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Platinum folate nanoparticles toxicity: Cancer vs. normal cells
Toxicology in Vitro 27 (2013) 882–889
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Platinum folate nanoparticles toxicity: Cancer vs. normal cells Tatsiana Mironava a,⇑, Marcia Simon b, Miriam H. Rafailovich c, Basil Rigas d a
Department of Medicine, Stem Cell Facility, Stony Brook University, Stony Brook, NY 11794, USA Department of Oral Biology and Pathology, School of Dental Medicine, Stony Brook University, Stony Brook, NY 11794, USA c Department of Materials Science and Engineering, Stony Brook University, Stony Brook, NY 11794, USA d Division of Cancer Prevention, Stony Brook University, Stony Brook, NY 11794, USA b
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Article history: Received 29 June 2012 Accepted 2 January 2013 Available online 11 January 2013 Keywords: Platinum folate nanoparticles Folic acid receptors Cancer Targeted drug delivery
a b s t r a c t Almost for two decades metallic nanoparticles are successfully used for cancer detection, imaging and treatment. Due to their high electron density they can be easily observed by electron microscopy and used in laser and radiofrequency therapy as energy releasing agents. However, the limitation for this practice is an inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue. To overcome this restraint we proposed to use folic acid coated metallic nanoparticles and determine whether they preferentially penetrate cancer cells. We developed technique for synthesizing platinum nanoparticles using folic acid as stabilizing agent which produced particles of relatively narrow size distribution, having d = 2.3 ± 0.5 nm. High resolution TEM and zeta potential analysis indicated that the particles produced by this method had a high degree of crystalline order with no amorphous outer shell and a high degree of colloidal stability. The keratinocytes and mammary breast cells (cancer and normal) were incubated with platinum folate nanoparticles, and the results showed that the IC50 was significantly higher for the normal cells than the cancer cells in both cases, indicating that these nanoparticles preferentially target the cancer cells. TEM images of thin sections taken from the two types of cells indicated that the number of vacuoles and morphology changes after incubation with nanoparticles was also larger for the cancer cells in both types of tissue studied. No preferential toxicity was observed when folic acid receptors were saturated with free folic acid prior to exposure to nanoparticles. These results confirm our hypothesis regarding the preferential penetration of folic acid coated nanoparticles to cancer cells due to receptor mediated endocytosis. Published by Elsevier Ltd.
1. Introduction Metallic nanoparticles such as gold, silver and platinum were successfully used for cancer detection, imaging and treatment for more than a decade (Arvizo et al., 2012; Bellah et al., 2012; Govender et al., 2012; Reddy et al., 2012; Veerasamy et al., 2011; Wang et al., 2012). Due to their high electron density they can be easily observed by electron microscopy and can be used in laser and radiofrequency therapy as energy releasing agents (Elliott et al., 2010; Elsherbini et al., 2011). This type of treatment induces coagulative necrosis to the cell by protein thermal denaturation and membrane lysis. However, inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue lead to the usage of single point source of thermal energy that is not uniform along the tumor resulting in uneven heating and tumor recurrences. To overcome this limitation metallic nanoparticles have to preferentially penetrate cancer cells. Even though numerous targeting ⇑ Corresponding author. Fax: +1 631 632 8052. E-mail address: [email protected] (T. Mironava). 0887-2333/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tiv.2013.01.005
strategies have been proposed (Gao et al., 2012; Glazer and Curley, 2010; Sanna and Sechi, 2012; Yang et al., 2012), most are fairly complicated, relying on the isolation of specific cell membrane receptors or their conjugates. This renders them not only costly, but useful only to one specific cell line. Here we present an alternative approach which is simple, relatively inexpensive, and promising. It has been reported by numerous groups (Basal et al., 2009; Kalli et al., 2008; Kularatne and Low, 2010; Liang et al., 2011; Low et al., 2008; Salazar and Ratnam, 2007; Tran et al., 2005) that cancer cells have significant up regulation of folic acid receptors. There are three folate receptor (FR) isoforms (FR-a, FR-b and FR-c) that have been identified in human tissues and tumors. FR-a and FR-b are known to be vastly over expressed in many human tumors, unlike normal tissues expressing insignificant levels of FR-a and low levels of FR-b (Ross et al., 1994; Vaitilingam et al., 2012). FR-c is only found in hematopoietic cells. Consequently, folate has been widely used as a targeting moiety of various anticancer drug systems (Chen et al., 2011; Huang et al., 2011; Ji et al., 2012; Low and Antony, 2004; Lu et al., 2012; Mishra et al., 2011; Werner et al., 2011). Since, it has been
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shown by numerous authors that folate-drug conjugates can quickly bind to the FR receptors and deliver their payload (Vlahov and Leamon, 2012; Xu et al., 2012). The FR-mediated drug delivery has been referred to as a molecular Trojan horse approach where drugs attached to folate are shuttled inside targeted-FRpositive cells in a stealth-like fashion. Despite the efficacy, folate is rarely used to coat metallic particles. Even though the synthesis can be accomplished in a simple two steps process, little is known about the parameters controlling the particle size. For example, the only other reported synthesis yielded 15 nm nanoparticles at room temperature (Teow and Valiyaveettil, 2010). Since it is well established (Freese et al., 2012; Mironava et al., 2010; Pan et al., 2007; Zhang et al., 2012) that the interaction of NP with cell is a sensitive function of particle diameter, the particles synthesized in this paper, which are 2 nm in diameter are expected to interact with cells quite differently than 15 nm particles. Furthermore, in order to employ these particles for therapeutic purposes, it is also critical to establish the differential toxicity between normal and cancer cells. In this paper we expose both cancer and normal cells of the same tissue to the folate stabilized platinum nanoparticle (Pt/folate NPs) and demonstrate that they are preferentially internalized in the cancer cells, where they have a significantly smaller IC50 level. Hence they can potentially be used for anti-cancer drug conjugation to be tested in cancer treatment protocols.
2. Materials and methods
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MCF10A – mammary breast cells, 36 year-old Caucasian female (Cat# CRL-10317, ATCC, Manassas, VA) cells were cultured with Lonza single kit medium containing 2.5 mM L-glutamine and supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone, 95% and 5% horse serum (Lonza, Allendale, NJ). Cells were plated at cell density 35,000 cells per well in a 96well dish for MTS assay or in a 24-well dish for the confocal microscopy respectively. Cells were treated with following concentrations of platinum/folate nanoparticles 0 lg/ml, 25 lg/ml, 50 lg/ ml, 75 lg/ml, 100 lg/ml, 125 lg/ml,150 lg/ml, 175 lg/ml, 200 lg/ ml, 225 lg/ml, 250 lg/ml and 300 lg/ml 24 h after platting. Samples were collected at specific time points (12 and 24 h) and were MTS assayed. All incubations were performed at 37 °C and 5% CO2. Following doubling times were determined for the cells used in this study: MCF7 – 29 h, MCF10A – 34 h, DO33 – 32 h, SCC13 – 18 h, SCC12B – 17 h. 2.3. MTS Cell mitochondrial activity was evaluated with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Biosciences, Madison, WI). In the typical experiment cells were platted at the initial density 35,000 cells per well in 96-well dishes. 20 ll of the MTS solution was added to the 100 ll of media, samples were incubated for 3 h at 37 °C. The absorbance was read at 490 nm by the automated microplate reader EL800.
2.1. Synthesis of Pt/folate nanoparticles
2.4. Cell staining for confocal microscopy
Pt/folate nanoparticles were synthesized utilizing 0.005 mol of the oxidant, potassium tetrachloroplatinate(II) (K2PtCl4) and 0.005 mol of the stabilizer, folic acid (C19H19N7O6) in 20 ml MilliQ deionized water. The Erlenmeyer flask containing this solution was heated up to 95 °C for about 20 min to fully dissolve potassium tetrachloroplatinate and folic acid. Over a couple of minutes the color of solution changed from yellow to brownish and after that the solution was allowed to naturally cool down. 20 ll of 0.019 M of sodium tetrahydridoborate (NaBH4) was gradually added up to the cooled solution, until solution color changed from brownish to black indicating reaction completion. The reaction differs from the previously reported by Teow et al. where alternative precursor was used and the synthesis was performed at room temperature (Teow and Valiyaveettil, 2010).
Cell area and overall morphology as a function of time and concentration was monitored using Leica confocal microscope. For these experiments, cells grown on cover slip glass in 24-wells dishes were fixed with 3.7% formaldehyde for 15 min following exposure to Pt/folate NPs for 12 and 24 h. Alexa Fluor 488–Phalloidin was used for actin fiber staining and Propidium Iodide for nuclei staining.
2.2. Cell culture Primary human epidermal keratinocytes were obtained from Stony Brook University Living Skin Bank. Normal Keratinocytes DO33 and two phenotypically distinct squamous carcinoma keratinocytes SCC13 and SCC12B were grown in the keratinocytes growth media KGM-2 supplemented with bullet kit (Cat# CC-3107, Lonza, Allendale, NJ) containing 0.4% bovine pituitary extract, 0.1% human recombinant epidermal growth factor, 0.1% insulin, 0.1% hydrocortisone, 0.1% transferrin, 0.1% epinephrine and 0.1% gentamicin sulfate amphotericin-B. SCC12 and SCC13 were initially isolated by Rheinwald and Beckett (1981). The cells used were derived from these lines through growth and passage in culture. MCF7 – mammary breast cancer cells, 69 years-old Caucasian female (Cat# HTB-22, ATCC, Manassas, VA) were cultured with ATCC-formulated Eagles’ Minimum Essential Medium (Cat# 302003, ATCC, Manassas, VA) with 0.01 mg/ml insulin and 10% of fetal bovine serum (Lonza, Allendale, NJ).
2.5. TEM TEM analysis was used to assess the size distribution of the Pt/ folate NPs as well as the fate of internalized particles. One drop of the original Pt/folate NPs solution was placed on 300 mesh copper grip, which was coated with formvar film. The sample was then dried out at room temperature. Gaussian distributions of diameters were calculated from the samples with more than 170 nanoparticles. After exposure to Pt/folate NPs for 2 days the cells were fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Phosphate Buffered Saline (PBS), stained in 2% uranyl acetate, dehydrated with ethanol, and then embedded in Propylene oxide. The specimen was cut into ultrathin sections (90 nm) with Reichart UltracutE ultramicrotome and stained on the grid with uranyl acetate and lead citrate. The samples were imaged using a FEI Tecnai12 BioTwinG2 transmission electron microscope. Digital images were acquired with an AMT XR-60 CCD Digital Camera System and compiled using Adobe Photoshop program. 2.6. pH pH-meter calibrated with standard buffers was used to measure pH value of the freshly prepared Pt/folate NPs colloidal suspensions. pH electrode was immersed in the NPs suspension for 30 s or until pH value stabilized. All measurements were performed in triplicates at 25 °C.
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2.7. Apoptosis induction and quantification Induction of apoptosis was done by 24 h treatment with 1 mM Actinomycin D (Sigma–Aldrich, St. Louis, MO) dissolved in Dimethyl sulfoxide (Sigma–Aldrich, St. Louis, MO). Cells treated with Pt/folate NPs for 24 h at different concentrations were stained using the Dead Cell Apoptosis Kit with Annexin V Alexa FluorÒ 488 & PI (Life Technologies, Grand Island, NY). Flowcytometry experiment was performed to determine whether cells underwent apoptosis or necrosis after exposure to Pt/folate NPs. 2.8. Statistical analysis Statistical analysis was performed via T-tests using mean, standard deviations and number of measurements with GraphPad software. 3. Results and discussion 3.1. Particle characteristics The TEM images of Pt/folate NPs are shown in Fig. 1, together with a histogram of the particle size distribution. From the figure we find that this synthesis method yields relatively small but fairly monodispersed particles with an average diameter, d = 2.3 ± 0.5 nm. An HRTEM image of the particles is shown in Fig. 1b where we can see well defined lattice plains with highly ordered atomic structures. Using the reciprocal space measurement method, we calculated the lattice spacing to be 3.93 ± 0.02 Å in good agreement with the value for bulk Pt FCC structure, d = 3.92 Å. From the image we can also see that the high degree of order in the lattice plains extends across the entire particle, and all the way to the surface. Hence no surface distortions are created from this method of synthesis in contrast to the citrate method recently reported (Sun et al., 2006) where a distinct amorphous region was observed around the perimeter of each particle. The synthesis procedure was repeated multiple times and results appeared to be statistically indistinguishable from batch to batch. 3.2. Pt/folate NPs stability The stability of Pt/folate NPs suspension depends upon the balance of repulsive and attractive forces that exist between particles as they execute random Brownian motion in solution. The folic acid is adsorbed on the positively charged surface of platinum particle. The overall surface charge of the Pt/folate NPs is negative. The
value of this negative charge is important for maintaining the stability of the colloidal solution and prevents settling due to particle agglomeration. To estimate the stability of the Pt/folate NPs zeta potential measurements were performed. The zeta potential is a measure of surface charge and hence provides information of the repulsion between nanoparticles, and the stability of the colloidal solution. A decrease of the Zeta potential over time is an indication that the colloidal suspension is becoming unstable, as the particles are becoming more prone to flocculation. In Table 1 we show experimental data for Pt/folate NPs colloidal solution, immediately after synthesis and after storage at 4 °C for three and 6 months. In this case the negative charge observed on the surface of the nanoparticles arises from the ionization of folic acid functional groups and indicates the presence of the folic acid coating. Consistent to Teow et al. observation, freshly prepared Pt/folate NPs have a surface charge with zeta potential of 43.73 ± 1.27 mV (Teow and Valiyaveettil, 2010). Particles stored for 3 and 6 months have zeta potential values of 41.49 ± 1.53 mV and 37.14 ± 1.39 mV respectively. Hence, no significant change in the stability of the Pt/folate NPs occurs during the first 3 months. After 6 months though a change of 15% is detected which may indicate the onset of degradation, and hence the maximum acceptable storage time for Pt/folate NPs should be no longer than 6 months. The Pt/folate NPs colloidal solution, was slightly acidic with a pH value of 6.4 ± 0.1. 3.3. Cell viability assay In order to determine whether the folate coating was effective in targeting cancer cells, we first measured the cell viability, as a function of particle concentration using an MTS assay in order to determine whether a differential existed between the IC50 concentrations of normal and cancer cells of different tissue. In Fig. 2 we plot the IC50 concentration after 12 and 24 h of exposure, where we can see that the values are significantly different between the cancer and normal cells in both types of tissue. From the figure we can see that after 12 h of exposure the IC50 of the normal cells is higher relative to that of the cancerous SCC13 or SCC12B and is 260% or 160% respectively. After 24 h we find a small decrease in the IC50 values for both the normal and the SCC12B cells, with the ratio between them remaining constant. On the other hand the decrease appears smaller for the SCC13 cells, decreasing the difference with the normal cells to 200%. Since the doubling time between these cells is comparable, we attribute these observed differences to increased internal accumulation of NPs with time.
Fig. 1. Pt/folate NPs imaged by TEM, (a) HRTEM, (b and c) (Pt/folic acid ratio 100/1), and particles Gaussian size distribution histogram (d).
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Table 1 Zeta potential and mobility values of the Pt/folate NPs. Pt/folate NPs Freshly prepared Stored for 3 months Stored for 6 month
Zeta Potential (mV) 43.73 ± 1.27 41.49 ± 1.53 37.14 ± 1.39
Fig. 3. Apoptosis-Necrosis assay (Annexin-V and PI staining) of MCF10A, MCF7, DO33, SCC12B and SCC13 treated with Pt/folate NPs for 24 h.
Fig. 2. IC 50 of Pt/folate NPs for different incubation time in the (a) cancerous keratinocytes SCC12B, SCC13 and normal keratinocytes DO33, and (b) in the breast cells normal MCF10A and cancerous MCF7.
A similar phenomenon is also observed for endothelial breast cells where the IC50 for the normal cells relative to the cancer is 160% after 12 h. Eventhough the absolute values in all cases decrease, the ratio between cancer and normal cells remains approximately the same after 24 h. Hence the IC50 for both the SCC13 and the MCF7 does not vary significantly during the observation time of 24 h whereas the IC50 for the SCC12B decreases significantly within the latter 12 h of observation period. Since the doubling time of these cells is close to 18 h in both cases, this difference may be related to varying sensitivity in the cell cycle for SCC12B. It is interesting to note that after 24 h the IC50 of the normal epidermal keratinocytes and endothelial breast cells is comparable, while the value for the cancer cells seems to be dependent on the tissue from which the carcinoma was isolated. It is also interesting to note that the observed IC50 concentration of Pt-folate NPs after 24 h of MCF7 cells exposure (86 lg/ml) is approximately identical to IC50 previously reported (Teow and Valiyaveettil, 2010). It has been previously reported that nanoparticles toxicity is a function of particle size (Mironava et al., 2010), however in our case we found that there is no size effect for the Pt-folate NPs in the particle range 2–15 nm.
respective strain, which in this case were significantly higher for normal cell lines: 106 lg/ml – normal keratinocytes DO33, 51 lg/ml – cancerous keratinocytes SCC13, 61 lg/ml – cancerous keratinocytes SCC12B, 124 lg/ml – normal epithelial breast cells, 86 lg/ml – cancerous epithelial breast cells. From the Fig. 3 we can see that all cells treated with Pt/folate NPs exhibited a decrease in the percentage of viable cells as compared to the untreated control, as expected. However, the mode of cell death is different is cell type specific for the normal cells and non-specific for the cancer cells. After 24 h of exposure to Pt/folate NPs MCF10A cells showed a 23% decrease in viable cells, a slight decrease in apoptotic cells, which constituted only 7% of control population, but a four folds increase from 11% to 39.3% in the necrotic cell fraction. The MCF7 cell culture showed a similar 20% decrease of viable cells after 24 h of exposure, but in this case the apoptotic fraction increased more than tenfold, while the necrotic fraction had a four folds increase. Significantly smaller increase in apoptosis and necrosis after exposure to Pt/folate NPs was recently reported (Teow and Valiyaveettil, 2010), however their initial fraction of apoptotic cells was much higher than reported in this paper, making a direct comparison difficult. DO33 keratinocytes after 24 h of exposure to Pt/folate NPs exhibited a 20% decrease in cell viability due to three folds increase in cell apoptosis process (28%).
3.4. Cell apoptosis and necrosis rates due to Pt/folate NPs exposure In order to investigate the mode of cell death upon exposure to Pt/folate NPs, the cells were stained using Dead Cell Apoptosis Kit with Annexin V Alexa FluorÒ 488 and PI. Flow cytometry was performed according to the procedures recommended by the manufacturer. Unstained cells were considered viable, cells stained with only red (PI) dye and cells stained with both red (PI) and green (Annexin V) dyes were recognized as necrotic cells, and cells that were stained with only green (Annexin V) dye were recognized as apoptotic. In order to have comparable amounts of cells experiencing toxicity after 24 h of exposure, the studies were performed using the IC-50 Pt-folate NP concentration for the
Fig. 4. Cell viability of the cells treated with IC50 concentrations of Pt/folate NPs for 24 h (compared to untreated control) with and without folic acid pre-treatment.
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Cancerous keratinocytes SCC13 had a 16% decrease in cell viability after 24 h of Pt/folate NPs exposure that resulted from the 5 and 1.5 folds increases in both apoptosis and necrosis, respectively. On the other hand Pt/folate NPs had a greater impact on the other cancerous keratinocytes SCC12B, were the decrease in cell viability was 15% after 24 h of exposure. The SCC12B cell death was caused by 9 folds increase in apoptosis and 3 folds increase in necrosis. We therefore found that the mode of the cell death, due to the Pt-folate NPs exposure, in normal cell lines was cell type specific; normal epidermal keratinocytes, DO33, decreased cell viability predominantly by apoptosis, while normal epithelial breast cells, MCF10A, decreased viability predominantly by necrosis. All the cancer cells studied on the other hand, decreased viability via both modes. The specificity to cell type in the reaction to nanoparticle exposure has been reported by multiple groups (Greulich et al., 2011; Hanley et al., 2009; Sohaebuddin et al., 2010). Our results are in agreement with those of Teow et al. who studied the effects of the larger 15 nm Pt folate NPs, where they observed that the
mode of cell death for the population occurred via both apoptosis and necrosis for the cancerous MC-7 cells, while the mode of death for their normal cells, which in this case were IMR90 fibroblasts, was mostly necrotic (Teow and Valiyaveettil, 2010). It is well established that necrosis is usually related to the loss of lysosomal membrane integrity (Vanlangenakker et al., 2008) while apoptosis is caused by caspase activation, calcium overload or death-inducing signals (Ghosh et al., 2009; Mattson et al., 1998). Therefore, the mechanism of Pt/folate NPs and its subsequent processing in our experiments seems to be cell line specific. That fact might be further employed for the anti-cancer drug formulations. 3.5. Pt/folate NPs penetration pathway To prove our hypothesis about receptor mediated endocytosis of the Pt/folate NPs we decided to block folic acid receptors in epithelial and epidermal cell lines by overloading them with 16 lg/ml of free folic acid and follow the exposure to Pt/folate NPs. From the
Fig. 5. Confocal microscopy pictures of the cell treated with Pt/folate NPs for 24 h and controls. (a–c) MCF7; (d–f) MCF10A; (g–i) DO33; (j–l) SCC12B; (m–o) SCC13 cells.
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Fig. 4 we can see, that addition of folic acid prior to exposure to IC50 concentration of the Pt/folate NPs resulted in the significantly higher cell survival rate (80%) compared to the cells exposed to NPs without pretreatment with folic acid (50%) for all cell types. Addition of the free folic acid to the cultures in the absence of the NPs exposure had no result on cell viability (results are not shown). Hence, the major penetration pathway of the Pt/folate NPs to the cell is the receptor mediated endocytosis. It is well known that folic acid is required for essential cell function, namely it is necessary for the synthesis of purines and pyrimidines (Davis and Nicol, 1988) it is also known that folate receptor is overexpressed in ovarian, lung, brain, head, neck, breast cancers and has a very high affinity to the folic acid and all sorts of folic acid conjugates (Elnakat and Ratnam, 2004; Shmeeda et al., 2006). Therefore any cargo linked to the folic acid will be retained within an endocytic vesicle or released into the cytoplasm.
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(Fig. 6). In the case of the keratinocytes, we find that the SCC cells are not able to extend philopodia which are observed only for the normal cells. Hence even though more than 50% of the cells have died at this concentration, even the surviving cells have sustained serious damage. 3.7. Intracellular fate of internalised Pt/folate NPs We also imaged the cells with transmission electron microscopy in order to determine the location of the particles once they are sequestered within the cells. The images of thin sections are shown in Fig. 7. Comparing the appearance of the normal and cancerous cells for both keratinocyes and breast cancer cells, we find that the cells exposed to nanoparticles have far more vacuoles. Magnification of the interior of the vacuoles shows the presence of electron opaque regions, which are particle agglomerates. Hence
3.6. Changes in cell morphology due to the Pt/folate NPs Confocal microscopy was also performed in order to determine if abnormal changes occur to the cell morphology due to the nanoparticles exposure. The images obtained for cells incubated with two concentrations, the IC50 concentration and twice this concentration are shown in Fig. 5, together with images of the control samples that were not exposed to nanoparticles. Following IC50 concentrations of Pt/folate NPs were used: MCF10A – 124 lg/ml, MCF7 – 86 lg/ml, DO33 – 106 lg/ml, SCC12B – 61 lg/ml, SCC13 – 51 lg/ml. From the figure we can see that the in vitro morphology of the keratinocytes and the breast endothelial cells is similar both for the normal and cancerous cells. In each case the cells that are not exposed to nanoparticles are adherent to each other and have approximately the same area. After exposure to the corresponding IC50 concentration for the particular cell type, we begin to see signs of damage. In all cases we find that the tissue has bare patches where dying cells have been lifted. The keratinocytes and the SCC cells behave similarly in that they extend philopodia to bridge the bare areas. More careful examination of the images shows that the SCC cells have fewer philopodia per cell than the normal ones. Similar bare patches are observed for the breast cells, but neither cancer nor normal cells develop philopodia. Hence eventhough 50% of the cells have died, minimal damage to the morphology of the remaining cells is observed. When the concentration is increased to twice the IC50 dose more serious damage is observed. We find that the actin is damaged and the cell area is decreased for all cell types
Fig. 6. Cell area of the cell treated with Pt/folate NPs for 24 h and controls. Fig. 7. TEM images of the cells exposed to the PtNPs for 24 h and control. (a and b) MCF7; (c and d) MCF10A; (e and f) DO33; (g and h) SCC12B; (i and j) SCC13 cells.
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images showed that the number of vacuoles, an indicator of NP uptake, was also significantly larger for the cancer cells in both types of tissue studied, consistent with the hypothesis that the folic acid coating was effective in enhancing the preferential penetration. References
Fig. 8. Number of vacuoles per cell area in the cultures treated with Pt/folate NPs and control.
the particles are sequested into these vacuoles after penetration. One can also count the number of vacuoles per unit cell area, and the results are plotted in Fig. 8. From the figure we can see that in all cases, the number of vacuoles is larger for the cancer cells than the normal cells, even though the absolute particle concentration corresponding to IC50 is lower. This confirms that the difference in cell sensitivity is due to the fact that the uptake is higher in the cancer cells than in the normal cells, which we postulate is correlated to the preferential delivery of the particles due to the enhanced expression of folic acid receptors in the cancer cells. Platinum-based anticancer agents such as cisplatin have been used for more than 30 years in chemotherapy for the treatment of solid tumors. However it is known that such therapy has severe side effects because of its low selectivity. One of the most researched options to overcome the disadvantage of cisplatin is the improvement of the drug accumulation via drug targeting. In recent years metallic nanoparticles were used as highly effective targeted delivery systems for the platinum-based therapy. Lately it was reported (Bhattacharyya et al., 2011) that cisplatin loaded onto gold nanoparticles that targeted cancer cell receptors exhibited enhanced therapeutic efficacy forward lung and ovarian cancers. On the other hand, Porcel et al. showed the results indicating that 3 nm PtNPs can significantly amplify the effect of fast ion irradiation therapy due to its high atomic mass (Porcel et al., 2010). They reported that irradiated PtNPs has a direct effect on DNA through the emittion of Auger electrons and indirect effect by production of water radicals that further damage DNA. However the main limitation for such combined therapy is the low selectivity of the PtNPs. Therefore, Pt-folate NPs can provide the selectivity which enables their use in existing therapies to decrease their side effects and improve their therapeutic index.
4. Conclusion In summary we have reported a technique for synthesizing Pt/ folate NPs of relatively narrow size distributions, high degree of crystalline order, with particle diameter of d = 2.3 ± 0.5 nm. The toxicological impact of these particles on normal and cancerous epithelial breast and epidermal keratinocytes were tested, and they were found to be significantly more toxic to the cancer cells for both cell types. The mode of cell death was found to be predominantly necrotic for the normal epidermal keratinocytes and apoptotic for the normal epithelium breast cancer cells, while mode of cell death was a combination of similar fractions of apoptosis and necrosis for all the cancer cells, regardless of phenotype. TEM
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Platinum folate nanoparticles toxicity: Cancer vs. normal cells Tatsiana Mironava a,⇑, Marcia Simon b, Miriam H. Rafailovich c, Basil Rigas d a
Department of Medicine, Stem Cell Facility, Stony Brook University, Stony Brook, NY 11794, USA Department of Oral Biology and Pathology, School of Dental Medicine, Stony Brook University, Stony Brook, NY 11794, USA c Department of Materials Science and Engineering, Stony Brook University, Stony Brook, NY 11794, USA d Division of Cancer Prevention, Stony Brook University, Stony Brook, NY 11794, USA b
a r t i c l e
i n f o
Article history: Received 29 June 2012 Accepted 2 January 2013 Available online 11 January 2013 Keywords: Platinum folate nanoparticles Folic acid receptors Cancer Targeted drug delivery
a b s t r a c t Almost for two decades metallic nanoparticles are successfully used for cancer detection, imaging and treatment. Due to their high electron density they can be easily observed by electron microscopy and used in laser and radiofrequency therapy as energy releasing agents. However, the limitation for this practice is an inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue. To overcome this restraint we proposed to use folic acid coated metallic nanoparticles and determine whether they preferentially penetrate cancer cells. We developed technique for synthesizing platinum nanoparticles using folic acid as stabilizing agent which produced particles of relatively narrow size distribution, having d = 2.3 ± 0.5 nm. High resolution TEM and zeta potential analysis indicated that the particles produced by this method had a high degree of crystalline order with no amorphous outer shell and a high degree of colloidal stability. The keratinocytes and mammary breast cells (cancer and normal) were incubated with platinum folate nanoparticles, and the results showed that the IC50 was significantly higher for the normal cells than the cancer cells in both cases, indicating that these nanoparticles preferentially target the cancer cells. TEM images of thin sections taken from the two types of cells indicated that the number of vacuoles and morphology changes after incubation with nanoparticles was also larger for the cancer cells in both types of tissue studied. No preferential toxicity was observed when folic acid receptors were saturated with free folic acid prior to exposure to nanoparticles. These results confirm our hypothesis regarding the preferential penetration of folic acid coated nanoparticles to cancer cells due to receptor mediated endocytosis. Published by Elsevier Ltd.
1. Introduction Metallic nanoparticles such as gold, silver and platinum were successfully used for cancer detection, imaging and treatment for more than a decade (Arvizo et al., 2012; Bellah et al., 2012; Govender et al., 2012; Reddy et al., 2012; Veerasamy et al., 2011; Wang et al., 2012). Due to their high electron density they can be easily observed by electron microscopy and can be used in laser and radiofrequency therapy as energy releasing agents (Elliott et al., 2010; Elsherbini et al., 2011). This type of treatment induces coagulative necrosis to the cell by protein thermal denaturation and membrane lysis. However, inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue lead to the usage of single point source of thermal energy that is not uniform along the tumor resulting in uneven heating and tumor recurrences. To overcome this limitation metallic nanoparticles have to preferentially penetrate cancer cells. Even though numerous targeting ⇑ Corresponding author. Fax: +1 631 632 8052. E-mail address: [email protected] (T. Mironava). 0887-2333/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tiv.2013.01.005
strategies have been proposed (Gao et al., 2012; Glazer and Curley, 2010; Sanna and Sechi, 2012; Yang et al., 2012), most are fairly complicated, relying on the isolation of specific cell membrane receptors or their conjugates. This renders them not only costly, but useful only to one specific cell line. Here we present an alternative approach which is simple, relatively inexpensive, and promising. It has been reported by numerous groups (Basal et al., 2009; Kalli et al., 2008; Kularatne and Low, 2010; Liang et al., 2011; Low et al., 2008; Salazar and Ratnam, 2007; Tran et al., 2005) that cancer cells have significant up regulation of folic acid receptors. There are three folate receptor (FR) isoforms (FR-a, FR-b and FR-c) that have been identified in human tissues and tumors. FR-a and FR-b are known to be vastly over expressed in many human tumors, unlike normal tissues expressing insignificant levels of FR-a and low levels of FR-b (Ross et al., 1994; Vaitilingam et al., 2012). FR-c is only found in hematopoietic cells. Consequently, folate has been widely used as a targeting moiety of various anticancer drug systems (Chen et al., 2011; Huang et al., 2011; Ji et al., 2012; Low and Antony, 2004; Lu et al., 2012; Mishra et al., 2011; Werner et al., 2011). Since, it has been
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shown by numerous authors that folate-drug conjugates can quickly bind to the FR receptors and deliver their payload (Vlahov and Leamon, 2012; Xu et al., 2012). The FR-mediated drug delivery has been referred to as a molecular Trojan horse approach where drugs attached to folate are shuttled inside targeted-FRpositive cells in a stealth-like fashion. Despite the efficacy, folate is rarely used to coat metallic particles. Even though the synthesis can be accomplished in a simple two steps process, little is known about the parameters controlling the particle size. For example, the only other reported synthesis yielded 15 nm nanoparticles at room temperature (Teow and Valiyaveettil, 2010). Since it is well established (Freese et al., 2012; Mironava et al., 2010; Pan et al., 2007; Zhang et al., 2012) that the interaction of NP with cell is a sensitive function of particle diameter, the particles synthesized in this paper, which are 2 nm in diameter are expected to interact with cells quite differently than 15 nm particles. Furthermore, in order to employ these particles for therapeutic purposes, it is also critical to establish the differential toxicity between normal and cancer cells. In this paper we expose both cancer and normal cells of the same tissue to the folate stabilized platinum nanoparticle (Pt/folate NPs) and demonstrate that they are preferentially internalized in the cancer cells, where they have a significantly smaller IC50 level. Hence they can potentially be used for anti-cancer drug conjugation to be tested in cancer treatment protocols.
2. Materials and methods
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MCF10A – mammary breast cells, 36 year-old Caucasian female (Cat# CRL-10317, ATCC, Manassas, VA) cells were cultured with Lonza single kit medium containing 2.5 mM L-glutamine and supplemented with 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin and 500 ng/ml hydrocortisone, 95% and 5% horse serum (Lonza, Allendale, NJ). Cells were plated at cell density 35,000 cells per well in a 96well dish for MTS assay or in a 24-well dish for the confocal microscopy respectively. Cells were treated with following concentrations of platinum/folate nanoparticles 0 lg/ml, 25 lg/ml, 50 lg/ ml, 75 lg/ml, 100 lg/ml, 125 lg/ml,150 lg/ml, 175 lg/ml, 200 lg/ ml, 225 lg/ml, 250 lg/ml and 300 lg/ml 24 h after platting. Samples were collected at specific time points (12 and 24 h) and were MTS assayed. All incubations were performed at 37 °C and 5% CO2. Following doubling times were determined for the cells used in this study: MCF7 – 29 h, MCF10A – 34 h, DO33 – 32 h, SCC13 – 18 h, SCC12B – 17 h. 2.3. MTS Cell mitochondrial activity was evaluated with CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Biosciences, Madison, WI). In the typical experiment cells were platted at the initial density 35,000 cells per well in 96-well dishes. 20 ll of the MTS solution was added to the 100 ll of media, samples were incubated for 3 h at 37 °C. The absorbance was read at 490 nm by the automated microplate reader EL800.
2.1. Synthesis of Pt/folate nanoparticles
2.4. Cell staining for confocal microscopy
Pt/folate nanoparticles were synthesized utilizing 0.005 mol of the oxidant, potassium tetrachloroplatinate(II) (K2PtCl4) and 0.005 mol of the stabilizer, folic acid (C19H19N7O6) in 20 ml MilliQ deionized water. The Erlenmeyer flask containing this solution was heated up to 95 °C for about 20 min to fully dissolve potassium tetrachloroplatinate and folic acid. Over a couple of minutes the color of solution changed from yellow to brownish and after that the solution was allowed to naturally cool down. 20 ll of 0.019 M of sodium tetrahydridoborate (NaBH4) was gradually added up to the cooled solution, until solution color changed from brownish to black indicating reaction completion. The reaction differs from the previously reported by Teow et al. where alternative precursor was used and the synthesis was performed at room temperature (Teow and Valiyaveettil, 2010).
Cell area and overall morphology as a function of time and concentration was monitored using Leica confocal microscope. For these experiments, cells grown on cover slip glass in 24-wells dishes were fixed with 3.7% formaldehyde for 15 min following exposure to Pt/folate NPs for 12 and 24 h. Alexa Fluor 488–Phalloidin was used for actin fiber staining and Propidium Iodide for nuclei staining.
2.2. Cell culture Primary human epidermal keratinocytes were obtained from Stony Brook University Living Skin Bank. Normal Keratinocytes DO33 and two phenotypically distinct squamous carcinoma keratinocytes SCC13 and SCC12B were grown in the keratinocytes growth media KGM-2 supplemented with bullet kit (Cat# CC-3107, Lonza, Allendale, NJ) containing 0.4% bovine pituitary extract, 0.1% human recombinant epidermal growth factor, 0.1% insulin, 0.1% hydrocortisone, 0.1% transferrin, 0.1% epinephrine and 0.1% gentamicin sulfate amphotericin-B. SCC12 and SCC13 were initially isolated by Rheinwald and Beckett (1981). The cells used were derived from these lines through growth and passage in culture. MCF7 – mammary breast cancer cells, 69 years-old Caucasian female (Cat# HTB-22, ATCC, Manassas, VA) were cultured with ATCC-formulated Eagles’ Minimum Essential Medium (Cat# 302003, ATCC, Manassas, VA) with 0.01 mg/ml insulin and 10% of fetal bovine serum (Lonza, Allendale, NJ).
2.5. TEM TEM analysis was used to assess the size distribution of the Pt/ folate NPs as well as the fate of internalized particles. One drop of the original Pt/folate NPs solution was placed on 300 mesh copper grip, which was coated with formvar film. The sample was then dried out at room temperature. Gaussian distributions of diameters were calculated from the samples with more than 170 nanoparticles. After exposure to Pt/folate NPs for 2 days the cells were fixed in a solution of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Phosphate Buffered Saline (PBS), stained in 2% uranyl acetate, dehydrated with ethanol, and then embedded in Propylene oxide. The specimen was cut into ultrathin sections (90 nm) with Reichart UltracutE ultramicrotome and stained on the grid with uranyl acetate and lead citrate. The samples were imaged using a FEI Tecnai12 BioTwinG2 transmission electron microscope. Digital images were acquired with an AMT XR-60 CCD Digital Camera System and compiled using Adobe Photoshop program. 2.6. pH pH-meter calibrated with standard buffers was used to measure pH value of the freshly prepared Pt/folate NPs colloidal suspensions. pH electrode was immersed in the NPs suspension for 30 s or until pH value stabilized. All measurements were performed in triplicates at 25 °C.
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2.7. Apoptosis induction and quantification Induction of apoptosis was done by 24 h treatment with 1 mM Actinomycin D (Sigma–Aldrich, St. Louis, MO) dissolved in Dimethyl sulfoxide (Sigma–Aldrich, St. Louis, MO). Cells treated with Pt/folate NPs for 24 h at different concentrations were stained using the Dead Cell Apoptosis Kit with Annexin V Alexa FluorÒ 488 & PI (Life Technologies, Grand Island, NY). Flowcytometry experiment was performed to determine whether cells underwent apoptosis or necrosis after exposure to Pt/folate NPs. 2.8. Statistical analysis Statistical analysis was performed via T-tests using mean, standard deviations and number of measurements with GraphPad software. 3. Results and discussion 3.1. Particle characteristics The TEM images of Pt/folate NPs are shown in Fig. 1, together with a histogram of the particle size distribution. From the figure we find that this synthesis method yields relatively small but fairly monodispersed particles with an average diameter, d = 2.3 ± 0.5 nm. An HRTEM image of the particles is shown in Fig. 1b where we can see well defined lattice plains with highly ordered atomic structures. Using the reciprocal space measurement method, we calculated the lattice spacing to be 3.93 ± 0.02 Å in good agreement with the value for bulk Pt FCC structure, d = 3.92 Å. From the image we can also see that the high degree of order in the lattice plains extends across the entire particle, and all the way to the surface. Hence no surface distortions are created from this method of synthesis in contrast to the citrate method recently reported (Sun et al., 2006) where a distinct amorphous region was observed around the perimeter of each particle. The synthesis procedure was repeated multiple times and results appeared to be statistically indistinguishable from batch to batch. 3.2. Pt/folate NPs stability The stability of Pt/folate NPs suspension depends upon the balance of repulsive and attractive forces that exist between particles as they execute random Brownian motion in solution. The folic acid is adsorbed on the positively charged surface of platinum particle. The overall surface charge of the Pt/folate NPs is negative. The
value of this negative charge is important for maintaining the stability of the colloidal solution and prevents settling due to particle agglomeration. To estimate the stability of the Pt/folate NPs zeta potential measurements were performed. The zeta potential is a measure of surface charge and hence provides information of the repulsion between nanoparticles, and the stability of the colloidal solution. A decrease of the Zeta potential over time is an indication that the colloidal suspension is becoming unstable, as the particles are becoming more prone to flocculation. In Table 1 we show experimental data for Pt/folate NPs colloidal solution, immediately after synthesis and after storage at 4 °C for three and 6 months. In this case the negative charge observed on the surface of the nanoparticles arises from the ionization of folic acid functional groups and indicates the presence of the folic acid coating. Consistent to Teow et al. observation, freshly prepared Pt/folate NPs have a surface charge with zeta potential of 43.73 ± 1.27 mV (Teow and Valiyaveettil, 2010). Particles stored for 3 and 6 months have zeta potential values of 41.49 ± 1.53 mV and 37.14 ± 1.39 mV respectively. Hence, no significant change in the stability of the Pt/folate NPs occurs during the first 3 months. After 6 months though a change of 15% is detected which may indicate the onset of degradation, and hence the maximum acceptable storage time for Pt/folate NPs should be no longer than 6 months. The Pt/folate NPs colloidal solution, was slightly acidic with a pH value of 6.4 ± 0.1. 3.3. Cell viability assay In order to determine whether the folate coating was effective in targeting cancer cells, we first measured the cell viability, as a function of particle concentration using an MTS assay in order to determine whether a differential existed between the IC50 concentrations of normal and cancer cells of different tissue. In Fig. 2 we plot the IC50 concentration after 12 and 24 h of exposure, where we can see that the values are significantly different between the cancer and normal cells in both types of tissue. From the figure we can see that after 12 h of exposure the IC50 of the normal cells is higher relative to that of the cancerous SCC13 or SCC12B and is 260% or 160% respectively. After 24 h we find a small decrease in the IC50 values for both the normal and the SCC12B cells, with the ratio between them remaining constant. On the other hand the decrease appears smaller for the SCC13 cells, decreasing the difference with the normal cells to 200%. Since the doubling time between these cells is comparable, we attribute these observed differences to increased internal accumulation of NPs with time.
Fig. 1. Pt/folate NPs imaged by TEM, (a) HRTEM, (b and c) (Pt/folic acid ratio 100/1), and particles Gaussian size distribution histogram (d).
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Table 1 Zeta potential and mobility values of the Pt/folate NPs. Pt/folate NPs Freshly prepared Stored for 3 months Stored for 6 month
Zeta Potential (mV) 43.73 ± 1.27 41.49 ± 1.53 37.14 ± 1.39
Fig. 3. Apoptosis-Necrosis assay (Annexin-V and PI staining) of MCF10A, MCF7, DO33, SCC12B and SCC13 treated with Pt/folate NPs for 24 h.
Fig. 2. IC 50 of Pt/folate NPs for different incubation time in the (a) cancerous keratinocytes SCC12B, SCC13 and normal keratinocytes DO33, and (b) in the breast cells normal MCF10A and cancerous MCF7.
A similar phenomenon is also observed for endothelial breast cells where the IC50 for the normal cells relative to the cancer is 160% after 12 h. Eventhough the absolute values in all cases decrease, the ratio between cancer and normal cells remains approximately the same after 24 h. Hence the IC50 for both the SCC13 and the MCF7 does not vary significantly during the observation time of 24 h whereas the IC50 for the SCC12B decreases significantly within the latter 12 h of observation period. Since the doubling time of these cells is close to 18 h in both cases, this difference may be related to varying sensitivity in the cell cycle for SCC12B. It is interesting to note that after 24 h the IC50 of the normal epidermal keratinocytes and endothelial breast cells is comparable, while the value for the cancer cells seems to be dependent on the tissue from which the carcinoma was isolated. It is also interesting to note that the observed IC50 concentration of Pt-folate NPs after 24 h of MCF7 cells exposure (86 lg/ml) is approximately identical to IC50 previously reported (Teow and Valiyaveettil, 2010). It has been previously reported that nanoparticles toxicity is a function of particle size (Mironava et al., 2010), however in our case we found that there is no size effect for the Pt-folate NPs in the particle range 2–15 nm.
respective strain, which in this case were significantly higher for normal cell lines: 106 lg/ml – normal keratinocytes DO33, 51 lg/ml – cancerous keratinocytes SCC13, 61 lg/ml – cancerous keratinocytes SCC12B, 124 lg/ml – normal epithelial breast cells, 86 lg/ml – cancerous epithelial breast cells. From the Fig. 3 we can see that all cells treated with Pt/folate NPs exhibited a decrease in the percentage of viable cells as compared to the untreated control, as expected. However, the mode of cell death is different is cell type specific for the normal cells and non-specific for the cancer cells. After 24 h of exposure to Pt/folate NPs MCF10A cells showed a 23% decrease in viable cells, a slight decrease in apoptotic cells, which constituted only 7% of control population, but a four folds increase from 11% to 39.3% in the necrotic cell fraction. The MCF7 cell culture showed a similar 20% decrease of viable cells after 24 h of exposure, but in this case the apoptotic fraction increased more than tenfold, while the necrotic fraction had a four folds increase. Significantly smaller increase in apoptosis and necrosis after exposure to Pt/folate NPs was recently reported (Teow and Valiyaveettil, 2010), however their initial fraction of apoptotic cells was much higher than reported in this paper, making a direct comparison difficult. DO33 keratinocytes after 24 h of exposure to Pt/folate NPs exhibited a 20% decrease in cell viability due to three folds increase in cell apoptosis process (28%).
3.4. Cell apoptosis and necrosis rates due to Pt/folate NPs exposure In order to investigate the mode of cell death upon exposure to Pt/folate NPs, the cells were stained using Dead Cell Apoptosis Kit with Annexin V Alexa FluorÒ 488 and PI. Flow cytometry was performed according to the procedures recommended by the manufacturer. Unstained cells were considered viable, cells stained with only red (PI) dye and cells stained with both red (PI) and green (Annexin V) dyes were recognized as necrotic cells, and cells that were stained with only green (Annexin V) dye were recognized as apoptotic. In order to have comparable amounts of cells experiencing toxicity after 24 h of exposure, the studies were performed using the IC-50 Pt-folate NP concentration for the
Fig. 4. Cell viability of the cells treated with IC50 concentrations of Pt/folate NPs for 24 h (compared to untreated control) with and without folic acid pre-treatment.
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Cancerous keratinocytes SCC13 had a 16% decrease in cell viability after 24 h of Pt/folate NPs exposure that resulted from the 5 and 1.5 folds increases in both apoptosis and necrosis, respectively. On the other hand Pt/folate NPs had a greater impact on the other cancerous keratinocytes SCC12B, were the decrease in cell viability was 15% after 24 h of exposure. The SCC12B cell death was caused by 9 folds increase in apoptosis and 3 folds increase in necrosis. We therefore found that the mode of the cell death, due to the Pt-folate NPs exposure, in normal cell lines was cell type specific; normal epidermal keratinocytes, DO33, decreased cell viability predominantly by apoptosis, while normal epithelial breast cells, MCF10A, decreased viability predominantly by necrosis. All the cancer cells studied on the other hand, decreased viability via both modes. The specificity to cell type in the reaction to nanoparticle exposure has been reported by multiple groups (Greulich et al., 2011; Hanley et al., 2009; Sohaebuddin et al., 2010). Our results are in agreement with those of Teow et al. who studied the effects of the larger 15 nm Pt folate NPs, where they observed that the
mode of cell death for the population occurred via both apoptosis and necrosis for the cancerous MC-7 cells, while the mode of death for their normal cells, which in this case were IMR90 fibroblasts, was mostly necrotic (Teow and Valiyaveettil, 2010). It is well established that necrosis is usually related to the loss of lysosomal membrane integrity (Vanlangenakker et al., 2008) while apoptosis is caused by caspase activation, calcium overload or death-inducing signals (Ghosh et al., 2009; Mattson et al., 1998). Therefore, the mechanism of Pt/folate NPs and its subsequent processing in our experiments seems to be cell line specific. That fact might be further employed for the anti-cancer drug formulations. 3.5. Pt/folate NPs penetration pathway To prove our hypothesis about receptor mediated endocytosis of the Pt/folate NPs we decided to block folic acid receptors in epithelial and epidermal cell lines by overloading them with 16 lg/ml of free folic acid and follow the exposure to Pt/folate NPs. From the
Fig. 5. Confocal microscopy pictures of the cell treated with Pt/folate NPs for 24 h and controls. (a–c) MCF7; (d–f) MCF10A; (g–i) DO33; (j–l) SCC12B; (m–o) SCC13 cells.
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Fig. 4 we can see, that addition of folic acid prior to exposure to IC50 concentration of the Pt/folate NPs resulted in the significantly higher cell survival rate (80%) compared to the cells exposed to NPs without pretreatment with folic acid (50%) for all cell types. Addition of the free folic acid to the cultures in the absence of the NPs exposure had no result on cell viability (results are not shown). Hence, the major penetration pathway of the Pt/folate NPs to the cell is the receptor mediated endocytosis. It is well known that folic acid is required for essential cell function, namely it is necessary for the synthesis of purines and pyrimidines (Davis and Nicol, 1988) it is also known that folate receptor is overexpressed in ovarian, lung, brain, head, neck, breast cancers and has a very high affinity to the folic acid and all sorts of folic acid conjugates (Elnakat and Ratnam, 2004; Shmeeda et al., 2006). Therefore any cargo linked to the folic acid will be retained within an endocytic vesicle or released into the cytoplasm.
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(Fig. 6). In the case of the keratinocytes, we find that the SCC cells are not able to extend philopodia which are observed only for the normal cells. Hence even though more than 50% of the cells have died at this concentration, even the surviving cells have sustained serious damage. 3.7. Intracellular fate of internalised Pt/folate NPs We also imaged the cells with transmission electron microscopy in order to determine the location of the particles once they are sequestered within the cells. The images of thin sections are shown in Fig. 7. Comparing the appearance of the normal and cancerous cells for both keratinocyes and breast cancer cells, we find that the cells exposed to nanoparticles have far more vacuoles. Magnification of the interior of the vacuoles shows the presence of electron opaque regions, which are particle agglomerates. Hence
3.6. Changes in cell morphology due to the Pt/folate NPs Confocal microscopy was also performed in order to determine if abnormal changes occur to the cell morphology due to the nanoparticles exposure. The images obtained for cells incubated with two concentrations, the IC50 concentration and twice this concentration are shown in Fig. 5, together with images of the control samples that were not exposed to nanoparticles. Following IC50 concentrations of Pt/folate NPs were used: MCF10A – 124 lg/ml, MCF7 – 86 lg/ml, DO33 – 106 lg/ml, SCC12B – 61 lg/ml, SCC13 – 51 lg/ml. From the figure we can see that the in vitro morphology of the keratinocytes and the breast endothelial cells is similar both for the normal and cancerous cells. In each case the cells that are not exposed to nanoparticles are adherent to each other and have approximately the same area. After exposure to the corresponding IC50 concentration for the particular cell type, we begin to see signs of damage. In all cases we find that the tissue has bare patches where dying cells have been lifted. The keratinocytes and the SCC cells behave similarly in that they extend philopodia to bridge the bare areas. More careful examination of the images shows that the SCC cells have fewer philopodia per cell than the normal ones. Similar bare patches are observed for the breast cells, but neither cancer nor normal cells develop philopodia. Hence eventhough 50% of the cells have died, minimal damage to the morphology of the remaining cells is observed. When the concentration is increased to twice the IC50 dose more serious damage is observed. We find that the actin is damaged and the cell area is decreased for all cell types
Fig. 6. Cell area of the cell treated with Pt/folate NPs for 24 h and controls. Fig. 7. TEM images of the cells exposed to the PtNPs for 24 h and control. (a and b) MCF7; (c and d) MCF10A; (e and f) DO33; (g and h) SCC12B; (i and j) SCC13 cells.
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images showed that the number of vacuoles, an indicator of NP uptake, was also significantly larger for the cancer cells in both types of tissue studied, consistent with the hypothesis that the folic acid coating was effective in enhancing the preferential penetration. References
Fig. 8. Number of vacuoles per cell area in the cultures treated with Pt/folate NPs and control.
the particles are sequested into these vacuoles after penetration. One can also count the number of vacuoles per unit cell area, and the results are plotted in Fig. 8. From the figure we can see that in all cases, the number of vacuoles is larger for the cancer cells than the normal cells, even though the absolute particle concentration corresponding to IC50 is lower. This confirms that the difference in cell sensitivity is due to the fact that the uptake is higher in the cancer cells than in the normal cells, which we postulate is correlated to the preferential delivery of the particles due to the enhanced expression of folic acid receptors in the cancer cells. Platinum-based anticancer agents such as cisplatin have been used for more than 30 years in chemotherapy for the treatment of solid tumors. However it is known that such therapy has severe side effects because of its low selectivity. One of the most researched options to overcome the disadvantage of cisplatin is the improvement of the drug accumulation via drug targeting. In recent years metallic nanoparticles were used as highly effective targeted delivery systems for the platinum-based therapy. Lately it was reported (Bhattacharyya et al., 2011) that cisplatin loaded onto gold nanoparticles that targeted cancer cell receptors exhibited enhanced therapeutic efficacy forward lung and ovarian cancers. On the other hand, Porcel et al. showed the results indicating that 3 nm PtNPs can significantly amplify the effect of fast ion irradiation therapy due to its high atomic mass (Porcel et al., 2010). They reported that irradiated PtNPs has a direct effect on DNA through the emittion of Auger electrons and indirect effect by production of water radicals that further damage DNA. However the main limitation for such combined therapy is the low selectivity of the PtNPs. Therefore, Pt-folate NPs can provide the selectivity which enables their use in existing therapies to decrease their side effects and improve their therapeutic index.
4. Conclusion In summary we have reported a technique for synthesizing Pt/ folate NPs of relatively narrow size distributions, high degree of crystalline order, with particle diameter of d = 2.3 ± 0.5 nm. The toxicological impact of these particles on normal and cancerous epithelial breast and epidermal keratinocytes were tested, and they were found to be significantly more toxic to the cancer cells for both cell types. The mode of cell death was found to be predominantly necrotic for the normal epidermal keratinocytes and apoptotic for the normal epithelium breast cancer cells, while mode of cell death was a combination of similar fractions of apoptosis and necrosis for all the cancer cells, regardless of phenotype. TEM
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