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The effect of surface charge on cellular uptake and inflammatory behavior of carbon dots
Colloid and Interface Science Communications 35 (2020) 100243
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Colloid and Interface Science Communications journal homepage: www.elsevier.com/locate/colcom
The effect of surface charge on cellular uptake and inflammatory behavior of carbon dots
T
Muhammad Usmana,b,c, Yumna Zaheera,b, Muhammad Rizwan Younisd, Ruken Esra Demirdogene, Syed Zajif Hussainf, Yasra Sarwara, Mubashar Rehmang, ⁎ Waheed S. Khana,b, Ayesha Ihsana,b, a
Nanobiotech group, Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad, Pakistan c Department of Biotechnology, IPBB, MNS-University of Agriculture, Multan, Pakistan d Marshall Laboratory of Biomedical Engineering, International Cancer Center, Laboratory of Evolutionary Theranostics (LET), School of Biomedical Engineering, Shenzhen University Health Science Center, Shenzhen 518060, China e Cankiri Karatekin University, Faculty of Science, Department of Chemistry, 18100 Cankiri, Turkey f Department of Chemistry and Chemical Engineering, Lahore University of Management Sciences (LUMS), Lahore 54792, Pakistan g Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan b
ARTICLE INFO
ABSTRACT
Keywords: Carbon dots Cytotoxicity Cellular uptake Surface charge Inflammatory cytokines
Synthesis of carbon dots (CDs) or carbon quantum dots (QCDs) from different carbon sources have attracted much attention due to their excellent potential to advance wide range of applications such as biosensing, bioimaging, photoscience and other biomedical usage. However, the interaction of CDs (without any complex surface functionalities) with different cell lines especially with respect to their surface charge still needs to be elaborated in certain phenomena i.e. cytotoxicity, cellular uptake and inflammatory profiling. In this study, two oppositely charged CDs were synthesized and compared for their surface charge dependent interaction with different cell lines. Positively charged CDs were synthesized by using bovine serum albumin (BSA) and negatively charged CDs were synthesized using citric acid (CA) by acidic hydrolysis and reflux methods, respectively. Using both CDs, a concentration dependent cytotoxicity was carried out on 3T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophage cell lines, while cellular uptake and inflammatory studies were conducted with RAW 264.7 macrophages cell line. Results showed that both BSA and CA-CDs were not cytotoxic up to 400 μg/mL. Flow-cytometry studies indicated that cellular uptake of negatively charged CA-CDs was quite higher (~6.25%) than that of positively charged BSA-CDs (~1.24%). On the other hand, positively charged BSACDs have significantly enhanced the inflammatory cytokines release by RAW 264.7 macrophages cells. A concentration dependent increase in tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was observed when RAW 264.7 macrophages cells were treated with BSA-CDs. In comparison, CA-CDs did not show any effect on these cytokines release. Therefore, it was concluded that the interaction of CDs with different cells is dependent on their surface charge which can also control their cellular influx and release of inflammatory cytokines.
1. Introduction Carbon dots (CDs) is a subclass of carbon based nanomaterials which have quasi-spherical morphology, photoluminescent (PL) property and sizes less than 10 nm were discovered in 2004 [1]. Compared to other nanomaterials, CDs exhibit quite unique properties such as ultra-small size, zero dimension, excellent biocompatibility, metal free
nature, high water dispersibility, chemical and photo-stability [2–4]. Number of methods have been developed for the facile synthesis of CDs from different carbon sources [5–12]. Due to their biocompatibility, fluorescence and colloidal stability as compared to metal quantum dots [13–15], CDs are considered ideal for various biological applications e.g. vaccine development [4,16], optical bioimaging [17–21], chemical [22,23], optical [24] and biological sensing [25–28], as a drug carrier
⁎ Corresponding author at: Nanobiotech group, Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan. E-mail address: [email protected] (A. Ihsan).
https://doi.org/10.1016/j.colcom.2020.100243 Received 29 November 2019; Received in revised form 27 January 2020; Accepted 27 January 2020 2215-0382/ © 2020 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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[29–31], nanomedicine [32] and other theranostic applications [33]. Due to the number of lucrative benefits offered by CDs i.e. tunable surface chemistry, ability to deliver multiple antigens simultaneously, high susceptibility to be phagocytosed by the immune cells, better tracing and imaging ability [4], there is much interest to use CDs in biomedical applications especially for cancer immunotherapy, tumor imaging and other clinical applications. Undoubtedly, CDs are biocompatible in a wide concentration range but there are elevated concerns about their toxicological implications and potential immune risks due to their vast use in biomedical discipline. Therefore, CDs interaction with biological system should be further investigated with regard to their safety issue in drug and vaccine development and in immunotherapy. For this purpose, dose dependent cellular toxicity, uptake and determination of immune regulatory cytokines of CDs are the basic key points to be investigated [34,35]. Apart from size, several other physicochemical features of nanomaterials having direct or indirect influence on biological systems, should be considered while working at nano-bio interface (in-vitro or in-vivo). For example, surface coating, concentration, aggregation tendency, and material composition [35–38] play a decisive role in determining the cellular toxicity, uptake and immunological role of nanomaterials. Many results indicated that surface passivation/surface coating/surface functionalization determines cellular toxicity and biological responses of CDs in different cells [39–41]. However, beside surface passivation, studies relating to cellular cytotoxicity, uptake, and immunological responses of CDs (without surface passivation/coating/functionalization) solely due to surface charge is surprisingly very rare. The nano–bio interface comprises of numerous dynamically interacting components, among which surface charge is an important one, which controls and contribute to the interaction of materials with biological systems, through promoting the adsorption of certain ions, biomolecules and minimize the surface energy. These are decisive phenomena to control the cellular toxicity, uptake, and immune response for a particular nanomaterial [42]. For many other nanomaterials surface charge has been found to be among the critical parameter which influences cellular uptake and cytotoxicity [38,42,43]. Surprisingly, the surface charge impact of CDs without complex surface coating/functionalities, on their inflammatory behavior, cellular uptake and cytotoxicity have not been addressed yet. In this scenario, this study was targeted to elucidate the surface potential-based interaction of CDs with in vitro grown cells and to determine their cellular toxicity and uptake. Moreover, an interdependence of surface charge and dose dependent inflammatory responses was also studied in RAW 264.7 macrophages cells. For this purpose, we synthesized two types of CDs, one from BSA, the positively charged CDs and the other one from Citric Acid (CA) the negatively charged CDs via acidic hydrolysis and reflux methods respectively. Multiple dose and time dependent studies were carried out to assess the potential toxicity of the designed CDs against three different cell lines (3 T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophages). Cellular uptake and inflammatory studies were carried out only with RAW 264.7 macrophages cells.
method, compared to the hydrothermal approach reported earlier [45]. Briefly, 1 g of anhydrous CA was dissolved into 10 mL of Milli Q water [deionized water (DI)]. Then 350 μL of Di-ethylenetriamine (DETA) was added to the solution which was then stirred and refluxed at 200 °C for 5 h. The pH of the thus obtained solution was adjusted to 7 with 1 M sodium hydroxide (NaOH). The neutralized solution was dialyzed by dialysis tube (SnakeSkin™ dialysis tubing, 10 k MWCO, 22 mm) having cut-off 10 kDa, for two hrs and then was lyophilized. 2.2. Synthesis of carbon dots from bovine serum albumin CDs from BSA were synthesized by slight modification of previously reported acidic hydrolysis method [46]. Briefly, 1 g of BSA was dissolved in 10 mL of Milli Q water and 30 mL concentrated sulfuric acid (95%) was added dropwise into the BSA solution under vigorous stirring. The glass vial containing the solution was then placed and kept in water bath at constant temperature of 50 °C for 2.5 h. Then pH of the solution was adjusted to 7 with 2 M NaOH. Following this, the solution was dialyzed and lyophilized. 2.3. Characterization The size of CDs was analyzed by using 500 μL of the prepared CDs dispersed in DI water. Samples were analyzed by Malvern Zetasizer Nano ZS with DTS Nano software. The emission spectra of the prepared CDs (400 μg/mL) dispersed in DI water and dulbecco modified eagle medium (DMEM) were taken at 365 nm. Samples were analyzed by Spectra Max M2 with SoftMax Pro software. For atomic force microscope (AFM) imaging, samples were prepared by taking 10 μL aliquots of the prepared samples and coated onto 0.5 cm2 glass slides. Three different areas of the samples were scanned and imaged by using the same tip at room temperature in contact mode. The samples were analyzed by AFM (SHIMADZU WET-SPM 9600), probe (OMCL-TR800PSA-1) with thickness of 100 μm and force constant of 0.57 Nm−1. Data was analyzed via SPM manager software. For transmission electron microscopic (TEM) studies, samples of 10% w/w CD solutions were prepared in Milli Q water. A small volume of the solution (5 μL) was then placed on 300 mesh copper grid (with formvar films) obtained from electron microscopy sciences (EMS FF300-Cu) and was allowed to dry overnight at ambient condition The samples were analyzed via a TEM JOEL 2000FX at an acceleration voltage of 200 kV. For functional group analysis, samples were analyzed by ATR-FTIR machine (Bruker, Alpha Platinum ATR) between 500 and 4000 cm−1. 2.4. Cytotoxicity studies Alamar Blue assay was performed to determine the cell viability percentage. For all the cell lines (3T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophages) cells were cultured in DMEM media supplemented with heat inactivated 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were seeded at density of 1× 104 to 1× 105 per well in 24 well culture plate separately (containing 500 μL of culture media in each well) at 37 °C in a 5% CO2 humified incubator. When confluence reached at 90%, cells were treated with CDs at different concentrations (6.25–400 μg/mL) and incubated (37 °C in a 5% CO2 humified incubator) for two different time periods i.e. for 24 and 72 h. All experiments were done in triplicates. The absorbance of plate was analyzed at 570 nm using Spectra Max M2 with SoftMax Pro software.
2. Material and methods All reagents and chemicals [Di-ethylenetriamine (DETA) reagent plus, bovine serum albumin (BSA), citric acid anhydrous reagent ACS and sulfuric acid certified ACS Plus] were purchased from Merck, Sigma Aldrich and Fisher Scientific and were used without further purification. Milli-Q water was obtained from Milli-Q system (Millipore, USA). SnakeSkin™ Dialysis Tubing, 10 K MWCO, 22 mm was purchased from ThermoFisher scientific, USA.
2.5. Inflammatory studies
2.1. Synthesis of carbon dots from citric acid
In all these tests (i.e. NO, TNF-α, IL-6) lipopolysaccharide (LPS 100 ng/mL) was used as positive control, while unstimulated cells were
CDs obtained from CA were synthesized by a quick and facile reflux 2
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Fig. 1. Schematic diagram of the synthesis of two different CDs, their cellular toxicity, uptake and inflammatory studies.
used as negative control.
absorbance from each well was recorded at 450 nm via Spectra Max M2 and the data was analyzed by SoftMax Pro software.
2.5.1. Nitric oxide test RAW 264.7 macrophages 1.4 × 104 cells were seeded in 12-well plate, treated with different concentrations (12.5 to 400 μg/mL) of CDs for 24 h. Each well contained 20 μL of Griess reagent [(N-(1-naphthyl) ethylenediamine dihydrochloride, 25 mL of a 0.1% (1 mg/mL) solution was purged with argon (Component A), sulfanilic acid 25 mL of a 1% (10 mg/mL) solution in 5% phosphoric acid (Component B)], 150 μL of nitrite containing sample (1 mL of 1 mM sodium nitrite in DI water) and 130 μL of DI was mixed and incubated for 30 min at room temperature. Plate absorbance from each well was collected at 548 nm using Spectra Max M2 with SoftMax Pro software.
2.5.3. Interleukin-6 (IL-6) For ELISA of interleukin 6 (IL-6), all solutions were freshly prepared as mentioned below. In detail, coating buffer (NaHCO3 (0.84 g) + Na2CO3 (0.356 g) in 100 mL), blocking buffer (1% BSA in PBS), assay diluents (1% (w/v) BSA + 0.05% (v/v) Tween PBS or 10% (v/v) FBS in PBS), washing buffer (0.05% (v/v) Tween in PBS). In step 1, Maxisorb plate was coated with 100 μL of antibody solutions and incubated at 4 °C overnight. In step 2, coated plate wells were blocked by adding 200 μL of blocking buffer and incubated for 2–3 h at room temperature (Plate A). Separately, Raw 264.7 macrophages cells were incubated with different concentrations of CDs (50–400 mg/μL) for 24 h (plate B). In step 3, 100 μL of supernatant from each well of plate B was aspirated and added to plate A and incubated for 2–4 h at room temperature. In step 4, Biotinylated antibody solution (100 μL) was applied to each well and plate was left for 1 h at room temperature. In step 5, 100 μL of Streptavidin HRP (1:2000 (v/v)) was added to each well of the plate and kept at room temperature for 1 h. washing of the plate with washing buffer was assured at each step. After washing, 100 μL of substrate solution (tetramethylbenzidine) was added in each well and incubated for 30 min in dark. Then stop solution was added. At the end, spectrophotometric analysis of each well was made, and the absorbance was recorded at 450 nm via Spectra Max M2 and the data was analyzed by SoftMax Pro software.
2.5.2. Tumor necrosis factor alpha (TNF-α) All solutions were freshly prepared for enzyme linked immunosorbent assay (ELISA) of tumor necrosis factor alpha (TNF-α). Briefly, all required buffers were prepared, coating buffer [NaHCO3 (0.84 g) + Na2CO3 (0.356 g) in water (100 mL), blocking buffer (1% (w/v) BSA in PBS), assay diluents (1% BSA + 0.05% (v/v), Tween in PBS or 10% FBS in PBS, washing buffer (0.05% (w/v) Tween in PBS]. In step 1, Maxisorb plate was coated with 100 μL antibodies solution and incubated at 4 °C overnight. In step 2, coated plate wells were blocked by adding 200 μL of blocking buffer and then incubated for 2–3 h at room temperature (Plate A). In a separate plate, Raw 264.7 macrophages cells were incubated with different concentrations of CDs (100 to 400 mg/μl) for 24 h (plate B). In step 3, 100 μL of the supernatant from the each well of Plate B, was aspirated and added into the wells of Plate A and left for 2–4 h at room temperature. In step 4, Biotinylated antibody solution (100 μL) was applied to each well and left for 1 h at room temperature. In step 5, Streptavidin HRP (1:2000 (v/v) 100 μL was added to each well and left for 1 h. For all above mentioned steps, plate was carefully washed with washing buffer. A 100 μL of substrate solution (tetramethylbenzidine was added to each well and incubated for further 30 min in dark. Finally stop solution was added and
2.6. Cell uptake study by flow cytometry RAW 264.7 macrophages 1.4 × 104 cells were seeded in 12-well plate and treated with two different concentrations of CDs; 200 and 400 μg/mL for 24 h. No dye was used for this experiment. For each sample, 3 × 104 events were analyzed using BD LSRFortessa Dual instrument with Flowjo software (version7.6.1). 3
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in the standard BSA scan) (Fig. S-5B). These peaks are attributed to NeH bending and NeH stretching which created an overall positive surface charge on the surface of BSA-CDs [49–51] which was further confirmed by a positive zeta potential value (+ 38.18) of BSA-CDs. In previous studies BSA coating had been reported to reduce the negative surface charged density and exerts overall positive charge to polystyrene NPs [52]. The emission spectra of the dialyzed and purified water dispersed and DMEM media dispersed CDs samples were analyzed at the excitation wavelength of 365 nm. To study the effect of dispersion media on the optical properties of CDs, the emission behavior of CDs was checked in both water and DMEM cell culture media. When dispersed in water, it was observed that CA-CDs had high PL intensity emission peak at 455 nm (Fig. S-6A) compared to the BSA-CDs which showed peak at 435 nm (Fig. S-6B). Compared with CDs dispersed in water, CDs dispersed in DMEM, emitted low PL intensity and in both cases, there was a slight shift in the peaks too (Fig. S-6C, S-6D). The rich composition of DMEM (minerals, vitamins and amino acids) might enhance the electrostatic interactions between CDs and surrounding media which ultimately attenuated the PL intensity of CDs in DMEM, as compared to water. PL intensity of CDs can be influenced by a wide range of factors such as concentration, doping agent, precursor and surface coating [1,2,12,23]. In both, water and DMEM medium the emission intensity of CA-CDs was found to be higher than that of BSACDs. BSA contain two residues of tryptophan (fluorescent amino acids) in its structure [46], which might also be responsible to delocalize the free electron density on the surface of CDs. The emission intensity of CA-CDs was higher than that of BSA-CDs, which is possibly due to the high density of carboxyl groups present on its surface. Earlier it was reported that the number of carboxyl groups on the outer surface of CDs is directly linked with enhanced PL [11,17,45]. This may be the reason for the high luminescence observed with the CA-CDs. After the synthesis and characterization of CDs, the first step was to check the surface potential-based biocompatibility or cellular cytotoxicity. Three cell lines were opted namely 3T3 fibroblast, MDA-MB231 breast cancer and RAW 264.7 macrophages. Alamar blue assay was performed to assess the cellular viability percentage within the concentration range of 0–400 μg/mL for CDs. As shown in (Fig. 3A, S-8C) and (Fig. 3B, S-8D), it was observed that after 24 and 72 h of incubation neither the CA-CDs nor the BSA-CDs were found to cause any toxicity to 3T3 fibroblast cells at any concentration range 0–400 μg/mL respectively. Similarly, for MDA-MB-231 breast cancer cells, no cell death was observed at 24 and 72 h, when incubated with CDs of both CA (Fig. S7A, S-8E) and BSA (Fig. 7B, S-8F). However, at concentration of 400 μg/ mL, CDs of CA caused significant reduction in the cell viability (~75%) of MDA-MB-231 breast cancer cells at 72 h (Fig. S-8. Similarly, upto 400 μg/mL of CA-CDs and BSA-CDs, no cytotoxicity was observed with RAW 264.7 macrophages (Fig. 4). It was reported earlier that CDs caused decrease in the viability and showed toxicity with RAW 264.7 macrophages cells at of 62.5 μg/mL and 250 μg/mL
Fig. 2. Transmission electron micrographs of CDs, A. CA-CDs, B. BSA-CDs.
3. Results and discussion Overall this study was designed to elucidate the impact of surface charge on CDs by investigating their cellular toxicity, uptake and inflammatory effects. In this regard, two oppositely charged CDs were separately studied for their interaction with different cell lines (Fig. 1). Both CDs exhibit an intense blue color under UV-light (λex: 365 nm) (Fig. S-1). The average diameter and zeta potential of CDs were estimated by dynamic light scattering (DLS). Results showed that the average hydrodynamic radius of the both CA-CDs (Fig. S-2A) and BSACDs (Fig. S-2B) was ~2–3 nm. The charge on the CDs was estimated via zeta potential measurements carried out at neutral pH in phosphate buffer saline (PBS). Overall charge found on BSA-CDs (Fig. S-3B) was positive i.e. +38.18 mV, while on CA-CDs a negative charge was found i.e. -30.75 mV (Fig. S-3A). Moreover, the size of CDs was confirmed via transmission electron microscopy (Fig. 2) and atomic force microscopy (AFM) (Fig. S-4). The results showed that both CA-CDs and BSA-CDs (Fig. 2) were uniformly dispersed and clearly in the size range from 2 to 3 nm, which is in accordance with DLS results and the previous reports [46–48]. The type of functional groups on the prepared CDs were analyzed and confirmed by FTIR spectroscopy. The IR peaks appearing in spectrum of CA-CDs at 1072, 1219 are due to the CeO stretching and bending vibrations while the peak between 2500 and 3200 cm−1 are attributed to the presence of OeH moiety in carboxylic (–COOH) groups. The peak at 1369 cm−1 is due to CeN stretching vibrations of amine (–NH2) group. The peaks observed at 1549 and 1742 cm−1 are due to and NeH bending and CeO stretching vibrations respectively. The amide peaks in the citric acid CDs scan is inserted due to the diethylenetriamine, which is absent in the citric acid scan (Fig. S-5A). The strong peak of carboxyl (1072 cm−1) moiety [48] in the scan, depicts their abundance on CA-CDs and assumed to create an overall negative charge on their surface, which was further confirmed by zeta potential (−30.75 mV) measurements. In case of BSA-CDs, which were prepared by the breakdown of complex BSA molecule in presence of sulfuric acid, the intense peaks were observed at 1521, 1642 and 3283 cm−1 (absent
Fig. 3. Cell viability of 3T3 Fibroblast cells A. with CA-CDs B. with BSA-CDs. in the concentrations 0–400 μg/mL of CDs after 24 h incubation. 4
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Fig. 4. Cell viability of RAW 264.7 macrophages cells A. with CA-CDs B. with BSA-CDs. in the concentrations 0–400 μg/mL of CDs after 24 h incubation. Table 1 A comparison of different biocompatible concentrations of CDs in previous studies. Sr.
Carbon Dots
Biocompatible concentrations range
Cell type
Reference
1 2 3 4 5 6 7 8 9 10 11
N-doped carbon dots Nitrogen and sulfur co-doped carbon dots Mesoporous silica nanoparticles carbon dots (MSNs-CDs) Metal-doped carbon dots Manganese-doped carbon quantum dots Carbon dot-ovalbumin (CD-OVA) Metal ions doped carbon quantum dots Citric acid-based carbon dots Glucose based carbon dots, carbon dots-Ricin toxin binding subunit B (CDs-RTB) Aspirin-based carbon dots Citric acid and BSA based carbon dots
10–200 μg/mL 25–400 μg/mL 0–100 μg/mL 10–500 μg/mL 0–500 μg/mL 0–1000 μg/mL 0–200 μg/mL 0–600 μg/mL 0–100 μg/mL 0–100 μg/mL 0–400 μg/mL
HeLa, 16HBE HeLa HeLa, MCF-7, A549, L929 PC12 HeLa DCs HeLa C6 RAW 264.7 RAW 264.7 3T3, MDA-MB-231, RAW 264.7
[31] [17] [30] [13] [14] [4] [15] [21] [53] [54] Our research
concentrations [39]. Zheng et [34] and Li et al [53] reported that CDs of citric acid origin caused no cellular toxicity upto 500 μg/mL concentration. In our perception, presence of carboxyl, hydroxyl, amine on the outer surface of CDs and absence of any complex molecule might have been the reason for high cell viability observed with these CDs. It is now a well-known fact that the surface functionalities/surface coating on the CDs play a critical role in determining the cellular toxicity [44]. Up till now, our results indicated that surface charges had no negative effect on cell viability in any of the three cell lines studied at 24 h time period. Our results are in accordance with previous reports (Table 1). However, depending on the surface passivation/functionalization and doping, the lethal/cytotoxic concentrations of CDs may be different in each case. For the reference, a comparison of cytotoxic concentrations of CDs, from previous studies is compiled in Table. 1. Besides the toxicity of CDs, a rather neglected aspect of CDs, is to elaborate the relation between the surface charge of CDs and the immune response against the CDs. Macrophages engage in the regularization of immune system by releasing inflammatory cytokines such as NO, TNF-α and IL-6 [48]. Therefore, these markers were used in these studies to investigate the surface potential based immunological role of CDs. Nitric oxide (NO) test was conducted to check initial inflammatory behavior of CDs in RAW 264.7 macrophages. It is evident that after 24 h of incubation, no nitric oxide (NO) production was observed neither against the CA-CDs (Fig. S-9A) nor the BSA-CDs (Fig. S-9B) upto 400 μg/mL concentration. Further pro-inflammatory behavior of both CDs was evaluated through ELISA of tumor necrosis factor alpha (TNF-α). Interestingly, no release of TNF-α was observed against any concentration of negatively charged CA-CDs (Fig. 5A), while, in case of BSA-CDs, the results indicated a concentration dependent (100–400 μg/mL) increase in the release of TNF-α (Fig. 5B). To further validate charge based pro-inflammatory response of CDs, interleukin 6 (IL-6) ELISA was performed. Similar to TNF-α results no IL-6 release was observed at any concentrations of CA-CDs (Fig. 6A),
while at 400 μg/mL, BSA-CDs caused a significant high release of IL-6 as compared to negative control (Fig. 6B). In a previous report, dose dependent increase in the inflammatory factors (i.e. NO, TNF-α and IL-6) was observed with Ricin toxin binding subunit B (RTB) attached CDs, which depicted a correlation between the release of inflammatory cytokines (TNF-α and IL-6) and the toxic surface functionality on CDs [53]. Those findings were strongly linked to the proved role of RTB to induce cell mediated immunity and not due to carbon dots. In current work, we used CA and BSA for CDs synthesis. Both were biocompatible molecules, which broke down to give carbon dots bearing small surface functionalities with no toxicity. As evident from our studies, only positively charged CDs (BSA-CDs) boosted TNF-α and IL-6 level. The positively charge CDs when interact with negatively charged cell membrane, may get agglomerated/bound with cell membrane which caused inflammation. It was also reported earlier that positively charged nanoparticles taken up by macrophages via micropinocytosis caused disruption in the plasma membrane which led to the up regulation of TNF-α and IL-6 and ultimately showed inflammatory responses [55,56]. For CA-CDs, no inflammatory cytokines were released (Fig. 5A, 6A). Generally, negatively charged particles are internalized through clathrin and dynamin dependent endocytosis in macrophages [55]. Moreover, similar charges on CA-CDs and cell membrane, may not cause any agglomeration and therefore, no inflammatory response was triggered. This finding is strongly supported by a pervious study that when lipo-polysaccharide (LPS) stimulated macrophages cells were treated with negatively charged particles, they rather suppressed inflammation [57]. In order to correlate and further elaborate the link between surface charge of CDs and inflammation it was necessary to check their cellular uptake or internalization by RAW 264.7 macrophages cells. Flow cytometry is considered the best technique to quantify the internalized materials within cell [58]. To analyze cellular uptake, two concentrations of CDs i.e. 200 μg/mL and 400 μg/mL were incubated with RAW 264.7 macrophages cells for 24 h. At 400 μg/mL 5
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Fig. 5. The expression level of inflammatory biomarker TNF-⍺ secreted by RAW 264.7 macrophages cells incubated with CDs for 24 h A. CA-CDs, B. BSA-CDs.
Fig. 6. The expression level of inflammatory biomarker IL-6 secreted by RAW 264.7 macrophages cells incubated with CDs for 24 h A. CA-CDs, B. BSA-CDs.
Fig. 7. Flow cytometric cell uptake studies of CDs for 24 h incubation, Where X. is untreated sample of RAW 264.7 macrophages used as negative control A. CA-CDs 200 μg/mL treated cells uptake, B. BSA-CDs 200 μg/mL treated cells uptake.
concentration, cellular uptake of CA-CDs was noted to be ~20.3% (Fig. 8A) which was higher than that of observed with untreated cells (negative control) (Fig. 7X) and BSA-CDs (~0.628%) (Fig. 8B). At 200 μg/mL concentration, CA-CDs showed a cellular uptake of ~6.25% (Fig. 7A), while for BSA-CDs it was ~1.24% (Fig. 7B). The flow cytometry data support our previous findings. Overall, negatively charged structure of the cell membrane allowed the negatively charged materials to pass through easily. It was reported
earlier that negatively charged particles showed higher cellular uptake [59,60]. The cell membrane hardly allowed positively charged materials to pass through due to the agglomeration of material with cell membrane. In the light of these facts, the cellular uptake of positively charged BSA-CDs was less, this might be due to the composition of cell membrane which has pivotal role in cellular uptake. For this reason positively charged CDs, caused macrophages to show inflammatory response [61]. The extent of cellular uptake of CDs is also related with 6
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Fig. 8. Flow cytometric cell uptake studies of CDs for 24 h incubation A. CA-CDs 400 μg/mL treated cells uptake, B. BSA-CDs 400 μg/mL treated cells uptake.
inflammation, but it may be different for different surface passivation/ functionalization [62]. [6]
4. Conclusion
[7]
The results of this study showed that an overall surface potential of CDs has a fundamental role to determine their cellular uptake and inflammatory response. The CDs obtained from CA and BSA were not toxic upto 400 μg/mL. The negatively charged CA-CDs showed no inflammatory response but a high cellular uptake as compared to BSACDs. By taking these into account CA-CDs can be used as carrier to enhance the uptake of drug/vaccine/therapeutic protein. The positively charged BSA-CDs caused increase in the release of TNF-α and IL-6 level and thus may be considered for immuno-stimulating activities. However, to use CDs in clinical applications, further studies are mandatory to investigate their persistence, degradation, and excretion, long term toxicological and associated immunological responses.
[8] [9] [10] [11] [12] [13]
Declaration of Competing Interest
[14]
None.
[15]
Acknowledgement
[16]
We are thankful to Prof. Vincent M. Rotello for providing lab space and expertise to conduct this research at department of Chemistry, University of Massachusetts, Amherst, USA. MU and AI thanks to Higher Education Commission (HEC), Pakistan, for financial support (HEC NRPU 7684).
[17] [18]
Appendix A. Supplementary data
[19]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.colcom.2020.100243.
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The effect of surface charge on cellular uptake and inflammatory behavior of carbon dots
T
Muhammad Usmana,b,c, Yumna Zaheera,b, Muhammad Rizwan Younisd, Ruken Esra Demirdogene, Syed Zajif Hussainf, Yasra Sarwara, Mubashar Rehmang, ⁎ Waheed S. Khana,b, Ayesha Ihsana,b, a
Nanobiotech group, Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad, Pakistan c Department of Biotechnology, IPBB, MNS-University of Agriculture, Multan, Pakistan d Marshall Laboratory of Biomedical Engineering, International Cancer Center, Laboratory of Evolutionary Theranostics (LET), School of Biomedical Engineering, Shenzhen University Health Science Center, Shenzhen 518060, China e Cankiri Karatekin University, Faculty of Science, Department of Chemistry, 18100 Cankiri, Turkey f Department of Chemistry and Chemical Engineering, Lahore University of Management Sciences (LUMS), Lahore 54792, Pakistan g Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan b
ARTICLE INFO
ABSTRACT
Keywords: Carbon dots Cytotoxicity Cellular uptake Surface charge Inflammatory cytokines
Synthesis of carbon dots (CDs) or carbon quantum dots (QCDs) from different carbon sources have attracted much attention due to their excellent potential to advance wide range of applications such as biosensing, bioimaging, photoscience and other biomedical usage. However, the interaction of CDs (without any complex surface functionalities) with different cell lines especially with respect to their surface charge still needs to be elaborated in certain phenomena i.e. cytotoxicity, cellular uptake and inflammatory profiling. In this study, two oppositely charged CDs were synthesized and compared for their surface charge dependent interaction with different cell lines. Positively charged CDs were synthesized by using bovine serum albumin (BSA) and negatively charged CDs were synthesized using citric acid (CA) by acidic hydrolysis and reflux methods, respectively. Using both CDs, a concentration dependent cytotoxicity was carried out on 3T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophage cell lines, while cellular uptake and inflammatory studies were conducted with RAW 264.7 macrophages cell line. Results showed that both BSA and CA-CDs were not cytotoxic up to 400 μg/mL. Flow-cytometry studies indicated that cellular uptake of negatively charged CA-CDs was quite higher (~6.25%) than that of positively charged BSA-CDs (~1.24%). On the other hand, positively charged BSACDs have significantly enhanced the inflammatory cytokines release by RAW 264.7 macrophages cells. A concentration dependent increase in tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) was observed when RAW 264.7 macrophages cells were treated with BSA-CDs. In comparison, CA-CDs did not show any effect on these cytokines release. Therefore, it was concluded that the interaction of CDs with different cells is dependent on their surface charge which can also control their cellular influx and release of inflammatory cytokines.
1. Introduction Carbon dots (CDs) is a subclass of carbon based nanomaterials which have quasi-spherical morphology, photoluminescent (PL) property and sizes less than 10 nm were discovered in 2004 [1]. Compared to other nanomaterials, CDs exhibit quite unique properties such as ultra-small size, zero dimension, excellent biocompatibility, metal free
nature, high water dispersibility, chemical and photo-stability [2–4]. Number of methods have been developed for the facile synthesis of CDs from different carbon sources [5–12]. Due to their biocompatibility, fluorescence and colloidal stability as compared to metal quantum dots [13–15], CDs are considered ideal for various biological applications e.g. vaccine development [4,16], optical bioimaging [17–21], chemical [22,23], optical [24] and biological sensing [25–28], as a drug carrier
⁎ Corresponding author at: Nanobiotech group, Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan. E-mail address: [email protected] (A. Ihsan).
https://doi.org/10.1016/j.colcom.2020.100243 Received 29 November 2019; Received in revised form 27 January 2020; Accepted 27 January 2020 2215-0382/ © 2020 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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[29–31], nanomedicine [32] and other theranostic applications [33]. Due to the number of lucrative benefits offered by CDs i.e. tunable surface chemistry, ability to deliver multiple antigens simultaneously, high susceptibility to be phagocytosed by the immune cells, better tracing and imaging ability [4], there is much interest to use CDs in biomedical applications especially for cancer immunotherapy, tumor imaging and other clinical applications. Undoubtedly, CDs are biocompatible in a wide concentration range but there are elevated concerns about their toxicological implications and potential immune risks due to their vast use in biomedical discipline. Therefore, CDs interaction with biological system should be further investigated with regard to their safety issue in drug and vaccine development and in immunotherapy. For this purpose, dose dependent cellular toxicity, uptake and determination of immune regulatory cytokines of CDs are the basic key points to be investigated [34,35]. Apart from size, several other physicochemical features of nanomaterials having direct or indirect influence on biological systems, should be considered while working at nano-bio interface (in-vitro or in-vivo). For example, surface coating, concentration, aggregation tendency, and material composition [35–38] play a decisive role in determining the cellular toxicity, uptake and immunological role of nanomaterials. Many results indicated that surface passivation/surface coating/surface functionalization determines cellular toxicity and biological responses of CDs in different cells [39–41]. However, beside surface passivation, studies relating to cellular cytotoxicity, uptake, and immunological responses of CDs (without surface passivation/coating/functionalization) solely due to surface charge is surprisingly very rare. The nano–bio interface comprises of numerous dynamically interacting components, among which surface charge is an important one, which controls and contribute to the interaction of materials with biological systems, through promoting the adsorption of certain ions, biomolecules and minimize the surface energy. These are decisive phenomena to control the cellular toxicity, uptake, and immune response for a particular nanomaterial [42]. For many other nanomaterials surface charge has been found to be among the critical parameter which influences cellular uptake and cytotoxicity [38,42,43]. Surprisingly, the surface charge impact of CDs without complex surface coating/functionalities, on their inflammatory behavior, cellular uptake and cytotoxicity have not been addressed yet. In this scenario, this study was targeted to elucidate the surface potential-based interaction of CDs with in vitro grown cells and to determine their cellular toxicity and uptake. Moreover, an interdependence of surface charge and dose dependent inflammatory responses was also studied in RAW 264.7 macrophages cells. For this purpose, we synthesized two types of CDs, one from BSA, the positively charged CDs and the other one from Citric Acid (CA) the negatively charged CDs via acidic hydrolysis and reflux methods respectively. Multiple dose and time dependent studies were carried out to assess the potential toxicity of the designed CDs against three different cell lines (3 T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophages). Cellular uptake and inflammatory studies were carried out only with RAW 264.7 macrophages cells.
method, compared to the hydrothermal approach reported earlier [45]. Briefly, 1 g of anhydrous CA was dissolved into 10 mL of Milli Q water [deionized water (DI)]. Then 350 μL of Di-ethylenetriamine (DETA) was added to the solution which was then stirred and refluxed at 200 °C for 5 h. The pH of the thus obtained solution was adjusted to 7 with 1 M sodium hydroxide (NaOH). The neutralized solution was dialyzed by dialysis tube (SnakeSkin™ dialysis tubing, 10 k MWCO, 22 mm) having cut-off 10 kDa, for two hrs and then was lyophilized. 2.2. Synthesis of carbon dots from bovine serum albumin CDs from BSA were synthesized by slight modification of previously reported acidic hydrolysis method [46]. Briefly, 1 g of BSA was dissolved in 10 mL of Milli Q water and 30 mL concentrated sulfuric acid (95%) was added dropwise into the BSA solution under vigorous stirring. The glass vial containing the solution was then placed and kept in water bath at constant temperature of 50 °C for 2.5 h. Then pH of the solution was adjusted to 7 with 2 M NaOH. Following this, the solution was dialyzed and lyophilized. 2.3. Characterization The size of CDs was analyzed by using 500 μL of the prepared CDs dispersed in DI water. Samples were analyzed by Malvern Zetasizer Nano ZS with DTS Nano software. The emission spectra of the prepared CDs (400 μg/mL) dispersed in DI water and dulbecco modified eagle medium (DMEM) were taken at 365 nm. Samples were analyzed by Spectra Max M2 with SoftMax Pro software. For atomic force microscope (AFM) imaging, samples were prepared by taking 10 μL aliquots of the prepared samples and coated onto 0.5 cm2 glass slides. Three different areas of the samples were scanned and imaged by using the same tip at room temperature in contact mode. The samples were analyzed by AFM (SHIMADZU WET-SPM 9600), probe (OMCL-TR800PSA-1) with thickness of 100 μm and force constant of 0.57 Nm−1. Data was analyzed via SPM manager software. For transmission electron microscopic (TEM) studies, samples of 10% w/w CD solutions were prepared in Milli Q water. A small volume of the solution (5 μL) was then placed on 300 mesh copper grid (with formvar films) obtained from electron microscopy sciences (EMS FF300-Cu) and was allowed to dry overnight at ambient condition The samples were analyzed via a TEM JOEL 2000FX at an acceleration voltage of 200 kV. For functional group analysis, samples were analyzed by ATR-FTIR machine (Bruker, Alpha Platinum ATR) between 500 and 4000 cm−1. 2.4. Cytotoxicity studies Alamar Blue assay was performed to determine the cell viability percentage. For all the cell lines (3T3 fibroblast, MDA-MB-231 breast cancer and RAW 264.7 macrophages) cells were cultured in DMEM media supplemented with heat inactivated 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were seeded at density of 1× 104 to 1× 105 per well in 24 well culture plate separately (containing 500 μL of culture media in each well) at 37 °C in a 5% CO2 humified incubator. When confluence reached at 90%, cells were treated with CDs at different concentrations (6.25–400 μg/mL) and incubated (37 °C in a 5% CO2 humified incubator) for two different time periods i.e. for 24 and 72 h. All experiments were done in triplicates. The absorbance of plate was analyzed at 570 nm using Spectra Max M2 with SoftMax Pro software.
2. Material and methods All reagents and chemicals [Di-ethylenetriamine (DETA) reagent plus, bovine serum albumin (BSA), citric acid anhydrous reagent ACS and sulfuric acid certified ACS Plus] were purchased from Merck, Sigma Aldrich and Fisher Scientific and were used without further purification. Milli-Q water was obtained from Milli-Q system (Millipore, USA). SnakeSkin™ Dialysis Tubing, 10 K MWCO, 22 mm was purchased from ThermoFisher scientific, USA.
2.5. Inflammatory studies
2.1. Synthesis of carbon dots from citric acid
In all these tests (i.e. NO, TNF-α, IL-6) lipopolysaccharide (LPS 100 ng/mL) was used as positive control, while unstimulated cells were
CDs obtained from CA were synthesized by a quick and facile reflux 2
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Fig. 1. Schematic diagram of the synthesis of two different CDs, their cellular toxicity, uptake and inflammatory studies.
used as negative control.
absorbance from each well was recorded at 450 nm via Spectra Max M2 and the data was analyzed by SoftMax Pro software.
2.5.1. Nitric oxide test RAW 264.7 macrophages 1.4 × 104 cells were seeded in 12-well plate, treated with different concentrations (12.5 to 400 μg/mL) of CDs for 24 h. Each well contained 20 μL of Griess reagent [(N-(1-naphthyl) ethylenediamine dihydrochloride, 25 mL of a 0.1% (1 mg/mL) solution was purged with argon (Component A), sulfanilic acid 25 mL of a 1% (10 mg/mL) solution in 5% phosphoric acid (Component B)], 150 μL of nitrite containing sample (1 mL of 1 mM sodium nitrite in DI water) and 130 μL of DI was mixed and incubated for 30 min at room temperature. Plate absorbance from each well was collected at 548 nm using Spectra Max M2 with SoftMax Pro software.
2.5.3. Interleukin-6 (IL-6) For ELISA of interleukin 6 (IL-6), all solutions were freshly prepared as mentioned below. In detail, coating buffer (NaHCO3 (0.84 g) + Na2CO3 (0.356 g) in 100 mL), blocking buffer (1% BSA in PBS), assay diluents (1% (w/v) BSA + 0.05% (v/v) Tween PBS or 10% (v/v) FBS in PBS), washing buffer (0.05% (v/v) Tween in PBS). In step 1, Maxisorb plate was coated with 100 μL of antibody solutions and incubated at 4 °C overnight. In step 2, coated plate wells were blocked by adding 200 μL of blocking buffer and incubated for 2–3 h at room temperature (Plate A). Separately, Raw 264.7 macrophages cells were incubated with different concentrations of CDs (50–400 mg/μL) for 24 h (plate B). In step 3, 100 μL of supernatant from each well of plate B was aspirated and added to plate A and incubated for 2–4 h at room temperature. In step 4, Biotinylated antibody solution (100 μL) was applied to each well and plate was left for 1 h at room temperature. In step 5, 100 μL of Streptavidin HRP (1:2000 (v/v)) was added to each well of the plate and kept at room temperature for 1 h. washing of the plate with washing buffer was assured at each step. After washing, 100 μL of substrate solution (tetramethylbenzidine) was added in each well and incubated for 30 min in dark. Then stop solution was added. At the end, spectrophotometric analysis of each well was made, and the absorbance was recorded at 450 nm via Spectra Max M2 and the data was analyzed by SoftMax Pro software.
2.5.2. Tumor necrosis factor alpha (TNF-α) All solutions were freshly prepared for enzyme linked immunosorbent assay (ELISA) of tumor necrosis factor alpha (TNF-α). Briefly, all required buffers were prepared, coating buffer [NaHCO3 (0.84 g) + Na2CO3 (0.356 g) in water (100 mL), blocking buffer (1% (w/v) BSA in PBS), assay diluents (1% BSA + 0.05% (v/v), Tween in PBS or 10% FBS in PBS, washing buffer (0.05% (w/v) Tween in PBS]. In step 1, Maxisorb plate was coated with 100 μL antibodies solution and incubated at 4 °C overnight. In step 2, coated plate wells were blocked by adding 200 μL of blocking buffer and then incubated for 2–3 h at room temperature (Plate A). In a separate plate, Raw 264.7 macrophages cells were incubated with different concentrations of CDs (100 to 400 mg/μl) for 24 h (plate B). In step 3, 100 μL of the supernatant from the each well of Plate B, was aspirated and added into the wells of Plate A and left for 2–4 h at room temperature. In step 4, Biotinylated antibody solution (100 μL) was applied to each well and left for 1 h at room temperature. In step 5, Streptavidin HRP (1:2000 (v/v) 100 μL was added to each well and left for 1 h. For all above mentioned steps, plate was carefully washed with washing buffer. A 100 μL of substrate solution (tetramethylbenzidine was added to each well and incubated for further 30 min in dark. Finally stop solution was added and
2.6. Cell uptake study by flow cytometry RAW 264.7 macrophages 1.4 × 104 cells were seeded in 12-well plate and treated with two different concentrations of CDs; 200 and 400 μg/mL for 24 h. No dye was used for this experiment. For each sample, 3 × 104 events were analyzed using BD LSRFortessa Dual instrument with Flowjo software (version7.6.1). 3
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in the standard BSA scan) (Fig. S-5B). These peaks are attributed to NeH bending and NeH stretching which created an overall positive surface charge on the surface of BSA-CDs [49–51] which was further confirmed by a positive zeta potential value (+ 38.18) of BSA-CDs. In previous studies BSA coating had been reported to reduce the negative surface charged density and exerts overall positive charge to polystyrene NPs [52]. The emission spectra of the dialyzed and purified water dispersed and DMEM media dispersed CDs samples were analyzed at the excitation wavelength of 365 nm. To study the effect of dispersion media on the optical properties of CDs, the emission behavior of CDs was checked in both water and DMEM cell culture media. When dispersed in water, it was observed that CA-CDs had high PL intensity emission peak at 455 nm (Fig. S-6A) compared to the BSA-CDs which showed peak at 435 nm (Fig. S-6B). Compared with CDs dispersed in water, CDs dispersed in DMEM, emitted low PL intensity and in both cases, there was a slight shift in the peaks too (Fig. S-6C, S-6D). The rich composition of DMEM (minerals, vitamins and amino acids) might enhance the electrostatic interactions between CDs and surrounding media which ultimately attenuated the PL intensity of CDs in DMEM, as compared to water. PL intensity of CDs can be influenced by a wide range of factors such as concentration, doping agent, precursor and surface coating [1,2,12,23]. In both, water and DMEM medium the emission intensity of CA-CDs was found to be higher than that of BSACDs. BSA contain two residues of tryptophan (fluorescent amino acids) in its structure [46], which might also be responsible to delocalize the free electron density on the surface of CDs. The emission intensity of CA-CDs was higher than that of BSA-CDs, which is possibly due to the high density of carboxyl groups present on its surface. Earlier it was reported that the number of carboxyl groups on the outer surface of CDs is directly linked with enhanced PL [11,17,45]. This may be the reason for the high luminescence observed with the CA-CDs. After the synthesis and characterization of CDs, the first step was to check the surface potential-based biocompatibility or cellular cytotoxicity. Three cell lines were opted namely 3T3 fibroblast, MDA-MB231 breast cancer and RAW 264.7 macrophages. Alamar blue assay was performed to assess the cellular viability percentage within the concentration range of 0–400 μg/mL for CDs. As shown in (Fig. 3A, S-8C) and (Fig. 3B, S-8D), it was observed that after 24 and 72 h of incubation neither the CA-CDs nor the BSA-CDs were found to cause any toxicity to 3T3 fibroblast cells at any concentration range 0–400 μg/mL respectively. Similarly, for MDA-MB-231 breast cancer cells, no cell death was observed at 24 and 72 h, when incubated with CDs of both CA (Fig. S7A, S-8E) and BSA (Fig. 7B, S-8F). However, at concentration of 400 μg/ mL, CDs of CA caused significant reduction in the cell viability (~75%) of MDA-MB-231 breast cancer cells at 72 h (Fig. S-8. Similarly, upto 400 μg/mL of CA-CDs and BSA-CDs, no cytotoxicity was observed with RAW 264.7 macrophages (Fig. 4). It was reported earlier that CDs caused decrease in the viability and showed toxicity with RAW 264.7 macrophages cells at of 62.5 μg/mL and 250 μg/mL
Fig. 2. Transmission electron micrographs of CDs, A. CA-CDs, B. BSA-CDs.
3. Results and discussion Overall this study was designed to elucidate the impact of surface charge on CDs by investigating their cellular toxicity, uptake and inflammatory effects. In this regard, two oppositely charged CDs were separately studied for their interaction with different cell lines (Fig. 1). Both CDs exhibit an intense blue color under UV-light (λex: 365 nm) (Fig. S-1). The average diameter and zeta potential of CDs were estimated by dynamic light scattering (DLS). Results showed that the average hydrodynamic radius of the both CA-CDs (Fig. S-2A) and BSACDs (Fig. S-2B) was ~2–3 nm. The charge on the CDs was estimated via zeta potential measurements carried out at neutral pH in phosphate buffer saline (PBS). Overall charge found on BSA-CDs (Fig. S-3B) was positive i.e. +38.18 mV, while on CA-CDs a negative charge was found i.e. -30.75 mV (Fig. S-3A). Moreover, the size of CDs was confirmed via transmission electron microscopy (Fig. 2) and atomic force microscopy (AFM) (Fig. S-4). The results showed that both CA-CDs and BSA-CDs (Fig. 2) were uniformly dispersed and clearly in the size range from 2 to 3 nm, which is in accordance with DLS results and the previous reports [46–48]. The type of functional groups on the prepared CDs were analyzed and confirmed by FTIR spectroscopy. The IR peaks appearing in spectrum of CA-CDs at 1072, 1219 are due to the CeO stretching and bending vibrations while the peak between 2500 and 3200 cm−1 are attributed to the presence of OeH moiety in carboxylic (–COOH) groups. The peak at 1369 cm−1 is due to CeN stretching vibrations of amine (–NH2) group. The peaks observed at 1549 and 1742 cm−1 are due to and NeH bending and CeO stretching vibrations respectively. The amide peaks in the citric acid CDs scan is inserted due to the diethylenetriamine, which is absent in the citric acid scan (Fig. S-5A). The strong peak of carboxyl (1072 cm−1) moiety [48] in the scan, depicts their abundance on CA-CDs and assumed to create an overall negative charge on their surface, which was further confirmed by zeta potential (−30.75 mV) measurements. In case of BSA-CDs, which were prepared by the breakdown of complex BSA molecule in presence of sulfuric acid, the intense peaks were observed at 1521, 1642 and 3283 cm−1 (absent
Fig. 3. Cell viability of 3T3 Fibroblast cells A. with CA-CDs B. with BSA-CDs. in the concentrations 0–400 μg/mL of CDs after 24 h incubation. 4
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Fig. 4. Cell viability of RAW 264.7 macrophages cells A. with CA-CDs B. with BSA-CDs. in the concentrations 0–400 μg/mL of CDs after 24 h incubation. Table 1 A comparison of different biocompatible concentrations of CDs in previous studies. Sr.
Carbon Dots
Biocompatible concentrations range
Cell type
Reference
1 2 3 4 5 6 7 8 9 10 11
N-doped carbon dots Nitrogen and sulfur co-doped carbon dots Mesoporous silica nanoparticles carbon dots (MSNs-CDs) Metal-doped carbon dots Manganese-doped carbon quantum dots Carbon dot-ovalbumin (CD-OVA) Metal ions doped carbon quantum dots Citric acid-based carbon dots Glucose based carbon dots, carbon dots-Ricin toxin binding subunit B (CDs-RTB) Aspirin-based carbon dots Citric acid and BSA based carbon dots
10–200 μg/mL 25–400 μg/mL 0–100 μg/mL 10–500 μg/mL 0–500 μg/mL 0–1000 μg/mL 0–200 μg/mL 0–600 μg/mL 0–100 μg/mL 0–100 μg/mL 0–400 μg/mL
HeLa, 16HBE HeLa HeLa, MCF-7, A549, L929 PC12 HeLa DCs HeLa C6 RAW 264.7 RAW 264.7 3T3, MDA-MB-231, RAW 264.7
[31] [17] [30] [13] [14] [4] [15] [21] [53] [54] Our research
concentrations [39]. Zheng et [34] and Li et al [53] reported that CDs of citric acid origin caused no cellular toxicity upto 500 μg/mL concentration. In our perception, presence of carboxyl, hydroxyl, amine on the outer surface of CDs and absence of any complex molecule might have been the reason for high cell viability observed with these CDs. It is now a well-known fact that the surface functionalities/surface coating on the CDs play a critical role in determining the cellular toxicity [44]. Up till now, our results indicated that surface charges had no negative effect on cell viability in any of the three cell lines studied at 24 h time period. Our results are in accordance with previous reports (Table 1). However, depending on the surface passivation/functionalization and doping, the lethal/cytotoxic concentrations of CDs may be different in each case. For the reference, a comparison of cytotoxic concentrations of CDs, from previous studies is compiled in Table. 1. Besides the toxicity of CDs, a rather neglected aspect of CDs, is to elaborate the relation between the surface charge of CDs and the immune response against the CDs. Macrophages engage in the regularization of immune system by releasing inflammatory cytokines such as NO, TNF-α and IL-6 [48]. Therefore, these markers were used in these studies to investigate the surface potential based immunological role of CDs. Nitric oxide (NO) test was conducted to check initial inflammatory behavior of CDs in RAW 264.7 macrophages. It is evident that after 24 h of incubation, no nitric oxide (NO) production was observed neither against the CA-CDs (Fig. S-9A) nor the BSA-CDs (Fig. S-9B) upto 400 μg/mL concentration. Further pro-inflammatory behavior of both CDs was evaluated through ELISA of tumor necrosis factor alpha (TNF-α). Interestingly, no release of TNF-α was observed against any concentration of negatively charged CA-CDs (Fig. 5A), while, in case of BSA-CDs, the results indicated a concentration dependent (100–400 μg/mL) increase in the release of TNF-α (Fig. 5B). To further validate charge based pro-inflammatory response of CDs, interleukin 6 (IL-6) ELISA was performed. Similar to TNF-α results no IL-6 release was observed at any concentrations of CA-CDs (Fig. 6A),
while at 400 μg/mL, BSA-CDs caused a significant high release of IL-6 as compared to negative control (Fig. 6B). In a previous report, dose dependent increase in the inflammatory factors (i.e. NO, TNF-α and IL-6) was observed with Ricin toxin binding subunit B (RTB) attached CDs, which depicted a correlation between the release of inflammatory cytokines (TNF-α and IL-6) and the toxic surface functionality on CDs [53]. Those findings were strongly linked to the proved role of RTB to induce cell mediated immunity and not due to carbon dots. In current work, we used CA and BSA for CDs synthesis. Both were biocompatible molecules, which broke down to give carbon dots bearing small surface functionalities with no toxicity. As evident from our studies, only positively charged CDs (BSA-CDs) boosted TNF-α and IL-6 level. The positively charge CDs when interact with negatively charged cell membrane, may get agglomerated/bound with cell membrane which caused inflammation. It was also reported earlier that positively charged nanoparticles taken up by macrophages via micropinocytosis caused disruption in the plasma membrane which led to the up regulation of TNF-α and IL-6 and ultimately showed inflammatory responses [55,56]. For CA-CDs, no inflammatory cytokines were released (Fig. 5A, 6A). Generally, negatively charged particles are internalized through clathrin and dynamin dependent endocytosis in macrophages [55]. Moreover, similar charges on CA-CDs and cell membrane, may not cause any agglomeration and therefore, no inflammatory response was triggered. This finding is strongly supported by a pervious study that when lipo-polysaccharide (LPS) stimulated macrophages cells were treated with negatively charged particles, they rather suppressed inflammation [57]. In order to correlate and further elaborate the link between surface charge of CDs and inflammation it was necessary to check their cellular uptake or internalization by RAW 264.7 macrophages cells. Flow cytometry is considered the best technique to quantify the internalized materials within cell [58]. To analyze cellular uptake, two concentrations of CDs i.e. 200 μg/mL and 400 μg/mL were incubated with RAW 264.7 macrophages cells for 24 h. At 400 μg/mL 5
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Fig. 5. The expression level of inflammatory biomarker TNF-⍺ secreted by RAW 264.7 macrophages cells incubated with CDs for 24 h A. CA-CDs, B. BSA-CDs.
Fig. 6. The expression level of inflammatory biomarker IL-6 secreted by RAW 264.7 macrophages cells incubated with CDs for 24 h A. CA-CDs, B. BSA-CDs.
Fig. 7. Flow cytometric cell uptake studies of CDs for 24 h incubation, Where X. is untreated sample of RAW 264.7 macrophages used as negative control A. CA-CDs 200 μg/mL treated cells uptake, B. BSA-CDs 200 μg/mL treated cells uptake.
concentration, cellular uptake of CA-CDs was noted to be ~20.3% (Fig. 8A) which was higher than that of observed with untreated cells (negative control) (Fig. 7X) and BSA-CDs (~0.628%) (Fig. 8B). At 200 μg/mL concentration, CA-CDs showed a cellular uptake of ~6.25% (Fig. 7A), while for BSA-CDs it was ~1.24% (Fig. 7B). The flow cytometry data support our previous findings. Overall, negatively charged structure of the cell membrane allowed the negatively charged materials to pass through easily. It was reported
earlier that negatively charged particles showed higher cellular uptake [59,60]. The cell membrane hardly allowed positively charged materials to pass through due to the agglomeration of material with cell membrane. In the light of these facts, the cellular uptake of positively charged BSA-CDs was less, this might be due to the composition of cell membrane which has pivotal role in cellular uptake. For this reason positively charged CDs, caused macrophages to show inflammatory response [61]. The extent of cellular uptake of CDs is also related with 6
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Fig. 8. Flow cytometric cell uptake studies of CDs for 24 h incubation A. CA-CDs 400 μg/mL treated cells uptake, B. BSA-CDs 400 μg/mL treated cells uptake.
inflammation, but it may be different for different surface passivation/ functionalization [62]. [6]
4. Conclusion
[7]
The results of this study showed that an overall surface potential of CDs has a fundamental role to determine their cellular uptake and inflammatory response. The CDs obtained from CA and BSA were not toxic upto 400 μg/mL. The negatively charged CA-CDs showed no inflammatory response but a high cellular uptake as compared to BSACDs. By taking these into account CA-CDs can be used as carrier to enhance the uptake of drug/vaccine/therapeutic protein. The positively charged BSA-CDs caused increase in the release of TNF-α and IL-6 level and thus may be considered for immuno-stimulating activities. However, to use CDs in clinical applications, further studies are mandatory to investigate their persistence, degradation, and excretion, long term toxicological and associated immunological responses.
[8] [9] [10] [11] [12] [13]
Declaration of Competing Interest
[14]
None.
[15]
Acknowledgement
[16]
We are thankful to Prof. Vincent M. Rotello for providing lab space and expertise to conduct this research at department of Chemistry, University of Massachusetts, Amherst, USA. MU and AI thanks to Higher Education Commission (HEC), Pakistan, for financial support (HEC NRPU 7684).
[17] [18]
Appendix A. Supplementary data
[19]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.colcom.2020.100243.
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