RETRACTED: Enhanced cellular uptake and in vivo pharmacokinetics of rapamycin loaded cubic phase nanoparticles for cancer therapy

Acta Biomaterialia 7 (2011) 3656–3669

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Enhanced cellular uptake and in vivo pharmacokinetics of rapamycin-loaded cubic phase nanoparticles for cancer therapy Priyambada Parhi, Chandana Mohanty, Sanjeeb Kumar Sahoo ⇑ Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa 751 023, India

a r t i c l e

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Article history: Received 9 February 2011 Received in revised form 8 June 2011 Accepted 10 June 2011 Available online 15 June 2011 Keywords: Apoptosis Bioavailability Biocompatibility Rapamycin Cubic phase nanoparticles

a b s t r a c t To date cancer is considered as one of the most devastating diseases due to its high rate of mortality. Preclinical studies have demonstrated that the Akt/mTOR (mammalian target of rapamycin) pathway is activated in cancers and inhibition of this pathway has great potential in anti-cancer therapy. Rapamycin, one of the most potent anti-cancer drugs, blocks Akt/mTOR function and has anti-proliferative activity in several cancers. To circumvent problems associated with rapamycin due to its poor water solubility, poor oral bioavailability, low accessibility to cancer tissues and systemic toxicity, rapamycin-loaded cubic nanoparticles (NP) were formulated with vitamin E D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS) as an emulsifier for oral delivery. Cubic NP were characterised and these particles demonstrated better cytotoxicity and apoptosis compared with native rapamycin under in vitro conditions due to their enhanced cellular uptake. The molecular impact of particulate systems on the Akt/mTOR pathway were elucidated by immunoblotting. Down-regulation of different anti-apoptotic genes of this pathway indicates activation of apoptotic signals leading to MIA PaCa cell death. An in vivo study demonstrated enhanced bioavailability of rapamycin in cubic NP in comparison with native rapamycin in a mouse model with no toxicity and good biocompatibility of void cubic NP at a higher dose of oral administration. Thus, rapamycin-loaded cubic NP can be used as an effective drug delivery system to produce better rapamycin therapeutics for the treatment of cancers. Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Rapamycin, a macrolide fungicide first isolated from Streptomyces hygroscopicus, has recently been shown to have potent anti-tumour activity, blocking the translation of proteins required for cell cycle progression from G1 to S phase, and it potentially arrests the growth of a number of tumours, including pancreatic cancer [1,2]. Rapamycin manifests its anti-tumour activity by inhibiting the mammalian target of rapamycin (mTOR) pathway, a central controller of eukaryotic cell growth and proliferation [3]. mTOR inhibition blocks signals which induce phosphorylation of the 70 kDa 40S ribosomal protein kinase (p70S6 K) and the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), leading to G1 arrest of most cell types [4,5] and to p53-independent apoptosis in others [6,7]. However, despite the anti-tumour potency of rapamycin in preclinical studies, clinical development of rapamycin has failed due to its poor solubility in water (2.6 lg ml1) [8]. Moreover, limited accessibility of the drug to the tumour tissues and the consequent requirement for high doses leads to unacceptable cytotoxicity, development of multiple drug resistance and adverse ⇑ Corresponding author. Tel.: +91 674 2302094; fax: +91 674 230072. E-mail address: [email protected] (S.K. Sahoo).

side-effects on normal tissues [9]. One possible way to overcome the problems of conventional rapamycin delivery/therapy is through nanoparticle (NP)-based therapeutics [10–12]. Current trends in rapamycin research have concentrated on the development of convenient and non-invasive home-based forms of administration of the drug, in the form of a pill. These pills may serve as a better way to administer the drug in both early and later stage cancer for those who are unable to accept other forms of treatment due to debility. Randomised trials showed that oral delivery reduces the undesirable drug-related side-effects (such as diarrhoea, nausea, vomiting, etc.) without compromising efficacy [13,14]. It provides an appropriate concentration of drug in the blood circulation and sustained release of the drug to the target tissue and shows much greater efficacy than current clinical forms of administration, such as injection and infusion [15]. The nanocrystal product RapamuneÒ, an oral tablet form of rapamycin, is able to increase the bioavailability of the drug to some extent by increasing the exposed surface area and simultaneously enhances its absorption in the gastrointestinal tract [16,17]. In a similar approach Bisht et al. described a method of preparation of amphiphilic NMA622 and NVA622 polymeric NP loaded with rapamycin for oral delivery and demonstrated the superior efficacy of their formulation over commercial oral rapamycin (Rapamune) [18]. Use of these oral

1742-7061/$ - see front matter Ó 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2011.06.015

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delivery systems certainly represents a promising approach to increasing the bioavailability of rapamycin [16–18]. However, cubic phase NP systems have recently received much more attention for sustained release of the delivered drug (both hydrophilic and hydrophobic). This phase has the ability to solubilise hydrophilic, hydrophobic and amphiphilic molecules [19]. Glyceryl monooleate (GMO), a synthetic lipid amphiphilic molecule approved by the Food and Drug Administration, has been used for controlled drug delivery in the form of cubic phase NP [20–23]. The cubic phase formed in water is a three-dimensional (3-D) network of curved lipid bilayers separated by an intricate network of water channels [21]. Many types of polymer have been used for NP formulation, but the requirements of biocompatibility and biodegradability have limited the choice of polymers used in clinical applications [24]. Representative materials are the non-ionic block co-polymer Pluronic F-127 and vitamin E D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS). These are of interest as they provide stability and chemical functionalisation of NP as drug delivery systems. Previous studies supported the fact that Pluronic F-127 can enhance drug transport by passive targeting to cancerous tissues, as well as sensitising multi-drug resistant tumours to various neoplastic agents by inhibiting the drug efflux transporter [22,25]. Similarly, vitamin E TPGS is a well-known biocompatible natural polymer that can act as an absorption enhancer and bioavailability promoter facilitating the transport of drug-loaded NP across the gastrointestinal barrier [15,26]. Besides serving as an oral enhancer, in recent years it was reported to be one of the most effective P-glycoprotein inhibitors amongst the surfactants [27,28]. In this study we describe a simple method of preparation and characterisation of rapamycin-loaded cubic NP (based on GMO) blended with the amphiphilic block co-polymer pluronic F-127 and a natural emulsifier vitamin E TPGS, which would enhance the oral bioavailability of the hydrophobic drug rapamycin. We have studied the anti-proliferative activities of the above formulation in different cancer cell lines by MTT assay. Cellular uptake, subcellular localisation and blocking of the AKT/mTOR pathway were studied in MIA PaCa cells, and the results confirm that rapamycin-loaded cubic NP were more effective in inhibiting tumour cell proliferation due to their higher uptake (as demonstrated by flow cytometry and confocal microscopy). Further, we report the pharmacokinetic profile of orally administered rapamycin encapsulated in cubic NP and its biocompatibility was tested in an animal model using Balb/c mice. 2. Materials and methods 2.1. Materials Rapamycin was procured from Fujian Kerui Pharmaceutical (Fujhou City, Peoples Republic of China). Pluronic F-127, potassium bromide, Tween-80, propidium iodide (PI), Annexin-V, 6-coumarin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO). GMO was obtained from Eastman (Memphis, TN). Dimethyl sulphoxide was obtained from Qualigen Fine Chemicals (Mumbai, India). Acetonitrile was purchased from Merck (Mumbai, India). TPGS was obtained from Sigma Aldrich (Steinheim, Germany). All other chemicals used were purchased from Sigma Aldrich (St. Louis, MO) without further purification.

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The above mixture was emulsified with 10 ml of Pluronic F-127 solution (10% w/v) by sonication, using a microtip probe sonicator (Vibra-Cell VCX750, Sonics and Materials Inc., Newtown, CT) at an amplitude of 30% for 2 min over an ice bath. The resultant solution was further emulsified with 10 ml of vitamin E TPGS (5% w/v) and sonicated for 2 min at an amplitude of 30% over an ice bath. The resultant NP emulsion was frozen (80 °C) and lyophilised by freeze drying for 6 days (80 °C and <10 mm Hg pressure, Lyphlock, Labconco, Kanas City, MO) to obtain a lyophilised powder for further use. To determine the cellular uptake of NP 6-coumarin-loaded cubic NP were prepared using the above procedure except that 100 lg of the dye was added to GMO prior to emulsification instead of rapamycin. 6-Coumarin incorporated in NP acts as a probe and offers a sensitive method to determine the qualitative and quantitative intracellular uptake of NP, as is evident from our previous studies [29,30]. 2.3. Physico-chemical characterisation of rapamycin-loaded cubic NP 2.3.1. Particle size and zeta (f) potential measurements Particle size and size distribution measurements were carried out using a Malvern Instruments Zetasizer Nano ZS (Malvern, UK) based on dynamic light scattering. Briefly, 1 mg ml1 rapamycin-loaded cubic NP solution was prepared in double distilled water and sonicated for 30 s in an ice bath (Vibra-Cell VC 505, Sonics and Materials, Newton, CT). Size measurements were performed on a NP suspension diluted in MilliQ water at 25 °C (100 ll diluted to 1 ml). The same suspension was used to measure the f potential of NP. All measurements were performed in triplicate. 2.3.2. Atomic force microscopy (AFM) The shape and surface morphology of rapamycin-loaded cubic NP were investigated by AFM (Nanoscope III A, Vecco, Plainview, NY). One drop of 1 mg ml1 rapamycin-loaded cubic NP solution was placed on freshly cleaved mica and incubated for 5 min. To remove unbound NP the surface was rinsed with DI water. The sample was air dried and mounted in a microscope scanner. The shape was observed and imaged in non-contact mode at a frequency of 312 kHz and scan speed of 2 Hz. 2.3.3. Transmission electron microscopic (TEM) studies The internal morphology of NP was examined by TEM. Briefly, 1 mg of cubic NP was suspended in distilled water and sonicated for 30 s. A drop of this suspension was placed on a carbon coated copper TEM grid (150 mesh, Ted Pella Inc., Rodding, CA), negatively stained with 1% uranyl acetate (w/v) for 10 min and allowed to air dry. The samples were imaged using a Philips 201 transmission electron microscope (Philips/FE Inc., Barcliff, Manor, NY) at 120 keV. The TEM photographs were obtained using NIH Image software.

2.2. Preparation of rapamycin-loaded cubic NP

2.3.4. Fourier transform infrared (FTIR) spectroscopy This experiment was performed to investigate the chemical interactions between rapamycin and the polymer matrix. Different samples (void cubic NP, native rapamycin and rapamycin-loaded cubic NP) were individually mixed with KBr (at a ratio of 1:10) and ground into a fine powder using an agate mortar. The mixture was pressed into a pellet at a pressure of 150 kg cm2. The KBr pellet was subjected to FTIR spectrometry (Spectrum RX 1, Perkin Elmer, Waltham, MA) by averaging 32 interferograms with a resolution of 2 cm1 in the range 800–4000 cm1.

Rapamycin-loaded cubic NP were prepared by a minor modification of our previous protocol [22]. Briefly, 200 mg of rapamycin were incorporated into the fluid phase of GMO (1.75 ml at 40 °C).

2.3.5. Differential scanning calorimetry (DSC) The physical status of rapamycin encapsulated in NP was characterised by DSC thermogram analysis (STA 6000 simultaneous

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thermal analyser, Perkin Elmer, Waltham, MA). Each sample of 7 mg (native rapamycin, void cubic NP or rapamycin-loaded cubic NP) was placed in a standard aluminium pan and the samples were purged with pure dry nitrogen gas at a flow rate of 10 ml min1. The temperature was increased at a rate of 10 °C min1 and the heat flow recorded from 30 to 300 °C. 2.3.6. X-ray diffraction (XRD) study XRD analysis was carried out to determine the crystallinity of the NP formulation. The spectra were obtained using a Bruker D8 Advance X-ray diffractometer (Bruker AXS, Madison, WI). The measurements were carried out at a voltage of 40 kV and 25 mA. The scanned angle was set from 3° 6 2h 6 40° and the scan rate was 0.9 min1. 2.3.7. Quantification of entrapment efficiency of rapamycin by high performance liquid chromatography (HPLC) method The efficiency of entrapment of rapamycin was determined as per our previous published protocol [31]. Rapamycin-loaded cubic NP were dissolved in acetonitrile (1 mg ml1). The sample was then sonicated for 2 min at an amplitude of 30% in an ice bath (Vibra-Cell VCX750, Sonics and Materials Inc., Newtown, CT) and centrifuged at 13,800 r.p.m. for 10 min at 4 °C (Sigma 1–15 K, Osterode am Harz, Germany). The obtained supernatant was analysed using a Waters™ 600 reverse phase isocratic mode (RP) system (Milford, MA). To quantify the drug 20 ll of supernatant was manually injected into the injection port and analysed using a mobile phase of acetonitrile:water in the ratio 80:20 vol.%. The flow rate was 1 ml min1 with a quaternary pump (Waters M600E™) at 20 °C with a C18 column (Nova-Pac, 150  4.6 mm internal diameter). The rapamycin level was quantified by UV detection at 278 nm with a dual wavelength detector (M2489). The amount of rapamycin in NP was obtained from the peak area correlated against a standard curve. All analysis was performed in triplicate. The entrapment efficiency was calculated from the equation: entrapment efficiency ð%Þ ¼ ðamount of rapamycin in NP=amount of rapamycin used in formulationÞ  100

2.3.8. In vitro release kinetics The in vitro release kinetics of rapamycin from rapamycinloaded cubic NP were determined by dissolving 10 mg of rapamycin-loaded cubic NP in 3 ml of PBS (0.01 M, pH 7.4) containing 0.1% Tween-80. Tween-80 was used to increase the solubility of rapamycin in the buffer solution as well as to maintain the sink conditions [29]. The resultant suspension was sonicated for 2 min in an ice bath at an amplitude of 30% and divided between three tubes containing 1 ml each. The tubes were kept in a shaker at 37 °C at 150 r.p.m. (Wadegati Labequip, Mumbai, India). At redetermined time intervals these tubes were removed and centrifuged at 13,800 r.p.m., 25 °C for 10 min (Sigma 1–15 K, Osterode am Harz, Germany). The supernatants were collected and lyophilised for 24 h. One milliliter of fresh PBS containing Tween 80 (0.1%) was added to the pellet obtained after centrifugation and the tubes were replaced in the shaker before the next readings. The lyophilised powder was dissolved in 1 ml of acetonitrile for dissolution of the drug and 20 ll of this solution was injected into the HPLC column to quantify the rapamycin released over time. 2.4. Cellular uptake study The cellular uptake study was performed using 6-coumarin and 6-coumarin-loaded cubic NP [31]. Briefly, MIA PaCa cells were seeded at a density of 1  105 cells per well in a 24-well plate (Corning, NY) and allowed to attach for 24 h at 37 °C in a CO2 incubator (Hera Cell, Thermo Scientific, Waltham, MA). The medium

was replaced with either 100 ng ml1 native 6-coumarin or 6-coumarin-loaded cubic NP and incubated for different time periods (1, 4 and 24 h) at 37 °C in a CO2 incubator. After incubation the cells were collected and washed twice with cold PBS (0.1 M, pH 7.4). FACS readings of the treated cells were taken to quantify intracellular 6-coumarin uptake by the MIA PaCa cells. Ten-thousand gated cells were analysed using a FACScan flow cytometer (FACSCalibur, Becton–Dickinson, San Jose, CA) and Cell Quest™ software (Becton–Dickinson, San Jose, CA). 2.5. Subcellular localisation of cubic NP by confocal microscopy We have further studied the in vitro subcellular localisation of 6-coumarin-loaded cubic NP in MIA PaCa cells by confocal microscopy. Briefly, cell monolayers were cultured in BioptakeÒ tissue culture plates (Bioptechs Inc., Butler, PA) at a density of 10,000 cells per plate and incubated at 37 °C in a CO2 incubator. The next day the cell monolayers were treated with 6-coumarin-loaded cubic NP (100 ng ml1) for different time periods (5, 15, 30 and 60 min). At the end of the incubation period the cell monolayers were washed three times with ice cold PBS (0.1 M, pH 7.4), followed by 10% buffered formaldehyde fixation for 15 min and finally staining with PI for 1 h. The plates were washed with PBS (0.1 M, pH 7.4) and the cells imaged in a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems GmbH, Wetzlar, Germany) equipped with an argon laser using a FITC filter (excitation 488 nm, emission 525 nm). The images were processed using Leica Application Suite software. 2.6. Cell proliferation assay Cell viability was evaluated by MTT assay as described previously [32]. Briefly, different pancreatic cell lines (PANC-1 and MIA PaCa-2), a leukemic cell line (K-562) and a human colon cancer cell line (HCT-116) were seeded at a density of 2000 cells per well in 96well plates (Corning, Corning, NY). The next day the cells were treated with different concentrations (0.1, 1, 5, 10, 50, and 100 ng ml1) of either native rapamycin dissolved in DMSO or the equivalent concentration of rapamycin-loaded cubic NP. The concentration of DMSO was kept below 0.1% (w/v) so that it did not have any significant effect on cell proliferation [31]. Medium treated cells and void NP treated cells were used as respective controls. After 5 days incubation 10 ll of MTT reagent was added, followed by incubation for 3 h in a cell culture incubator at 37 °C (Hera Cell, Thermo Scientific, Waltham, MA). The intracellular formazon crystals formed were dissolved in DMSO and the colour intensity was measured at 540 nm using an ELISA plate reader (Synergy HT, BioTekÒ Instruments Inc., Winooski, VT). The anti-proliferative effect was measured as a percentage of cell growth with respect to the respective control. The IC50 value was calculated by non-linear regression analysis using the sigmoid plot equation. 2.7. Mitochondrial depolarisation study using JC1 dye The changes in the mitochondrial membrane potential (MMP) were evaluated by flow cytometry using JC1 dye [33]. In brief, MIA PaCa cells at a density of 2  105 were seeded in 6-well plates (Corning, NY). The next day the cells were treated with native rapamycin or rapamycin-loaded cubic NP at a concentration of 100 ng ml1 and incubated for 48 h. After predetermined times of incubation the cells were collected by trypsinization and centrifugation (3000 r.p.m., 5 min). The cell pellets were then washed twice with PBS (0.01 M, pH 7.4) and incubated with 3 ll of JC1 at 37 °C for 30 min in the dark. Then each sample was examined on a FL-1 versus FL-2 dot plot in a Becton Dickinson FACSCalibur. Ten-thousand gated cells were analysed using a FACScan flow

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cytometer (FACSCalibur, Becton–Dickinson, San Jose, CA) and Cell Quest™ software (Becton–Dickinson, San Jose, CA). 2.8. Apoptosis study by flow cytometry The induction of apoptosis by native rapamycin and rapamycinloaded cubic NP was analysed by flow cytometry [34]. MIA PaCa cells were seeded at a density of 2  105 cells per well in 6-well plates (Corning, NY) containing 2 ml of growth medium and incubated overnight for attachment at 37 °C. The next day 2 ml of medium containing 50 ng ml1 native rapamycin or the equivalent concentration of rapamycin-loaded cubic NP was added to the cells, followed by incubation for 48 h in a CO2 incubator (Hera Cell, Thermo Scientific, Waltham, MA). Cells treated with medium and void cubic NP were used as controls. After 48 h the cells were washed twice with PBS (0.01 M, pH 7.4) and collected by trypsinization. The pelleted cells were resuspended in 500 ll of 1 binding buffer, 1 ll of Annexin V–FITC and 2 ll of PI (Sigma Annexin V–FITC apoptosis detection kit) and incubated at room temperature in the dark for 20 min. Stained cells were analysed by FACScan flow cytometry using Cell Quest software (FACSCalibar, Becton– Dickinson, San Jose, CA). All experiments were carried out in triplicate. 2.9. Western blot analysis The molecular mechanism of apoptosis was demonstrated by Western blotting [22]. In brief, MIA PaCa cells (1  106 cells ml1) were treated with 100 ng ml1 rapamycin (both native rapamycin and rapamycin-loaded NP) for 48 h. Cells not treated with the drug but treated with void cubic NP were used as controls. After 48 h

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protein lysates and cell extracts were prepared. Cells were collected by scraping, washed with 1 PBS, followed by detergent lysis with 50 mM L1 Tris–HCl (pH 8.0), 150 mM L1 NaCl, 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS, containing protease and phosphatase inhibitors (Sigma, St. Louis, MO). The protein concentration was calculated using a Pierce BCA protein assay kit (Pierce, Rockford, IL). Protein detection was done after electrophoretic transfer of SDS polyacrylamide gel electrophoresis separated proteins to polyvinylidene fluoride (PVDF) membranes (GE Healthcare). The PVDF membranes were incubated with primary antibodies recognising p-p70S6K1, 4E-BP1, p-4E-BP1 and c-myc (Imgenix Corp., San Diego, CA), p70S6K1, Akt, p-Akt and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA) and Bcl-2 (Cell Signalling Technology, Danvers, MA.) at 1:1000 dilution either overnight at 4 °C or for 1 h at room temperature with gentle shaking, followed by incubation with goat anti-mouse/rabbit IgG–horseradish peroxidase conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:5000 dilution for 40 min at room temperature. The protein of interest was detected by incubation with Enhanced Chemiluminescence Plus (Amersham Biosciences, Little Chalfont, UK).

2.10. In vivo biocompatibility study of void cubic NP In order to confirm the non-toxic nature of the void cubic NP an in vivo toxicity study was conducted [18]. Void cubic NP were administered to BALB/c mice (n = 3) at 500 mg kg1 via oral gavage. The mice were observed daily for behavioural abnormalities and total body weights were taken at different time intervals. After 30 days the treated mice were killed and the major visceral organs were observed for histological abnormalities.

(a)

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Intensity (%)

20 15 10 5 0

1

10

100

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Size (d.nm)

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Fig. 1. (a) Mean particle size of rapamycin-loaded cubic NP determined using a particle size analyser. Hydrodynamic diameter = 88 ± 6 nm (n = 3). (b) Size distribution of rapamycin-loaded cubic NP determined by AFM. (c) A representative picture of rapamycin-loaded cubic NP obtained by transmission electron microscopy (bar = 0.2 lm).

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2.11. In vivo pharmacokinetics study The main objective of this experiment was to compare the pharmacokinetics of native rapamycin with rapamycin-loaded cubic NP after oral drug administration. This experiment was carried out with the permission of the Institutional Animal Ethics Committee of the Institute of Life Sciences, Bhubaneswar, India. For this study male BALB/c mice weighing 20–25 g were used. These mice were divided into two groups (n = 3): group 1 mice were administered native rapamycin dissolved in MilliQ water containing 1 vol.% Tween-20; group 2 mice were given rapamycin-loaded cubic NP dissolved in distilled water at an equivalent dose of 15 mg kg1 to native rapamycin by oral gavage. At different time intervals peripheral blood from the retro-orbital plexus was collected and the serum separated. The rapamycin concentration in the blood was measured by HPLC analysis as described earlier [35]. 2.12. Cell culture All cell culture experiments were performed using PANC-1, MIA PaCa-2, HCT-116 and K562 cancer cell lines from the American Type Culture Collection (Manassas, VA) and were cultured using DMEM (Pan Biotech GmbH, Aidenbach, Germany) with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin–streptomycin. The cells were maintained at 37 °C in a humidified, 5% CO2 atmosphere in an incubator (Hera Cell, Thermo Scientific,

Waltham, MA). All other chemicals were procured from Himedia Laboratories (Mumbai, India). 2.13. Statistical analysis Student’s t-test was used to conduct statistical analyses. Data are expressed as means ± standard deviation and values of P < 0.05 were taken to be indicative of significant differences and P < 0.005 were considered very significant differences. 3. Results 3.1. Physicochemical characterisation of rapamycin NP We have successfully prepared aqueous dispersed rapamycinloaded cubic NP with an average diameter of 88 ± 6 nm and polydispersity index of 0.09 ± 0.02, as determined by dynamic light scattering (Fig. 1a). The average f potential of the NP was 31.5 ± 3.5, which is certainly likely to increase the stability of the NP in dispersion. The studies also suggest that particles with such a high negative surface charge are more attracted to the mucosal surface, thus preventing elimination of oral drug formulations via the alimentary canal [36]. TEM and AFM studies were performed to further confirm the size distribution and shape of the rapamycin-loaded cubic NP. The images show that the average

Fig. 2. (a) FTIR spectra of (i) native rapamycin, (ii) rapamycin-loaded cubic NP and (iii) void cubic NP. (b) DSC thermogram of (i) native rapamycin, (ii) rapamycin-loaded cubic NP and (iii) void cubic NP. (c) XRD study of (i) native rapamycin (ii) rapamycin-loaded cubic NP (iii) void cubic NP. (d) In vitro release kinetics of rapamycin from cubic NP in PBS (0.01 M, pH 7.4) at 37 °C.

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sizes of the particles were 100 nm and 70 nm, respectively (Fig. 1b and c). The images further show that the particles were discrete with a spherical outline and monodisperse size distribution. Moreover, from the HPLC analysis it was observed that rapamycin was efficiently loaded in cubic NPs, reaching a high encapsulation efficiency of 92 ± 3.4%. This implies that 92% of the drug used in our formulation was entrapped inside the NP (184 mg of drug entrapped out of 200 mg of native drug added). Further, FTIR studies were used to characterise any chemical interaction that occurred in the polymer due to addition of the drug during NP formulation. The FTIR spectra of rapamycin, rapamycin-loaded cubic NP and void cubic NP are shown in Fig. 2a. Characteristic peaks due to different functional groups in native rapamycin, appeared at 3418 cm1, due to O–H stretching vibrations, and 2875 and 2932 cm1, due to C–H stretching vibrations, while the peak 1718 cm1 corresponded to C@O carbonyl stretching, whereas the peak at 1377 cm1 was due to –CH bending/deformation. However, the bands which appeared in void cubic NP were almost identical to the rapamycinloaded cubic NP, with the addition of some peaks due to native rapamycin. The peak at 3418 cm1 in native rapamycin, due to O–H stretching vibrations of intermolecularly bonded O–H groups, also appeared at 3420 cm1 in rapamycin-loaded cubic NP. Moreover, intensified C–H stretching vibration bands and C@O carbonyl stretching bands were obtained at the nanoparticle surface compared with void cubic NP, indicating the presence of rapamycin in rapamycin-loaded cubic NP [10].

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The physical status of the drug formulated in the cubic NP was compared with native rapamycin by DSC analysis. The DSC thermograms of native rapamycin, rapamycin-loaded cubic NPs- and void cubic NPs- are shown in Fig. 2b. Rapamycin in its natural state exists as crystals, which are characterised by a high peak at the melting point (180 °C). However, when encapsulated in cubic NP the peak at this original melting point disappears. To further understand the nature of rapamycin in our cubic NP formulations XRD studies were undertaken. The characteristics peaks of native rapamycin, as shown in Fig. 2ciii, demonstrates the traits of a highly crystalline structure. However, no characteristic rapamycin peaks were observed when the drug was entrapped in NP (Fig. 2cii). This indicates that the drug was molecularly dispersed or in an amorphous state, which favours easy diffusion of drug molecules through the polymeric matrix, resulting in sustained release of the drug from the NP [29,31]. Sustained release of the drug from NP is an important factor in achieving successful NP formulations. The in vitro release profile showed a biphasic release pattern of entrapped rapamycin from rapamycin-loaded cubic NP, as shown in Fig. 2d. Approximately 32.6 ± 1.18% of the entrapped rapamycin was released in 24 h, followed by a sustained release of the drug, i.e. about 77.9 ± 1.95% of the drug was released after 30 days. The observed initial burst release could be due to diffusion of surface adsorbed drug present just beneath the surface of the NP, followed by sustained release of rapamycin entrapped inside the NP [32]. Similar trends of release was observed by Trickler et al. in

Fig. 3. (a) Quantitative cellular uptake of native 6-coumarin (green) and 6-coumarin-loaded cubic NP (pink) by cell line MIA PaCa determined by flow cytometry. (b) Histogram of quantitative cellular uptake of native 6-coumarin and 6-coumarin-loaded cubic NP. Data are means ± SEM (n = 3). ⁄⁄P < 0.005.

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chitosan/GMO NP, showing an initial burst release of 10% and 45% for paclitaxel and dexamethasone, respectively, in 1 h [37]. 3.2. Subcellular localisation by confocal microscopy The cubic NP formulation was further assessed by cellular uptake studies using the cell line MIA PaCa. The quantitative cellular uptake results demonstrated that 6-coumarin-loaded cubic NP had a higher cellular uptake in comparison with native 6-coumarin (Fig. 3a). 6-Coumarin-loaded cubic NP showed 2.04, 2.43 and 1.54 times more cellular uptake than native 6-coumarin after 1, 4 and 24 h incubation, respectively (Fig. 3b). Similarly, in another study nuclear co-localisation of 6-coumarin-loaded cubic NP was observed qualitatively by confocal microscopy (Fig. 4a). By combining the images of 6-coumarin-loaded cubic NP formulations with the orange-red PI stained nuclear material we could demonstrate the nuclear co-localisation of our formulation in MIA PaCa cells (Fig. 4b). At early times of incubation (up to 30 min) the free intracellular green fluorescence of 6-coumarin was restricted to the cytoplasm, with no nuclear co-localisation of the formulation. Interestingly, after 1 h treatment 6-coumarin green fluorescence was seen in the intranuclear region of MIA PaCa cells, suggesting time-dependant intracellular localisation as well as nuclear co-localisation of our nanoparticulate formulation. 3.3. In vitro mitogenic assay The therapeutic efficiency of drug-loaded NP depends on their uptake, intracellular distribution and, more importantly, on the

dose of drug that is released from the internalised NP inside the cell. So, to investigate the therapeutic efficiency of our formulation different cancer cell lines were treated with void NP, native rapamycin and rapamycin-loaded cubic NP at different concentrations and cell proliferation was measured by a standard MTT colorimetric assay on the fifth day (Fig. 5). All the cell lines studied showed a typical dose-dependant sigmoidal anti-proliferative effect. The IC50 values of native and rapamycin-loaded cubic NP, obtained by MTT assay in different cell lines, are shown in Table 1. These results demonstrate that rapamycin-loaded cubic NP were 1.55, 1.31, 2.27 and 2.76 times more effective than native rapamycin in the K562, HCT-116, PANC-1 and MIA PaCa-2 cell lines, respectively. Hence, the results obtained reveal comparable inhibition of cell proliferation, with rapamycin-loaded NP being more effective than native rapamycin in solution in controlling in vitro cancer cell growth. 3.4. Mitochondrial depolarisation study using JC1 dye Mitochondria play a pivotal role in apoptosis, and loss of MMP (an event in early apoptosis) is associated with a permeability transition. In the early stages of apoptosis the cationic dye JC1 is used to detect MMP as it exhibits potential-dependent accumulation in mitochondria leading to the formation of red fluorescent aggregates [38,39]. Here we have compared the loss of MMP in cells treated with 100 ng ml1 native rapamycin or rapamycin-loaded cubic NP, shown in Fig. 6a. We found enhanced apoptosis indices for rapamycin-loaded cubic NP, 48.77% compared with 21.25% for native rapamycin. We anticipate that native rapamycin and

Fig. 4. (a) At various time intervals the subcellular localisation of 6-coumarin-loaded cubic NP were studied in the cell line MIA PaCa using propidium iodide (PI) (for nuclear staining) by confocal microscopy. (b) Nuclear localisation of 6-coumarin-loaded cubic NP was observed by combining green fluorescent dye-labelled 6-coumarin with PI staining for 1 h in cell line MIA PaCa.

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Fig. 5. Dose-dependent cytotoxicity study of native rapamycin (d), rapamycin-loaded cubic NP (N) and void cubic NP (j) in different cancer cell lines. Cytotoxicity was measured at 5 days by MTT assay. Percentage inhibition was determined with respect to controls. Data are means ± SEM (n = 6). ⁄⁄P < 0.005; ⁄P < 0.05.

Table 1 IC50 values of native rapamycin and rapamycin-loaded cubic NP in different tumour cells as observed by cytotoxicity assay. Tumour cells

IC50 values (ng ml1) Native rapamycin

Rapamycin-loaded cubic NP

K562 HCT-116 PANC-1 MIA PaCa-2

73.42 42.75 57.49 95.87

47.24 32.57 25.32 34.71

tive for Annexin and are placed in the lower right quadrant. The FACS results demonstrated that rapamycin-loaded cubic NPs treated cells showed more greater numbers of cells in early apoptosis, i.e. 13.8% compared with 1.74% for native rapamycin treated cells. This result suggests that cells treated with rapamycin-loaded cubic NP were able to induce greater apoptosis in the MIA PaCa cell line in comparison with the native drug. Thus the NP treated cells showed 7.93 times more apoptosis than those treated with native rapamycin. 3.6. Western blot analysis

rapamycin-loaded cubic NP might induce apoptosis through a mitochondrial pathway, with the induction of apoptosis being higher in the case of rapamycin-loaded cubic NP than native rapamycin.

3.5. Apoptosis analysis by flow cytometry The induction of apoptosis by inhibition of mTOR is considered to be one of the principle mechanisms by which rapamycin results in tumour regression [5,31]. Accordingly, we have investigated the ability of rapamycin to induce apoptosis in pancreatic cancer cells by incubating MIA PaCa cells with equivalent concentrations of 50 ng ml1 native rapamycin and rapamycin-loaded cubic NP for 48 h. The FACS results showed the presence of early apoptotic, advanced apoptotic and necrotic cell populations in all treated cells (Fig. 6b). The fractions of cells that are in early apoptosis were posi-

Rapamycin induces apoptosis mostly through inhibition of the Akt/mTOR/p70S6K1 pathway, a central pathway of protein translation involved in the regulation of cell proliferation, growth, differentiation and survival [40]. To determine the ability of rapamycin to promote apoptosis in the MIA PaCa cell line we investigated the signal transduction pathway induced by rapamycin in MIA PaCa cells by Western blot analysis (Fig. 7). As is evident from Fig. 7, pAkt (the key modulator in the upstream pathway), p-p70S6K1 and p-4E-BP1 (the downstream signalling targets) were highly expressed in control cells and void cubic NP treated cells. In contrast, the band intensity decreased more in rapamycin-loaded cubic NP treated cells. This is due to the fact that rapamycin inhibits phosphorylation of Akt, p70S6K1 and 4E-BP1 in the mTOR signalling pathway. As a result, a smaller activated band of anti-apoptotic BCL-2 was obtained in NP treated as compared with native rapamycin treated cells. However, there was no significant

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difference in c-Myc expression in the native rapamycin and rapamycin-loaded cubic NP cases. From the above results it is evident that our formulation inhibited p-Akt by decreasing the activation of p-p70S6K1 and p-4E-BP1, inducing apoptotic signals in the mTOR pathway, leading to enhanced cell death compared with native rapamycin. 3.7. In vivo biocompatibility study of void cubic NP The biocompatibility study was conducted by administering 500 mg kg1 void cubic NP to BALB/c mice by oral gavage, followed by killing the animal after a period of 30 days. As can be seen in Fig. 8a, despite a relatively large dosage, the mice receiving void cubic NP demonstrated no evidence of weight loss and no gross tissue changes as seen by histological observation. Similarly, no behavioural changes were observed in the mice during the follow-up observation in the study time period.

3.8. In vivo pharmacokinetics study We have compared the bioavailability of native rapamycin and that in NP formulations by administering a dose of 15 mg kg1 by oral gavage to BALB/c mice. The mean rapamycin concentration in the mice serum after different time intervals was quantified by HPLC (Fig. 8b). The pharmacokinetic parameters taken into consideration are listed in Table 2, with Cmax values (maximum drug concentration encountered after oral administration) of 890 ng ml1 for rapamycin-loaded cubic NP and 152 ng ml1 for native rapamycin, and the corresponding Tmax values (at which Cmax is reached) of 2 h for both. Similarly, the mean area under the concentration– time curve (AUC0–infinity) was found to be 31219.8 ng h1 ml1 for rapamycin-loaded cubic NP and 2566.427 ng h1 ml1 for native rapamycin. The half-life (T1/2) of rapamycin-loaded cubic NPs was increased by 1.75-fold in comparison with native rapamycin, indicating a maximum residence time (MRT) in the systemic

Fig. 6. (a) Loss of mitochondrial membrane potential (MMP) in MIA PaCa cells due to the effect of native rapamycin and rapamycin-loaded cubic NP (100 ng ml1) determined by flow cytometry after 48 h treatment. (Top right) Live cells; (bottom right) cells with loss of membrane potential. (b) Induction of apoptosis in the cell line MIA PaCa by native rapamycin and rapamycin-loaded cubic NP (50 ng ml1) determined by flow cytometry after 48 h treatment. (Top left) Necrotic cells; (top right) late apoptotic cells; (bottom left) live cells; (bottom right) early apoptotic cells.

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1

2

3

4

p70S6K1 p- p70S6K1 Akt p- Akt 4E-BP1 p- 4EBP1 BCL-2 c-Myc β-actin Fig. 7. Inhibition of p-Akt and downstream targets of mTOR expression induce apoptosis in MIA PaCa cells as confirmed by immunoblotting analysis. Lane 1, whole cell lysate; lane 2, void cubic NP treated cells; lane 3, native rapamycin treated cells; lane 4, rapamycin-loaded cubic NP treated cells.

circulation of rapamycin in rapamycin-loaded cubic NP 1.8 times greater than native rapamycin after oral administration. 4. Discussion The main aim of our work was to develop a rapamycin-loaded cubic nanoparticulate system to overcome the problems at present associated with rapamycin delivery, which may allow clinical application of this potential drug candidate as aqueous formulations. The particle size and size distribution of NP always plays an important role in determining the drug release kinetics, cellular uptake and biodistribution of the NP, thus influencing the in vitro as well as in vivo therapeutic effects of the drug-loaded NP against malignancies [15,41]. Studies conducted by Feng et al. reported that NP below 500 nm in size with a high f potential can be efficiently taken up via the lymphatic system (M cells of Peyer’s patches) and can cross the membrane of epithelial cells by endocytosis [15,36]. In the light of this we anticipate that our nanoparticles, having a small size (88 nm) and high surface charge (–31 mV), would favour intestinal uptake and enhanced circulation half-lives, as well as being able to evade the reticuloendothelial system (RES). Furthermore, FTIR, DSC and XRD analyses have clearly demonstrated the chemical integrity of rapamycin and its interaction with the polymer. While assessing the in vitro release profile of our formulation we observed a typical biphasic release profile (i.e. diffusion and degradation) of rapamycin from rapamycin-loaded cubic NP. The observed initial burst release is due to discharge of surface bound drug molecules present on the polymeric matrix of rapamycin-loaded cubic NP, followed by sustained release of the drug by partitioning in the hydrophobic core of GMO and subsequent diffusion/erosion of the polymeric matrix, enabling the drug to be released from NP into the aqueous medium [20,37]. To demonstrate the increased cytotoxicity of the rapamycinloaded cubic NP intracellular uptake of our formulation was investigated. Here we have used 6-coumarin, a fluorescent marker widely used as a probe in intracellular uptake experiments,

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because of its biocompatibility, high fluorescence activity, low dye loading (<0.5 wt.%) and low leaching rate [30,31,42]. Taking advantage of its intrinsic green fluorescence we have studied the cellular uptake of 6-coumarin-loaded cubic NP by MIA PaCa cells by flow cytometry. The cellular uptake results demonstrate a trend of increasing 6-coumarin (native and nanoparticle form) uptake with time in MIA PaCa cells. However, the cellular uptake found for the nanoparticle formulation was 2.04-, 2.43- and 1.54-fold more than native 6-coumarin after 1, 4 and 24 h treatment. This higher uptake may be due to the cellular entry of the 6-coumarin-loaded NP via endocytosis, while internalisation of the native dye is by passive diffusion [22,31]. Through the process of endocytosis enough NP could be internalised within the cell to release the entrapped dye in a sustained manner over a period of time. It can be seen from Fig. 4a that the incubation time is also an important factor determining the cellular uptake efficiency: the longer the incubation time, the higher the cell uptake efficiency of the NP. Interestingly, in our study the green fluorescence intensity of 6-coumarin-loaded NP was observed around and inside the nuclei (red, stained with PI) of MIA PaCa cells after 1 h treatment, which indicates successful nuclear co-localisation of the formulation with increasing period of treatment (Fig. 4b). Previous studies demonstrated that NP of 100 nm or less have been shown to co-localise in the nuclear compartment [43,44]. Recently Costanzo et al. reported that NP with sizes around or below 100 nm showed optimum cellular and nuclear uptake in epithelial and smooth muscle cells [45]. Hence, we could anticipate that our formulated cubic NP (which are smaller than 100 nm) would show enhanced cellular uptake, owing to the nanoscale size and, in addition, mucoadhesive properties. Besides the above mechanism, other reasons may also exist which could explain higher uptake of our formulated NP, such as the mucoadhesive properties of our delivery system. It has earlier been reported that both TPGS and GMO incorporation in NP formulations can significantly enhance cellular adhesion, adsorption and bioavailability of delivered drugs in different cancer cells [22,36]. The MTT assay demonstrated that the rapamycin-loaded cubic NP formulation showed a comparatively lower IC50 value than the native drug, in all the different cancer cell lines (Table 1). This result is in accord with the higher cellular uptake of cubic NP in MIA PaCa cells. In general cellular uptake of the native drug is by diffusion [41]. Studies have shown that after diffusion and intracellular saturation, further internalisation of the native drug is prevented. Thus the small fraction of intracellular native drug available during the initial period of drug diffusion was responsible for eliciting the anti-proliferative effect of native rapamycin in various cancer cell lines. Conversely, enough NP could be internalised into cancer cells by endocytosis to result in sustained drug release from NP on continuous exposure to cancer cells, resulting in significant cell toxicity [46]. Apoptotic regulation has been studied by the observation of mitochondria and the changes in MMP. During apoptosis large conductance channels in the inner mitochondrial membrane, known as mitochondrial permeability transition pores, open, which is thought to trigger the loss of MMP, a fundamental characteristics of apoptosis in all model systems of programmed cell death [39,47,48]. In our study MIA PaCa cells treated with rapamycinloaded cubic NP showed a profound loss of MMP, compared with those treated with native rapamycin for 48 h, consequently leading to apoptosis (Fig. 6a). Further, we have studied apoptosis by Annexin V–FITC labelling and observed that in native rapamycin treated cells 1.74% of the populations were apoptotic, while 0.19% was necrotic. In contrast, for rapamycin-loaded cubic NP 13.8% were apoptotic and 0.43% were necrotic. The results confirm that cells treated with rapamycin-loaded cubic NP showed 7.93 times more apoptosis than those treated with native rapamycin. However, the

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Fig. 8. (a) Toxicity profile of void cubic NP administered at a dose of 500 mg kg1 to BALB/c mice after a period of 30 days. (i) Oral administration of void cubic NP revealed no weight loss by BALB/c mice. (ii) Thirty days after administration animals were killed and histological profiles were taken. Representative photomicrographs of lung, kidney, intestine and liver by H&E staining. (b) In vivo pharmacokinetics study of native rapamycin (j) and rapamycin-loaded cubic NPs (d) (15 mg kg1) in Balb/c mice after oral administration. The inserted bar diagram shows the bioavailability of native rapamycin and rapamycin-loaded cubic NP 24 h after drug administration. Data are means ± SEM (n = 3). ⁄⁄P < 0.005.

Table 2 Pharmacokinetic parameter in serum of BALB/c mice after oral administration. Parameter 1

Cmax (ng ml ) Tmax (h) AUC0–infinity (ng h1 ml1) T½ (h) MRT (h)

Native rapamycin

Rapamycin-loaded cubic NP

152.0 2 2537.992 16.2 21.85

890 2 31054.88 28.3 40.29

overall percentage apoptosis (early and late apoptosis) was 4.83% in cells treated with native and 27.55% in cells treated with rapamycin-loaded NP (Fig. 6b). It is worth mentioning that at the

concentration of 50 ng f ml1 (either in solution or encapsulated in NP) used in our experiments negligible cells underwent unprogrammed cell death (necrosis) and more underwent programmed cell death pattern (apoptosis). In this regard, Kenerson et al. have demonstrated that with decreasing doses of rapamycin the degree of necrosis decreased in renal tumours [49]. However, Acharya et al. observed a higher population of cells undergoing necrosis compared with cells undergoing apoptosis at higher concentrations of rapamycin in MCF7 cells [31]. The negligible percentage of necrotic cells observed in the current study could be due to the low dose of rapamycin. However, at the same concentration better uptake of rapamycin-loaded cubic NP resulted in greater

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accumulation of the delivered drug inside tumour cells, accompanied by its sustained release, thereby resulting in a greater percentage of cells in the apoptotic phase and with time, resulting in a reduction in cell viability. Our Western blot analysis results confirmed that apoptosis was associated with the down-regulation of p-Akt, p70S6K1 and p-4EBP1, key proteins involved in the mTOR pathway. Among the various signalling pathways, the Akt/mTOR pathway is our focus, owing to its involvement in both the inhibition of apoptosis and the promotion of cell proliferation by affecting the phosphorylation status of cell survival and apoptosis inducing proteins like BAD [3] (Fig. 9). It was previously reported that Akt, a target of PI3 K, is phosphorylated and activated in a variety of cancer cell lines [22,50]. Our Western blot results also demonstrate that rapamycin inhibits the phosphorylation of Akt, which is the major regulator of the mTOR signalling pathway (Fig. 7). It was previously reported that mTOR inhibits two separate downstream pathways that control the translation of specific subsets of mRNA, including 4E-BP1 and p70S6K1 [3,51]. Our findings also demonstrate that rapamycin inhibits the phosphorylation of 4E-BP1 and p70S6K1 in MIA PaCa cells. Further, the inhibition of phosphorylation is more pronounced in cells treated with rapamycin-loaded NP compared with those treated with native rapamycin. mTOR has been found to be localised in both the cytoplasm and nucleus. Earlier reports suggest that the mTOR/raptor complex is mostly cytoplasmic, whereas the mTOR/rictor complex is abundant in both the cytoplasm and nucleus, although the molecular mechanism of the effect of rapamycin on mTOR/rictor remains elusive. In this regard, Rosner et al. have shown that rapamycin regulates cytoplasmic and nuclear mTORC2 differently in non-transformed, nonimmortalised primary human fibroblasts [52]. In our studies we

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may anticipate that after the uptake of rapamycin encapsulated in our formulation greater amounts of NP accumulate in the cytoplasm of cancer cells, while a small proportion is accumulated in the nucleus. This results in greater down-regulation of mTOR and, consequently, its downstream targets p70S6K1 and 4E-BP1, in comparison with native drug treated cells (Fig. 9). As a result the expression of BCL-2, which plays a major role in cell survival, preventing apoptosis in different cell types, was reduced in cells treated with rapamycin-loaded NP, in contrast to those treated with native rapamycin. Further, we found that expression of c-Myc (pro-apoptotic and anti-apoptotic functions) showed no significant change in rapamycin treated (in both native and in formulation forms) cells when compared with control cells. Hence, it can be assumed that the induction of apoptosis is due to another pathway involving mTOR other than c-Myc. Thus it is noteworthy that, compared with native rapamycin, the drug in the NP formulation efficiently inhibits downstream targets of the mTOR signalling pathway, which in turn activate apoptosis, leading to cell death, as observed in the MTT assay. One of the greatest challenges of drug delivery involves defining the bioavailability and accessibility to cancerous tissue. In this view various water soluble analogues of the hydrophobic drug rapamycin (temsirolimus (CCI-779), deforolimus (AP23573) and everlimus (RAD 001)) sirolimus NanoCrystal Rapamune, nanoparticle albumin-bound (nab) rapamycin (ABI-009) and amphiphilic NMA622 and NVA622 polymeric nanoparticles loaded with rapamycin have been developed which show anti-tumour activity in xenograft models [16–18,53–55]. Our main aim in this study was to develop an aqueous NP formulation which could enhance the oral bioavailability of the hydrophobic anti-cancer drug rapamycin. In this regard we have developed a muco-adhesive nanocarrier

Fig. 9. Schematic representation of the molecular mechanism of apoptosis induced by rapamycin. Earlier reports implied that the Akt/mTOR pathway contributes to MIA PaCa tumour cell survival. Activated Akt mediates oncogenesis through phosphorylation/activation of mTOR, which regulates cell growth by phosphorylation of p70S6K1 and 4E-BP1 and inactivates pro-apoptotic proteins (BAD), preventing apoptosis. We hypothesize that internalisation of cubic NP by endocytosis results in accumulation of NP in the cytoplasm, while a smaller proportion of particles accumulate in the nucleus. The sustained release of rapamycin from the NP blocks the phosphorylation of mTOR protein present in both the cytoplasm and nucleus, thereby blocking downstream targets in the mTOR pathway involved activating pro-apoptotic proteins (BAD).

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and, as anticipated, the pharmacokinetics results certainly explain the enhanced bioavailability of the delivered rapamycin. The in vivo study demonstrated that rapamycin-loaded cubic NP were 6-, 7.5- and 9.6-fold more bioavailable than native rapamycin after 2, 4 and 8 h oral administration, respectively. It is worth mentioning that the drugs released from NP were still detected in plasma 24 h after oral administration, and showed 11-fold more bioavailability than native rapamycin (Fig. 8b). Bisht et al. illustrated the pharmacokinetic parameters of Rapamune (a commercially available oral formulation) administration to mice and showed that the mean area under the concentration–time curve (AUC0–infinity) was 9536.7 ng h1 ml1 [18]. Comparatively, we observed an increase in AUC0–infinity of 31219.8 ng h1 ml1 for rapamycin-loaded cubic NP in our mice model. Our results therefore demonstrate superior pharmacokinetic disposition of rapamycinloaded cubic NP over commercial a formulation of rapamycin, i.e. Rapamune. Here we anticipate that the small size (100 nm) and enhanced cellular uptake due to the muco-adhesive character of our formulation rendered it more bioavailable, compared with native rapamycin. Further, the toxicity study revealed that the polymeric NP were non-toxic even at high doses (500 mg kg1) of oral administration in BALB/c mice. Thus our studies clearly demonstrate the significance of cubic phase NP delivery systems (based on GMO and vitamin E TPGS) in comparison with native rapamycin, owing to the enhanced cellular uptake, resulting in a reduction in cell viability by apoptosis induction in pancreatic cancer cell lines under in vitro condition. 5. Conclusion The rapamycin-loaded cubic NP formulation under discussion consists of vitamin E TPGS and GMO, an absorption enhancer and a bioavailability promoter, which improve the therapeutic index of drug-loaded NP compared with native rapamycin. The results show that rapamycin-loaded cubic NP were comparatively more effective than native rapamycin under in vitro conditions with time due to greater cellular uptake, sustained intercellular drug retention and enhanced anti-proliferative effects, by inducing apoptosis. Most importantly, the enhanced cellular internalisation and sustained release of entrapped rapamycin in our formulation results in its enhanced systemic in vivo bioavailability. Thus, in the light of the currently still unmet medical need for strategies that focus on enhanced bioavailability of administered drug, the formulated delivery system holds much promise for the treatment of cancer in near future. Acknowledgement Financial support to the Institute in the form of a TATA Innovative Fellowship from the Department of Biotechnology to S.K.S. is gratefully acknowledged. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 3, 4, 6 and 8, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/ j.actbio.2011.06.015. References [1] Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev Cancer 2004;4:335–48. [2] Easton JB, Houghton PJ. MTOR and cancer therapy. Oncogene 2006;25: 6436–46. [3] Hidalgo M, Rowinsky EK. The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene 2000;19:6680–6.

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