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Improving efficacy, oral bioavailability and delivery of paclitaxel using protein-grafted solid lipid nanoparticles Deep Pooja, Hitesh Kulhari, Madhusudana Kuncha, Shyam Sunder Rachamalla, David J. Adams, Vipul Bansal, and Ramakrishna Sistla Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00691 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016
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Improving efficacy, oral bioavailability and delivery of paclitaxel using protein-grafted solid lipid nanoparticles Deep Pooja,¥,†, # Hitesh Kulhari,¥,†, ‡ Madhusudana Kuncha,¥ Shyam S. Rachamalla,# David J. Adams,‡, ǁ Vipul Bansal,†, ‡,* Ramakrishna Sistla,¥,* ¥
Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad, Telangana 500007, India ‡
Health Innovations Research Institute, RMIT University, Melbourne, VIC 3083 Australia
†
Ian Potter NanoBioSensing Facility, NanoBiotechnology Research Laboratory, School of
Science, RMIT University, Melbourne, VIC 3001, Australia ǁ
Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong,
Wollongong, NSW 2522, Australia. #
Faculty of Pharmacy, College of Technology, Osmania University, Hyderabad, Telangana
500007, India
*Corresponding authors: Ramakrishna Sistla, PhD Principal Scientist, Medicinal Chemistry & Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad-500007, India Phone: +91-40-27193753 (office) Email:
[email protected] Vipul Bansal, PhD Director, Ian Potter NanoBiosensing Facility RMIT University Melbourne 3001, Australia Phone:+61-3-99252121 Email:
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Abbreviations A549 cell, adenocarcinomic human alveolar basal epithelial cell; AO, Acridine orange; AUC, Area under the curve; Cl, Total clearance; CNS, Colloidal nanocarrier systems; Cmax, Peak plasma concentration; CSN, Coumarin-6 loaded solid lipid nanoparticles; DMSO, Dimethyl sulfoxide; PSN, paclitaxel loaded solid lipid nanoparticles; EB, Ethidium bromide; EDC, 1Ethyl-3-(3-dimethylaminopropyl)carbodiimide; EE: Encapsulation efficiency; FBS, Fetal bovine serum; FITC, Fluorescein isothiocyanate; FTIR,Fourier transform infrared spectroscopy; GMS, Glyceryl monostearate; HPLC, High performance liquid chromatography; IC50, Half-maximal inhibitory concentration; LPSN, WGA-conjugated, paclitaxel loaded solid lipid nanoparticles; MRT, Mean residence time; MTT, 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; NAG, N-acetyl-D-glucosamine; NHS, N-hydroxysuccinimide; PI, Propridium iodide; PK, Pharmacokinetic; PLGA, poly (lactic-co-glycolic acid); PTX, Paclitaxel; SD, Standard deviation; SLN, Solid lipid nanoparticles; t1/2, Plasma half-life; tmax, Time to reach peak plasma concentration; TPGS, d-alpha tocopheryl polyethylene glycol 1000 succinate; TPSA, Succinoyal TPGS; RPMI, Roswell Park Memorial Institute medium 1640; WGA, Wheat germ agglutinin.
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Abstract Oral delivery of anticancer drugs remains challenging despite the most convenient route of drug administration. Hydrophobicity and non-specific toxicities of anticancer agents are major impediments in the development of oral formulation. In this study, we developed wheat germ agglutinin (WGA)-conjugated, solid lipid nanoparticles to improve the oral delivery of the hydrophobic anticancer drug,paclitaxel (PTX). This study was focused to improve the PTX loading in biocompatible lipid matrix with high bioconjugation efficiency. WGA-conjugated, PTX-loaded solid lipid nanoparticles (LPSN) exhibited enhanced anticancer activity against A549 lung cancer cells after internalization through N-acetyl-D-glucosamine receptors than free PTX. Biodistribution studies in rats revealed that LPSN significantly improved the oral bioavailability and lung targetability of PTX which could be due to cumulative bioadhesive property of the nanocarrier system and the targeting ligand WGA. Keywords: Wheat-germ agglutinin; Solid lipid nanoparticles; Paclitaxel; Bioavailability; Lung cancer
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INTRODUCTION At present, chemotherapy is one of the primary and standard treatments for all types of cancer. Paclitaxel (PTX) is to date, one of the most potent antineoplastic agents andis effective against a variety of cancers including breast, ovarian, non-small cell lung cancer, head and neck, colon cancer and AIDS related Kaposi’s sarcoma. PTX inhibits the cellular growth by stabilizing the microtubule assembly through interaction with tubulin, followed by block of cell replication in the late G2 mitotic phase of cell cycle.1-6 Despite having wide spectrum activity, its clinical impact is minimal due to its low aqueous solubility, poor oral bioavailability (< 10%) and nonspecific cytotoxicity.7-12 The commercial formulation of PTX (Taxol®)causes several unwanted side effects such as hypersensitivity reactions, nephrotoxicity, and neurotoxicity. These side effects are largelydue to the presence of cremophor EL (polyethoxylated castor oil) and ethanol (1:1, v/v) in PTX formulation and also due to non-specific interaction of PTX with normal cells.13,14 Additionally, cremophor EL can also cause leaching of plasticizer from infusion bags and polyethylene tubing. To overcome these drawbacks of PTX, different drug delivery systems such as liposomes,15 micelles,16 nanoparticles,17,18 and polymer-drug conjugate19 have been attempted. PTX formulations such as Opaxia® (PTX-polyglutamate conjugate) and Abraxane® (PTXalbumin nanoparticle conjugate, are approved by FDA for intravenous (IV) treatment of breast cancer. However, IV administration is inconvenient and painful to patients and it also requires regular hospital visits which lead to a decrease in patient compliance. Oral administration would offer the advantages over IV administration such asimproved patient compliance, reduced administration costs and enable the chronic treatment of cancer.20 Therefore, various approaches
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have been investigated for oral formulation of PTX.21-24 The oral administration of PTX is limitedbecause of its low bioavailability which is mainly due to low aqueous solubility, effects of multidrug efflux transfer P-glycoprotein (P-gp) and pre-systemic metabolism in the liver by cytochrome P450 metabolic enzymes. P-gp and cytochrome P450 are abundantly co-localized in the gastrointestinal tract and act synergistically to first pass metabolism. To enhance oral bioavailability, different PTX analogues have been developed such as BMS-275183 and ortotaxel. Both analogues exhibited asignificant increase in bioavailability but clinical use is limited by their toxicological profile and reduced anti-tumoral activity by ortotaxel.25-27 Other approaches such as co-administration of PTX with either a P-gp inhibitor or cytochrome P450 inhibitor (verapamil, flavonoids and cyclosporine)28-30 have also been used to improve the bioavailability of PTX but non-specificity of PTX remains inevitable. The clinical success and potential of a therapeutic compound is dependent on the selection of an optimum drug delivery system which can provide biocompatibility, high drug payload, targetability, controlled drug release, improved bioavailability and pharmacokinetic profile. The primary objective of this study was to develop a cremophor and ethanol free, oral formulation of PTX using solid lipid nanoparticle as a carrier system. Secondly, PTX-loaded nanoparticles were conjugated to wheat germ agglutinin (WGA), a plant lectin, for site-specific delivery to lung tumor cells. Lectins are proteins that recognize and bind to the carbohydrate moiety of glycoproteins and glycolipids. These glycoconjugates are the integral part of human enterocyte membrane and are over-expressed incancer cells.31 After oral administration, the hydrophobic drugs are solubilized with the help of pancreatic fluid and biliarylipid in the upper part of gastro-intestinal tract. Therefore, these molecules are absorbed from the small intestine but shorter transit time (3.5−4.5 h) through thesmall intestine limits their oral bioavailability.32
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WGA binds specifically to N-acetyl-D-glucosamine (NAG) and sialic acid that are present on cell surfaces throughout the entire intestine.33 The interaction between WGA lectin-grafted nanoparticles and carbohydrate moieties results in the anchoring of molecules in the intestine for a longer time. Thus, conjugation of lectin to nanocarriers helps to increase the oral bioavailability of encapsulated drugs34,35 and site-specific delivery of anticancer drugs. MATERIALS AND METHODS Materials. Glyceryl monostearate was purchased from Alfa Aesar (Hyderabad, India). Glyceryl tripalmitate, glyceryl trimyristate, glyceryl tristearate and stearic acid, fluorescein isothiocyanate (FITC)-labeled lectin from Triticum vulgaris (WGA), bovine serum albumin, N(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), stearyl amine, d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and dialysis tubing (molecular weight cut off 12000-14000) were obtained from Sigma Aldrich (St. Louis, MO, USA). Lecithin soy was a product of Hi Media Labs. (Mumbai, India). Bradford reagent was purchased from Biomatik Corp. (Hyderabad, India). Tween®80, poloxomer 188 and poly(vinyl) alcohol were purchased from Sd Fine-Chem. Ltd. (Mumbai, India). A549 human lung cancer cells were obtained from American Type Culture Collection (ATCC, Manassas, USA). Roswell Park Memorial Institute (RPMI) 1640, 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco, USA. Dead cell apoptosis kit with Annexin V FITC and propidium iodide (PI) was purchased from Invitrogen. Paclitaxel was received as gift sample from TherDose Pharma Pvt. Ltd. (Hyderabad, India). Optimization of paclitaxel-loaded colloidallipid nanostructures (PSN). Paclitaxelloaded solid lipid nanoparticles were prepared by solvent emulsification and evaporation method 6 ACS Paragon Plus Environment
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as reported previously with minor modifications.36 Briefly, PTX, lipids and lecithin soy were dissolved in 2 ml chloroform and poured in to 10 mL deionised water containing different concentrations (1%, 1.5% and 2% w/v) of surfactants (poloxomer 188, Tween®80 and polyvinyl alcohol). The dispersion was homogenized at 13528 ×g for 5 min and sonicated for 20 min. The mixture was stirred for 3 h to evaporate chloroform. The nanoparticle dispersion was centrifuged at 25155×g for 45 min. Supernatant was collected for the estimation of entrapment efficiency andnanoparticles were obtained as a pellet at the bottom of tubes. Nanoparticles were washed thrice with distilled water and lyophilized using trehalose dehydrate as a cryoprotectant. For the optimization of lipid, surfactant and co-surfactant concentrations, one factor-one time strategy was used and its effect on particle size, surface charge and PTX encapsulation efficiency was evaluated. PTX encapsulation efficiency. After 100 times dilution, the supernatant was analyzed for PTX concentration using HPLC instrument (Waters, USA) equipped with an octadecylsilane column (Water Reliant, 250 × 4.6 mm, 5 µm) and photodiode array detector. The mobile phase, acetonitrile (55%) and water (45%), was pumped at a flow rate of 1.0 mL/min and peaks were monitored at a wavelength of 229 nm. The calibration curve was prepared in the range of 0.1-10 µg/mL concentrations of PTX. The regression equation was y= 76525x+6241 with correlation coefficient of 0.999.The inter/intra-day accuracy and precision were within a relative standard deviation of ≤5%. Drug encapsulation efficiency was determined as follows: % Encapsulation efficiency = 1 − (
) × 100
Where Ds is amount of drug in supernatant, and Di is initial amount of drug added.
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Bioconjugation of WGA to PSN. WGA lectin was conjugated to PSN surface by EDC/NHS reaction and conjugation efficiency was determined by Bradford protein assay.37 Lyophilized nanoparticles were dispersed in PBS and incubated with EDC and NHS (1 mL of 0.1M EDC/NHS) for 2 h. Nanoparticles were centrifuged at 11180 ×g for 15 min to remove EDC and NHS. Nanoparticles were redispersed in PBS and incubated with WGA lectin overnight. LPSN were collected by centrifugation at 11180 ×g for 30 min. The supernatant was diluted 50 times and mixed with Bradford reagent. After 30 min incubation, the absorbance was measured at 595 nmusing a microplate reader (BioTek Synergy 4, USA). The amount of WGA in supernatant was determined by using bovin serum albumin standard curve. Physicochemical characteristics of nanoparticles. The mean particle diameter, size distribution and zeta potential of lipid nanoparticles were determined by dynamic light scattering technique using a particle size analyzer Nano-ZS (Malvern Instruments Ltd., Malvern, UK). Samples were diluted appropriately with MilliQ water and analyzed at 25 °C with a backscattering angle of 173°. Surface morphology of LPSN was observed using transmission electron microscope (FEI Tecnai™, G112, Philips, Japan). LPSN were dispersed in distilled water and a drop of the suspension was cast onto a copper grid. An excess of staining solution was removed with blotting paper and samples were air-dried before observing under the TEM. X-ray diffraction patterns of GMS, PTX and PSN were obtained using Siemens/Bruker D-5000 X-ray diffractometer (Germany) equipped with Cu K α radiation generated at 45 kV and 20 mA. The diffraction patterns run over the 2θ range from 2 to 60°. Physical state of PTX, before and after encapsulation in nanoparticles was determined by Differential Scanning Calorimeter (DSC) analysis. DSC scans of free PTX and PSN were performed on a DSC1 Star System (Mettler Toledo, Switzerland). The samples were scanned from 40 °C to 270 °C at a speed of 10 °C/min,
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under nitrogen environment. Bioconjugation of WGA to PSN was confirmed by Fourier transform infrared spectroscopy (FTIR) analysis. In vitro release of PTX from PTX suspension, PSN and LPSN was studied in phosphate buffer saline pH 7.4. PTX solution, PSN or LPSN, equivalent to 0.5 mg PTX, were placed in dialysis tubes (12–14 kD molecular weight cutoff). These dialysis bags were incubated in 150 mL PBS at 37 °C and 100 rpm. At predetermined time intervals, 1 mL samples were collected and replaced with the same volume of fresh media. The amount of PTX in the collected samples was measured by HPLC at 229 nm. LPSN formulation was stored at refrigeration condition (4 °C) for a period of 90 days. The mean particle size, polydispersity, zeta potential, and drug content were analyzed to determine the effect of storage conditions on the stability of nanoparticles. In vitro cytotoxicity assay. To determine the cytotoxicity of PTX, PSN and LPSN cell proliferation assay was carried out using the MTT reagent. Briefly, 1×104 A549 cells (counted by CountessTM automated cell counter) were seeded in a 96-well plate for 24 h. Next day, cells were incubated with PTX, PSN or LPSN formulations in a dose-dependent manner for 48 or 72 h. The media was replaced with fresh 100 µL serum-free RPMI 1640 media containing MTT reagent (concentration 0.5 mg/mL) and incubated for further 4 h. To solubilize the violet formazan crystals, the RPMI-MTT mixture was replaced by 150 µL DMSO. Finally, the optical density or absorbance of the samples was measured at 574 nm using a microplate reader (BioTek Synergy4). Each experiment was carried out in triplicate and the results are expressed as % cell viability versus time.
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Competitive binding and cytotoxicity assay. Competitive binding studies were performed in the presence of NAG. Cells were incubated with 25 µM and 50 µM NAG before treatment with PTX, PSN and LPSN. Rest of the procedure was similar to that described earlier. In vitro cellular uptake studies. A549 cells were seeded in 24-well plates at 5×104 cells/well and cultured at 37 °C for 24 h. Coumarin 6-loaded solid lipid nanoparticles (CSN) and lectin conjugated CPN (LCSN) were added and cells incubated for different time intervals (0.5, 1 and 3 h). Cells were washed with cold PBS and fixed with 4% paraformaldehyde for 10 min. Cells were again washed with PBS and incubated with Hoechst 33342 (2 µg/mL). Images were captured using a fluorescence microscope (Nikon, Japan) after washing the cells with PBS. For competitive binding assay, cells were pre-incubated with 50 µM of NAG for 1 h before adding CSN and LCSN. Apoptosis studies. PTX-induced apoptosis in A549 cells was studied qualitatively and quantitatively by acridine orange/ethidium bromide(AO/EB) assay and Annexin V FITC/PI assay, respectively. For AO/EB assay, cells were seeded (1×105 cells/well) in a 24-well plate and cultured for 24 h. Cells were treated with PTX, PSN or LPSN equivalent to 50 ng/mL PTX. After 24 h of treatment, media was removed and cells were incubated with a mixture of AO/EB containing 10 µg/mL of each, for 15 min. Cells were washed and observed using a fluorescence microscope. For Annexin V FITC/PI assay, cells were seeded at a density of 1×106 cells/well in a 6well plate and incubated overnight. Cells were incubated with PTX, PSN or LPSN equivalent to 50 ng/mL PTX. After 24 h of treatment, the cells were collected by trypsinization and cell suspensions were washed with cold PBS. Cells were resuspended in 100 µL binding buffer and to each sample, 5 µL Annexin V-FITC and 2 µL PI (PI working solution of 50 µg/mL) were
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added. Samples were incubated 15 min in the dark and then analyzed using a flow cytometer (BD FACS Canto™ II, CA, USA). Pharmacokinetic and tissuedistribution studies in rats. Plasma PTX profile: In vivo pharmacokinetics of PTX suspension, PSN or LPSN were evaluated in Wistar rats. The animal experimental protocol (IICT/PHARM/SRK/FEB/2013/10; 20th March 2013) for this study was approved by the Institutional Animal Ethics Committee (IAEC) of the CSIR-Indian Institute of Chemical Technology, Hyderabad. The studies performed in animals were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Government of India). The animals were acclimatized for one week prior to experimentation. Animals were housed in Biosafe which is maintained at temperature of 22±2 °C and relative humidity of 50–60% under 12 h:12 h light/dark cycle for a week before the experiment. Animals were fasted overnight before the experiment and water was made available ad libitum. Seventy two animals were divided into 3 groups, each containing 24 animals. PTX suspension was prepared by dispersing PTX in 2% w/v gum acacia. PSN or LPSN were dispersed in normal saline. Three groups of rats were administered PTX suspension or PSN or LPSN orally using an oral gavage at a dose of PTX (25 mg/kg body weight). Blood samples (approximately 0.3 mL) were collected at predetermined time intervals (0.5, 1, 1.5, 2, 4, 8, 12 and 24 h) from the retro-orbital plexus into microcentrifuge tubes containing EDTA. Blood samples were centrifuged at 1789 ×g for 10 min and supernatant (plasma) was collected. Tissue-distribution studies: For the determination of PTX distribution in various organs, animals were sacrificed at 1, 2, 4, 8, 12 and 24 h after administration of PTX suspension, PSN or
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LPSN. After sacrificing the animals, organs (liver, spleen, lung, kidney, heart and brain) were carefully excised, washed with normal saline, weighed and stored at−80 °C until analysis. Analysis of PTX in plasma and tissue samples. Plasma and tissue samples were analyzed for PTX content. Weighed organ samples were finely minced with surgical scalpel blades and then homogenized in normal saline to form 0.2 g/mL tissue homogenate. A volume of 200 µL tissue homogenate was extracted with 500 µL of a mixture of acetonitrile and methanol (1:1), vortexed for 90 s and centrifuged at 11180×g for 15 min. The supernatant was collected, filtered through a 0.22 µm filter and analyzed for PTX content using a HPLC system. The mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid (48:52% v/v) was delivered at a flow rate of 1 ml/min. The temperature for the column was set at 25±5 °C and injection volume was 20 µL. Docetaxel was used as internal standard. Statistical analysis. In vitro cytotoxicity and in vivo studies data were presented as mean ± SEM (standard error of mean) and statistically analyzed by one way ANOVA test followed by Bonferroni post hoc test. The difference was considered significant when p < 0.05. RESULT AND DISCUSSION Optimization of paclitaxel loaded colloidal lipid nanostructures (PSN). Paclitaxelloaded solid lipid nanoparticles (PSN) were prepared by single emulsification and evaporation method. PSN were prepared with four different lipids namely, viz. glyceryl trimyristate (GTM), glyceryl tripalmitate (GTP), glyceryl monostearate (GMS) and glyceryl tristearate (GTS). Table S1 represents the physicochemical parameters of different PSN formulations. The mean particle diameter was 198.2, 185.6, 152.9 and 149.7 nm for PSN prepared with GTM, GTP, GMS and GTS, respectively. Thus the minimum size of nanoparticles was obtained with GTS and GMS. However, nanoparticles prepared with GMS had a higher negative zeta potential (−24.5±1.38 12 ACS Paragon Plus Environment
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mV) and entrapment efficiency (91.34±1.24%) than those prepared with GTS (−17.7±2.15 mV, 75.47±0.93 %). Therefore, GMS was selected for further formulation of PSN. The physiochemical parameters of nanoparticles are significantly affected by the nature and concentration of surfactants in the aqueous phase. In this study, three different types of surfactants, viz.Tween®80, poloxamer 188 (Pluronic®F-68), and polyvinyl alcohol (PVA) were used at three different concentrations (1%, 1.5% and 2% w/v). At all three concentrations, the minimum particle size was observed with Tween®80 followed by poloxamer188 and PVA (Table S2). As the concentration of Tween®80 was increased, the nanoparticle size was decreased significantly. However, the encapsulation efficiency (EE) was also decreased (74.19±2.05%) at a high concentration (2% w/v) of Tween®80. The maximum EE (91.34±1.24%) was observed with 1.5% w/v of Tween®80 and therefore this concentration was selected for further optimization. In the next step, lecithin soy was used as internal emulsifier and the effects of its amount on formulation parameters were studied. Lecithin soy acts as a co-surfactant and is used to improve the stability and reduce the size of nanoparticles. The results in Table S3 show that as the amount of soya lecithin was increased from 15 to 25% w/w, the nanoparticle size decreased (from 169.5 to 149.8 nm) whereas EE was increased from 86.29% to 92.42%. Therefore, the optimized PSN formulations contained the GMS as primary lipid and were emulsified using Tween®80 (1.5% w/v) and soya lecithin (25% w/w). Bioconjugation of WGA to the PSN. For conjugation between two molecules, free functional groups should be present on each of the molecule.WGA contains both amine and carboxylic functional groups. However, the lipid matrix used in this study does not contain any free functional group. Therefore, successful conjugation between WGA and lipid carrier warranted use of an additional component containing either free –COOH or –NH2 group. To 13 ACS Paragon Plus Environment
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achieve this, 15% of GMS was replaced with stearyl amine or stearic acid to give either free amine or carboxylic group, respectively on nanoparticle surface (Table S4). When stearyl amine containing PSN were conjugated with WGA using EDC/NHS chemistry, size of nanoparticles was increased to 239.2 nm and zeta potential was changed to −16.1 mV (Table S5). However, the conjugation efficiency was substantially less (11.45%) which can be explained by the basic mechanism of EDC/NHS reaction, in which carboxylic group is activated with EDC and the formed intermediate is stabilized by NHS. This intermediate (activated WGA in this case) interacts with the free amine on other molecule (stearyl amine containing PSN in this case) to form an amide bond. Given that a WGA molecule contains both free carboxylic and amine groups, there is high possibility of intramolecular cross-linking of WGA molecules than conjugation between WGA and nanoparticles. In contrast, activation of –COOH group of stearic acid followed by conjugation with WGA may offer a better strategy to obtain WGA-conjugated PSN. As evident from Tables S4 and S5, conjugation increased to 74.1%, whereas size was retained at197.4 nm. Recently, TPGS is receiving attention as an excipient in many pharmaceutical formulations. TPGS is a water-soluble derivative of Vitamin E and acts as emulsifier and solubilizer. Moreover, TPGS inhibits the P-gp mediated drug efflux and enhances oral absorption of drug molecules.38,39 As discussed earlier, P-gp mediated efflux is one of the reasons for low oral bioavailability of PTX. However, TPGS itself does have free functional groups. Therefore, we used succinoyl-TPGS (TPSA) having free carboxylic group. TPSA was synthesized as reported previously.40 We incorporated TPSA (15% w/w) in the lipid matrix. Nanoparticles prepared with TPSA exhibited smaller particle size, lower polydispersity and higher conjugation efficiency than nanoparticles prepared with stearic acid or stearyl amine.
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The surface charge of WGA lectin conjugated PSN (LPSN) was −16.5 mV which was lower than unconjugated formulations prepared with TPSA (−29.4 mV). WGA is cationic at physiological pH and the isoelectric point of WGA is pH 9.Therefore, a significant decrease in surface potential confirmed the conjugation of WGA on PSN surface. Physicochemical characteristics of PNP and LPNP.TEM analysis showed that LPSN were spherical in shape (Figure1a) with size ranging 50-100 nm. FTIR analysis confirmed the surface group interaction between PSN and WGA (Figure 1b). FTIR spectrum of PSN showed characteristic signature of carboxylic group at 1736 cm-1whereas LPSN showed amide bond peak at 1687 cm-1 confirming the conjugation of LPSN.X-ray diffraction patterns of pure PTX showed characteristic sharp features between 5 to 30°, confirming the crystalline state of PTX (Figure 1c). These sharp peaks of PTX were not observed in the pattern of PSN suggesting that, in PSN, PTX was present in its amorphous form rather than crystalline. The physical state of PTX as a pure compound and after encapsulation in the nanoparticles was also determined by DSC analysis (Figure 1d). The melting, endothermic peak of pure PTX appeared at 215°C. However this peak was not observed in PSN confirming the conversion of crystalline phase of PTX to amorphous phase in nanoparticles. One additional peak at 95°C was also observed in PSN thermogram which correspond to cryoprotectant (trehalose dihydrate) used for lyophilization of nanoparticles. In vitro release of PTX from PTX suspension, PSN and LPSN was evaluated in PBS. Figure S1 demonstrates the cumulative percentage of drug released from three formulations as a function of time. A slow and incomplete release of PTX was observed with PTX suspension whereby approximately 15.2% of drug was released after 96 h. The release behavior of PTX from PSN and LPSN exhibited a biphasic pattern. Approximately 27.9% of PTX was released
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from PSN and 30.1% from LPSN in the initial 24 h followed by a more sustained release of drug up to 96 h. There was insignificant (p>0.05) difference in the release of PTX from PSN and LPSN. We observed a similar pattern of drug release previously with hydrophobic drugs loaded into SLN.36,37 There was no significant change in the particle size (191.3 versus 195.9), zeta potential (−18.3 versus −18.9 mV) and drug content (92.3% versus 91.5%) of LPNP after 90 days of storage at refrigerated condition (4 °C), suggesting long-term stability of the nanoparticles. In vitro anticancer activity. Comparative cytotoxicity of different formulations, PTX, PSN and LPSN, were assessed against A549 human lung carcinoma cells. For the evaluation of dose-dependent effects, cells were incubated with different doses in the range of 1 ng/mL to 400 ng/mL. Figure 2 shows the % cell viability of A549 incubated with PTX formulations. The halfmaximal inhibitory concentration (IC50) values for PTX, PSN and LPSN were 164.56, 100.26 and 41.04 ng/mL, respectively after 48 h of treatment (Table 1). The results indicated that LPSN substantially inhibited A549 cell growth compared with free PTX or PSN. The IC50 values for all three formulations were further decreased after 72 h treatment. LPSN had 5.4 times lower IC50 value than free PTX. As expected, in comparison to PSN, LPSN showed significantly higher efficacy and lower IC50 values (p0.0005) higher for the LPSN-treated cells (34.5%) than free PTX-treated cells (9.8%). Apoptosis assay results revealed that LPSN-treated cells lost their membrane integrity which allowed the nuclear material staining dyes i.e. EB(in AO/EB assay) and PI (in Annexin V FITC/PI assay), which are generally unable to cross the intact cell membrane, to enter the cells. Although PSN incubation had similar effects, the extent of cell membrane damage was lower than LPSN. Pharmacokinetic and tissue-distribution of LPSN. The mean plasma PTX concentration versus time profiles after a single oral administration of PTX, PSN or LPSN at a
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dose of 25 mg/kg body weight, in Wistar rats are shown in Figure5a. The pharmacokinetic parameters for PTX, PSN and LPSN were calculated using non-compartmental analysis with Ramkin software. As shown in Figure5a and Table 2, LPSN markedly increased peak plasma concentration (3.81 µg/mL) in comparison to free PTX (1.75 µg/mL). Comparatively, free PTX concentration was decreased rapidly and PTX levels were not detectable after 8 h. For both PSN and LPSN, PTX concentration was detected up to 24 h which suggested the sustained release of drug from the nanoparticles. The area under the curve (AUC), which is considered an indicator of the extent of absorption, was also significantly higher for LPSN (30.03 µg/mL/h) than PSN (16.1 µg/mL/h) or PTX (7.78 µg/mL/h). Given at the same concentration, LPSN was retained in the blood for longer durations indicating an increase in oral bioavailability of PTX after administration as LPSN. The clearance (Cl) was decreased for LPSN in comparison to PSN or PTX. The decrease in Cl led to prolonged blood circulation of LPSN, as also reflected in the AUC. This led to doubling of the mean residence time (MRT) for LPSN (8.94h) in contrast to PTX (4.99h). Furthermore, the plasma half-life (t1/2) for LPSN increased to 1.78 times greater than PTX. The increase in t1/2 of LPSN would help reduce the dosing frequency. The pharmacokinetic data reveals a marked improvement in the bioavailability of PTX after administration of the proposed oral formulation of PTX compared to that of conventional drug. The enhanced oral bioavailability could also be explained by enhanced interaction of LPSN with microfold cells (M cells) at absorption site. M cells are present on intestinal Peyer’s patches and are involved in absorption transportation of nanoparticles after oral administration.43 M cells can keep absorbed nanoparticles intact and efficiently transport them to lymphoid tissues. Both SLN and lectins
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have the ability to bind M cells which not only enhance bioavailability but also bypass the liver and avoid first pass metabolism. Further to assess the targetability of our oral formulation, we evaluated the PTX concentrations in the different body tissues including heart, liver, spleen, kidney and lung (Figure 5b). Free PTX was distributed mainly to the liver followed by kidney, spleen, brain and lung. The distribution of LPSN was lungs>spleen> brain> liver and kidney. Accumulation of LPSN in the lung tissues shows the importance of the developed formulation because it will not only help to deliver most of the drug molecules to the diseased tissue but also reduce the non-specific toxicity of the PTX. As the liver is the major site for metabolism of PTX, the low accumulation of LPSN in the liver could be responsible for prolonged blood circulation of LPSN. To better understand the accumulation of formulations in lungs, the target tissue, pharmacokinetic parameters were calculated for LPSN, PSN and PTX (Table 3). The results clearly demonstrated the high specificity of LPSN towards the lungs. The LPSN showed 12.4 times increase in the amount of PTX delivered to the lungs than free PTX which could be attributed to prolonged residence time (12.64 h versus 5.77 h) and decreased clearance (15.04 mL/h versus 1.27 mL/h) of PTX from the lung tissues. These results can be explained by the physicochemical properties and composition of LPSN. The solid lipid nanoparticles carrier systems have high bioadhesion property which results in longer residence time at the site of absorption and increased bioavailability. RussellJones et al45 demonstrated that lectin conjugation to the polymeric nanoparticles enhanced the trans-cellular transport of nanoparticles. Such glyco-targeting systems bind to the mucins present on the surface of mucous layer present in the small intestine. Both M cells and enterocytes are the primary sites for the cyto-invasion of these systems. Therefore the combined favorable
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properties of SLN and WGA at the absorption site i.e. small intestine, enhanced the oral bioavailability of LPSN and WGA-conjugation and further helped to take the nanoparticles to the lungs. Therefore, the combination of WGA as a targeting ligand and solid lipid nanoparticles as a nanocarrier system could be one of the most favourable platforms for the oral delivery of drugs to the lungs. CONCLUSIONS Targeting of cancer cells using various ligands has substantially advanced drug delivery systems. In the present study, PTX-loaded, lectin-conjugated solid lipid nanoparticles (LPSN) were developed with maximum possible pay load with nanoscale size and high conjugation efficiency. LPSN were rapidly taken up by the A549 cells through receptor-mediated endocytosis which resulted in higher cellular toxicity and PTX-induced apoptosis. The competitive binding assays performed during cytotoxicity and cellular uptake studies suggested the role of WGA conjugation in the intracellular delivery of nanoparticles. Pharmacokinetic data confirmed the improved performance of LPSN over free PTX. In tissue distribution studies, LPSN were preferentially accumulated in the lungs whereas the free PTX was accumulating in the liver. Although the biodistribution studies were performed in normal, healthy rats, the results revealed the effectiveness of LPSN to deliver PTX to the lungs and at the same time bypass nontargeted tissues. ACKNOWLEDGEMENT Authors thank the Director, Indian Institute of Chemical Technology, Hyderabad for providing the necessary facilities and support. Authors are grateful to M/s Therdose Pharma for providing PTX. H.K. acknowledges the IICT-RMIT Research Centre for PhD scholarship. D.P. thanks the Council of Scientific and Industrial Research (CSIR), New Delhi and Department of 21 ACS Paragon Plus Environment
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Education, Australian Government for awarding a Senior Research Fellowship and Endeavour Research Fellowship, respectively. This work was financially supported by CSIR grant under project Advanced Drug Delivery (ADD-CSC 0302).V.B. acknowledges the Australian Research Council (ARC) for a Future Fellowship (FT140101285) and the ARC for funding support through an ARC Linkage (LP130100437) scheme. Competing financial interests. The authors declare no competing financial interests. ASSOCIATED CONTENT Supporting Information Supporting information is available free of charge on the ACS Publications website at DOI: Optimization for formulation parameters including type of lipid, type and concentration of surfactant, effect of co-surfactant and surface charge modifier. Optimization of WGAconjugation to the nanoparticles. In vitro drug release profiles of different PTX formulations. Time-dependent cellular uptake studies of fluorescent dye loaded nanoparticles. REFERENCES (1) Horwitz, S. B. Mechanism of action of taxol. Trends Pharmacol. Sci. 1992, 13, 134-136. (2) Sparano, J. A.; Wang, M.; Martino, S.; Jones, V.; Perez, E. A.; Saphner, T.; Wolff, A. C.; Sledge, G. W.; Wood, W. C.; Davidson, N. E. Weekly paclitaxel in the adjuvant treatment of breast cancer. New Engl. J. Med. 2008, 358, 1663-1671. (3) Montana, M., Ducros, C.; Verhaeghe, P.; Terme, T.; Vanelle, P.; Rathelot, P. Albuminbound paclitaxel: the benefit of this new formulation in the treatment of various cancers. J. Chemother. 2011, 23, 59-66.
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(20) O’ neill, V. J.; Twelves, C. J. Oral cancer treatment: developments in chemotherapy and beyond. Br. J. Cancer 2002, 87, 933-937. (21) Lian, H.; Zhang’ T.; Sun, J.; Liu, X.; Ren, G.; Kou, L.; Zhang, Y.; Han, X.; Ding, W.; Ai, X.; Wu, C.; Li, L.; Wang, Y.; Sun, Y.; Wang, S.; He, Z. Enhanced oral delivery of paclitaxel using acetylcysteine functionalized chitosan-vitamin E succinate nanomicelles based on a mucus bioadhesion and penetration mechanism. Mol. Pharm. 2013, 10(9), 3447-3458. (22) Joshi, N.; Saha, R.; Shanmugam, T.; Balakrishnan, B.; More, P.; Banerjee, R. Carboxymethyl-chitosan-tethered lipid vesicles: hybrid nanoblanket for oral delivery of paclitaxel. Biomacromolecules 2013, 14(7), 2272-2282. (23) Xu, W.; Fan, X.; Zhao, Y.; Li, L. Cysteine modified and bile salt based micelles: preparation and application as an oral delivery system for paclitaxel. Colloids Surf. B Biointerfaces 2015, 128, 165-171. (24) Groo, A. C.; Bossiere, M.; Trichard, L.; Legras, P.; Benoit, J. P.; Lagarce, F. In vivo evaluation of paclitaxel-loaded lipid nanocapsules after intravenous and oral administration on resistant tumor. Nanomedicine (Lond) 2015, 10(4), 589-601. (25) Broker, L. E.; Veltkamp, S. A.; Heath, E. I.; Kuenen, B. C.; Gall, H.; Astier, L.; Parker, S.; Kayitalire, L.; Lorusso, P. M.; Schellens, J. H.; Giaccone, G. A phase I safety and pharmacologic study of a twice weekly dosing regimen of the oral taxane BMS-275183. Clin. Cancer Res. 2007, 13 (13), 3906–3912. (26) Mastalerz, H.; Cook, D.; Fairchild, C. R.; Hansel, S.; Johnson, W.; Kadow, J. F.; Long, B. H.; Rose, W. C.; Tarrant, J.; Wu, M. J.; Xue, M. Q.; Zhang, G.; Zoeckler, M.; Vyas, D. M. The discovery of BMS-275183: an orally efficacious novel taxane. Bioorg. Med. Chem. 2003, 11 (20), 4315–4323.
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(27) Brooks, T. A.; Minderman, H.; O'Loughlin, K. L.; Pera, P.; Ojima, I.; Baer, M. R.; Bernacki, R. J. Taxane-based reversal agents modulate drug resistance mediated by Pglycoprotein, multidrug resistance protein, and breast cancer resistance protein. Mol. Cancer Ther. 2003, 2 (11), 1195–1205. (28) Wang, F.; Zhang, D.; Zhang, Q.; Chen, Y.; Zheng, D.; Hao, L.; Duan, C.; Jia, L.; Liu, G.; Liu, Y. Synergistic effect of folate-mediated targeting and verapamil-mediated P-gp inhibition with paclitaxel-polymer micelles to overcome multi-drug resistance. Biomaterials 2011, 32(35), 9444-9456. (29) Sarisozen, C.; Vural, I.; Levchenko, T.; Hincal, A. A.; Torchilin, V. P. PEG-PE-based micelles co-loaded with paclitaxel and cyclosporine A or loaded with paclitaxel and targeted by anticancer antibody overcome drug resistance in cancer cells. Drug Deliv. 2012, 19(4), 169-176. (30) Malingre, M. M.; Beijnen, J. H.; Schellens, J. H. Oral delivery of taxanes. Invest. New Drugs 2001, 19, 155-162. (31) Gorelik, E.; Galili, U.; Raz, A. On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis. Cancer Metastasis Rev. 2001, 20, 245–277. (32) Porter, C. J.; Pouton, C. W.; Cuine, J. F.; Charman, W. N. Enhancing intestinal drug solubilisation using lipid-based delivery systems. Adv. Drug Deliv. Rev. 2008, 60(6), 673-691. (33) Pusztai, A.; Ewen, S. W.; Grant, G.; Brown, D. S.; Stewart, J. C.; Peumans, W. J.; Van Damme, E. J.;
Bardocz, S. Antinutritive effects of wheat-germ agglutinin and other N-
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(35) Liu, Y.; Wang, P.; Sun, C.; Zhao, J.; Du, Y.; Shi, F.; Feng, N. Bioadhesion and enhanced bioavailability by wheat germ agglutinin-grafted lipid nanoparticles for oral delivery of poorly water-soluble drug bufalin. Int. J. Pharm. 2011, 419(1-2), 260-265. (36) Pooja, D.; Kulhari, H.; Tunki, L.; Chinde, S.; Kuncha, M.; Grover, P.; Rachamalla, S. S.; Sistla, R. Nanomedicines for targeted delivery of etoposide to non-small cell lung cancer using transferrin functionalized nanoparticles. RSC Adv. 2015, 5, 49122-49131. (37) Pooja, D.; Tunki, L.; Kulhari, H.; Reddy, B. B.; Sistla, R. Characterization, biorecognitive activity and stability of WGA grafted lipid nanostructures for the controlled delivery of rifampicin. Chem. Phys. Lipids 2015, 193, 11-17. (38) Dintaman, J. M.; Silverman, J. A. Inhibition of P-glycoprotein by D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Pharm. Res. 1999, 16(10), 1550-1556. (39) Pooja, D.; Kulhari, H.; Singh, M. K.; Mukherjee, S.; Rachamalla, S. S.; Sistla, R. Dendrimer-TPGS mixed micelles for enhanced solubility and cellular toxicity of taxanes, Colloids Surf. B Biointerfaces, 2014, 121, 461-468. (40) Kulhari, H.; Pooja, D.; Shrivastava, S.; Telukutala, S. R.; Barui, A. K.; Patra, C. R.; Naidu, V. G. M.; Adams, D. J.; Sistla, R. Cyclic-RGDfK peptide conjugated succinoyl-TPGS nanomicelles for targeted delivery of docetaxel to integrin receptor over-expressing angiogenic tumours. Nanomed. Nanotech. Biol. Med. 2015, 11(6), 1511-1520. (41) Shah, R.M.; Rajasekaran, D.; Ludford-Menting, M.; Eldridge, D.S.; Palombo, E.A.; Harding, I.H. Transport of stearic acid-based solid lipid nanoparticles (SLNs) into human epithelial cells. Colloids Surf. B Biointerfaces 2016, 140, 204-212. (42) Mo, Y.; Lim, L. Y. Mechanistic study of the uptake of wheat germ agglutinin-conjugated PLGA nanoparticles by A549 cells. J. Pharm. Sci. 2004, 93(1), 20-28.
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Figure legends Figure 1. Physicochemical characterizations of different paclitaxel (PTX) formulations. (a) TEM micrograph of WGA-conjugated, PTX-loaded solid lipid nanoparticles (LPSN), (b) Fourier transform infrared spectra of PTX, PTX-loaded solid lipid nanoparticles (PSN) and LPSN,(c) Xray diffraction patterns of PTX and PSN, and (d) Differential scanning calorimetry scans of PTX, PSN and LPSN. Figure 2. Cell viability versus concentration curve for A549 human lung cancer cells incubated with paclitaxel (PTX), PTX-loaded solid lipid nanoparticles (PSN) and WGA-conjugated PSN (LPSN) for (a) 48 h and (b) 72 h. Figure 3.Cellular uptake of coumarin-6 loaded solid lipid nanoparticles (CSN) and lectin conjugated CSN (LCSN) by A549 human lung cancer cells. Cell nucleus was stained with Hoechst 33342 dye. LCSN+NAG represents the uptake of LCSN in the presence of N-acetyl-Dglucosamine. Figure 4. Apoptosis studies.(a) Fluorescent microscopic images of A549 cells after 24 h incubation with 50 ng/mL of paclitaxel (PTX), PTX-loaded solid lipid nanoparticles (PSN) and WGA-conjugated PSN (LPSN) followed by staining with ethidium bromide and acridine orange. (b) Bar graph showing the quantitative apoptosis levels determined by annexin V FITC/PI assay. Figure 5. Pharmacokinetic studies. (a) Plasma profile (b) Tissue distribution profile of paclitaxel (PTX), PTX-loaded solid lipid nanoparticles (PSN) and WGA conjugated PSN (LPSN) after oral administration in Wistar rats at a dose of 25 mg/Kg body weight. The data represents mean ± SD; n=4.
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FIGURES
b) Transmittance (a.u.)
a)
PTX
PSN
LPSN
4000 3500 3000 2500 2000 1500 1000
c)
PSN
Wavenumber (cm-1)
PTX
Endothermal
d)
Intensity (a.u.)
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PSN LPSN
PTX
10
20
30
40
80
50
120
160
200 o
240
Temperature ( C)
2θ (degree)
Figure 1
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b)
100
80
100
PTX PSN LPSN
PTX PSN LPSN
80
Cell viability (%)
a)
Cell viability (%)
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60
40
20
60
40
20
0
0 50 100 150 200 250 300 350 400
50 100 150 200 250 300 350 400
Concentration (ng/mL)
Concentration (ng/mL)
Figure 2
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Figure 3
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Figure 4a
% Live cells % Apoptosis
100
% Apoptosis
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80 60 ###
40
*** ***
20 0
*** trol Con
PTX
PSN
N LPS
Figure 4b
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PTX Concentration (µg/mL)
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PTX PSN LPSN
4
3
2
1
0 0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)
Figure 5a
Figure 5b
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TABLES
Table 1. IC50 values for paclitaxel (PTX), PTX loaded solid lipid nanoparticles (PSN) and WGA conjugated PSN (LPSN) after 48 h and 72 h incubation of A549 human lung cancer cells (Mean±SEM; N=4)
Formulations
IC50 values (ng/mL) 48 h
*
72 h
PTX
164.56±1.57
60.27±1.15
PSN
100.26±1.06***
32.51±1.19***
LPSN
41.04±1.42***,###
11.06±0.61***, ###
25 µM NAG+LPSN
57.19±1.74
17.56±1.15
50 µM NAG+ LPSN
96.61±1.07a
30.44±1.34a
indicates comparison between pure PTX Vs PSN and LPSN, #indicates comparison between
PSN and LPSN, aindicates comparison with PSN. ***
P
