Nanodiamonds (ND) are carbon nanoparticles that are about 2–8 nm in diameter (1,2) and represent an emerging class of materials with important clinical applications in medicine (3,4) . Their consistent dimensions, unique surface properties, facile processing parameters and scalability (5,6) innate biocompatibility and applicability as imaging/diagnostic tools (7–9) make them a suitable candidate for therapeutic and diagnostic approaches (10–15). With the increasing interest in ND for in vivo bio-medical applications, it became important to study its possible interactions with the cells of the immune system.
Different studies have demonstrated the interaction of ND with immune system. NDs can cause immune-stimulation (16–19) or immunosuppression which could reduce the resistance of organism towards infections (20). In vitro treatment with NDs did not show toxicity towards leukocytes and erythrocytes (21). NDs are taken up by NK cells and monocytes in a dose dependent manner without causing a reduction in cell viability (22). Induction of apoptosis has been observed in both normal and cancer cells at higher doses of 200–1000 μg/ml (23).
The mechanisms of uptake of different nanoparticles especially carbon nanotubes by lymphocytes and their modulatory effects on the immune response have been reported (24–30). Pristine carbon nanotubes are as such highly agglomerated and insoluble in aqueous media and therefore do not interact efficiently with cells. Acid functionalized carbon nanotubes with carboxyl groups attached to carbon atoms become negatively charges, disperse easily in aqueous media and interact efficiently with various kinds of cells in vitro and in vivo (31–33). We have previously shown the efficient interactions of acid functionalized single-walled carbon nanotubes (AF-SWCNTs) with lung epithelial cells (34), erythrocytes (35,36), T and B lymphocytes (37–39) macrophages (40) and NK cells (41). In the present study, we evaluated the uptake of carboxylated fluorescent NDs (cFND) with the B and T lymphocytes and compared it with the uptake of AF-SWCNTs. We found that there was low uptake of cFNDs by resting B cells, T cells, macrophages and granulocytes. Also, the cFNDs uptake by B and T cells did not increase with time. We further studied the uptake of cFND by resting and activated B and T cells as previously done for AF-SWCNTs. Here, we found that after activation, uptake of cFND was increased in B and T cells. But this increase in uptake was still much lower than uptake of AF- SWCNTs. Confocal microscopy results indicated that in resting cells, cFNDs were localized mainly around the cells membrane but in activated B and T cells they were localized inside the cells.
Materials and Methods
Inbred C57BL/6 mice (8-15 weeks old, 20-25g body weight) were used throughout the study. Animals were maintained in the animal house facility at South Asian University (SAU), New Delhi, and obtained from the National Institute of Nutrition (NIN), Hyderabad. Animals were housed in positive pressure air-conditioned units (250C, 50 % relative humidity) and on a 12- hour light/dark cycle. Water and chow were provided ad libitum. All the experimental protocols were conducted strictly in compliance with the guidelines notified by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Environment and Forest (www.envfor.nic.in/divisions/awd/ cpcsea_laboratory.pdf). The study was duly approved by SAU Institutional Animal Ethics Committee.
Reagents and other supplies
Lipopolysaccharide (LPS) from Escherichia coli O26:B6, staphylococcal enterotoxin B (SEB) from Staphylococcus aureus, Carboxylated fluorescent nanodiamonds [cFNDs] (ND900NV- 100nm-COOH) were from Adamas Nanotechnologies Inc. (Raleigh, NC, USA). RPMI-1640 medium (with 2mM glutamine, 1mM sodium pyruvate, 4.5 gm glucose/litre, 10 mM HEPES, 1.5 gm/litre sodium bicarbonate and 20μg/ml gentamycin) and fetal bovine serum (FBS) were from Gibco (Carlsbad, CA, USA). Anti-mouse CD16/32 purified, FITC anti-mouse CD3, PE-anti- mouse CD19, APC-anti-mouse F4/80, PE-CY7 anti-mouse Gr1 and 7AAD were purchased from eBiosciences (San Diego, CA, USA).
Isolation of Spleen cells.
Mice were euthanized using carbon dioxide, dissected and spleens were harvested. For isolation of splenocytes, spleen was meshed using 70µm strainer in a Petri-dish containing RPMI complete medium (5ml). Content was transferred to 15 ml conical tube and cells pelleted down at 1500 rpm for 5 minutes. Supernatant was discarded and the pellet was resuspended in 1 ml ACK lysis buffer and kept on ice for 5 minutes with intermittent shaking. One ml of RPMI complete medium (RPMI 1640 media with 15% heat inactivated Fetal Bovine Serum, 2mM L- Glutamine, 4.5gms Glucose per litre, 10mM HEPES, 1.5g per litre Sodium bicarbonate, 1mM Sodium pyruvate and 80mg/ litre gentamycin) was added to the tube and cells centrifuged at 1500 rpm for 5 minutes. Pellet was washed twice and was finally suspended in 1 ml RPMI complete medium and counted using a hemocytometer.
Cells were cultured without and with LPS/SEB and incubated with carboxylated fluorescent nanodiamonds (cFNDs). Cells were harvested, washed with PBS +2% FBS, Fc receptors were blocked using anti-mouse CD16/32 and B and T cells were stained with anti-mouse CD19 or CD3 antibody respectively. Cells were fixed with 4% Paraformaldehyde (PFA) for 15 minutes, samples were washed thrice to remove residuals of PFA and pellet was resuspended in 500µl PBS + 2% FBS. B and T lymphocytes were sorted using FACS aria III (purity > 96%) and cells were collected in 5ml round bottom tube precoated with RPMI complete medium. After collection, cells were pelleted down by centrifugation at 1500 rpm for 5 minutes and resuspended in 100µl PBS. Cells were adhered onto poly-l-lysine (0.01%) coated coverslips, dried to remove excess PBS (presence of PBS can cause formation of crystals in the sample) and mounted onto glass slide using prolong gold antifade reagent (prevents photobleaching). A coverslip was placed on glass slide, the slide sealed with transparent nail-paint, dried and visualized under an A1R Laser Confocal Scanning Microscope (Nikon, Japan).
Spleen cells from C57BL/6 mice were cultured (106 cells/ml in RPMI-1640 complete medium in 24 well culture plate. Cells were either unstimulated (resting) or stimulated in vitro with LPS (5µg/ml) and SEB (5 µg/ml) for 24 and 72 h respectively in sterile environment. Thereafter, the cells were incubated with cFND (10 µg/ml) for 4, 12 and 24 h after each mentioned time point and were harvested, washed with PBS three times and incubated with anti-mouse CD16/32 (0.5µg/106 cells in 100 µl PBS + 2% FBS) for 30 minutes on ice to block Fc receptors followed by staining with anti-mouse CD19/ CD3/ F4/80 or Gr-1 antibody (1 µg/106 cells) respectively for 30 minutes on ice in dark. Separately, equal number of cells were stained with Isotype control antibodies to set flow cytometry gates. Samples were washed twice to remove excess antibody and were resuspended in 500µl PBS. 7AAD (2µg/ml) were added to the samples and incubated for 15 minutes on ice to identify dead cell population. After incubation, cells were analysed on a BD aria-III flow cytometer and analysed using FACS Diva software. (BD Biosciences, San Jose, CA, USA.)
Each experiment was repeated three times. Statistical Analysis was performed using Sigma Plot software (Systat software, San Jose, CA). Student’s t-test was used to calculate the significance level between groups and ANOVA was used to calculate significance between and within groups.
Characterization of cFNDs
Diamond comprises a crystal lattice of sp3 hybridized carbon atoms and as such in this form diamonds are not fluorescent. If the lattice is bombarded with high energy helium atoms, few nitrogen vacant (NV) spaces are created in the lattice in place where carbon atoms existed before. Nanodiamonds with these vacant spaces have fluorescence properties. Fluorescent nanodiamonds with an average size of 100 nm containing >900 ppm of NV centres were used in the study. Using a laser particle analyzer (Malvern DLS zeta sizer) pristine FNDs were found to have a zeta potential of 1.1 mV whereas the zeta potential of carboxylated FNDs (cFNDs) was - 43.3 mV (Figure 1). The cFNDs could be excited by 488 nm laser and had an emission maximum of 785 nm that could be read in the PE-Cy7 window on the flow cytometer (BD aria III).
Figure 1: : Measurement of Zeta potential of fluorescenated nanodiamonds (FND) and carboxylated fluorescenated nanodiamonds (cFND). Zeta potential values of non- carboxylated fluorescenated nanodiamonds of size 90 nm and carboxylated fluorescenated nanodiamonds of size 100 nm has been measured using Malvern DLS zeta sizer.
Uptake of cFND by subpopulations of spleen cells.
In order to assess the uptake of cFND, spleen cells (106 cells/ml) were cultured in 24 well culture plate with cFND (10µg/ml) for 4, 12 and 24 h. Cells were harvested, stained with anti-mouse CD19, CD3, F4/80 and Gr-1 antibody to stain B cells, T cells, macrophages and granulocytes respectively and were analysed for cFND uptake by different cell populations. Results are presented as cFND percent positive cells (closed circles) and mean fluorescence intensity (MFI) of cFND uptake (open circles) in spleen subpopulations (Figure 2). The percentage of cFND positive cells are around 6% (B and T cells) and 12% (granulocytes and macrophages). This percent uptake in all the cell types are consistent throughout different time points. However, MFI of splenic granulocytes was maximum (350 at 24 h) amongst all four cell types. These results show that the uptake of cFNDs by resting spleen B and T lymphocytes is relatively poor.
Figure 2:Uptake of Carboxylated Fluorescenated nanodiamonds (cFND) by spleen subpopulations in vitro. Spleen cells were isolated from C57BL/6 mice and (1 x106/ml) were cultured with cFND (10 µg/ml) for 4, 12 and 24 h. After incubation, cells were washed, stained with anti-mouse CD19, CD3, F4/80, Gr-1 antibody and 7AAD. Samples were analyzed for uptake of cFND by different live spleen cell subpopulations using FACSaria III. Left Y axis shows percent uptake of cFND by different subpopulations of spleen ± SEM of three independent experiments. Number of events acquired on flow cytometer were 50,000.
Uptake of cFND by resting and activated B and T cells by flow cytometry:
Spleen cells were activated with LPS (5 µg/ml) and SEB (5 µg/ml) for 48 and 72 h respectively, washed and incubated with cFND for 4, 12 and 24 h to study the uptake of cFND. Results of flow cytometric analysis of these cells are shown in Figure 3. These results show that cFND positive cells increased after B and T cell activation, though
Figure 3:Uptake of cFND by resting and LPS/SEB activated B and T lymphocytes. Spleen cells were isolated from C57BL/6 mice and 1 x 106 cells/ml were cultured in 24 well culture plate without and with LPS (5 µg/ml) for 48 h and without and with SEB (5 µg/ml) for 72 h and incubated thereafter with cFND for 4, 12 and 24 h. Harvested cells were washed twice with PBS and stained with anti-mouse CD19 and anti-mouse CD3 antibody to stain B and T cells respectively, cells were counterstained with 7AAD to gate out 7AAD+ dead cells. Data analysed using FACSaria III and analysed using FACS diva software. In each case 10,000 events were analysed.
Uptake of cFND by confocal microscopy
To determine the localization of cFND in resting and activated B and T cells, confocal microscopy was performed. For this purpose, resting and activated spleen cells were incubated with cFND for 4 h followed by staining with anti CD19 and CD3 antibodies. Pure (>96%) T and B cells were isolated using FACS aria III cell sorter and were examined for cFND uptake under confocal microscope. Results in Figure 4A show the confocal microscopy images of resting and LPS activated B cells and Figure 4B shows similar data for resting and SEB activated T cells. These results show that in resting B cells the cFNDs appear to be localized on cell membrane whereas in activated B and T cells some penetration of cFNDs into cytoplasmic space was apparent. Localization of cFNDs was also examined in cell sections by z-sectioning on confocal microscope. Results in Figure 5A and 5B shows the z-sectioning data that confirmed the membrane localization of cFNDs in resting B and T cells and some cytoplasmic localization in activated B and T cells.
Figure 4:cFND uptake by resting and LPS and SEB activated B and T cells respectively.Spleen cells were isolated from C57BL/6 mice and were cultured in 24 well culture plate with and without LPS (5 µg/ml) for 48 h and with and without SEB (5ug/ml) for 72 hours. After that cells were incubated with cFND (10 µg/ml) for 4 h, harvested and stained with anti mouse CD19 and CD3 antibody. Pure CD19+ B cells and CD3+ T cells were obtained by cell sorting on a FACSaria III cell sorter. Cells were collected and adhered onto poly-l-lysine coated coverslips and were mounted on glass slide using prolong gold antifade reagent. Slides were allowed to dry and analysed using Nikon Confocal Microscope. Magnification 100 X.
Figure 5: : Uptake of carboxylated Fluorescenated Nanodiamonds (cFND) by resting and activated B and T cells (Z-sectioning images). Spleen cells were isolated from C57BL/6 mice and were cultured with and without LPS and SEB (5 µg/ml), incubated with cFND for 4 h, harvested and stained with anti-mouse CD19/CD3 antibody. CD19/CD3 positive cells were sorted using FACS aria III, mount cells onto coverslip and analysed by Confocal Microscopy. Magnification 100X.
Nanodiamonds (NDs) are nanoparticles that have high biocompatibility and low toxicity (42– 44). Further chemical inertness of diamond core with highly tailorable and fully accessible surface, capable of acquiring a number of functional groups that can be used for non-covalent or covalent attachment of drugs or biomolecules (45,46). NDs are thus especially suitable for use in targeted drug delivery (47). A special feature of fluorescent nanodiamonds is that their bright fluorescence does not fade even on extended exposure to incident light (48). This is unlike various fluorescent molecular probes that show a rapid fading of the fluorescence when continuously exposed to incident laser light. This property of FNDs make them specially suitable for in vitro and in vivo imaging studies (49–51). Not much information is available about the uptake characteristics of fluorescent nanodiamonds by different types of cells. We have presented data in this communication about the uptake of cFNDs by leukocytes, especially the B and T cells.
There was relatively poor uptake of cFNDs by resting B and T cells. Confocal data further shows that the observed uptake of cFNDs in resting B and T cells was essentially due to the binding of these nanoparticles with the cell membrane of B and T cells. Since we have previously examined the uptake of AF-SWCNTs by resting and activated B and T cells (40), it was of interest to compare the uptake of these two types of nanoparticles by B and T cells. Comparative data of the uptake of cFNDs and AF-SWCNTs by resting and activated B and T cells is given in Table 1.
Table 1: : comparison of FAF-SWCNT and cFND uptake by resting and activated B and T cellsData shows values for uptake at maximum activation time point i.e. 48 h for B cells treated with LPS (5 µg/ml) and 72 hours for T cells with SEB (5 µg/ml) and FAF-SWCNT/cFND incubation of 4 hours.
These results clearly show that in the case of AF-SWCNTs, the activation process resulted in a substantial increase in the percentage of both B and T cells positive for fluorescenated AF-SWCNTs (FAF-SWCNTs) as well as in the mean fluorescence intensity that is a measure of average per cell uptake of the nanoparticles. In case of cFNDs also, the percentage of cells positive for cFNDs increased significantly in activated B and T cells but the increase was significantly lower than that seen for the uptake of FAF-SWCNTs (Table 1). Further, there was no change in the MFI values of cFND uptake by activated B and T cells. Confocal data further showed that the cFND uptake characteristics were qualitatively different for resting and activated B and T cells. In resting cells the uptake essentially reflected some membrane binding of cFNDs whereas in activated B and T cells, the particles were internalized in the cells. Thus, no change in the MFI of cFNDs in resting and activated B and T cells seem to reflect a change in localization of these particles in resting and activated cells.
The non-fading fluorescence of fluorescent nanodiamonds makes them highly suitable for imaging work. The fact that there is higher internalization of cFNDs in activated B and T cells opens up the possibility of in vivo imaging of B and T cell tumors by using cFNDs. High uptake of AF-SWCNTs by activated B and T cells also results in killing of activated cells (38). We found that the cFNDs are not significantly toxic to lymphocytes (data not shown). Relative low toxicity of nanodiamonds as compared to AF-SWCNTs may further support the proposition of use of cFNDs for imaging activated B and T cells in vivo. This proposition would however require further testing.