One of the biggest challenges in oncology is the treat-ment of brain cancer. Brain cancer is the leading cause of cancer-related death in the US in patients under 35. Anaplastic astrocytomas (Grade III) and gliomblasto-mas (Grade IV) are most aggressive brain cancers with survival period of 24 and 9 months, respectively (Kemper et al., 2004). Currently available systemic chemotherapy is less effective due to presence of the blood–brain bar-rier (BBB) which restricts the penetration of most drugs into the brain.
Paclitaxel is active against various cancers and has also shown its efficacy against malignant gliomas and brain metastatses (Glantz et al., 1999; Brandes et al., 2000; Fellner et al., 2002; Li et al., 2003). However, its effi-cacy is variable and low in the treatment of brain tumors due to its limited entry into the brain. In addition, low concentration of paclitaxel has been found in the normal brain as well as in tumor brain tissues after intravenous administration (Eiseman et al., 1994; Heimans et al.,
1994; Sparreboom et al., 1996; Gallo et al., 2003). The low uptake of paclitaxel by the brain is due to expression of P-glycoprotein (P-gp) at the apical membrane of the brain endothelial cells (Breedveld et al., 2006). Several strategies have been investigated to increase the delivery of paclitaxel including invasive implant, nanoparticles (NPs), conjugate with brain delivery vector Angiopep-2 (Koziara et al., 2004; Nikanjam et al., 2007; Tanner et al., 2007). Several approaches such as local invasive deliv-ery by direct injection, induction of enhanced perme-ability and physiological targeting strategies have been reported for brain targeting (Begley, 2004; Pardridge, 2004; Gaillard et al., 2005). Among all various strategies, NP delivery for brain targeting has been successful and widely investigated. Various NPs have been reported for the brain-targeted delivery including Tween® 80-coated NP (Kreuter, 2001; Xie et al., 2010), transferrin receptor-specific antibody-conjugated NP (Olivier et al., 2002; Olivier, 2005; Carroll et al., 2010) and thiamine-coated NP (Lockman et al., 2003). Glutathione is a water-soluble
RESEARCH ARTICLE
Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers
Werner Geldenhuys, Thomas Mbimba, Thong Bui, Kimberly Harrison, and Vijaykumar Sutariya
Department of Pharmaceutical Sciences, Northeast Ohio Medical Unviersity (NEOMED), Rootstown, OH, USA
AbstractPaclitaxel is not effective for treatment of brain cancers because it cannot cross the blood–brain barrier (BBB) due to efflux by P-glycoprotein (P-gp). In this work, glutathione-coated poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) of paclitaxel were developed for brain targeting for treatment of brain cancers. P-gp ATPase assay was used to evaluate the NP as potential substrates. The NP showed a particle size suitable for BBB permeation (particle size around 200 nm) and higher cellular uptake of the NP was demonstrated in RG2 cells. The P-gp ATPase assay suggested that the NP were not substrate for P-gp and would not be effluxed by P-gp present in the BBB. The in vitro release profile of the NP exhibited no initial burst release and showed sustained drug release. The proposed coated NP showed significantly higher cytotoxicity in RG2 cells compared with uncoated NP (p ≤ 0.05). Tubulin immunofluorescent study showed higher cell death by the NP due to increased microtubule stabilization. In vivo brain uptake study in mice showed higher brain uptake of the NP containing coumarin-6 compared with solution. The proposed brain-targeted NP delivery of paclitaxel could be an effective treatment for the brain cancers.Keywords: paclitaxel, brain-targeted delivery, PLGA NP, tubulin immunofluorescent, brain cancer
Address for Correspondence: Vijaykumar Sutariya, Department of Pharmaceutical Sciences, Northeast Ohio Medical Univesity (NEOMED), Rootstown, OH 44272, USA. E-mail: [email protected]
(Received 27 August 2010; revised 19 April 2011; accepted 01 May 2011)
antioxidant that helps protect cells from reactive oxygen species such as free radicals and peroxides. The con-sideration of glutathione-coated NP as a brain-targeted delivery can be rationalized since large number of glu-tathione transporters is present at the BBB (Wang et al., 2007; Liang et al., 2008; Gaillard., 2010). Glutathione ligand should bind to the BBB glutathione transporters, subsequently increasing the number of NP at the BBB interface. We hypothesize that the glutathione-coated NP will give enhanced drug transport via brain by either a facilitated transport or increased passive diffusion due to an increased concentration gradient at the BBB interface. Figure 1 shows schematic diagram of glutathione-coated poly-(lactide-co-glycolide)-polyethyleneglycol (PLGA-PEG) NP containing paclitaxel (PLGA-PEG-glutathione NP).
NP made from PLGA-b-PEG appear to be an excellent drug carrier due to its low tissue toxicity, few side effects and a controllable drug release rate (Carroll et al., 2010). Furthermore, the PLGA-PEG NP can reduce the uptake by reticulo-endothelial system (RES) in the blood in vivo compared with the unmodified PLGA.
In the present study, we report for the first time a novel drug delivery system using glutathione-coated PLGA-PEG NP aimed at crossing the BBB. These pro-posed paclitaxel-loaded NP are suitable for treatment of brain cancer in in vivo animal models such as brain tumor-bearing mouse model. The initial characterization of the PLGA-PEG-glutathione NP such as particle size, cellular uptake, in vitro drug release, cytotoxicity, in vivo brain uptake study and in vivo biodistribution has been described.
Materials and methods
MaterialsPLGA-PEG-COOH (RESOMER® RGP d 50105, copo-lymer ratio 50:50, PEG content 8%) was obtained from Boehringer Ingelheim Chemicals, Inc., Petersburg, VA, USA. Paclitaxel was purchased from A.G. Scientific, Inc, San Diego, CA, USA. Glutathione (reduced) was obtained from MP Biomedicals, LLC, Solon, OH. Coumarin-6 was received from Sigma- Aldrich Co, St. Louis, MO, USA.
Phosphate buffered saline (PBS) and 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum and penicillin–streptomycin were purchased from Hyclone, Logan, UT, USA. Acetone and acetonitrile were purchased from VWR International, Batavia, IL, USA. HEPES, NaCl and EDTA were received from VWR Internationals, West Chester, PA, USA. All other chemi-cals used in the study were of analytical grade and were used without any further purification.
Preparation of glutathione-coated PLGA-PEG NPPaclitaxel-loaded NPNP made of PLGA-PEG-COOH were prepared using a nanoprecipitation method as described elsewhere (Gu et al., 2008; Carroll et al., 2010). Briefly, PLGA-PEG-COOH (120 mg) and paclitaxel (4 mg) were dissolved in 3 mL of acetone and mixed together dropwise into 6 ml of water, giving a final NP concentration of 20 mg/mL. The NP were stirred for 2 h at 40°C and were centrifuged at 12,000 × g for 45 min. After discarding the supernatant, NP were resuspended in 0.5 mL of 0.01 M HEPES, 0.15 M NaCl and 0.1 mM EDTA, pH 7.0. In order to get 1% w/v, 2%w/v or 3% w/v glutathione coating per 1 mL NP solu-tion, 10, 20 or 30 mg glutathione was added to 1 mL of NP solution (20 mg/mL) and incubated at room temperature for 30 min before use. Here, 1% w/v, 2% w/v or 3% w/v value suggests the amount of glutathione per 1 mL of NP solution and is not the amount of glutathione per weight of NP. The amount of glutathione adsorbed on the NP sur-face was measured by Promega GSH-GloTM Glutathione Assay kit (Promega Corporation, Madison, WI, USA).
Coumarin-loaded NPThe 6-courmarin-loaded NP were prepared following the same protocol that was described above. However, 15 µL of 6-coumarin (1mg/mL stock solution in acetone) was used instead of paclitaxel during the procedure.
Effect of glutathione coating on NP sizeThe particle size and polydispersity index of uncoated and glutathione-coated PLGA-PEG NP (1, 2 or 3% w/v glutathione per 1 mL NP solution) were measured using PSS-NICOMP particle sizing system (NICOMP™ 380, Santa Barbara, CA, USA). The effect of glutathione coating at different concentration (1, 2 or 3% w/v glutathione per 1 mL NP solution) on particle size of the NP was studied.
Determination of entrapment efficiency (%)Paclitaxel was dissolved in methanol and the solution was diluted to obtain different concentrations of paclitaxel. The solutions of paclitaxel were measured by UV spec-trophotometer (Shimadzu, UV-2101 PC, Kyoto, Japan) at a wavelength of 230 nm (λ
max) to obtain the calibration
curve of the drug. The NP-containing paclitaxel were dis-solved in methanol and the concentration of paclitaxel was determined by UV spectrophotometer 230 nm. The absorbance values were converted to concentrations
Figure 1. Schematic diagram of brain-targeted nanoparticle (NP) showing paclitaxel encapsulated in NP containing hydrophilic corona of polyethyleneglycol (PEG) and glutathione coating.
by extrapolation from a calibration standard curve (r2 = 0.9996) and the entrapment efficiency was deter-mined. All experiments were repeated three times and average values were used. The entrapment efficiency (%) was calculated by the following equation:
Entrapment
efficiency (%) =
Weight of the drug in NP
Weight of the feeding drug×100
(1)
where, drug in NP is the amount of drug loaded in the NP and feeding drug is the total amount of drug added during preparation of NP.
P-gp ATPase assayTo evaluate NP as potential substrates or inhibitors for P-gp, we utilized our previously published method using recombinant protein with minor modifications (Carroll et al., 2010). Briefly, recombinant human P-gp was purchased from BD Genetest, San Jose, CA as a mem-brane preparation. These membranes were prepared from insect high five cells that were transfected with the cDNA of human P-gp using a baculovirus vector. To test for P-gp-ATPase activity, 2 µL of the NP solution in buffer was added to 8 µL (total of 40 µg of protein) of membrane preparation in a white clear bottom 96-well plate. Verapamil, a known substrate of P-gp was used as a control at 20 µM final concentration. The ATPase reaction was initiated with the addition of 90 µL of reac-tion buffer (50 mM Tris-MES, 2 mM EDTA, 50 mM KCl, 2 mM dithiothreitol, and 5 mM sodium azide and 2 µM MgATP, pH 6.8). This assay mixture was incubated in an Eppendorf 96-well plate heater/shaker for 30 min at 37°C. The reaction was stopped with the addition of 100 µL of a Kinase-glo solution (Promega Corporation, Madison, WI 53711, USA) which reacts with ATP not used in by P-gp. After 10 min incubation at room temperature, lumines-ence was evaluated in a BioTek® Synergy 4 plate reader (BioTek, Winooski, VT 05404, USA). To determine if the NP were inhibitors of P-gp-ATPase, the NP were co-incu-bated with 20 µM verapamil. All experiments were done in triplicate.
In vitro drug releaseThe paclitaxel release rate from the NPs was measured in PBS buffer (pH 7.4) medium by high-performance liquid chromatography (HPLC) method (Mu and Feng, 2003). The paclitaxel-loaded (10 mg) NPs were suspended in 10 mL PBS buffer in screw-capped tubes and the tubes were placed in an orbital shaker plus water bath (Sci-ERA Hot Shaker, Bellco Biotechnology, Vineland, New Jersey, USA) at 37°C and shaken horizontally at 120 rpm. The tubes were taken out of the water bath after a par-ticular time interval and centrifuged at 4000 rpm for 1 h. The separated NPs were resuspended in 10 mL fresh PBS buffer and then put back in the shaker bath. The super-natant was used for paclitaxel analysis by HPLC method. The HPLC system consisted of a Perkin Elmer HPLC, flexar binary pump, flexar autosampler and UV visible
detector. Chromatographic separations were achieved using Agilent ODS column (4.6 mm × 100 mm, 5 µm particle size, C
18) at 25°C. The mobile phase consisted of
deionized water and acetonitrile, with 50% acetonitrile in deionized water at 0 min and then linear gradients to 90% acetonitrile in 12 min and was delivered at a flow rate of 1.0 mL/min. UV detection was performed at 227 nm. The absorbance values were converted to concentrations by extrapolation from a standard curve (r2 = 0.9996). The cumulative percent drug release was then determined. All experiments were repeated three times and average values were used.
Cell cultureA rat glioma cell line (RG2) was obtained from ATCC, Manassas, VA 20108, USA. The RG2 cells were cultured in DMEM supplemented with 10% calf bovine serum and antibiotics (100 µg/mL of streptomycin and 100 unit/mL of penicillin) at 37°C in a humidified incubator with 5% CO
2. The cells were maintained in exponential growth
phase by periodic subcultivation.
Intracellular distribution of 6-coumarin-loaded NP in RG2 cellsThe RG2 cells were seeded with 500 µL DMEM containing 10% fetal bovine serum with penicillin (100 units/ mL), streptomycin (100 mg/mL) at 10,000 cells per well in an eight-well poly-D-lysine-coated chamber slide. Cells were then incubated in 5% CO
2 at 37°C for 24 h to achieve
70–90% confluent cells. The medium was aspirated and the cells were washed with PBS three times. Then 500 µL of DMEM media containing 50 µg of 6-coumarin-loaded NP 2% glutathione (2% w/v glutathione coating per 1 ml NP solution, concentration of NP: 20 mg/mL) was added to separate wells. The cultures were then incubated in 5% CO
2 at 37°C for 2 h. The media was aspirated after 2 h and
cells were washed three times with PBS. DAPI was added to each well for nuclear staining and a cover slip was placed on the chamber slide. The slide was then exam-ined by fluorescent microscopy with 20× magnification.
MTT assayRG2 cells were plated 50,000 cells/well in a 96-well plate in DMEM containing 10% fetal bovine serum, penicil-lin (100 units/mL) and streptomycin (100 mg/mL) and allowed to adhere at 37°C with 5% CO
2 for 24 h prior to
the assay. Cells were treated with various doses of blank NP, paclitaxel solution (P-solution), paclitaxel-loaded NP (P-NP) and glutathione-coated NP containing paclitaxel (P-NP-glutathione; 100 µM, 300 µM and 500 µM) for 48 h. After incubation, 50 µL of MTT dye (5 mg/mL in PBS, pH 7.4) was added to each well and the cells were incubated for 4 h at 37°C. The 96-well plate was spun for 3 min at 700 × g and the medium was withdrawn. To each well, 100 µL of dimethyl sulfoxide (DMSO) and 15 µL of 0.1 M gly-cine buffer (pH 10.5) were added. The plate was imme-diately read on a microplate reader (Spectramax-340PC, Bucher Biotec AG, Basel, Switzerland). Absorption was
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measured at 570 nm and obtained values were expressed as a percentage of untreated cells.
Immunofluorescence of tubulin and western blottingThe immunofluorescence of tubulin was carried out using RG2 cells. The cells were grown to 90% confluence on 24-well plates for 2 days in culture medium. The cells were then treated with P-solution (50 µM) or P-NP (50 µM) or P-NP-2% glutathione (50 µM) for 24 h. The cells were washed twice with cold PBS, fixed and permeabi-lized with cold methanol for 10 min at−20°C. The cells were then blocked with 2% bovine serum albumin (BSA) in PBS and were incubated with anti-β-tubulin monoclo-nal antibody (CP07; Calbiochem, Mississauga, ON, USA) in PBS containing 1% BSA for 60 min at room tempera-ture. The fluorescein isothiocyanate-conjugated anti-mouse IgG rabbit antibody (Jackson Immunoresearch, Westgrove, PA, USA) was added into each well after three washes. Pictures of cells were taken at 60× using an OLYMPUS® fluorescence microscopy. Western blot analysis of the RG2 cells treated with control, uncoated P-NP, P-NP-1% Tween and P-NP-2% glutathione NP was carried out in order to observe the acetylation of tubu-lin in the cells by paclitaxel released from the different formulations. Cells were washed twice with Dulbecco’s phosphate buffered saline and harvested in 1.5 eppen-dorf with RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 1.0% sodium deoxycholate 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, PH 8.0; TEKnova, 2290 Bert Dr. Hollister, CA 95023) and incubated on ice for 30 min. Samples were centri-fuged at 14,000 rpm for 45 min at 4°C, insoluble debris were discarded and the supernatant was transferred to new labeled tubes. Proteins estimation was done using Pierce BCA Protein Assay Kit (Thermo Scientific, 3747 N. Meridian Rd., Rockford, IL 61101). Samples were resolved in SDS-PAGE and electrotransferred to Immunblot polyvinylidene fluoride membrane (92008; Invitrogen, Carlsbad, CA). The membrane was blocked for 1 h into 5% non-fat dried milk in TBST. Antibody against acetylated-tubulin (1:2,000; Sigma) was applied at room temperature for 1 h. After three washes of 10 min each, the membrane was incubated with secondary anti-mouse secondary (1:1,000; Santa Cruz Biotechnology) for 1 h at room tem-perature. Chemiluminescence detection was carried out with Thermo SuoerSignal western Chemoluminescent Kit (Thermo-Pierce). The blot was reprobed with β-actin antibody, and the results were used as loading controls.
In vivo brain uptake and biodistribution studiesAn experimental protocol approved by NEOUCOM ani-mal care committee was used for the proposed animal study. Animal care was conducted with strict adherence to institutional guideline and to guidelines specified in the IACUC Guidebook 2002 (IACUC guideline, 2002). The male mice (C57BL/6 mice Harlan laboratories, IN, USA) at 2 months of age were used in the present study. The mice were housed individually in cages and given free
access to standard laboratory food and water. Coumarin-6-(a fluorescent dye) loaded uncoated NP, Coumarin-6-loaded NP-2% glutathione and coumarin-6 aqueous solution were injected to the C57BL/6 intraperitoneally and the mice were sacrificed and brains were dissected. The brain slices were observed using OLYMPUS® fluores-cent microscope (Model: Provis AX 70, Olympus America, Inc, Melville, NY, USA). Brain tissue was homogenized in PBS pH 7.4 and fluorescence intensity (excitation wave-length 387 nm, emission wavelength 470 nm) was mea-sured using Bioteck® microplate reader (Biotek Synergy 4, Bioteck Instruments,Winooski, VT, USA).
In vivo biodisirbution studyEach mouse was anesthetized and then injected intrap-eritoneally 5 mg/kg of coumarin-6 in NP-2% glutathione or solution. At 1 h post injection, the mice were sacrificed and the biodistribution of coumarin-6 in tissues such as brain, liver, lungs, kidneys and adiapose tissues (obtained from each mouse) was studied by measuring fluorescence intensity (excitation wavelength 387 nm, emission wave-length 470 nm) using the Bioteck® microplate reader.
Results and discussion
Preparation of glutathione-coated PLGA-PEG NPThe actual amount of glutathione adsorbed on the sur-face of NP-2% glutathione after 2% w/v glutathione coat-ing per 1 mL NP solution was measured using Promega GSH-Glo™ Glutathione Assay kit. The results of glutathi-one assay indicated that the amount of glutathione on the surface of NP-2% glutathione was 0.205 ± 0.013% w/w (n = 3).
Effect of glutathione coating on particle size of NPThe particle size and polydispersity index of blank NP, uncoated NP containing paclitaxel and glutathione-coated NP containing paclitaxel are shown in Table 1. Paired t-test analysis indicated that particle size of NP-containing paclitaxel was significantly higher than blank NP (p ≤ 0.05) which was due to drug loading of NP. Glutathione-coated NP showed slight increase in particle size due to adsorption of glutathione on the NP surface. Particle size of NP slightly decreased with increase in glu-tathione coating from 1% to 3% w/v glutathione per 1 mL NP solution which may be due to hydrophilic nature of glutathione. The particle size of NP-2% glutathione was suitable for BBB permeation (Carroll et al., 2010). The
Table 1. Effect of glutathione coating on NP size.
polydispersity index of NP-2% glutathione was less than 1 which indicated the normal distribution or Gaussian distribution of NP size.
Entrapment efficiency (%)The entrapment efficiency was found to be 64.82 ± 3.72% in the case of paclitaxel-loaded uncoated NP, whereas NP-2% glutathione containing paclitaxel had an entrap-ment efficiency of 60.73 ± 2.23%. The entrapment effi-ciency depended mainly on paclitaxel’s hydrophobic interactions with the hydrophobic core of the NP (the PLGA).
P-gp ATPase assayAs paclitaxel is a substrate for the BBB efflux pump P-gp, we investigated the effect of the encapsulated paclitaxel NPs with human P-gp. The results of the assay are shown in Figure 2. Verapamil is used as control substrate for P-gp. As expected, verapamil (20 µM) caused P-gp to utilize ATP, and this use of ATP was significant, when compared with the vehicle control (p < 0.05). Similarly, when human recombinant P-gp were co-incubated with the uncoated and glutathione-coated NPs, there was an increase in ATP use by P-gp (p < 0.05 compared with vehicle control). This suggests that the NPs may act as substrate for P-gp, although when comparing the NPs’ ATP use by P-gp with that of verapamil, no statistical significant difference was seen. The results of this assay suggest that the NPs, either coated or uncoated, may be substrate for P-gp and that the glutathione coating does not increase efflux by P-gp. To test whether the NPs could possibly inhibit vera-pamil efflux by P-gp, we co-incubated verapamil with glutathione-coated NPs in the presence of P-gp. As can be seen, the ATP use was similar to that of the NPs alone. Unfortunately, the decrease in ATP use as compared with verapamil alone was not statistically significant, suggesting that ATP use was essentially the same—i.e. no apparent competition or addition of the two as substrate was seen. Further studies will be needed to elucidate the exact interaction mechanism between the NPs and P-gp.
In vitro drug releaseThe release of drug from NP may depend on drug diffu-sion, PLGA surface and bulk erosion or swelling (Mu and Feng, 2003) which is attributed to the slow degradation
of PLGA polymer. No significant initial burst release was observed in NP-2% glutathione which may be due to the loss of drug on the surface of NP during the glutathione coating (Figure 3). Paired t-test analysis showed that the release behavior of NP-2% glutathione was not signifi-cantly different than uncoated NP (p ≤ 0.05). It was found that 11.24 ± 0.82% (n = 3) of paclitaxel was released within 250 h for NP-2% glutathione
Figure 2. Human recombinant P-glycoprotein (P-gp) assay which measures ATP (luminescent-assay) use in the efflux of substrates. Verapamil (20 µM) is a known substrate of P-gp and is used as a positive control. Data are represented as mean ± SD, where N = 4–5. Statistical significance is denoted by “*” (p < 0.05).
Figure 3. Effect of glutathione content on paclitaxel release from nanoparticle (NP). Values represent mean ± SD (n = 3).
Figure 4. Cellular uptake of nanoparticle-(NP) 2% glutathione in RG2 cells. Nuclei were stained with DAPI and are visible in blue (B). The uptake of coumarin-6-loaded NP is visible in green (A and C). Figure C displays overlaying images obtained combining the fluorescein isothiocyanate and the DAPI filters. Magnification: 20×.
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Intracellular uptakeFrom the fluorescence images, we can observe the green fluorescence of NP containing coumarin-6 in the cyto-plasm and blue fluorescence from the nucleus due to DAPI labeling (Figure 4). The uptake of NP by the cells is likely to have occurred via endocytosis. The fluorescence intensity of coumarin-6-loaded glutathione-loaded NP was more compared with uncoated NP using paired t-test analysis. This increase is likely attributed to carrier-mediated endocytosis of the NP via glutathione trans-porters. The higher fluorescence intensity in cells treated with coumarin-6-loaded NP was due to the slow release of coumarin-6 from the encapsulated NP while untreated cells did not show any auto fluorescence which was simi-lar to that observed by others (Sahoo and Labhasetwar, 2005; Carroll et al., 2010).
MTT assayMTT assay was used to evaluate the cytotoxic efficacy of P-NP and P-NP-glutathione in RG2 cells. Different concentrations of glutathione coating (1%, 2% and 3% w/v glutathione per 1 mL NP solution) were used to treat RG2 cells. The glutathione-coated NP of pacli-taxel showed increased cytotoxic effect with increas-ing amount of glutathione (data not shown). P-NP-2% glutathione showed optimum cytotoxic effect at both 50 µM and 100 µM concentrations. Figure 5 shows cytotoxic effect of paclitaxel on RG2 cells treated with P-solution in 0.5% DMSO, uncoated P-NP, P-NP-2% glutathione and P-NP-1% Tween (1% w/v Tween added to 1 mL NP solution; NP concentration 20 mg/mL) at 50 µM or 100 µM drug concentrations. Paired t-test analysis showed that cytotoxic effect of the glutathione-coated NP was significantly higher compared with uncoated
NP (p ≤ 0.05). We believe that higher cytotoxicity of glutathione-coated NP was due to increased cellular uptake of NP by the cells and the increased cellular uptake was due to carrier-mediated transport of NP via the glutathione transporters. In our previous studies, we have also found that blank PLGA NP did not alter RG 2 cells proliferation in MTT assay demonstrating that the PLGA NP would not influence our results (Caroll et al., 2010). P-NP-2% glutathione showed significantly higher cytotoxic effect compared with P-solution or uncoated NP at both 50 µM and 100 µM concentrations by paired t-test analysis (p ≤ 0.05). We also compared the cytotoxic effect of the P-NP-2% glutathione with P-NP-1% Tween which is a well-reported drug delivery system for brain targeting (Olivier et al., 1999; Sun et al., 2004; Das and Lin, 2005; Ren et al., 2009). The P-NP-2% glutathione showed almost similar cytotoxic effect (significantly not different as per paired t-test analysis) compared with P-NP-1% Tween at both 50 µM and 100 µM drug concentrations (p ≤ 0.05). These results indicated that the proposed P-NP-2% glutathione would give similar cytotoxic effect to the Tween-coated NP in future in vivo study. The disadvantage with the use of Tween-coated NP for brain targeting is the cytotoxic effect of Tween to the body while glutathione is an amino acid and is not a cytotoxic (Olivier et al., 1999).
Immunofluorescence of tubulin and western blottingPaclitaxel interferes with microtubule depolymerization resulting in the accumulation of acetylated α-tubulin and stabilized microtubule structures (Dowdy et al., 2006). Figure 6A shows immunofluorescence images of acelyated α-tubilin in the RG2 cells. (i) Control, (ii) Uncoated PX-NP, (iii) P-NP-1% Tween and (iv) P-NP-2% glutathione. The NP-2% glutathione caused a modest increase in acetylated tubulin compared with control and uncoated NP and almost same to 1% Tween-coated NP. This effect was corroborated by immunohistochem-istry. The microtubules of control cells were fine, dif-fused and evenly distributed cytoplasmic networks, whereas cells treated with uncoated NP showed rela-tively thicker microtubule bundles. Cells treated with NP-2% glutathione and NP-1% Tween showed even more prominent microtubule. These results indicate that NP-2% glutathione and NP-1% Tween showed more cell death by increased microtubule stabilization. Western blot analysis of the cells as described for immu-nohistochemistry was studied in order to confirm the acetylation of tubulin and microtubule stabilization by paclitaxel-loaded NP-2% glutathione. Figure 6B shows the western blot analysis for acetylated tubulin with (i) Control, (ii) Uncoated PX-NP, (iii) P-NP-1% Tween and (iv) P-NP-2% glutathione. The results of western blot analysis indicated that treatment with NP-2% glutathione of paclitaxel led to a significant increase in acetylated tubulin compared with control and uncoated P-NP. The acetylation of tubulin was almost similar to the P-NP-1% Tween. These results support
Figure 5. The cytotoxic effect of paclitaxel on RG2 cells treated with paclitaxel solution (P-solution) in 0.5% dimethyl sulfoxide, uncoated nanoparticle (NP) of paclitaxel (P-NP), P-NP-2% glutathione and P-NP-1% Tween at 50 µM or 100 µM (n = 5). Glutathione-coated NP of paclitaxel showed significant cytotoxic effect in cells compared with paclitaxel solution or uncoated NP at both 50 µM and 100 µM concentrations (p ≤ 0.05). P-NP-2% glutathione showed almost similar cytotoxic effect (significantly not different) compared with P-NP-1% Tween (well-reported brain-targeted delivery system) at both 50 µM and 100 µM concentrations (p ≤ 0.05).
our hypothesis that paclitaxel-loaded NP-2% glutathi-one will be equally cytotoxic to the well-documented brain-targeted 1% Tween-coated NP delivery system in future in vivo animal study. Moreover, the data sug-gest that glutathione coating does not interfere with the paclitaxel activity. As can be seen, coating the NP with glutathione did not interfere with the ability of the NP to
deliver and release their paclitaxel payload, indicating that these NP will release the drug inside cells.
In vivo brain uptake and biodistribution studiesIn a pilot study, the brain tissue uptake of glutathione-coated NP, coumarin 6- (a fluorescent dye) loaded NP-2% glutathione and coumarin-6 aqueous solutions were
Figure 7. (A) The glutathione-coated NP showed more accumulation in the brain than control. The control (coumarin-6 aqueous solution at the same dose) showed a more diffuse uptake distribution pattern as compared with the NP-solution. (B) Representative brain distribution of coumarin-6 in the mice after 1 h of 0.2 ml intraperitoneal administration of 5 mg/kg of coumarin-6 in glutathione-coated nanoparticle (NP-2% glutathione).
Figure 8. (A) Brain tissue uptake of coumarin-6 in the mice after 1 h of 0.2 ml intraperitoneal administration of 5 mg/kg of coumarin-6 in glutathione-coated nanoparticle (NP-2% glutathione). Mice treated with glutathione-coated NPs showed statistically significant increase in fluorescence intensity in brain tissue at 1 h, compared with control (coumarin-6 aqueous solution at same dose; n = 3; p ≤ 0.05). (B) In vivo biodistribution studies of coumarin-6-loaded NP-2% glutathione. Concentration of coumarin-6 in different tissues at 1 h post injection of 0.2 ml coumarin-6-loaded NP-2% glutathione or coumarin-6 solution. Each mouse was injected intraperitoneally 5 mg/kg of coumarin-6 in NP-2% glutathione or solution. Data represent the mean ± SD (n = 3).
Figure 6. Effect of paclitaxel on tubilin acetylation. (A) (i) Control, (ii) Uncoated PX-NP, (iii) Tween-coated NP containing paclitaxel (P-NP-1% Tween) and (iv) P-NP-2% glutathione. RG2 cells were treated with uncoated NP (50 µM) or P-NP-2% glutathione ( 50 µM) for 24 h. Visualization of microtubules before and after drug treatment was done under fluorescent microscopy. The microtubules of control cells were fine, diffuse and evenly distributed cytoplasmic networks, whereas cells treated with uncoated P-NP showed relatively thicker microtubule bundles. Cells treated with P-NP-2% glutathione and NP-1% Tween shows even more prominent microtubule surrounding the nucleus with brighter staining and exhibits more cells death compared with control and uncoated P-NP due to increased microtubule stabilization. Magnification: 20×. (B) Western blot of acetylated tubulin with (i) Control, (ii) Uncoated P-NP, (iii) P-NP-1% Tween and (iv) P-NP-2% glutathione. Data suggest that the glutathione coating does not interfere with paclitaxel activity. Protein loading is also shown with β-actin (molecular weight: 42 kDa)
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injected into the C57BL/6 male mice via intraperitone-ally. The mice were sacrificed and brains excised. The glutathione-coated NP showed accumulation near stria-tum after 1 h in the mice (Figure 7A) and the coumarin-6 uptake by brain was higher than the coumarin-6 aqueous solution at the same dose (Figure 7B). In addition, NP-2% glutathione containing coumarin-6 showed higher brain tissue uptake at 1 h compared with coumarin-6 aque-ous solution (Figure 8A). To further support our finding, similar results of brain tissue uptake of lactoferrin-con-jugated NP in mice have been reported (Hu et al., 2009). This result supports our hypothesis that glutathione-coated NP containing paclitaxel will target the brain and will provide the better cytotoxicity to the brain tumor in brain tumor animal model. The results of the biodistri-bution experiments are shown in Figure 8B. The results shown in Figure 8B indicate that the glutathione-coated NP showed distribution in liver, lung and kindey and adiapose tissues. NP-2% glutathione showed very lower levels of fluorescent intensity in other tissues such as the liver, lungs, kidneys and adiapose tissues compared with the coumarin-6 solution. The fluorescent intensity of coumarin-6 in brain tissues was lower than lever and kidney but was similar to lung in both NP-2% glutathione and solution. The fluorescent intensity of coumarin-6 in brain was higher than the adiapose tissues for both the formulations.
The results of brain tissue uptake of coumarin-6 in the mice after 1 h of 0.2 mL intraperitoneal administra-tion of 5 mg/kg of coumarin-6 in NP-2% glutathione and uncoated NP is shown in Figure 9. Mice treated with glutathione-coated NPs showed statistically significant increase in fluorescence intensity in brain tissue at 1 h compared with uncoated NP (n = 3) by paired t-test (p ≤ 0.05). These results indicate that the physically absorbed glutathione on the NP are not removed in vivo and the higher brain uptake of these glutathione-coated NP was due to the glutathione transporters present in the BBB.
Conclusions
Novel and promising glutathione-coated NP was developed for brain-targeted delivery of paclitaxel for treatment of brain cancer. Particle sizes obtained were around 200 nm, making them suitable for intravenous administration and able to penetrate to the brain. The NPs showed a sustained drug release behavior and were not substrates of P-gp and will not be effluxed by the P-gp pump across the BBB. Cellular uptake study in RG2 cells showed higher uptake of the glutathione-coated NP compared with uncoated NP. Cell prolifera-tion studies using MTT in RG2 cells indicated superior cytotoxic efficacy of the coated NP compared with uncoated NP or free paclitaxel in solution. The NP also showed cytotoxicity similar to the well-documented Tween-coated NP for brain targeting. The tubulin immunofluorescent study indicated higher microtu-bule stabilization in NP-2% glutathione compared with control. This may be due to increased levels of paclitaxel in the cells due to higher carrier-mediated transcyto-sis of coated NP into the cells. The NP-2% glutathione containing fluorescent dye showed higher brain uptake compared with dye solution in in vivo mouse model. Further evaluation of the proposed glutathione-coated NP in in vivo brain cancer animal model is needed to determine the efficacy of the proposed NP in treatment of brain cancer.
Declaration of interest
The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
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