0.5% drug loading efficiency was observed for DNL-PE. The percentage drug loading and loading efficiency for DNL-PE were slightly less than that of DNL-2, which again may be attributed to the presence of DSPE in the formulation.

FESEM photographs show that the experimental formulations had smooth surface. DNL-PE showed homogenous distribution pattern without the presence of any lumps or agglomerates in the lyophilized sample.

In our study we have adopted three different methods, DLS, FESEM and Cryo-TEM in order to precisely understand size and distribution of experimental NLs both in the lyophilized and hydrated states. Sizes of lyophilized samples observed using FESEM were smaller than those detected by DLS. The difference in size may be because of the DLS method which measures the hydrodynamic diameter of NLs in aqueous suspension. NLs might swell and increase in size during the sample preparation in double distilled (distilled twice) water. A similar observation has been reported.^18,19 Size analysis data show that the average size (Z-average) of DNL-PE was well below 100 nm. Further, PDI values obtained suggest a narrow size distribution of the experimental NLs.

The smaller is the size of the drug nanocarriers, the easier is to keep them suspended in a liquid. Larger drug carriers precipitate out more easily than the smaller drug carriers or nanosize drug carriers, as the precipitation phenomenon is governed by Stokes' law.²⁰ Further, a surface charge more negative than −30 mV or more positive than +30 mV is generally considered critical to form stable suspensions.^9,21 In the present work, DNL-PE had nanosize (Z-average, 82.1 nm) with negative surface charge (−60.7 mV), which predicts its prolonged stability in a suspended form. Further, in our body, positively charged drug carriers are eliminated more quickly than the negative charge drug carriers, claiming more blood residence time of the experimental formulations.²²

A proportional variation in weight% and atomic% of various elements (C, O, and P) was observed in DNL-PE as compared to DNL-2. The data suggest the presence of DSPE in the NLs varied the weight% and atomic% of the elements measured in the experimental formulation DNL-PE.

Cryo-TEM analysis is now becoming an essential tool for the evaluation of lipid based nano constructs. Due to their delicate nature, the lipid based membrane structures are more prone to get damaged under high vacuum in case of normal TEM (freeze fractured TEM). The Cryo-TEM analysis allows for direct imaging of nanosize lipid based membrane structures in their native state at a very low temperature condition. In our study, the experimental NLs were found to be mostly unilamellar including a very few bilamellar structures (<0.05%) below 100 nm size range. The formed NLs were spherical, with intact lamellarity and without any perforations on their membrane, justifying good and stable formation of structure. This may be attributed to the optimization of the manufacturing parameters of NLs done in our study.

In vitro drug release data for DNL-PE show that the drug released in a sustained manner from the formulations. A relatively higher cumulative amount of drug released from DNL-PE than DNL-2. Presence of DSPE might assist comparatively easier DTX diffusion by modifying drug diffusion pathways through the lipid carrier membrane. The kinetic data show that drug release from DNL-PE might follow complex mechanisms of diffusion as well as erosion from the experimental NLs as the data was best fitted to Korsmeyer–Peppas kinetic model.¹⁶

Effective delivery of drug to brain is often a herculean task for the formulation scientists. Most of the trials in recent years have failed to demonstrate a remarkable efficiency of drug to cross BBB.²³ Many studies have been reported about the improved brain delivery of DTX through lipid-based nanocarriers (e.g. liposome or solid lipid nanoparticles).^6,24 Li, et al.²⁴ has reported an increased brain delivery of DTX through glucose modified liposomes by using C6 glioma cell line as the in vitro model. Venishetty et al.⁶ investigated the brain uptake of DTX along with ketoconazole loaded in folate-grafted solid lipid nanoparticles in brain endothelial cells (bEnd.3). Both of the studies also reported in vivo brain pharmacokinetic data to show the brain uptake of DTX loaded lipid nanocarriers in comparison to the free drug. However, the present study is predominantly different from the other such reports. In this study DSPE incorporated phospholipid vesicular nanocarrier has shown its incredible potential of BBB permeation as documented by in vivo gamma scintigraphy imaging method. In our study, we have used three different techniques such as confocal microscopy of brain tissue, gamma scintigraphy imaging as well as evaluation of brain pharmacokinetic profile of the drug by LCMS/MS method to provide a concrete evidence of BBB crossing potential of the experimental NL (DNL-PE), which has not been reported before for any lipid based nanocarriers carrying DTX. DNL-PE is stable at its nanosize and was able to release the drug in a sustained manner. Further, better residence time in blood as well as in brain tissue as compared to the free drug has made this nanomedicine unique for its future clinical application. In our case, we have tried to provide enough evidence both qualitatively and quantitatively to confirm that the experimental DNL-PE was able to penetrate through BBB sufficiently and accumulated in the brain tissue for a longer period of time.

The confocal images of brain tissue showed extensive distribution of FITC labeled NLs throughout the organ. The NLs accumulated more in the granular portions of brain tissue than the agranular portions. The fluorescence intensity from the DNL-PE treated brain tissue was quite higher than that of the DNL-2 treated tissue for both 3 h and 5 h, supporting higher brain penetrability of the optimized DSPE-formulation over the non-DSPE ones. Further, in the picture, the normal brain architecture was found to remain unaltered, signifying absence of any severe detrimental effect of the formulation to normal brain cells within the experimental time period.

Gamma scintigraphy provides direct information on the localization of radiolabeled materials in the body. In the study, we used this imaging technique to provide a concrete evidence of BBB permeation potential of DNL-PE/DNL-2 with respect to the free drug suspension. The imaging was done at 3 h and 5 h post i.v. injection of ^99mTc labeled NLs and ^99mTc labeled free drug in different groups of rats. Both the radiolabeled NLs and the radiolabeled free drug were found to distribute in all the vital organs in the body, though the maximum radioactivity signal was observed in liver. Substantial radioactivity signal from the brain of rats treated with radiolabeled DNL-PE was clearly visible at 3 h. The signal was persisted at 5 h also, signifying longer retention of ^99mTc labeled DNL-PE in the brain tissue. However, very weak signals were exhibited in the brains of animal treated ^99mTc labeled DNL-2 and free drug at 3 h (at 5 h, no radioactivity signal was detected for them). The images proved a successful BBB permeation of ^99mTc labeled DNL-PE into the brain. By dint of the tiny size, high lipophilic nature and higher retention in blood, ^99mTc labeled DNL-PE could permeate through BBB. The cell membrane-mimicking property of the phospholipid carrier might further help it retaining in the brain tissue to release the drug for a prolonged period of time.

Biodistribution study of the radiolabeled ^99mTc labeled DNL-PE, ^99mTc labeled DNL-2 and ^99mTc labeled free DTX was carried out with the help of a well-type gamma scintillation counter to evaluate complete distribution pattern of radiolabeled complex inside the body of the experimental rats. Radiolabeled DNL-PE, DNL-2 and free drug were found to distribute into the tissues within 0.5 h. DNL-PE showed higher residence time in blood as compared to free drug. Highest amount of drug uptake was seen in liver for both DNL-PE and free drug, suggesting their hepatic clearance from the body. The radiolabeled complexes were also found to be distributed in other macrophage rich organs such as spleen and lungs. Accumulation of ^99mTc labeled NLs and free drug in brain was in the order of ^99mTc labeled DNL-PE > ^99mTc labeled DNL-2 > ^99mTc labeled free drug at all the time points of the investigation. Unlike ^99mTc labeled DNL-PE, the brain/blood biodistribution ratio of radiolabeled free drug was continuously less, supporting once again low brain permeating nature of the free drug. Significant drug accumulation in kidneys from DNL-PE/free drug indicates predominant renal excretion of them with a slower excretion of DNL-PE than the free drug.

The plasma and brain kinetic parameters of DTX in animals treated with DNL-PE showed a higher value of AUC, AUMC, V_d and a lower rate of clearance in comparison to free drug suspension. The plasma drug concentration after 24 h was significantly (p ≤ 0.05) higher for DNL-PE (98.35 ng ml⁻¹) whereas the same was non-detectable in case of free drug suspension. C_max of the free drug was 3833.4 ng ml⁻¹ at 1 h. Due to their much smaller size (<100 nm), the NLs might be able to escape from the macrophages and remained in the circulation for a prolonged period of time. The enhanced blood circulation property further helped NLs (DNL-PE) to get sufficient time to cross BBB and accumulate in brain tissue.

Concentration of DTX in brain was much higher for DNL-PE than for free drug suspension at all the time points of the study. For DNL-PE, DTX concentration increased from 0.5 h to 3 h after which it decreased in a controlled manner up to 24 h. Free drug suspension showed much lower drug kinetic profile (significantly lower AUC_0–∞, AUMC_0–∞, MRT) justifying inability of the drug to cross BBB efficiently. The optimized in vitro properties such as nanosize and high lipophilicity of DNL-PE may be responsible for BBB crossing. Further, a sustained release of the drug from the NLs might be responsible to maintain a higher drug concentration in brain, which overall led to its enhanced brain pharmacokinetic profiles.

Conclusion

The work has presented an optimized method to develop nanoscale size phospholipid based carrier system of DTX for successful brain delivery. Lyophilized DNL-PE has shown nanosize range (below 100 nm) with 7.8% drug loading along with a sustained DTX release profile in vitro. A significant accumulation of drug loaded NL was noticed in the brain. Effective BBB permeation, prolonged brain retention and sustained drug release property of the experimental NL favor their clinical application. The formulation procedure was kept simple optimizing the critical parameters, which would further help their smooth technology transfer for large scale production and subsequent clinical translation. The experimental NL may be tested for their in vivo effectiveness in rat brain tumor xenograft model, which is the future plan of this work.

Declaration of interest

The authors of this article have no conflicts of interest to declare.

List of abbreviations

AUC	Area under the curve
AUMC	Area under the first moment curve
BBB	Blood–brain barrier
BHT	Butylated hydroxyl toluene
CHL	Cholesterol
cm	Centimeter
Cmax	Peak plasma concentration
Clt	Total body clearance
DIC	Differential interference contrast
DLS	Dynamic light scattering
DTX	Docetaxel
DNL	Docetaxel loaded nanosize phospholipid-based carrier
DSPE	1,2-distearoyl-sn-glycero-3-phosphotidylethanolamine
DNL-PE	Docetaxel loaded DSPE incorporated nanosize phospholipid-based carrier
FTIR	Fourier transform infrared spectroscopy
FESEM	Field emission scanning electron microscopy
FITC	Fluorescein isothiocyanate
g	Gram
h	Hour
ID	Injected dose
L	Liter
LC/MS	Liquid chromatography/mass spectroscopy
min	Minutes
mg	Milligram
mg kg^−1	Milligram per kilogram
MRT	Mean residence time
ml	Milliliter
NL	Nanosize phospholipid-based carrier
NLs	Nanosize phospholipid-based carriers
nm	Nanometer
PDI	Polydispersity index
PTX	Paclitaxel
TEM	Transmission electron microscopy
t1/2	Half life
Vd	Apparent volume of distribution

Acknowledgements

The authors are highly thankful to the Department of Science and Technology (DST), Govt. of India for financial support to carry out the project (sanction no. DST/INSPIRE FELLOWSHIP/2012/237). Further, Indian Institute of Chemical Biology (IICB), Kolkata, Indian Association for the Cultivation of Science (IACS), Kolkata and Bose Institute, Kolkata are also being acknowledged for providing some instrumentation facilities.

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Footnote

† Mita Chatterjee Debnath has contributed significantly and has equal authorship in this paper.

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