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Keywords:

  • cadmium selenide;
  • quantum dots;
  • water soluble;
  • toxicity;
  • DNA damage;
  • red blood cell;
  • platelets

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cadmium selenide (CdSe) quantum dots have gained increased attention for their potential use in biomedical applications. This has raised interest in assessing their toxicity. In this study, water-soluble, cysteine-capped CdSe nanocrystals with an average size of 15 nm were prepared through a one-pot solution-based method. The CdSe nanoparticles were synthesized in batches in which the concentration of the capping agent was varied with the aim of stabilizing the quantum dot core. The effects of the CdSe quantum dots on DNA stability, aggregation of blood platelets, and reducing activity of iron were evaluated in vitro . DNA damage was observed at a concentration of 200 μg/mL of CdSe quantum dots. Furthermore, the CdSe nanocrystals exhibited high reducing power and chelating activity, suggesting that they may impair the function of haemoglobin by interacting with iron. In addition, the CdSe quantum dots promoted aggregation of blood platelets in a dose dependent manner. © 2012 IUBMB IUBMB Life, 64(12): 995–1002, 2012


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Nanostructured materials offer some attractive possibilities in the fields of biomedicine and bioanalysis. In particular, CdSe quantum dots have been identified as an important material because of their unique optical properties, which can be tuned across the visible spectrum. The potential use of Cd-based nanoparticles in bioapplications such as bioimaging and biosensors (1) demands that we understand their bioimpact. Human tissue is permeable to far infra-red light. CdSe nanoparticles may thus access the human body either through injured tissue or through various exposure routes such as inhalation and dermal contact (2, 3). CdSe quantum dots are currently under investigation for potential application in various fields, amongst them are the following: (i) diagnosis and treatment of diseases, such as cancer and Alzheimer's (4, 5); and (ii) drug delivery (6).

Although it is well known that bulk CdSe is cytotoxic, it has been suggested that CdSe nanoparticles are cytocompatible and safe for use (7). This postulate is based in part on the use of protecting groups or surface modifications around the CdSe core of the quantum dot. These coatings have been shown to be protective, but their long-term stability has not been confirmed (7). It has been observed that exposure of mice to CdSe nanoparticles led to their ready assimilation by various organs (8). The stability of Cd-based nanoparticles has been found to influence their toxicity (9). Using nanoparticles of 2- and 5 nm diameter, respectively, Pace et al. (9) demonstrated that toxicity of Cd-based nanoparticles in Daphnia magna was as a result of decomposition of the nanoparticle coating, leading to leakage of Cd2+ ions into the environment. This suggests that quantum dots can be rendered nontoxic initially for in vivo use when appropriately coated. It has been further noted that apart from release of Cd ions into the medium, the toxicity of even fairly stable CdSe quantum dots may still occur through other mechanisms (9). Altogether, this warrants the need to further explore the long-term stability of the coatings used both in vivo and in vitro (10, 11). Although there is evidence suggesting that CdSe quantum dots could be toxic (12), literature is fraught with contrasting views (2). In addition, the toxicity of Cd-based quantum dots is thought to depend on several factors such as charge, length of ligand, and size (13). Nagy et al. (13) further observed that the toxicity of Cd-based quantum dots is regulated by charge, length of ligand, and size. However, charge and ligand length appeared more important than size. Because the toxicity of a nanoparticle is a function of several of its physico-chemical features, it could be assumed that nanoparticles influence physiological events through several mechanisms. This makes it essential to identify the broad spectrum of the action of these species on biochemical pathways. A study by Liu et al. (8), reported that CdSe quantum dots induced liver damage primarily through oxidative stress. On the other hand, a study by Nagy et al. (13), suggest that the toxicity of CdSe quantum dots is independent of oxidative stress. This implies that whilst there is relative consensus that CdSe quantum dots are toxic, there is controversy regarding their mechanisms of action.

Various exposure routes such as inhalation, dermal, oral, injection, and ingestion during the synthesis and applications of CdSe quantum dots could possibly lead these nanoparticles to the blood circulatory system where interaction with blood cellular components will take place. This work reports the synthesis of water-soluble, cysteine-capped CdSe nanoparticles using a previous reported one-pot solution-based method (11). The approach involved the reduction of selenium by sodium borohydride forming sodium hydrogen selenide followed by the addition of the cadmium salt, and capping group, cysteine; under controlled reaction conditions. This route produces water-soluble and biologically capped CdSe quantum dots.

We then conducted a battery of in vitro assays to establish the effect of CdSe quantum dots on the stability of DNA, aggregation of blood platelets, and reducing activity of iron. Our findings suggest that CdSe quantum dots have the capability to damage DNA, promote platelet aggregation, and they influence the reducing activity of iron. This study further highlights the potential toxicity of CdSe nanoparticles and provides insights into some of the broad mechanisms of action by which these quantum dots influence physiological events.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Synthesis of CdSe Quantum Dots

Water-soluble, cysteine-capped CdSe quantum dots were synthesized following the procedures described by Oluwafemi et al. (11). Selenium powder was added to 30 mL of deionized water to a final concentration of 0.32 mM, and the solution was placed in a three-necked round-bottomed flask at room temperature. Sodium borohydride (0.81 mM) was added to the reaction mixture under inert conditions. After 2 h, 20 mL of 3.2 × 10−4 M solution of CdCl2 and L-cysteine ethyl ester hydrochloride were added to the reaction mixture in molar ratios of 1:10, 1:20, and 1:30. The reaction mixture was then heated at 90 °C for 6 h under nitrogen gas. The cysteine-capped CdSe particles were separated from the mixture by filtration techniques, and the sample was then concentrated by rotary evaporation. A brownish red to orange material was precipitated with acetone. This product was centrifuged to generate a pellet fraction that was then dried under vacuum and weighed out to give a material which was readily dispersible in water.

Morphological Characterization of CdSe Quantum Dots Using HRTEM and TEM

Samples for transmission electron microscopy (TEM) analysis from all three batches (ratios of 1:10, 1:20, and 1:30) were prepared by placing an aliquot of CdSe quantum dots solution onto an amorphous carbon substrate supported by a copper grid and then allowing the solvent to evaporate at room temperature. The morphology and particle sizes of the samples were characterized using a JEOL 1010 TEM at an accelerating voltage of 100 kV. Pictures were captured using a Megaview III camera and imaged using Soft Imaging Systems iTEM software. Detailed morphological and structural features were investigated using high-resolution transmission electron microscopy (HRTEM) analysis coupled to a JEOL 2010 transmission electron microscope operated at an accelerating voltage of 200 kV.

Optical Characterization of CdSe Quantum Dots Using UV and PL Spectroscopy

A Perkin-Elmer Lamda 20 UV–vis spectrophotometer was used to carry out optical measurements in the 200–1100 nm wavelength range at room temperature. Samples were placed in quartz cuvettes (1 cm path length) and the absorbance was recorded. Photoluminescence (PL) spectra were recorded at room temperature using a Perkin-Elmer LS 55 luminescence spectrometer with a Xenon lamp over a range of 400–800 nm. The samples were placed in quartz cuvettes, and the excitation peaks were analyzed and recorded.

DNA Damage Assay

This experiment was conducted by modifying the method described by Habib and Tabata (14). pGEM®-T Easy (Promega, USA) plasmid DNA was exposed to cysteine-capped CdSe quantum dots from different batch ratios (1:10, 1:20, and 1:30) at variable concentrations (200, 600, and 1000 μg/mL). The CdSe quantum dots (6 μL) were suspended in phosphate buffered saline in the presence of DNA (300 ng) at a final volume of 20 μL and incubated for 24 h at 37 °C. Titanium dioxide (TiO2), which is known to induce DNA damage (15), was used as a positive control at a final concentration of 1000 μg/mL. After incubation, an aliquot of 5 μL of sample was taken and diluted up to 1 mL for spectrophotometric analysis at 260 nm. A total of 5 μL of loading buffer was added to each remaining sample to stop the reaction, before running the samples on an ethidium bromide stained, 0.8% agarose gel at 70 V for 1 h 30 min. The agarose gels were viewed and imaged on the GENE Genius vacutec Bio imaging system.

Platelet Aggregation Assay

This experiment was conducted on platelets that were isolated from rat blood based on the method as described by Mekhfi et al. (16). The study was approved by the Ethics Research Committee of the University of Zululand. Diethyl ether was used to anaesthetize the rats, and this was followed by dissection to collect blood from the abdominal aorta. The collected blood was mixed with acid dextrose anticoagulant (5:1 v/v). The platelets were obtained from the blood through a series of centrifugation and wash steps. The blood was centrifuged at 1,200 rpm for 15 min, thereafter, at 2,200 rpm for 2 min before the supernatant was collected and centrifuged at 3,200 rpm for 15 min. The sediment containing the platelets was resuspended in 5 mL of washing buffer and centrifuged at 300 rpm for 15 min. The supernatant was discarded, and platelets were suspended in 10 mL of resuspension buffer. Calcium chloride was added to the prepared platelets in the ratio 0.4 mL platelet solution: 10 μL of CaCl2 (final concentration of CaCl2 was 1.3 mM). A platelet activating enzyme, thrombin (5 U/mL) was used as a positive control, and heparin (0.3 mg/mL) was used as a negative control in this experiment. A total of 150 μL of platelets and 150 μL of CdSe quantum dots of different concentrations and different batch ratios (ranging from 200, 400, 600, 800, and 1,000 μL/mL) were added in each well of the microplate. The samples were preincubated at 37 °C for 5 min. The reaction was monitored at 415 nm for 20 min at 30 s intervals using a Biotek plate reader mounted with Gen5 software. All methods were repeated in triplicate, and the results were represented as mean ± SEM. Students t-test was used to analyze statistical difference between controls and samples exposed to CdSe quantum dots.

Assessment of the Iron Reducing Power of CdSe Quantum Dots

This experiment was conducted by adopting the method described by Oyaizu (17). Test tubes were set up in triplicate with 10 μg/mL of CdSe quantum dots from different batch ratios (1:10, 1:20, and 1:30) at the various concentrations (200, 400, 600, 800, and 1000 μg/mL), 2.5 mL of phosphate buffer and 2.5 mL of 1% potassium ferricyanide were added in each corresponding test tube and incubated at 50 °C for 20 min, followed by the addition of 2, 5 mL of 10% trichloroacetic acid and left to stand for a further 5–10 min before being centrifuged at 1,000 rpm for 10 min. Thereafter, 2.5 mL of supernatant was diluted by adding 2.5 mL of distilled water. Finally, 0.5 mL of 0.1% ferric chloride was added to the solution, and the absorbance was read at 700 nm.

Assessment of the Chelation Activity of CdSe Quantum Dots on Iron

This experiment was conducted based on a method as described by Derker and Welch (18). Test tubes were set up in triplicate. A total of 3.75 mL of deionized water, 0.1 mL FeCl2, and 0.2 mL of ferrozine was added to 10 μg/mL of CdSe quantum dots from different batch ratios (1:10, 1:20, and 1:30) at various concentrations (200, 400, 600, 800, and 1000 μg/mL) and left to stand for 10 min. Citric acid (0.065 M) and 7.5 mM EDTA were used as positive and negative standards, respectively. Absorbance was read at 562 nm, and the percentage chelating activity in relation to concentration of the quantum dots was calculated using the following formula.

  • equation image

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

One-Pot Solution-Based Synthesis of Biocompatible CdSe Quantum Dots

Water-soluble, cysteine-capped CdSe quantum dots were successfully prepared using the one-pot solution-based route (11). Increasing molar ratios of cadmium salt to capping agent were used in this method, with the aim of creating a more stable or saturated capping layer that surrounds the quantum dot core. Molar ratios of 1:10, 1:20, and 1:30 were used. A color change was observed as the molar ratios between cadmium chloride and the capping group, cysteine increased. The batch ratio of 1:10 exhibited a light golden yellow color, batch ratio 1:20 produced a darker orange color, and batch ratio 1:30 had a dark red color (Fig. 1).

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Figure 1. CdSe quantum dots generated using various molar ratios of cadmium salt and coating agent. (A) Represents CdSe quantum dots of sample batch ratio 1:10, note golden yellow color. (B) Represents CdSe quantum dots of sample batch ratio 1:20, note orange color. (C) Represents CdSe quantum dots of sample batch ratio 1:30, note dark red color. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Morphological Characterization of CdSe Quantum Dots

The morphology of CdSe quantum dots was studied using TEM and HRTEM techniques. The TEM results showed that these quantum dots appeared to be spherically shaped and monodispersed, with an average particle size of 15 ± 2.12 nm (Fig. 2). The nanoparticles produced showed similar characteristics across each batch ratio. The crystallinity of the CdSe nanoparticles was evidenced by the presence of distinct lattice fringes (Fig. 3). The lattice d-spacing of 0.34 nm observed corresponds to the (111) plane of cubic CdSe (Fig. 3C).

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Figure 2. TEM images of CdSe quantum dots (A) shows mono dispersed, spherically shaped CdSe quantum dots. Scale bar used was 80 nm (B) shows CdSe particles that clump together to result in agglomeration of particles. Scale bar used was 50 nm.

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Figure 3. Morphological characterization using HRTEM. (A) HRTEM images from batch ratio 1:10 note the overlapping crystal lattices from particles that are overlapping each other. (B) The presence of clearly defined lattice fringes to confirm the crystallinity of the CdSe quantum dots. (C) Shows the interplanar distance of 0.34 nm which is indexed to the (111) plane of cubic CdSe.

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Optical Characterization of CdSe Quantum Dots

UV–vis absorption spectroscopy and photoluminescent spectroscopy techniques were performed simultaneously. All the batches of CdSe samples had similar optical properties. Upon excitation, each sample of the CdSe quantum dots emitted in the blue region and exhibited an emission maximum of 410 nm. The UV–vis absorption spectrum of aqueous solutions of CdSe quantum dots showed an excitonic peak at 290 nm (Table 1). The increase in ratios of the cadmium salt and capping agent, during the synthesis of CdSe quantum dots did not affect their optical properties; furthermore, these optical characteristics were consistent with the results reported by Oluwafemi et al. (11). The band gap of the as-prepared CdSe quantum dots was ∼300 nm for each batch. After the synthesis and characterization of the cysteine-capped CdSe quantum dots, the particles were purified using 0.2-μm microfilters, and the average yield was calculated to be ∼±3000 ug. All the methods were repeated in triplicate, and the results were presented as mean ± SEM.

Table 1. Optical characterization of CdSe quantum dots
Batch ratio of CdSe quantum dotsUV–vis a bsorption peakBand gapPhotoluminescence emission maxima
  1. The data represent optical features of each batch ratio of cysteine-capped CdSe quantum dots emitting at the same range.

1:10290 nm300 nm410 nm
1:20290 nm300 nm410 nm
1:30290 nm300 nm410 nm

CdSe Quantum Dots Induce Damage to Plasmid DNA

Figure 4 shows the results observed for the in vitro interaction of DNA and CdSe quantum dots. The single band of DNA observed in the negative control of untreated DNA, marked as supercoiled form (lane N; Fig. 4A), represents plasmid DNA that was not exposed to quantum dots. This DNA migrated on the agarose gel as a distinct band made up of the supercoiled form of the plasmid. Lane P represents DNA that was treated using titanium dioxide (positive control), which is known to damage DNA (15). In this lane, two distinct bands of DNA were observed (Fig. 4A). The first band represents supercoiled DNA running at approximately the same size as molecular markers of 3000 base pairs (bp). A second band, representing a linearized form of the plasmid DNA, was obtained in the sample that was exposed to titanium dioxide nanoparticles. The partial conversion of some DNA from supercoiled to relaxed form in the sample treated with titanium dioxide potentially represents DNA damage. The relaxed form of the plasmid DNA was similarly observed in DNA exposed to variable concentrations of CdSe quantum dots. However, the band representing the relaxed form of DNA appeared less bold in the samples of DNA exposed to CdSe quantum dots that were prepared at the highest concentration of cysteine coating agent (1:30 molar ratio). Because damaged DNA absorbs more light at A260 nm compared to undamaged DNA, spectrophotometric analysis further confirmed the damage of DNA (Fig. 4B). In general, the absorbance readings of DNA treated with CdSe quantum dots increased in a dose dependent manner for the CdSe quantum dots made at varying concentrations of cysteine capping agent (Fig. 4B; lanes 1–8). The absorbance readings obtained were thus in agreement with data obtained using agarose gel electrophoresis.

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Figure 4. Induction of DNA damage by CdSe quantum dots. (A) Ethidium bromide stain of 0.8% agarose gel. Lane M: Molecular marker, Lane P: Positive controltitanium dioxide, Lane N: Negative control- untreated DNA, Lane 1: DNA treated with 200 μg/mL of CdSe quantum dots from batch ratio 1:10. Lane 2: DNA treated with 600 μg/mL of CdSe quantum dots from batch ratio 1:10. Lane 3: DNA treated with 1000 μg/mL of CdSe quantum dots from batch ratio 1:10. Lane 4: DNA treated with 200 μg/mL of CdSe quantum dots from batch ratio 1:20. Lane 5: DNA treated with 600 μg/mL of CdSe quantum dots from batch ratio 1:20. Lane 6: DNA treated with 1000 μg/mL of CdSe quantum dots from batch ratio 1:20. Lane 7: DNA treated with 200 μg/mL of CdSe quantum dots from batch ratio 1:30. Lane 8: DNA treated with 600 μg/mL of CdSe quantum dots from batch ratio 1:30. Lane 9: DNA treated with 1000 μg/mL of CdSe quantum dots from batch ratio 1:30. (B) Confirmation of DNA damage by spectrophotometric analysis. DNA exposed to increased concentrations of CdSe quantum dots from a given batch ration was associated with proportional DNA damage as confirmed by increase in absorbance values. (The data were expressed as mean SEM, n = 3). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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CdSe Quantum Dots Promote Aggregation of Blood Platelets

Blood clotting is an important physiological process that is under the control of complex physiological reactions. The aggregation of blood platelets is one of the most prominent reactions that promote blood clotting. This experiment was aimed at exploring the effect of CdSe quantum dots on blood platelets aggregation. The aggregation of blood platelets results in the formation of a complex that absorbs more light than would be absorbed by unaggregated platelets. The effects of CdSe quantum dots on platelet aggregation were investigated by exposing blood platelets isolated from rats to increased concentrations of CdSe quantum dots. CdSe quantum dots synthesized at the three different molar ratios of cysteine capping agent were used. The positive control was constituted of platelets that were exposed to thrombin, whereas the negative control consisted of platelets that were exposed to heparin. As expected thrombin promoted platelet aggregation, whereas heparin suppressed aggregation (Fig. 5). In general, platelet aggregation increased in a dose dependent manner across all the batches.

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Figure 5. Effects of CdSe quantum dots on platelet function. Thrombin has a higher absorbance reading then heparin. Note batch ratio 1:30 exhibits the highest platelet aggregation activity in a dose dependent manner. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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CdSe Quantum Dots Exhibit Iron Reduction and Chelation Activities

This experiment sought to establish the reducing power and chelating activities of CdSe quantum dots on iron. The ability of CdSe quantum dots to suppress the formation of the ferrous iron–ferrozine complex, alternatively expressed as Fe2+ chelating activity (%), was assessed. EDTA was used as a positive control as it is a strong chelating agent. EDTA (at 1000 μg/mL) registered 75% chelating activity (Fig. 6). The chelating activity of the CdSe quantum dots was investigated. CdSe quantum dots (batch ration 1:10) had a distinctly powerful chelating activity that was concentration dependent. On the other hand, CdSe quantum dots from batch ratios of 1:20 and 1:30 had undetectable chelating activity that compared with that of the negative control (untreated sample). The reducing power of CdSe quantum dots on iron was observed. Citric acid was used as a positive control in this experiment, and registered 80% reduction of iron. It was observed that the various batch ratios of CdSe quantum dots were capable of reducing iron in vitro in a concentration dependent manner (Fig. 7). Interestingly, the most reactive quantum dots were from the batch ratio of 1:10. This further suggests that reactivity of the CdSe quantum dots was a function of the concentration of capping agent used during synthesis.

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Figure 6. Chelation activity of CdSe quantum dots on iron. Iron is chelated by CdSe quantum dots in a dose dependent manner for batch ratio 1:10. Batch ratio (1:20 and 1:30) exhibited poor chelation activity across all concentrations. All experiments were done in triplicates. The data are expressed as a mean SEM, n = 3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 7. Reducing power activity of CdSe quantum dots on iron. Iron is reduced by CdSe quantum dots in a dose dependent manner. The lowest Batch ratio (1:10) exhibited the highest reducing power. All experiments were done in triplicates. The data is expressed as a mean SEM, n = 3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Water-soluble, cysteine-capped CdSe quantum dots were prepared using the one-pot solution-based route. The cadmium salt (cadmium chloride) and the capping agent (L-cysteine ethyl ester hydrochloride) were added to the reduced selenium in molar ratios of 1:10, 1:20, and 1:30. It was anticipated that increasing the concentration of cysteine in the reaction, would result in a more saturated and structurally stable capping layer which surrounds the surface of CdSe quantum dot core. This would ensure the generation of a product that is stable, especially under physiological conditions. Oxidation can occur between pairs of cysteine side chains to form disulfide bonds. The presence of such bonds plays an important structural role in stabilizing the surface coating layer around the CdSe core. It is important that the core and the component ions remain stable, to ensure that CdSe quantum dots maintain their integrity during interaction with biological entities.

Capping these quantum dots with cysteine serves many additional functions. Previous studies show that cysteine is the most suitable thiol that is able to encapsulate the CdSe quantum dot without disturbing its optical and electronic features (19). Cysteine functions as an agent for solubilization in water, stabilization of the core complex within the quantum dot, and finally for the possible conjugation with biomolecules. The CdSe core is attached to cysteine through the mercapto group. This means that the mercapto group binds to the cadmium atom and the polar amino side groups of cysteine, making CdSe hydrophilic quantum dots. These features give rise to great potential in terms of bioapplications such as drug, molecular, or gene delivery as the free ammonium and carboxylate groups are available for covalent binding to many biomolecules such as amino acids, proteins, hormones, and nucleic acids. All the batches of the CdSe quantum dots that were made using varying ratios of the cysteine capping exhibited similar physical characteristics (Fig. 2). These features are important as they collectively contribute to the potential toxicity of CdSe quantum dots (3). HRTEM images confirmed the crystalline nature of the semiconducting quantum dots. In most samples, the lattice fringes were clearly observed. UV and PL readings also revealed similar excitation peaks and emitting ranges for each batch ratio, confirming that irrespective of the increase in capping agent concentration, CdSe quantum dots still exhibited previously reported optical properties (19).

Although the toxicity of CdSe quantum dots has been demonstrated previously, most of the studies were based on use of cell-lines and whole animal studies (9, 13). In this study, we used CdSe quantum dots of larger size (15 ± 2.12 nm in diameter) than those that were used by Pace et al. (9) and evaluated their toxicity using a cheaper in vitro approach. Furthermore, there is need to expand our knowledge of the broad spectrum of mechanisms in which CdSe quantum dots may affect physiological events.

The purified CdSe quantum dots were exposed to DNA, RBC components, and blood platelets to examine the in vitro interactions of CdSe quantum dots with these important biological components. The damage of DNA observed in the presence of CdSe quantum dots may infer that these materials could be carcinogenic. Indeed, there is evidence from independent analyses suggesting that Cd-based quantum dots may damage DNA based on cell line- and whole animal based-studies (9, 13). However, this study demonstrates DNA damage based on the direct exposure of DNA to quantum dots. On the other hand, Nagy et al. (13) proposed that DNA damage was driven largely by expression of proinflammation factors that promote DNA damage. We contend that it is possible that Cd-based quantum dots may also induce DNA damage through direct oxidative damage.

Because there is evidence that CdSe quantum dots are readily assimilated by various organs in the body, it must be observed that they inevitably interact with blood. The effect of CdSe quantum dots on the function of blood is not fully known. This prompted us to explore the effect of CdSe on the reducing function of iron and to investigate their effects on platelet aggregation. Data from the iron reducing power and chelating activity of CdSe nanoparticles suggest that CdSe nanoparticles may have reduced iron by donating an electron to Fe3+, converting it to Fe2+. The CdSe quantum dots from batch ratio 1:10 exhibited high iron chelation activity. Batch ratio 1:20 and 1:30 exhibited very poor chelating activity. The explanations for these findings are related to the oxidative and reductive abilities of the cadmium and selenium ions released from the quantum dot core. Pace et al. (9) proposed that the toxicity of Cd-based quantum dots in Daphnia magna was as a result of decomposition of the nanoparticle coating, leading to leakage of Cd2+ ions into the environment. As the particle size decreases, the band gap increases, allowing the component ions of the CdSe core to exhibit high oxidative and reductive properties. Data based on the effects of CdSe quantum dots from the batch ratios of 1:20 and 1:30 suggest that by increasing the concentration of cysteine which surrounds the CdSe surface, the quantum dots were possibly better stabilized. Altogether, findings from this study, suggest that CdSe quantum dots may interfere with the function of RBCs. Their iron chelating and reducing powers could influence the role of haemoglobin in transporting oxygen and carbon dioxide. This study further supports the notion that may be one of the primary mechanisms by which Cd-based quantum dots affect biochemical events. This finding is in contrast to the observation by Nagy et al. (13) in which it was observed that Cd-based nanoparticles did not rely on oxidation to manifest toxicity. However, Nagy et al. (13) relied on indirect evidence to come to this conclusion as their approach was to observe if toxicity of the nanoparticles decreased upon introduction of an inhibitor of oxidation.

An assessment of the effect of CdSe on the aggregation of blood platelets showed that at a concentration of 1000 μg/mL, the CdSe quantum dots (all batch ratios) promoted aggregation of platelets. This suggests that CdSe quantum dots may promote blood clotting. CdSe quantum dots prepared using the highest molar ratio of cysteine capping agent (batch ratio 1:30) were also capable of promoting platelet aggregation, particularly at the highest concentration used (1000 μg/mL). This may imply that the high cysteine coating did not stabilize the nanoparticles enough to prevent platelet aggregation. A previous study showed that carbon nanoparticles promote thrombosis in rat models (20). On the other hand, another study demonstrated that polystyrene nanoparticles were able to reduce the rate of aggregation of blood by interacting with specific clotting factors (21). Thus, it seems that the mechanism in which specific nanomaterials interact with some of the constituents of blood determines whether they would promote or slow down blood clotting. Based on this study, CdSe quantum dots may promote thrombosis. This compromises the prospects of using CdSe quantum dots in biomedical interventions.

Recently, there was a call to conduct broad based investigations that seek to understand the behavior of CdSe nanoparticles under varied experimental conditions to control their biosafety (22). This study suggests that CdSe quantum dots damage DNA and may interfere with the function of blood. Our findings have contributed toward broadening the scope of our understanding of the toxicity of CdSe quantum dots.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by the Department of Science and Technology (DST) and National Research Foundation (NRF) of South Africa through the DST/NRF South African Research Chairs Initiative (SARCHi) program. We wish to further acknowledge the DST/NRF for a Well-Founded Laboratory Equipment grant awarded to AS.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. EXPERIMENTAL PROCEDURES
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES