Intravenous administration of trans-resveratrol-loaded TPGS-coated solid lipid nanoparticles for prolonged systemic circulation, passive brain targeting and improved in vitro cytotoxicity against C6 glioma cell lines

Abstract

trans-Resveratrol (RSV), a natural molecule isolated from red wine, is widely known for several therapeutic potentials. RSV is proved for cardioprotective, vasodilation, anti-inflammatory, and anticancer effects. Recently, anticancer potential against glioma cells has also been reported. However, the clinical application of RSV in glioma treatment is largely limited because of its rapid metabolism and elimination from systemic circulation thereby exhibiting low biological half-life and poor brain distribution as well. Therefore, the main objective of this study was to enhance the circulation time, biological half-life and passive brain targeting of RSV using D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)-coated solid lipid nanoparticles (RSV-TPGS-SLN). RSV-TPGS-SLN formulations were prepared by a solvent emulsification evaporation method and evaluated for several nanoparticulate characteristics. In vitro anticancer potential and cellular internalization of nanoparticles were also investigated in C6 glioma cell lines. Pharmacokinetics and biodistribution studies were carried out following intravenous administration in healthy Charles Foster rats. RSV-TPGS-SLN showed significantly higher in vitro cytotoxicity against C6 glioma cell lines and excellent cellular internalization. RSV-TPGS-SLN showed 11.12 and 9.37 times higher area under the curve and plasma half-life than RSV solution, respectively. Moreover, brain distribution of RSV-TPGS-SLN was found to be 9.23 times higher in comparison to that of RSV alone. Thus, we anticipate that the RSV-TPGS-SLN formulation can be applied as a potential tool for improving circulation time, biological half-life and passive brain targeting of RSV, thereby being immensely useful in the treatment of glioma.

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Introduction

Cancer is one of the most important disorders causing morbidity and mortality all over the world. Amongst several cancer types, glioma is a grade IV neuroepithelial tumour, accounting for 80% of all primary central nervous system tumours.¹ Glioma is diffusely penetrating throughout the brain and extends far beyond the original tumour mass. As a consequence of such metastatic nature, unfortunately, every last tumour cell cannot be removed surgically. Despite combination treatment of surgery, radiotherapy and chemotherapy, glioma is an insidious and destructive type of brain cancer associated with poor prognosis, frequent recurrence and extremely high lethality.² Surgical resections of brain tumours led to a median survival of 3 months.³ Additional radiotherapy and chemotherapy successfully prolong median survival of the patient up to a year.⁴ Therefore, modern research is still trying to find new drug molecules and novel therapeutic delivery systems to improve the lifetime of glioma patients.

trans-Resveratrol (3,4,5-trihydroxystilbene) (RSV) is a natural non-flavonoid polyphenolic compound abundantly present in grapes, red wine, peanuts, berries, and in several materials of normal human diet. RSV was first isolated from the roots of Veratrum grandiflorum O. Loes (white hellebore) in 1940 and later from the roots of Polygonum cuspidatum (Japanese knotweed) in 1963.^5,6 RSV exists in cis and trans forms. Of these, the trans form of RSV was proved for several desirable biological actions such as cardioprotection, preventing platelet aggregation, vasodilation, prolongation of lifespan and cancer prevention.⁷ Recently, RSV has been proved for its anticancer potential against glioma.^8–10

Several molecular mechanisms have been proved for the chemopreventive and chemotherapeutic potential of RSV against glioma.¹¹ RSV initiated p53-dependent apoptosis in glioma cells through essential binding to plasma membrane integrin αVb3.¹² RSV induced both dose-dependent and time-dependent apoptosis in human glioma U251 and U87 cells by suppressing cyclin D1 expression in G0/G1 growth phase.⁹ In C6 glioma cell lines, RSV increased the expression of caspase-3 mRNA and caspase-3 activation, thereby inhibiting cell growth.⁸ Cancer cells of solid tumours stimulate the formation of new blood vessels for providing nutrients and oxygen to tumour cells for the development of the tumour. Malignant gliomas are vascular tumours that produce an important mediator of angiogenesis called vascular endothelial growth factor (VEGF). RSV suppressed VEGF expression in rat RT-2 glioma cells and inhibited the proliferation of human umbilical vein endothelial cells. Intraperitoneal administration of RSV to Fischer 344 rats implanted with RT-2 glioma cells showed anti-tumour and anti-angiogenesis efficacy. Survival rates of animals were significantly increased.¹³ RSV is also reported for suppression of tumour invasion by inhibiting matrix metalloproteinases (MMPs) which is a key factor involved in the degradation of extracellular matrix during invasion.¹⁰ Thus, RSV exerts cell cycle arrest by anti-angiogenesis and reduction of tumour invasion mechanisms. Though RSV shows strong efficacy against glioma cells and is associated with several desirable pharmacological effects, its therapeutic applications are limited because of short biological half-life, rapid metabolism and elimination.

Plasma half-life (t_1/2) of RSV after oral administration was found to be only 15 minutes.¹⁴ RSV undergoes glucuronidation by glucuronosyl transferase to form trans-resveratrol-C/O-diglucuronides and sulfonation by sulfotransferase to form trans-resveratrol-3-sulfate and trans-resveratrol-disulfates.¹⁵ About 22–44% of the administered dose or 31–63% of glucuronic acid conjugates and sulfate conjugates were excreted within 12 h in urine.^15,16 Intravenous administration of RSV also showed a short t_1/2 ranging from 7.8 to 33 minutes.^17,18 Short half-life and rapid metabolism of RSV require higher dose and frequent administration for achieving a therapeutic effect. Several attempts such as complexation with β-cyclodextrins, biodegradable polymeric nanoparticles, solid lipid nanoparticles, polymeric lipid-core nanocapsules and β-cyclodextrin nanosponges have been made in milieu for improving bioavailability and decreasing intensive metabolism.¹⁵ RSV nanocapsules were prepared by an interfacial polymer deposition approach and in vivo biodistribution studies were carried out in rats.¹⁹ RSV nanocapsules showed significantly higher concentrations in brain, liver and kidney in comparison to free RSV after i.v. and oral administration. Lesion indexes of nanocapsule-treated animals were found to be reduced up to 9-fold compared to free RSV-treated groups in the duodenum, jejunum and ileum following oral as well as i.p. treatment. RSV-loaded small oligolamellar liposomes prepared by a sonication/extrusion method showed sustained release, higher efficacy of resveratrol for cell proliferation, photoprotection and a cell-stress response against UV-B-induced oxidative damage.^20,21 Oral administration of RSV-curcumin co-encapsulated liposomes in prostate specific PTEN knockout mice showed higher bioavailability and improved anti-tumor effect against prostate cancer.²² Intravenous administration of RSV liposomes in nude Balb/c female mice with subcutaneous head and neck squamous cell carcinoma led to significant reduction (70%) of tumour volume.²³ RSV encapsulated in the amphiphilic block copolymer methoxy poly(ethylene glycol)-poly(caprolactone) showed better ability to penetrate cell membranes and higher efficacy against glioma cells than free drug.²⁴ Solid lipid nanoparticles prepared using compritol 888 ATO (3%, w/w), phospholipon 80H and poloxamer 188 showed higher stability and intracellular delivery of RSV.²⁵ An excellent review of Amri et al. provides further details on RSV nanoformulations for bioavailability enhancement.¹⁵ However, prolongation of systemic circulation has not yet been addressed to improve the therapeutic potential of RSV. Moreover, a major obstacle for glioma chemotherapy is the blood brain barrier (BBB). Therefore, brain targeting efficacy is also essential to attain the chemopreventive and anticancer potential of RSV against glioma.

Nanoparticulate drug delivery systems have been investigated for delivery of several therapeutic molecules across the BBB.²⁶ Among them, solid lipid nanoparticles (SLNs) have been proved to be potential drug delivery systems for brain targeting through passive mechanisms. SLNs are made up of lipids present in the human body and several foodstuffs. SLNs are highly suitable for brain-targeted chemotherapeutics due to their small diameter, spherical shape and favourable zeta potential, prolonged drug release, stability, rapid cellular uptake (internalisation) (5–10 min), avoidance of reticuloendothelial system, etc.²⁷ SLNs were also proved as safe and potential carriers for parenteral administration of chemotherapeutic agents.²⁸ Therefore, SLN was explored in the present investigation for passive brain targeting of RSV.

D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is a novel stabilizer showing higher entrapment efficiency and enhanced cellular uptake over traditional stabilizers such as polyvinyl alcohol, poloxamer 188, tween 80 etc.^29,30 TPGS is also reported for a wide variety of pharmaceutical applications such as bioavailability enhancer, emulsifier, and P-glycoprotein (P-gp) inhibitor.^31,32 However, the effect of TPGS on pharmacokinetics, brain accumulation potential and cytotoxic potential of SLN through intravenous administration has not been investigated. Therefore, the main objective of this study was to develop RSV-loaded TPGS-coated SLN (RSV-TPGS-SLN) formulation for prolonging the systemic circulation after intravenous (i.v.) administration in rats. Further, the passive brain-targeting potential of RSV-TPGS-SLN was also investigated by tissue distribution studies for proving potential utilization in the treatment of glioma. The proposed structure of RSV-TPGS-SLN is shown in Fig. 1. Systematic nanoparticulate characterizations were carried out extensively to evaluate the properties of RSV-TPGS-SLN. In vitro cytotoxicity and cellular internalization of fluorescent molecule-loaded SLN were also investigated to demonstrate the efficacy of the prepared SLN against glioma.

Fig. 1 Schematic diagram of RSV-TPGS-SLN showing molecular arrangement of drug and other SLN components.

Experimental

Materials

RSV was kindly provided by Cayman Chemical Company (E. Ellsworth Road, Ann Arbor, MI, USA). Tristearin, 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St Louis, MO, USA). Phosphatidylcholine from soybean (LIPOID S 100) and TPGS were kind gift samples of Lipoid GmbH (Ludwigshafen, Germany) and Antares Health Products Inc. (St Charles, Illinois, USA), respectively. Coumarin 6 was kindly provided by Parishi Chemicals (Surat, India). Analytical grade chloroform, ethanol and methanol were purchased from SD Fine-Chem Ltd (Mumbai, India). C6 glioma cells were obtained from National Centre for Cell Science (NCCS), Pune, India. Dulbecco's modified Eagle's medium (DMEM) with high glucose and foetal bovine serum albumin were purchased from Himedia (Mumbai, India) and Thermo Fisher Scientific – Gibco (Waltham, MA, USA). Buffer salts and other chemicals used in this study were of analytical grade.

HPLC analytical method

Reverse-phase high-performance liquid chromatography (HPLC) was used for RSV quantifications in this study as reported earlier.³³ The HPLC system consisted of inline degasser, 515 HPLC binary pump (Waters, USA), rheodyne 7725i manual injector (Waters, USA), C18 reverse-phase (250 × 4.6 mm, 5 μm) ODS2 column protected with a guard column (12 × 4.6 mm, 5 μm) of the same material (Waters Corp., Milford, MA, USA), and a photodiode array detector (Waters, USA). The mobile phase used was methanol : phosphate buffer, pH 6.8 (pH adjusted with 0.5% v/v orthophosphoric acid solution in Milli-Q water) (63 : 37%, v/v). The flow rate of mobile phase was set at 1.0 mL min⁻¹. The column was maintained at 25 ± 2 °C using a column oven (Waters, USA) and the effluent was detected at 306 nm. HPLC peak area and retention time measurements were determined using the operating software Empower Node 2054. Standard curves were plotted in the range of 10–5000 ng mL⁻¹.

Preparation of RSV-TPGS-SLN

RSV-loaded TPGS-coated SLN was prepared by a solvent emulsification evaporation method as reported elsewhere with slight modifications.³⁴ Briefly, RSV, tristearin, soya phosphatidylcholine (S-100) and TPGS were dissolved in 2 mL mixture of chloroform and methanol (2 : 1) by warming in a water bath. The warm organic phase was emulsified with 20 mL of aqueous phase (triple distilled water) maintained at 75 °C using ultraturrax (IKA, Germany) using a probe of 1 cm diameter, operated at 18 000 rpm for 10 min. The dispersion was sonicated at a frequency of 0.5 cycles and 60% amplitude using a probe-type ultrasonicator (Heilscher, Germany) and was stirred using a magnetic stirrer for 24 h for evaporation of organic solvent. Drug loading was 0.05% to the volume of formulation. The concentration of soya phosphatidylcholine and TPGS were 0.25% each to the volume of formulation (20 mL). The resultant nanoparticles were washed twice with triple distilled water by a centrifugation method using Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane, molecular-weight cut-off = 10 000 (Millipore, Billerica, MA, USA), at 4000 rpm for 20 min (C-24BL, Remi Centrifuge, India) to remove un-entrapped RSV. The filtered nanoparticles were re-suspended in required volume of 0.9% w/v sodium chloride (normal saline). Similarly coumarin-6 loaded fluorescent SLN (COU-TPGS-SLN) for cell internalization studies was prepared using the same procedure by replacing RSV with coumarin-6 (0.05% w/v). A placebo-TPGS-SLN was also prepared without drug. The drug to lipid ratio and sonication time (varied to get lower particle size and higher entrapment efficiency) of various batches of RSV-TPGS-SLNs are presented in Table 1.

Table 1 Drug to lipid ratio/sonication time of different batches of RSV-TPGS-SLNs and characterization results

Formulation code	Drug : tristearin ratio (mg)	Sonication time (min)	Polydispersity index	Zeta potential (mV)
RSV-TPGS-SLN 1	1 : 5	1	0.486 ± 0.12	−10.77 ± 1.02
RSV-TPGS-SLN 2	1 : 5	3	0.43 ± 0.09	−10.59 ± 0.94
RSV-TPGS-SLN 3	1 : 5	5	0.241 ± 0.11	−9.4 ± 1.24
RSV-TPGS-SLN 4	1 : 10	1	0.462 ± 0.15	−11.78 ± 1.03
RSV-TPGS-SLN 5	1 : 10	3	0.263 ± 0.12	−10.5 ± 2.94
RSV-TPGS-SLN 6	1 : 10	5	0.358 ± 0.76	−9.44 ± 1.59
RSV-TPGS-SLN 7	1 : 15	1	0.311 ± 0.14	−19.64 ± 2.03
RSV-TPGS-SLN 8	1 : 15	3	0.277 ± 0.09	−11.91 ± 0.97
RSV-TPGS-SLN 9	1 : 15	5	0.347 ± 0.15	−6.07 ± 1.49

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Characterization of RSV-TPGS-SLN

Particle size, polydispersity index and zeta potential. RSV-TPGS-SLN was placed in a sample cuvette made of polystyrene and the particle size and polydispersity index were measured using a Delsa™ Nano C particle analyzer (Beckman, USA). The mean particle size was determined from triplicate measurements of each sample. Similarly, samples were placed in a zeta cell and the zeta potential was measured.

Entrapment efficiency and morphology. The encapsulation efficiency of RSV-TPGS-SLN was determined after 24 hours of preparation by a direct method using HPLC. Briefly, 10 mg of lyophilized RSV-TPGS-SLN was dissolved in 1 mL of methanol and further diluted suitably with mobile phase. The diluted nanoparticle solution was filtered through a 0.45 μm syringe filter and injected into the HPLC system for quantification.
where W_N is the amount of drug encapsulated in RSV-TPGS-SLN and W_T is the total amount of drug used for the preparation of SLN.

The shape of optimized batch of RSV-TPGS-SLN was analysed by transmission electron microscopy (TEM, JEM 2010F, JOEL, Japan). Samples were prepared by placing one drop on a copper grid, dried under vacuum and examined.

In vitro drug release. In vitro release of RSV-TPGS-SLN was carried out using a dialysis membrane (12 000–14 000 Da molecular weights) in phosphate buffer saline (PBS) pH 7.4 containing 0.1% w/v of tween 80. RSV-TPGS-SLN equivalent to 2 mg of RSV was added to a dialysis tube and placed in 50 mL of release medium stirred at 100 rpm using a magnetic stirrer at 37 °C. Aliquots of sample (1 mL) were withdrawn at pre-determined time intervals (0, 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36 and 48 hours). The sink condition was maintained by replacing equal volume of fresh release medium. The samples were analyzed using HPLC for quantification. The percentage of drug release was plotted against time to assess the RSV release pattern. The in vitro release data were also plotted for zero-order, first-order, Higuchi and Korsmeyer–Peppas models to assess the kinetics and mechanism of drug release.

Drug–excipient compatibility studies. The chemical interaction of drug with other excipients was assessed by Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectra of RSV and lyophilized RSV-TPGS-SLN were obtained by conventional KBr disk/pellet method (Shimadzu, model 8400S, Tokyo, Japan). A small quantity of sample was ground gently with anhydrous KBr and compressed to form a thin pellet in the FT-IR sample holder. The FT-IR spectrum was obtained in the range 400 to 4000 cm⁻¹.

DSC analysis. DSC thermograms of RSV, tristearin, soya phosphatidylcholine, TPGS and lyophilized RSV-TPGS-SLN were obtained to study the drug–lipid interaction and crystalline nature of RSV using a DSC Q1000 (TA Instruments, USA). Samples (2 mg) were sealed in aluminium pans and scanned at a heating rate of 10 °C min⁻¹ over the temperature range of 4–300 °C, under nitrogen flow of 50 mL min⁻¹.

X-ray diffraction analysis. X-ray diffraction patterns of RSV and lyophilized RSV-TPGS-SLN were obtained with an X-ray diffractometer (Bruker D8 Discover), using Ni-filtered Cu-K radiation at 45 kV and 40 mA. The scattered radiation in crystalline regions of the sample was measured with a vertical goniometer. The diffraction patterns were obtained between 5° and 80° angle using a step size of 0.045° with a detector resolution in 2θ (diffraction angle) at 25 °C.

Cytotoxicity against C6 glioma cells. In vitro cytotoxicity of RSV, RSV-TPGS-SLN and placebo-TPGS-SLN in C6 glioma cells was screened by MTT assay. Briefly, C6 glioma cells were seeded onto 96-well microtitre plates at 1 × 10⁴ cells per well in complete DMEM and incubated at 37 °C in humidified CO₂ (5%) incubator (Galaxy® 170 S, Eppendorf, Germany) environment for 24 h. The cells were exposed to fresh DMEM culture medium containing different concentrations of test samples (RSV, RSV-TPGS-SLN 5, placebo-TPGS-SLN and equivalent lipid components of placebo-TPGS-SLN without TPGS) for 72 h at 37 °C in a humidified CO₂ (5%) incubator. After incubation, the medium was replaced with 20 μL of MTT (5 mg mL⁻¹ in PBS) solution and the cells were incubated for 4 h at 37 °C in a humidified CO₂ (5%) incubator. Culture medium and MTT were removed completely. The formed insoluble formazan crystals that are proportional to the number of viable cells were dissolved in 100 μL dimethyl sulfoxide. The plate was agitated for 10 min and absorption was measured at 570 nm using a multimode reader (Synergy H1 hybrid, Biotek, USA). The absorbance of control cells treated with equivalent quantity of nanoparticulate dispersion medium (0.9% w/v of sodium chloride, normal saline) was used to calculate the cytotoxicity. The percentage viability and cytotoxicity was calculated by the following equation:

Data are expressed as mean ± SD (n = 3).

Cellular uptake of COU-TPGS-SLN. C6 glioma cells in complete DMEM were seeded on a cover glass placed in 35 mm Petri dishes at 1 × 10⁴ viable cells/cover glass. The cells were incubated overnight at 37 °C in a humidified CO₂ (5%) incubator and subsequently incubated with COU-TPGS-SLN (100 μL per cover glass). After 2 h, the cells were washed thrice with cold PBS (pH 7.4) and fixed using paraformaldehyde for 20 min. The cells were again washed thrice with cold PBS. The nuclei were stained by incubating with 0.1 μg mL⁻¹ of DAPI for 10 min. Further, the cell monolayer was washed thrice with PBS, and the cover glass was taken from Petri dishes, mounted on microscope slides and observed by a confocal laser scanning microscope (CLSM) (Zeiss LSM510 Meta LSM with Plan-Apo 40X (1.3-NA) and Zeiss LSM Meta 510 software).³⁰

Haemocompatibility studies

Evaluation of haemolysis. As RSV-TPGS-SLN was intended for intravenous administration, the evaluation of haemolytic property was essential to prove the haemocompatibility. Haemolytic analyses of RSV, RSV-TPGS-SLN and placebo-TPGS-SLN were performed based on earlier publications.^35,36 Human blood samples were purchased from an authorized blood bank. 2 mL of blood was centrifuged at 1344 × g for 10 min at room temperature in a sterile graduated centrifuge tube. Plasma layer was removed carefully using a micropipette. Equal volume of normal saline solution was added to the erythrocyte pellet and the tube was mixed gently. The erythrocyte suspension was centrifuged again at 1344 × g for 10 min at room temperature. This washing protocol was repeated 3 times. Finally, the erythrocytes were diluted up to 10 mL using normal saline solution and resuspended gently. RSV, RSV-TPGS-SLN (equivalent to 10, 50 and 100 μg mL⁻¹ of RSV) and equivalent placebo-TPGS-SLN were mixed with 2 mL of erythrocyte suspension separately in sterilized Eppendorf tubes. Positive control (100% lysed erythrocytes) and spontaneous negative control samples were prepared by diluting equal volumes of erythrocyte suspension with 1% Triton X-100 and normal saline, respectively. All samples were incubated at 37 °C and mixed gently every 15 min. Aliquots (200 μL) were collected at predetermined time intervals (0.5, 1, 2, 4 and 8 h) and centrifuged at 1344 × g for 10 min. Supernatants (100 μL) were incubated for 30 min at room temperature for oxidation of haemoglobin in to oxyhaemoglobin. The absorbance was measured spectrophotometrically at 540 nm. The percentage of haemolysis was calculated using the following formula:
where A_Sample is the absorbance of supernatants of erythrocytes incubated with RSV/SLNs, A_{Spontaneous control} is the absorbance of supernatants of erythrocytes incubated with normal saline equivalent to the volume of samples and A_{Positive control} is the absorbance of supernatants of erythrocytes incubated with 1% Triton X-100 solution in normal saline. Triplicate experiments were performed and the data are expressed as mean ± SD (n = 3).

Platelet aggregation. Quantitative evaluation of platelet aggregation was carried out by counting the platelets using a haematological counter (Multisizer 4, Beckmann Coulter, USA) after incubation with test samples.^35,36 Briefly, RSV, RSV-TPGS-SLN (equivalent to 10, 50 and 100 μg mL⁻¹ of RSV) and equivalent placebo-TPGS-SLN were incubated at three different concentrations for 2 h at 37 °C with 1 mL of blood. Similarly, spontaneous control was prepared by incubating whole blood samples with PBS (equivalent to the volume of SLNs) at the same experimental conditions. After incubation, samples were diluted and analysed by a haematological counter (Multisizer 4, Beckmann Coulter, USA) after mixing. All samples were analyzed in triplicate and the values are expressed as mean ± SD (n = 3).

Qualitative analysis of platelet aggregation was carried out by microscopic observation of stained peripheral blood smears after incubating heparinised whole blood with test samples. After incubation, peripheral blood smears were prepared on clean glass slides. The slides were air dried for 2 min and stained by Leishman's stain for 5 min (Span Diagnostics, India). After rinsing the slides with distilled water, a cover glass was placed on each and analyzed with an optical microscope in immersion objective and images were captured using a digital camera.

Pharmacokinetic studies. The protocol for pharmacokinetic study was approved by the Central Animal Ethical Committee of the University (CPCSEA). Healthy Charles Foster rats (150–200 g) of either sex were obtained from Central Animal House, Institute of Medical Science, Banaras Hindu University, Varanasi, India. The rats were housed in polypropylene cages over dust-free husk for one week before experiments with 12 h light/dark cycle at 25 ± 2 °C and 40–70% relative humidity. The animals were fed with rat chow and water ad libitum. The animals were divided into 2 groups of 6 rats in each group. Free RSV solubilised in 0.3 M β-cyclodextrin in sterile water for injection and RSV-TPGS-SLN (equivalent to 2 mg kg⁻¹ of RSV) were administered via i.v. route through tail vein. Blood samples (200 μL) were collected through the retro-orbital vein at predetermined time intervals (0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h) under ether anaesthesia. Blood samples were collected in heparinised Eppendorf tubes.

The collected blood was centrifuged at 4000 rpm (C-24BL, Remi Centrifuge, India) for 15 min at room temperature. Plasma was separated and stored at −20 °C until analysis. The liquid–liquid extraction (protein precipitation) method was used for separation of RSV from plasma. The plasma was deproteinized by adding ethyl acetate and centrifuged at 15 000 rpm for 15 min. The supernatant was transferred to a fresh tube and evaporated to dryness at 45 °C in nitrogen gas atmosphere. The residue was reconstituted with mobile phase and centrifuged at 15 000 rpm for 10 min. Supernatant (20 μL) was injected into a HPLC system for quantification of RSV. Peak area of chromatograms of samples was calculated using Empower Pro® HPLC software and respective plasma concentration was calculated using linearity equation of calibration curve. The calibration curve was previously prepared by spiking various concentrations (10 to 5000 ng mL⁻¹) of RSV in plasma and analysed by HPLC using the same protocol. Pharmacokinetic analysis was carried out using WinNonlin 6.1 professional software (Pharsight Corporation, NC, USA). Noncompartmental intravascular analysis was employed for the calculation of pharmacokinetic parameters.

In vivo biodistribution studies. The complete protocol for tissue distribution studies was approved by the CPCSEA. Healthy Charles Foster rats (150–200 g) of either sex, obtained from Central Animal House, were randomly divided into 2 groups consisting of 3 rats in each group. RSV solubilised in 0.3 M β-cyclodextrin in sterile water for injection and RSV-TPGS-SLN (equivalent to 2 mg kg⁻¹ of RSV) were administered via i.v. route through tail vein in overnight-fasted animals. The animals were sacrificed at 90 min after injection by cervical dislocation. Brain, liver, kidney, lungs and spleen were rapidly excised and washed with sterile physiological saline solution. The tissue samples were wiped with filter paper, weighed and immediately stored in frozen condition until analysis. The tissue samples were finely minced with scissors and homogenised with 2 mL of ethyl acetate. Homogenized samples were centrifuged at 15 000 rpm for 15 min. The supernatant was collected in a clean tube. The residue was extracted again with 2 mL of ethyl acetate and combined with the first portion of supernatant. The supernatant was evaporated to dryness under nitrogen gas stream. The residue was reconstituted with mobile phase and analyzed by HPLC. The results are reported as the amount of RSV per gram of tissue.

Statistical analysis. Statistical analysis was carried out by one-way ANOVA followed by Bonferroni test using GraphPad Prism statistical software (GraphPad Software Inc., La Jolla, CA) to compare all data. p value < 0.05 was considered statistically significant.

Results and discussion

HPLC analytical method

The HPLC calibration curve of RSV was found to be linear from 10 to 5000 ng mL⁻¹ with a correlation coefficient (R²) value of 0.9998 for in vitro samples. As the HPLC method followed in our study is well established and published, we are reporting only the correlation coefficient (R²) value.³³

Characterization of RSV-TPGS-SLN

Particle size, polydispersity index and zeta potential. The mean particle sizes of various RSV-TPGS-SLN batches varied from 128.6 ± 16.11 nm to 429.1 ± 24.75 nm as shown in Fig. 2(a). As the drug to lipid ratio varied from 1 : 5 to 1 : 15, particle size was statistically increasing for all sets of sonication times (p < 0.05). For instance, particle size for drug to lipid ratio of 1 : 5, 1 : 10 and 1 : 15 at 1 min of sonication time (RSV-TPGS-SLN batches 1, 4 and 7) was found to be 196.3 ± 13.79, 265.4 ± 17.18 and 423.9 ± 25.09 nm, respectively, which are statistically increasing (p < 0.05). As the sonication time increased from 1 min to 3 min, the particle size decreased significantly for all sets of drug to lipid ratio (1 : 5, 1 : 10 and 1 : 15) (p < 0.05). The decrease in particle size is due to sonication energy which breaks down nanostructures during preparation. On further increasing sonication time from 3 to 5 min, particle size was found to be statistically increased (p < 0.05). The increase in particle size may be due to coalescence or aggregation of lipids resulting from excess sonication time.³⁷ Polydispersity index of RSV-TPGS-SLN batches varied from 0.241 ± 0.11 to 0.486 ± 0.12. Zeta potential varied from −6.07 ± 1.49 to −19.64 ± 2.03 mV.

Fig. 2 (a) Particle size and (b) entrapment efficiency of various batches of RSV-TPGS-SLNs. (c) Transmission electron micrograph and (d) in vitro drug release profile of RSV-TPGS-SLN 5. Values are represented as mean ± SD (n = 3).

Entrapment efficiency and morphology. Entrapment efficiency of RSV-TPGS-SLN formulations varied from 38.75 ± 5.64 to 74.42 ± 7.67% as shown in Fig. 2(b). As the drug to lipid ratio was varied from 1 : 5 to 1 : 10, the entrapment efficiency was found to be increased for all sets of sonication times (p < 0.05). For instance, when the drug to lipid ratio was varied from 1 : 5 to 1 : 10, the entrapment efficiency was found to be increased from 49.28 ± 2.67 to 70.18 ± 5.19% at 3 min sonication time. Further increase in concentration of lipid (drug to lipid ratio varied from 1 : 10 to 1 : 15) did not show such an increase in entrapment efficiency. For example, entrapment efficiency was changed from 70.18 ± 5.19 to 71.75 ± 5.67% at 3 min of sonication. These results suggested that a drug to lipid ratio of 1 : 10 is sufficient to accommodate the maximum number of RSV molecules in the SLN matrix. The higher entrapment efficiency may be due to the high lipid concentration that resulted in housing of more RSV molecules (lipophilic in nature) at the interspaces of the hydrophobic lipid matrix. The lesser entrapment efficiency of RSV-TPGS-SLN formulations having 1 : 5 drug to lipid ratio may be due to saturation of lipid matrix interspaces with fewer RSV molecules. The formulations having a drug to lipid ratio of 1 : 15 failed to improve the partition of RSV molecules during formation of nanoparticles which may be a reason for the insignificant increase in entrapment efficiency from the formulations having drug to lipid ratio of 1 : 10. The RSV-TPGS-SLN 5 having lower particle size of 203.1 ± 14.91 nm, narrow polydispersity index of 0.263 ± 0.12 and higher entrapment efficiency of 70.18 ± 5.19% was selected as optimized formulation for further investigations. TEM micrographs of RSV-TPGS-SLN 5 showed particles of spherical shape (Fig. 2(c)). As the RSV-TPGS-SLN was proposed to be used in the treatment of brain cancer, the mean particle size was optimized to be suitable for passive brain targeting. The intercellular gap between the cancer cells in most solid tumours was found to be between 380 and 780 nm. Accordingly, nanoparticles of 100 to 300 nm in size can easily extravasate through these intercellular gaps.³⁸ The size of optimized RSV-TPGS-SLN 5 will be suitable for extravasation through these passageways and accumulate at tumour sites via the enhanced permeability and retention effect. Therefore, RSV-TPGS-SLN 5 formulation having optimum size can be applied for passive accumulation at tumour sites.

In vitro drug release. In vitro drug release profile of RSV-TPGS-SLN 5 is shown in Fig. 2(d). The formulation showed sustained release without any burst release. The cumulative percentage of drug released was found to be 69.87 ± 6.97% after 48 h. Absence of burst release may be due to a lack of free RSV molecules at the surface of SLN or in the dispersion medium of the formulation. As the lipophilic RSV molecules are housed at intermolecular spaces of the SLN matrix, the drug release may be sustained which depends on strong drug–lipid affinity, concentration gradient and depletion of drug molecules from the lipid matrix. Higuchi kinetics was shown to be the best fit model for RSV-TPGS-SLN 5 (R² = 0.9854) (Table 2). Therefore, the drug release mechanism was found to be diffusion. The release exponent (n) value calculated by the Korsmeyer–Peppas model was found to be 0.6307. Therefore, the mechanism of release was found to be anomalous transport.

Table 2 Correlation coefficient (R²) and release exponent (n) values of in vitro drug release kinetics from RSV-TPGS-SLN

Model of release kinetics	Correlation coefficient (R^2)	Release exponent (n)
Zero order	0.8282	—
First order	0.9422	—
Higuchi model	0.9854	—
Korsmeyer–Peppas model	0.9511	0.6307

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Drug–excipient compatibility studies. FT-IR spectra of RSV and RSV-TPGS-SLN 5 are shown in Fig. 3(a) and (b). RSV showed its characteristic absorption bands at 3288.7 cm⁻¹ for O–H stretching of alcoholic group, 1606.7 cm⁻¹ for C–C stretching of olefinic group, 1442.8 cm⁻¹ and 1587.4 cm⁻¹ for C=C stretching of aromatic ring, 1153.4 cm⁻¹ for C–O stretching and 964.4 cm⁻¹ for trans olefinic bond (Fig. 3(a)). The RSV-TPGS-SLN 5 spectrum showed all the characteristic peaks of RSV with minor shifts in absorption bands (Fig. 3(b)). Alcoholic O–H stretching at 3184.58 cm⁻¹, C–C stretching of olefinic group at 1635.69 cm⁻¹, C=C stretching of aromatic ring at 1464.02 cm⁻¹ and 1587.47 cm⁻¹, C–O stretching at 1147.68 cm⁻¹ and trans olefinic band at 966.37 cm⁻¹ were observed. The presence of all characteristic absorption bands of RSV in the RSV-TPGS-SLN 5 spectrum confirmed that there is no potential chemical interaction between RSV and other formulation excipients.

Fig. 3 Fourier transform infrared (FT-IR) spectra of (a) RSV and (b) RSV-TPGS-SLN 5.

DSC analysis. DSC thermograms of RSV, phosphatidylcholine, tristearin, TPGS and RSV-TPGS-SLN 5 are shown in Fig. 4(a). RSV showed a sharp endothermic peak at 267.71 °C which corresponds to its melting point. Phosphatidylcholine showed two blunt peaks at 68.12 and 79.04 °C. Tristearin and TPGS showed sharp endothermic peaks at 59.23 and 35.11 °C, respectively, corresponding to their respective melting points. RSV-TPGS-SLN 5 showed a sharp peak at 69.71 °C which corresponds to the merged peaks of tristearin and phosphatidylcholine with minor shift in their positions. Absence of RSV melting peak at 267.71 °C in the chromatogram of RSV-TPGS-SLN 5 suggested that RSV is dispersed in amorphous form.

Fig. 4 (a) Differential scanning calorimetric thermograms of RSV, phosphatidylcholine, tristearin, TPGS and RSV-TPGS-SLN 5. (b) X-ray diffraction patterns of RSV and RSV-TPGS-SLN 5.

X-ray diffraction (XRD) analysis. XRD patterns of RSV and RSV-TPGS-SLN 5 are shown in Fig. 4(b). RSV showed sharp diffraction peaks at 6.62°, 13.2°, 16.36°, 19.18°, 22.28°, 23.54°, 25.18°, 28.26°, 31.6°, 38.32° and 45.18° in 2θ scale. These results showed the crystalline nature of RSV. In contrast, RSV-TPGS-SLN 5 showed only two small peaks at 23.48° and 24.62° and did not show other characteristic intense sharp peaks. These results suggested that RSV is converted to amorphous form in the lipid matrix of RSV-TPGS-SLN 5. XRD results correlate well with the DSC results, confirming the conversion of RSV from crystalline form to amorphous form.

Cytotoxicity against C6 glioma cells. Cytotoxicity study was performed to assess the efficacy of RSV and RSV-TPGS-SLN 5 against C6 glioma cell lines. Cytotoxicity of pristine RSV, RSV-TPGS-SLN 5 and placebo-TPGS-SLN is shown in Fig. 5(a). RSV-TPGS-SLN 5 showed significantly higher cytotoxicity than RSV in all individual concentrations (p < 0.05). The additional cytotoxic potential can be explained from the cytotoxicity of placebo formulation. Placebo-TPGS-SLN showed statistically significant increase in cytotoxicity in each increment of its concentration (p < 0.05) whereas equivalent concentration of lipid components of placebo-TPGS-SLN dispersed in water without TPGS did not show any cytotoxicity (not shown in figure). These results evidently indicate that the cytotoxicity of placebo-TPGS-SLN is due to TPGS. The cytotoxic potential of TPGS has been proved for several types of cancer types both in vitro and in vivo.^39–42 TPGS was more effective at 40 μM concentration by inducing apoptosis in A549 cells as measured by DNA fragmentation.⁴² TPGS induced apoptosis in androgen receptor negative (AR⁻) DU145, PC3, and androgen receptor positive (AR+) LNCaP prostate cancer cells.³⁹ TPGS induced G1/S cell cycle arrest and apoptosis in breast cancer cell lines MCF-7 and MDA-MB-231 at 20 μM. The induction of G1/S phase cell cycle arrest by TPGS was associated with upregulation of P21, P27Kip1 proteins, and phospho-AKT and downregulation of antiapoptotic proteins survivin and Bcl-2.⁴¹ Essentially, TPGS did not affect the non-tumorigenic cells (MCF-10A and MCF-12F) suggesting its efficacy specifically against cancer cells. Therefore, the additive cytotoxicity of RSV-TPGS-SLN 5 formulation in our study is due to the presence of TPGS. The added cytotoxic potential of RSV-TPGS-SLN 5 will be more beneficial in the treatment of glioma without any toxicity to normal cells.

Fig. 5 (a) In vitro anticancer potential (cytotoxicity) of RSV, RSV-TPGS-SLN 5 and placebo-TPGS-SLN. Values are represented as mean ± SD (n = 3), p < 0.05. Cellular internalization of coumarin-6 loaded TPGS-coated SLN in C6 glioma cancer cells assessed by CLSM: (b) FITC channel (green colour showing coumarin-6 loaded SLNs localized within the cytoplasm); (c) DAPI channel (blue colour showing stained nucleus); (d) superimposed image of FITC channel and DAPI channel.

Cellular uptake of coumarin-6 loaded SLN. Fig. 5 shows the CLSM images of C6 glioma cells after treatment with COU-TPGS-SLN and counter stained by DAPI to visualize the nucleus. Fig. 5(b) and (c) show CLSM images of FITC and DAPI channels captured using appropriate filters. The cellular uptake of images of COU-TPGS-SLN (green colour) was visualized by superimposing the FITC channel and DAPI channel (blue colour) (Fig. 5(d)). COU-TPGS-SLN showed excellent cell internalization in C6 glioma cells after 2 h of incubation. The nanoparticles were found to be concentrated at the cytoplasm. According to earlier reports, molecular mechanisms of RSV such as increased expression of caspase-3 mRNA and caspase-3 activation, inhibiting MMPs and upregulation of p53, Bax and Bak, downregulation of Bcl-2 for release of cytochrome c and deactivation of IAP take place at the cytoplasm.^8,10,43 Therefore, the present design of RSV-TPGS-SLN 5 will be more beneficial for exerting its cytotoxicity at the cytoplasm.

Haemocompatibility studies

Evaluation of haemolysis. Nanoparticle formulations should not cause haemolysis during and after intravenous infusion. As per international standards, spontaneous haemolysis limit is 1% to preserve the integrity and functionality of erythrocytes in blood.^35,44 In our study, the haemolysis of RSV and RSV-TPGS-SLN 5 formulation was within the recommended limit in all concentrations throughout the study period of 8 h (Fig. 6). Placebo-TPGS-SLN showed less than 1% haemolysis at 10 μg mL⁻¹ whereas the limit was exceeded with the samples treated with 50 μg mL⁻¹ at 8 h and 100 μg mL⁻¹ at 4 and 8 h. This may be due to the interaction of SLN components with blood cells at these concentrations. However, RSV-TPGS-SLN 5 of similar concentrations (50 and 100 μg mL⁻¹) showed haemolysis within the recommended limits. This can be explained by the protective effect of RSV on erythrocytes by decreasing malondialdehyde level and protein carbonyl group content.⁴⁵ Moreover, RSV is also reported for other antioxidative mechanisms such as reduction of glutathione and membrane sulphydryl groups (–SH) in erythrocytes which thereby exhibit a strong protective effect.⁴⁶ All these protective mechanisms of RSV prevent degradation of erythrocytes in RSV-TPGS-SLN 5 treated samples even at higher concentrations.

Fig. 6 Percentage of haemolysis at different time intervals in whole blood samples after addition of RSV, placebo-TPGS-SLN and RSV-TPGS-SLN at (a) 10, (b) 50 and (c) 100 μg mL⁻¹. (d) Number of platelets after addition of PBS, RSV, placebo-TPGS-SLN and RSV-TPGS-SLN at 10, 50 and 100 μg mL⁻¹. Values are represented as mean ± SD (n = 3).

Platelet aggregation. Excessive or inappropriate aggregation of platelets upon intravenous infusion of SLN formulations can lead to thrombus formations in blood vessels that result in transient ischaemia, myocardial infarction or stroke. Therefore, assessment of platelet aggregation upon i.v. administration of SLN formulations is essential. RSV-treated samples did not show any significant difference in platelet count compared to PBS-treated samples for all three concentrations. These results suggested that RSV is not involved in platelet aggregation. Placebo-TPGS-SLN showed significantly lower (p < 0.05) platelet count than PBS-treated samples. The lower platelet count observed in placebo-TPGS-SLN may be due to the effect of SLN components. Though all components of placebo-TPGS-SLN are present in RSV-TPGS-SLN 5, it showed higher platelet count than placebo-TPGS-SLN and similar platelet count to PBS-treated samples. This may be due to antiplatelet aggregation property of RSV. Systemic administration of RSV is proved to block platelet aggregation in rabbits.⁷ Moreover, RSV is also shown to decrease the size of thrombus generated by laser-induced damage to endothelium in genetically hypercholesterolaemic mice.⁷ Furthermore, RSV is also proved for an antiplatelet aggregation effect in vitro. Thromboxane A2 (TxA2) is a potent inducer of platelet aggregation. TxA2 is synthesized by COX1 in platelets. Prostacyclin is an antiplatelet aggregator which is synthesized by COX2 in vascular endothelial cells. RSV is proved for selective inhibition of COX1 which causes a decrease in TxA2 and thereby prevents platelet aggregation.⁷

In addition to platelet count, the platelet aggregation in whole blood was observed by optical microscopy after incubation. Erythrocytes, leucocytes and platelets were visualized and the platelets are indicated by arrow marks on the microphotographs (Fig. 7). Supportively, no platelet aggregation was seen in all samples (RSV, RSV-TPGS-SLN and placebo-TPGS-SLN) at all concentrations. Though a slight decrease in platelet count was observed with placebo-TPGS-SLN in quantitative measurements, platelets were distributed evenly throughout the blood smears upon microscopic observations. All these observations indicated that RSV-TPGS-SLN 5 and RSV are nontoxic, haemocompatible and safe for intravenous administration.

Fig. 7 Light microscopy images of Leishman's stained whole blood samples after treating with (a) PBS equivalent volume to 10 μg mL⁻¹ of test samples, (b) PBS equivalent volume to 50 μg mL⁻¹ of test samples, (c) PBS equivalent volume to 100 μg mL⁻¹ of test samples, (d) 10 μg mL⁻¹ RSV, (e) 50 μg mL⁻¹ RSV, (f) 100 μg mL⁻¹ RSV, (g) 10 μg mL⁻¹ placebo-TPGS-SLN, (h) 50 μg mL⁻¹ placebo-TPGS-SLN, (i) 100 μg mL⁻¹ placebo-TPGS-SLN, (j) 10 μg mL⁻¹ RSV-TPGS-SLN, (k) 50 μg mL⁻¹ RSV-TPGS-SLN and (l) 100 μg mL⁻¹ RSV-TPGS-SLN. Images were captured at a magnification of 100×.

Pharmacokinetic studies. Comparative plasma concentration time profiles of RSV and RSV-TPGS-SLN 5 up to 48 h and parts of the curves up to 2 h are shown in Fig. 8(a) and (b). The pharmacokinetic parameters are presented in Table 3. Plasma concentration time curve of RSV solution showed a rapid decline up to 0.5 h, slight increase at 1 h (a small second peak), declined again up to 2 h and remained undetectable after 2 h. Similarly, RSV-TPGS-SLN 5 showed a marked decline at 1 h, again rose to give a second peak concentration at 2 h and declined up to 8 h. The plasma concentration increased again to a small extent at 12 h and continued to decline up to 24 h, after which the concentration became undetectable. The appearance of a second peak and observation of a slight increase in plasma concentration in RSV and RSV-TPGS-SLN 5 may be due to enterohepatic circulation of drug. Enterohepatic circulation phenomenon of RSV was demonstrated by conversion of drug into glucuronide/sulfate conjugates and secreted to small intestine via breast cancer resistance protein and specific proteins multidrug resistance protein 2.⁴⁷ The observed enterohepatic circulation of drug in our study was well correlated with the earlier findings. Initial plasma concentration (C₀) of RSV (2625.33 ± 366.74 ng mL⁻¹) after i.v. administration was found to be significantly higher than that of RSV-TPGS-SLN 5 formulation (2162.39 ± 193.21 ng mL⁻¹) (p < 0.05). Area under the curve (AUC) and plasma half-life (t_1/2) after i.v. administration of RSV-TPGS-SLN 5 were found to be approximately 11.12 and 9.37 times higher than those of RSV, respectively. The higher AUC and t_1/2 values revealed that RSV-TPGS-SLN 5 had significantly enhanced systemic availability, long circulation and half-life compared to RSV. The improved pharmacokinetic profile of RSV-TPGS-SLN 5 formulation may be due to the presence of TPGS at the surface of nanoparticles. The plasma half-life (t_1/2) of RSV obtained in this study was higher than an earlier reported value (0.13 ± 0.02 h) at 15 mg kg⁻¹ and well correlated with the value (0.55 h, calculated by Bayesian estimations) in a population pharmacokinetic study at 2, 10 and 20 mg kg⁻¹ of RSV after i.v. administrations in rats.^17,18 Short t_1/2 of RSV is well correlated with its higher clearance (CL) value (1679.48 ± 460.73 mL h⁻¹ kg⁻¹). In contrast, the longer half-life of RSV-TPGS-SLN 5 is due to lower CL value (151.99 ± 11.44 mL h⁻¹ kg⁻¹). The volumes of distribution (V_L) of RSV (1383.18 ± 177.36 mL kg⁻¹) and RSV-TPGS-SLN 5 (1212.87 ± 159.04 mL kg⁻¹) were found to be the same (p > 0.05). At the same time, V_L of both RSV and RSV-TPGS-SLN 5 were found to be higher than the total body water of rat (150 mL kg⁻¹ for a body weight of 0.25 kg).¹⁷ These results suggested that both RSV and RSV-TPGS-SLN 5 undergo extensive tissue binding. Mean residence time (MRT) of RSV-TPGS-SLN 5 was found to be approximately 11.4 times higher than that of RSV solution which confirmed its long circulation potential. TPGS present at the surface of RSV-TPGS-SLN 5 prevents the adhesion of plasma proteins (by stealth nature) and thereby avoids recognition of SLNs by the reticuloendothelial system which ultimately resulted in prolonged systemic circulation. The overall observation of higher AUC, t_1/2 and MRT and lower CL of RSV-TPGS-SLN 5 than pristine RSV clearly indicates that the RSV-TPGS-SLN 5 design will be a potential tool for enhancing systemic availability and prolonged systemic circulation of RSV.

Fig. 8 Comparative plasma concentration time profile of RSV and RSV-TPGS-SLN 5: (a) 48 hours and (b) initial 2 hours after i.v. administration of 2 mg kg⁻¹ dose; each data point is the representation of mean ± SD (n = 6), p < 0.05. (c) Comparative in vivo biodistribution of RSV and RSV-TPGS-SLN 5 in brain, lungs, liver, spleen and kidney after i.v. administration of 2 mg kg⁻¹ dose; values are represented as mean ± SD (n = 3), p < 0.05. (*) Statistically significant difference from RSV; (^#) statistically insignificant difference from RSV.

Table 3 Pharmacokinetic parameters of RSV and RSV-TPGS-SLN 5 after intravenous administration of 2 mg kg⁻¹ dose^a

Pharmacokinetic parameters	RSV	RSV-TPGS-SLN-5
a Data are presented as mean ± SD (n = 6). (*) Statistically significant difference from RSV; (^#) statistically insignificant difference from RSV (p < 0.05).
Initial plasma drug concentration, C0 (ng mL^−1)	2625.33 ± 366.74	2162.39 ± 193.21*
Area under curve, AUC (ng h mL^−1)	1130.96 ± 245.50	12 574.95 ± 1074.95*
Plasma half-life, t1/2 (h)	0.59 ± 0.11	5.53 ± 0.55^#
Clearance, CL (mL h^−1 kg^−1)	1679.48 ± 460.73	151.99 ± 11.44*
Volume of distribution, VL (mL kg^−1)	1383.18 ± 177.36	1212.87 ± 159.04^#
Mean residence time, MRT (h)	0.60 ± 0.04	6.84 ± 0.23*

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In vivo biodistribution studies. The biodistribution of RSV and RSV-TPGS-SLN 5 is shown in Fig. 8(c). RSV-TPGS-SLN 5 showed the highest brain accumulation of 7.48 ± 1.69 μg g⁻¹, which is approximately 9.23 times higher than that of RSV solution (0.81 ± 0.48) (p < 0.05). Colloidal nanocarriers having sizes of 200 and 250 nm were well reported some time ago for crossing the BBB.^26,48,49 Passive brain-targeting efficiency of such nanoparticles is proved in healthy and diseased conditions in both in vitro and in vivo experiments.^26,48–50 Polydispersity of RSV-TPGS-SLN 5 was found to be 0.263 ± 0.12. Therefore, our RSV-TPGS-SLN 5 having a particle size of 203.1 ± 14.91 nm can be expected to effectively cross the BBB to deliver RSV in the brain. Kidney is the major elimination organ of RSV. Total excretion of RSV after i.v. administration was calculated to be 42.3 to 83.2%.¹⁶ The distribution of RSV-TPGS-SLN 5 in kidney (0.83 ± 0.13 μg g⁻¹) was found be approximately half that of RSV solution (1.540 ± 0.27 μg g⁻¹) (p < 0.05). The lower distribution of RSV-TPGS-SLN 5 in kidney would be expected to decrease the elimination of RSV. The difference in distribution of RSV-TPGS-SLN 5 and RSV in the lungs, liver and spleen was found to be statistically insignificant (p > 0.05). The overall review of biodistribution results showed that the brain distribution RSV-TPGS-SLN 5 was significantly (9.23 times) higher than that of RSV solution. Therefore, RSV-TPGS-SLN 5 can be applied as an effective tool for potential brain targeting of RSV which is useful in the treatment of glioma.

Conclusions

RSV-loaded TPGS-coated SLN formulations were successfully prepared and evaluated by state of the art techniques. The optimised RSV-TPGS-SLN 5 formulation showing average particle size of 203.1 ± 14.91 nm, narrow polydispersity and higher entrapment efficiency was selected as optimized formulation. RSV-TPGS-SLN 5 showed sustained drug release up to 48 h which is beneficial for prolonged action of a drug. RSV-TPGS-SLN 5 showed significantly higher cytotoxicity than RSV against C6 glioma cells (p < 0.05). Moreover, COU-TPGS-SLN showed excellent cellular internalization in C6 glioma cells. COU-TPGS-SLN was found to be concentrated at the cytoplasm of cancer cells which is the major site of action of RSV for anticancer activity. Therefore, the anticancer efficacy may be expected to be higher. Pharmacokinetic studies showed higher AUC, t_1/2 and MRT and lower CL of RSV-TPGS-SLN 5 than RSV solution. These results strongly suggested that RSV-TPGS-SLN 5 will be the best suitable tool for improving systemic availability as well as prolonged circulation. The brain distribution of RSV-TPGS-SLN 5 was found to be 9.23 times higher than that of RSV solution, indicating the passive brain-targeting potential of SLN. The objective of prolonged systemic circulation and passive brain accumulation was achieved using RSV-TPGS-SLN 5. Therapeutic application of RSV-TPGS-SLN 5 against glioma and other proven diseases may be possible after evaluation of in vivo anticancer potential.

Acknowledgements

This research work was financially supported by University Grants Commission in the form of Junior and Senior Research Fellowships under Rajiv Gandhi National Fellowship Scheme, grant no. F.14-2 (SC)/2010 (SA-III). Special Assistance Programme of University Grants Commission (UGC-SAP) and Central Instrument Facility Centre (CIFC) of IIT (BHU), Varanasi provided analytical instrumentation facility for carrying out various nanoparticle characterizations. The authors kindly acknowledge Prof. Sitaram Velaga, Department of Health Science, Lulea University of Technology, Sweden for carrying out X-ray diffraction studies and differential scanning calorimetry analysis. The authors also acknowledge Prof. S. C. Lakhotia, Emeritus Professor & DST/DAE Ramanna Fellow, Department of Zoology, Banaras Hindu University, Varanasi for providing confocal laser scanning microscope facility. The first author gratefully acknowledges Dr C. Periasamy, Assistant Professor, Malaviya National Institute of Technology, Jaipur for carrying out X-ray diffraction studies.

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