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Intracellular Delivery of Etoposide Loaded. Biodegradable Nanoparticles: Cytotoxicity and Cellular Uptake Studies. Khushwant S. Yadav1, Sheeba Jacob2, ...

Copyright © 2011 American Scientific Publishers All rights reserved Printed in the United States of America

Journal of Nanoscience and Nanotechnology Vol. 11, 6657–6667, 2011

Intracellular Delivery of Etoposide Loaded Biodegradable Nanoparticles: Cytotoxicity and Cellular Uptake Studies Khushwant S. Yadav1 , Sheeba Jacob2 , Geetanjali Sachdeva2 , and Krutika K. Sawant1 ∗ † 1

TIFAC–Centre of Relevance and Excellence in NDDS, Pharmacy Department, The M. S. University of Baroda, Fatehgunj, Vadodara 390002, Gujarat, India 2 Primate Biology, National Institute for Research in Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai 400012, India

RESEARCH ARTICLE

The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGAMPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 M and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 M for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.

Keywords: Etoposide, Biodegradable Nanoparticles, Cytotoxicity, Cellular Uptake.

1. INTRODUCTION Intracellular delivery of the therapeutic agents refers to the delivery of the drug within the cell. The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. The cytotoxicity of the anticancer drugs would be enhanced if they are within the cancerous cells.1 Such a delivery would minimize the drug distribution and maximize the destruction of the cancerous cells. The drug loaded polymeric nanoparticles (NPs) have demonstrated an increase in the cellular uptake and increased therapeutic ∗

Author to whom correspondence should be addressed. Present address: Pharmacy Department, Faculty of Technology and Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara 390001, Gujarat, India. †

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activity in the past.2–3 The drug delivery systems based on biodegradable polymers can be applied for preparation of NPs. The most widely used polymers for biodegradable nanoparticles have been poly(lactide-coglycolide) (PLGA). PLGA is known for its biocompatibility and resorbability through natural pathways in the body.4 PLGA based polymeric NPs have been extensively studied for encapsulation of anticancer drugs.5–6 Polyethyleneglycol (PEG) modified biodegradable polymer is one of the most popular materials to prepare stealth nanoparticles which would be used for controlled release.7 Poly(lactide-co-glycolide)methoxy-poly(ethylene glycol) (PLGA-MPEG) copolymer finds wide applications in drug delivery for preparation of nanoparticles.8 The PEG layer provides a steric barrier to the particle and its opsonization is reduced. Poloxamers (Pluronic F68) are tri-block copolymers of

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polypropylene oxide (PPO) and polyethylene oxide (PEO) that have been employed for surface modification of PLGA based NPs.9 The cellular uptake of the drug-loaded NPs would depend on internalization and retention of the NPs by the diseased cells and this can be visualized by confocal microscopy. Confocal microscopy has been proved useful for determining the fate of the NPs containing fluorescent dyes or drugs within the cells. 6-coumarin has been used as a fluorescent marker for studying the cellular uptake of PLGA nanoparticles in the cell lines. The cellular uptake of the PLGA nanoparticles containing 6-coumarin can be quantified by measuring the fluorescence intensity of the nanoparticles within the cells. Etoposide (ETO) is one of the most commonly used drugs in the chemotherapy of leukemia. The conventional oral therapy of ETO has drawbacks of low bioavailabilty and parenteral therapy causes inconvenience and pain to the patients as it has to be given through a continuous IV infusion over 24–34 h.10 ETO loaded biodegradable nanoparticles would overcome these drawbacks by providing a sustained release. The authors have previously developed etoposide loaded PLGA nanoparticles,11 etoposide loaded PLGA-MPEG NP and etoposide loaded PLGA-Pluronic NP.12 All the three developed nanoparticles showed sustained release of etoposide. In the present study we evaluated the cytotoxicity and cellular uptake of the three etoposide loaded PLGA based nanoparticles (ETO-PLGA NP, ETO-PLGA-MPEG NP and ETOPLGA-Pluronic NP) on two cancer cell lines, L1210 and DU145.

2. MATERIALS AND METHODS 2.1. Materials Etoposide was obtained as a gift sample from Biocon Ltd., Bangalore, India; PLGA was obtained as a gift sample from Boehringer Ingelheim Limited, Germany; Pluronic F-68 (BASF) was obtained as a gift sample from Alembic Ltd, Vadodara, India; Chloroform, Methanol, Glacial acetic acid, Potassium dihydrogen phosphate, Potassium dihydrogen phosphate, Sodium chloride and Acetone were obtained from SD Fine Chemicals, Mumbai, India; 6-Coumarin was obtained from Polysciences Inc., USA; 3-(4,5-dimethylthiaol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, USA), Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 100 U/mL penicillin and 100 g/mL streptomycin with 10% fetal bovine serum (Sigma, USA) were obtained as gift samples from NIRRH, Mumbai, India; L1210 cell line and DU 145 cell lines were obtained as gift samples from National Center for Cell Sciences, Pune, India; Fluoromount-G was obtained from Southern Biotech Associates, USA and Polycarbonate membranes (sizes 6658

0.2, 0.45, 2 m and 25 mm) were obtained from Whatman, USA. 2.2. Maintenance and Subculturing of Cell Lines L1210 mouse leukemia cell line was maintained in DMEM media supplemented with 100 U/mL penicillin and 100 g/mL streptomycin with 10% fetal bovine serum at 37  C in a 5% CO2 humidified atmosphere. L1210 was grown exponentially as a suspension culture. DU 145 prostatic cancer cells were maintained in DMEM media supplemented with 100 U/mL penicillin and 100 g/mL streptomycin with 10% fetal bovine serum at 37  C in a 5% CO2 humidified atmosphere. DU 145 cells were grown exponentially as monolayer adherent culture. 2.3. Preparation and Characterization of Nanoparticles Etoposide loaded PLGA nanoparticles (ETO-PLGA NP), PLGA-Pluronic nanoparticles (ETO-PLU NP) and Etoposide loaded PLGA-mPEG nanoparticles (ETOPLGA-MPEG NP) were prepared by solvent evaporation technique using high pressure homogenizer, as per method described earlier.11–12 Fluorescent NPs were prepared by using 6-coumarin (0.01% w/w), using solvent evaporation technique to investigate the in vitro cellular uptake of NP. A solution of 6-coumarin and PLGA in chloroform was emulsified into distilled water containing Pluronic F-68 (0.1% w/v). This primary emulsion was passed through high pressure homogenizer (Emulsiflex, C5, Avestin, Canada) for 4 cycles at 10000 psi pressure. The homogenized O/W emulsion was immediately added drop-wise to an aqueous solution of Pluronic F-68 and the contents were stirred overnight with a magnetic stirrer (Remi Equipments, Mumbai) to evaporate the chloroform. Nanoparticles were recovered by centrifugation for 30 min at 25000 rpm, washed and lyophilized (Heto Dry Winner, Denmark) for 24 hrs to yield freeze dried nanoparticles. Samples were frozen at −70  C and placed immediately in the freeze-drying chamber. 6-coumarin loaded PLGAMPEG NP and PLGA-PLURONIC NP were prepared similarly by using PLGA-MPEG and PLGA-PLURONIC copolymers. The freeze dried nanoparticles were dispersed in distilled water for particle size analysis using Malvern Zetasizer 3000 (Malvern Instruments, UK). The measurement of nanoparticle size was based on photon correlation spectroscopy (PCS). 2.4. Cytotoxicity Assay on L1210 and DU145 Cells Cytotoxicity was determined by the use of 3-(4,5dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay.13 Briefly, 100 l of L1210 or DU145 cells J. Nanosci. Nanotechnol. 11, 6657–6667, 2011

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(5 × 104 cells/mL) were seeded onto each well of 96-well plate (Corning Incorporated Life Sciences, Acton, MA, USA) and incubated for 24 h at 37  C in a humidified CO2 atmosphere. After incubation, 100 L of DMSO medium containing test sample (pure drug, Blank NP or drug loaded NP at a concentration of 5, 10, 20, 50 and 100 M) or complete medium for untreated controls were distributed in the 96-well plates and plates were then incubated at 37  C for 24 h. The culture medium was subsequently removed and medium containing 20 l MTT reagent (5 mg/mL) was added to each culture well. After 4 h of incubation, the cells were washed carefully with PBS and the crystals were dissolved by the addition of 10% SDS for 20 min with occasional shaking. Finally, absorbance, at 570 test wavelength and 630 reference wavelength, was measured using an automated microplate reader (Labsystem Multiskan, Helsinki, Finland). In each experiment, the test sample was analyzed in six individual wells. Cell survival was estimated as a percentage of the corresponding control. 2.5. Cytotoxicity Study of the Polymers Used

at 430 nm and emission wavelength at 485 nm. The fluorescent intensity was used for quantitative study of the uptake.14 Results obtained were expressed as a percentage of the total intensity of fluorescent labeled nanoparticles found in the solubilized cells to the total intensity of fluorescent labeled nanoparticles in the cells. 2.8. Confocal Microscopy Confocal microscopy was used to localize and quantify the uptake of particles by the cancer cells.15 After initial passage in tissue culture flasks, cells were grown to semi-confluence in DMEM supplemented media in 6-well tissue culture plates on Corning’s circular glass cover-slips at 37  C and 5% CO2 atmosphere. After filtration, the nanoparticle suspension was incubated with the cells at 37  C for a period of 1, 2, 3, 4 and 24 h. The media was then removed and the plates were washed thrice with sterile PBS. After the final wash, the cells were fixed with 4% (v/v) paraformaldehyde in PBS for 1.0 h at room temperature and were washed four times with PBS. Individual cover-slips were then mounted cell side up on clean glass slides with fluorescence-free glycerol based mounting medium Fluoromount-G. Differential interference contrast (DIC) and fluorescence images were acquired with a confocal microscope (Zeiss Confocal LSM 410, USA) at an excitation wavelength of 495 nm and an emitting wavelength of 520 nm. Z-stack Images were also taken to give a visual presentation of the NP uptake in each section of the cell at different cell depths. 2.9. Statistical Methods

2.6. IC50 Determination The concentration of drug or NP formulation required to inhibit cell proliferation by 50% (IC50  was determined by plotting the percentage of cell growth inhibition versus the concentration of pure drug or NP formulation. 2.7. Cell Uptake Efficiency by L1210 Cells L1210 cells were plated in Falcon 96-well plates at a density of 5 × 103 cells/well and after the cells reached 80% confluence (cells were counted by hemacytometer using Light microscope) the culture medium (DMEM supplemented with 100 U/mL penicillin and 100 g/mL streptomycin with 10% fetal bovine serum) was changed with that containing 6-courmarin loaded NPs. The particles were dispersed in the DMSO medium at concentration of 50, 100, 200 and 250 mg/ml for 2 h. After incubation, the suspension was removed and the wells were washed three times with 50 ml of PBS to eliminate traces of NPs left in the wells. After that, 50 ml of 0.5% Triton X-100 in 0.2 N NaOH was added to the sample wells to lyse the cells. The amount of fluorescence present in each well was then measured by microplate reader with excitation wavelength J. Nanosci. Nanotechnol. 11, 6657–6667, 2011

All data were processed and analyzed by Sigma-Plot 8.0 software (SPSS, IL). The statistical significances were evaluated by t-test of the software and p value less than 0.05 was accepted as statistically significant.

3. RESULTS AND DISCUSSION 3.1. Characterization of Nanoparticles Etoposide loaded PLGA based nanoparticles were prepared by oil-in-water single-emulsion solvent evaporation method using high pressure homogenization. The NPs formed were uniform, discrete and less than 200 nanometers in size. The mean particle size of PLGA NP ranged from 155 ± 54 to 172 ± 55 nm, PLGA-MPEG NPs were in the size range of 13402 ± 34 nm to 1629 ± 113 nm and PLGA-PLURONIC NP were in the size range of 1480 ± 21 nm and 1699 ± 63 nm. 3.2. Cytotoxicity Studies Cell cytotoxicities of ETO, ETO-PLGA NP, ETO-PLGAMPEG NP and ETO-PLGA-PLU NP for L1210 and 6659

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The three different polymers PLGA, PLGA-MPEG and PLGA-PLURONIC used in the NP formulations were also tested for their cytotoxicities at two concentrations, actual concentration (AC) used in the NP formulation and double concentration (DC) used in the NP formulation. The polymers were dissolved in DMSO and then filtered by 0.22  filter before carrying out the MTT assay as explained above.

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Fig. 1. (a) % Viability of L1210 cells by MTT Assay using Plain drug etoposide (ETO), and Etoposide loaded NPs ETO-PLGA NP, ETOPLGA-MPEG NP and ETO-PLGA-PLU NP. (b) IC50 values of ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGAPLU NP for L1210 cells.

Fig. 2. (a) % Viability of DU145 cells by MTT Assay using Plain drug etoposide (ETO), and Etoposide loaded NPs ETO-PLGA NP, ETOPLGA-MPEG NP and ETO-PLGA-PLU NP. (b) IC50 values of ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGAPLU NP for DU145 cells.

DU145 cells were investigated. The % viability obtained for L1210 cells shown in Figure 1(a) revealed that pure drug ETO showed lower cytotoxicity compared to its nanoparticulate formulations. PLGA based NP formulations played an important role in enhancing the cytotoxic effect of ETO, which was probably due to increase in their intracellular uptake. MTT assay on L1210 viability showed that the cytotoxicity of ETO and ETO loaded NP was concentration depended and there was decrease in the viability of the L1210 cells as the concentration was increased from 5 to 100 M. Concentrations above 100 M were not tested as the values of IC50 were determinable within the range tested. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 M for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The IC50 values significantly decreased (p > 0005) in the nanoparticulate formulations than the free drug (Fig. 1(b)). The IC50 values decreased 2.9 times for ETO-PLGA NP, 3.7 and 3.3 times for ETO-PLGA-MPEG NP and ETO-PLGAPLU NP respectively compared with free drug. The order of cytotoxicity was ETO-PLGA-PLU NP > ETO-PLGAMPEG NP > ETO-PLGA NP > ETO.

Similarly in DU145 cells, the cytotoxicity of the drug loaded NP was more than the free drug ETO. Figure 2(a) shows % Viability of DU145cells by MTT assay using Plain drug etoposide (ETO), and Etoposide loaded NPs ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGAPLU NP.. The IC50 values for DU145 cells were 98.4, 75.1, 60.1 and 71.3 M for ETO, ETO-PLGA NP, ETOPLGA-MPEG NP and ETO-PLGA-PLU NP respectively as shown in Figure 2(b). The IC50 values significantly decreased (p > 0005) in the nanoparticulate formulations than the free drug. The IC50 values decreased 1.3 times for ETO-PLGA NP, 1.6 and 1.4 times for ETOPLGA-MPEG NP and ETO-PLGA-PLU NP respectively compared with free drug. The order of cytotoxicity was ETO-PLGA-MPEG NP > ETO-PLGA-PLU NP > ETOPLGA NP > ETO. It was seen that the IC50 values of pure drug etoposide was decreased when it was loaded in NPs. The results are in comparison to study by Patlolla and Venkateswarlu,16 which showed that the IC50 value of the pure drug etoposide (33 M) was decreased when it was encapsulated in lipid nanosphere (5 M). Comparing the results of the cytotoxicity studies of ETO for the two cell lines, L1210 and DU145, it was seen

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that both ETO and ETO loaded NP had lower IC50 values for L1210 cells. Hence lower doses of ETO or ETO loaded NP would be required in the therapy of leukemia than in prostate cancer. The study indicates the probable reason why etoposide is generally recommended for treating leukemia rather than prostate cancer.

Table II. Cytotoxicity study of polymers on L1210 cells and DU145 cells by MTT Assay.

3.3. Long Term Cytotoxicity Study on L1210 Cells

PLGA-Pluronic

It has been reported that a relatively short incubation period used in the MTT-based cytotoxicity assay (24 or 48 h) was not enough to determine long-term cytotoxicity of the nanoparticulate formulations.17 In the present study time based cytotoxicity on L1210 cell lines were carried out for 1, 3 and 5 days. Table I shows time based cytotoxicity study of ETO and ETO loaded NP on L1210 cells by MTT Assay. The study showed that the cytotoxicity of ETO pure drug solution on L1210 cells did not have any significant change in the IC50 values on the 3rd and 5th day. But the IC50 values significantly decreased on 3rd and 5th day for all the three nanoparticulate formulations. This was probably due to the fact that pure drug solution exerted its maximum cytotoxic effect within 24 hours and hence there was no further significant decrease in the viability of the L1210 cells on the 3rd and 5th day. The three NP formulations had a significant decrease in the viability of the L1210 cells on the 3rd and 5th day. The nanoparticles showed their cytotoxic effect once the drug was released from the NPs and solubilized either in the cytoplasm or in the nucleus. Among the three NP formulations, ETO-MPEG-PLGA NP had a comparatively lower IC50 value on the 5th day, indicating its better efficiency in inhibiting cells for a sustained period.

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99.5 ± 0.17 99.1 ± 0.12 97.7 ± 0.21 97.1 ± 0.19 99.4 ± 0.09 98.9 ± 0.20

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due to the release of the entrapped drug from the NP and not because of the polymers used. 3.5. Cellular Uptake Efficiency The time and concentration dependent cellular uptake of the NPs was determined by quantifying the percentage of fluorescence. The uptake of fluorescent NPs by

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Table II gives % Viability values for PLGA, PLGAMPEG and PLGA-PLURONIC polymers on L1210 cells and DU145 cells after 48 h. It was seen that viability was more than 99% in AC and DC for PLGA and more than 97% for PLGA-MPEG and PLGA-PLURONIC. This confirmed that the used polymers did not have their own cytotoxicity effect on the cells in the concentrations used. The study concluded that the cytotoxicity was observed for the drug loaded NP on L1210 and DU145 cells was

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Fig. 3. (a) DU145 Cell uptake efficiency (%) of PLGA, PLGA-MPEG and PLGAPLURONIC NP at different time of 0.5, 1, 2 and 4 h at NP concentration = 100 g/ml. Data represent mean ± SD, n = 6. (b) DU145 Cell uptake efficiency (%) of PLGA, PLGA-MPEG and PLGAPLURONIC NP at different NP Concentration of 50, 100, 200 and 250 g/ml. Incubation time is 4 h. Data represent mean ± SD, n = 6.

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DU145 cells over an interval of 0.5, 1, 2 and 4 h is shown in Figure 3(a). It was seen that the uptake of the NPs was increased with time from 28 to 38% for PLGA NP, 32 to 47% for PLGA-MPEG NPs and 33 to 50% for PLGA-PLURONIC NPs in 30 min to 4 h respectively. Figure 3(b) shows concentration dependent uptake for NPs in DU145. Uptake of PLGA NP increased to 38% for 100 g/ml but was reduced with further increase in concentration. Similarly, there was a reduction in the uptake of PLGA-MPEG NP and PLGA-PLURONIC NP at higher concentration of 200 g/ml and 250 g/ml. Highest uptake of 47% and 50% was achieved at 100 g/ml concentration for PLGA-MPEG NP and PLGA-PLURONIC NP respectively. Similarly for L1210 cells (Fig. 4(a)), the uptake increased from 15 to 50% for PLGA NP, from 23 to 65 for PLGA-MPEG NPs and 25 to 66% for PLGA-PLURONIC NPs in 30 min to 4 h respectively. The effect of was also similar in the L1210 cells (Fig. 4(b)) and the highest uptake of all the three types of NPs was seen at 100 g/ml concentration (45% for PLGA NP, 55% for PLGA-MPEG NP and 58% for PLGA-PLURONIC NP). (a) 80 Cell uptake efficiency (%)

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Fig. 5. Confocal microscope image showing cellular uptake of coumarin loaded PLGA NP at different time interval on L1210 cell lines at 37  C at 63× oil immersion magnification. (C) is the control, (1 h) image taken at 1 h, the NP have been uptaken inside the cell and are inside both the cytoplasam and in nucleus, (2 h) image taken after 2 h, NP are seen in both cytoplasm and nucleus, intensity of fluroscence is increased (4 h) image taken after 4 h, the NP are seen especially in nucleus alone and very few particles are seen in cytoplasm, fluroscence intensity is maximum indicating maximum uptake.

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Fig. 4. (a) L1210 Cell uptake efficiency (%) of PLGA, PLGA-MPEG and PLGA-PLURONIC NP at different time of 0.5, 1, 2 and 4 h at NP concentration = 100 g/ml. Data represent mean ± SD, n = 6. (b) L1210 Cell uptake efficiency (%) of PLGA, PLGA-MPEG and PLGA-PLURONIC NP at different NP Concentration of 50, 100, 200 and 250 g/ml. Incubation time is 4 h. Data represent mean ± SD, n = 6.

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Confocal microscopy of the cells exposed to PLGA nanoparticles showed fluorescence activity in the cells within 30 min which increased with time for both the cell lines L1210 (Fig. 5) and DU145 (Fig. 6). The control experiment performed by incubating cells with 6-coumarin solution (Figs. 5(C) and 6(A)) showed that intracellular fluorescence was insignificant compared to that of cells incubated with nanoparticles. Hence, it was concluded that the fluorescence observed inside the cells was only due to the presence of nanoparticles. The outline of DU145 cell was clearly seen in Figure 6, in which the cell membrane (cm), cytoplasm (cp) and nucleus (nu) were seen distinctively as indicated by the arrows. At 1 h, (Fig. 6(B)) the NPs were seen particularly inside the cytoplasm of the cells. In the 2 h (Fig. 6(C)) the NPs were seen migrating towards the nucleus and were visible in both cytoplasm and nucleus. At 4 h (Fig. 6(D)), the NPs were prominently seen in the nucleus where as only a small fraction of NPs were seen in the cytoplasm. The nanoparticles as compared to free drug would take more time to reach the nucleus as the NPs would take more time to be present in a solubilized form within the cells.18 Figure 7 shows Z-stack Confocal images of PLGA NP showing uptake in DU 145 cells. Serial z-sections of the cells were taken and each section demonstrated J. Nanosci. Nanotechnol. 11, 6657–6667, 2011

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Fig. 7. Z stack confocal microscopy images of PLGA NP showing uptake in DU 145 cells. A = 72.3 m, B = 68.1 m, C = 65.3 m, D = 61.0 m, E = 58.1 m, F = 54.3 m, G = 50.1 m, H = 45.3 m, I = 32.1 m, J = 28.2 m, K = 21.6 m, L = 15.1 m.

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Fig. 6. Confocal microscope image showing cellular uptake of coumarin loaded PLGA NP at different time interval on DU145 cell lines at 37  C at 63× oil immersion magnification. (A) is the control, (B) image taken at 1 h, the NP have been uptaken inside the cell and are inside the cytoplasam, no NP seen in nucleus, (C) image taken after 2 h, NP are seen in both cytoplasm and nucleus, (D) image taken after 4 h, the NP are seen especially in nucleus alone and very few particles are seen in cytoplasm.

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Fig. 8. Z stack Confocal microscopy images of PLGA-mPEG NP showing uptake in DU 145 cells. A = 53.3 m, B = 62.1 m, C = 67.3 m, D = 69.3 m, E = 63.1 m, F = 54.3 m, G = 51.1 m, H = 44.3 m.

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Fig. 9. Z stack confocal microscopy images of PLGA-Pluronic NP showing uptake in DU 145 cells. A = 12.8 m, B = 24.1 m, C = 28.3 m, D = 32.6 m, E = 48.1 m, F = 52.1 m, G = 55.1 m, H = 65.3 m, I = 68.4 m.

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Fig. 10. Z stack confocal microscopy images of PLGA-PLURONIC NP showing uptake in L1210 cells. a = 123 m, b = 142 m, c = 160 m, d = 170 m, e = 181 m, f = 223 m, g = 261 m, h = 314 m, i = 331 m.

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Fig. 11. Z stack confocal microscopy images of PLGA-mPEG NP showing uptake in L1210 cells. a = 623 m, b = 642 m, c = 610 m, d = 570 m, e = 681 m, f = 683 m, g = 721 m, h = 753 m, i = 581 m, j = 482 m, k = 366 m, l = 251 m, m = 128 m, n = 105 m, o = 92 m, p = 52 m.

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Fig. 12. Comparison of Fluorescence image (F), Differential image (D) and overlap of Fluorescence Difference image (F-D) in L1210 cells for uptake of PLGA-mPEG NP. The images 1, 2 and 3 were taken at 62.3 m and 4, 5 and 6 at 48.2 m.

fluorescence activity in between 15.1 m and 72.3 m from the surface of the cells (Fig. 7). Figure 8 shows Z-stack Confocal images of PLGA-MPEG NP showing uptake in DU 145 cells. Serial z-sections of the cells were taken and each section demonstrated fluorescence activity in between 44.3 m and 69.3 m from the surface of the cells. Figure 9 shows Z-stack Confocal images of PLGA-PLURONIC NPs showing uptake in DU 145 cells. Serial z-sections of the cells were taken and each section demonstrated fluorescence activity in between 12.8 m and 68.4 m from the surface of the cells. Figure 10 shows Z-stack Confocal images of PLGA-PLURONIC NPs showing uptake in L1210 cells. Serial z-sections of the cells were taken and each section demonstrated fluorescence activity in between 12.3 m and 33.1 m from the surface of the cells, indicating that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Figure 11 shows Z stack confocal microscopy images of PLGA-mPEG NP showing uptake in L1210 cells. A total of 16 sections were taken ranging from 5.2 m (image 11(p)) to 75.3 m (image 11(h)) from the surface of the cells, indicating that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Figure 12 shows a comparison of Fluorescence image (F), Differential image (D) and overlap of Fluorescence Difference image (F-D) in L1210 cells for uptake of PLGA-mPEG NP. The Fluorescence images of L1210 cells does not show the clear outline of the cells and differential image gives a clear picture of the cell, but does not show 6666

the fluorescent NP. Therefore overlap of Fluorescence and Differential image (F-D) was used to give a clear picture both the cell and the fluorescent NPs. Images 13(1, 2 and 3) were taken at 62.3 m and Images 13(4, 5 and 6) at 48.2 m. The Fluorescence Differential images F-D3 and F-D6 show that the NPs were clearly within the L1210 cells and not merely adsorbed onto the cell.

4. CONCLUSION Cytotoxicity studies of the Etoposide loaded NPs on L1210 and DU 145 cells showed that developed NP had increased cytotoxicities due to their better uptake by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-PLURONIC NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Among the two NP formulations; Eto-PLGA-Pluronic NP had the highest cytotoxicity effect on L1210 cells and ETO-PLGA-MPEG NP on DU145 cells. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Cytotoxicity study on the polymers used showed that all the three polymers used (PLGA, PLGA-MPEG and PLGA-PLURONIC) did not have their own cytotoxicity effect on the cells in the concentrations used. The cellular uptake of ETO loaded NPs was both time and concentration dependent and was highest at a concentration of 100 g/ml and at 4 hours duration for J. Nanosci. Nanotechnol. 11, 6657–6667, 2011

Yadav et al.

both the cell lines. Confocal microscopy of the cells exposed to PLGA nanoparticles showed that the fluorescence observed inside the L1210 and DU145 cells was concluded to be only due to the presence of nanoparticles. Z-stack Confocal images of NP showing uptake in DU 145 and L1210 cells were taken and each section demonstrated fluorescence activity indicating that the nanoparticles were internalized by the cells and not simply bound to their surface. It was concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug. The surface modified NPs, ETO-MPEG NP and ETOPLGA-PLU NP, had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.

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Intracellular Delivery of Etoposide 4. M. S. Shive and J. M. Anderson, Adv. Drug Deliv. Rev. 28, 5 (1997). 5. M. L. Hans and A. M. Lowman, Curr. Opin. Solid State Mater. Sci. 6, 319 (2002). 6. Y. Mo and L. Lim, J. Controlled Release 108, 244 (2005). 7. R. Gref, Y. Minamitake, M. T. Peracchia, V. Trubetskoy, V. Torchilin, and R. Langer, Science 263, 1600 (1994). 8. K. Georgia and A. Konstantinos, J. Nanosci. Nanotechnol. 6, 3080 (2006). 9. M. J. Santander-Ortega, A. B. Jódar-Reyes, N. Csaba, D. BastosGonzález, and J. L. Ortega-Vinuesa, J. Colloid Interface Sci. 302, 522 (2006). 10. J. M. Henwood and R. N. Brogden, Drugs 39, 438 (1990). 11. K. S. Yadav and K. K. Sawant, Curr. Drug Del. 7, 51 (2010). 12. K. S. Yadav, K. Chuttani, A. K. Mishra, and K. K. Sawant, Drug Dev. Res. 71, 228 (2010). 13. R. I. Freshney, Measurement of Viability and Cytotoxicity, Culture of Animal Cells, 3rd edn., Wiley-Liss, New York (1994), p. 287. 14. K. Y. Win and S. S. Feng, Biomaterials 26, 2713 (2005). 15. J. Panyam, S. K. Sahoo, S. Prabha, T. Bargar and V. Labhasetwar, Int. J. Pharm. 262, 1 (2003). 16. R. R. Patlolla and V. Venkateswarlu, J. Drug Target. 16, 269 (2008). 17. Z. Zhang and S. S. Feng, Biomaterials 27, 4025 (2006). 18. H. S. Yoo and T. G. Park, J. Controlled Release 100, 247 (2004).

Received: 30 July 2010. Accepted: 23 December 2010.

RESEARCH ARTICLE

J. Nanosci. Nanotechnol. 11, 6657–6667, 2011

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