Surface functionalization of highly luminescent carbon nanodots from Dioscorea hispida with polyethylene glycol and branched polyethyleneimine and their in vitro study

2.1 Materials

All chemicals were of reagent grade and used without further purification. BPEI, PEG_1500N, and quinine sulfate were purchased from Sigma-Aldrich (USA & UK). Ultrapure water (∼18.2 MΩ·cm, 25 °C) was obtained from a water purifying system (Sartorius, Model Arium®611DI). Embryos were obtained from the Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia.

2.2 Modification of the carbon nanodots

Starch nanoparticles were prepared from Gadong tubers using the method described by Kim et al. (2012). The starch nanoparticles were converted to C-dots following the method of Chin et al. (2012). The synthesized C-dots were modified according to the method outlined by Mao et al. (2010). Approximately 0.2 g of O; O-bis(3-aminopropyl)polyethylene glycol 1500 (PEG_1500N) was mixed with 50 mL of a C-dot solution (0.5 mg/mL), and the mixture was heated to 120 °C for 72 h under nitrogen flow. After the reaction, the PEG_1500N-passivated C-dots were cooled to room temperature followed by dialysis (cutoff molecular weight 8000–14,000) and ultrafiltration (cutoff = 30 kDa). The same procedure was used to modify the C-dots with BPEI.

2.3 Morphology and average particle size

The morphology and particle size of the C-dots and surface-passivated C-dots were analyzed using a high-resolution transmission electron microscope (HR-TEM) (JEOL, JEM-200F) with accelerating voltage 200 kV, and a transmission electron microscopy (TEM) (Philips) operated at 300 kV. A drop of the C-dots suspension was deposited on a carbon-coated microscopy grid and dried at room temperature. The modified C-dots average particle sizes were determined using the IMAGE-J and iTEM programs (Borse et al., 2017; Mao et al., 2010).

2.4 Particle size distribution and zeta potential analysis

The particle size distribution and zeta potential of both the C-dots and modified C-dots were determined using Dynamic Light Scattering (DLS) and Zeta analyzer (Malver Instrument Ltd.). The diluted aqueous C-dots solutions were sonicated for 15 min before analysis. The experiment was carried out three times to validate the data.

2.5 UV-visible absorbance measurements

UV-vis absorbance measurements of the sample were obtained using a UV-vis spectrophotometer (UV-2450 SHIMADZU). The suspension was diluted with ultra-pure water in a ratio of 1:2 (mL) and its absorbance was monitored between 200 and 800 nm (Mao et al., 2010). Ultrapure water was used as a reference.

2.6 Fluorescence of the modified C-dots

Fluorescence emission spectra were recorded using Perkin Elmer LS 55-Luminescence Spectrophotometer using path length of 1 cm slit width relative to excitation and emission were both 2.5 nm. The suspension was diluted with ultrapure water in a ratio of 1:2 (mL) and scanned from 450 to 800 nm, while excitation was tested from 200 to 500 nm (Mao et al., 2010). Gadong starch was used as a control.

2.7 Fourier transform infra-red spectroscopy

Fourier transform infra-red (FTIR) spectroscopy was performed to analyze the functional groups present on the surface of modified C-dots. The modified C-dots were prepared in a sodium chloride pellet, and the spectra were collected between 400 and 4000 cm⁻¹.

2.8 Ultraviolet lamp (UV-lamp)

The fluorescence properties of the modified C-dots were further confirmed using a UV-lamp. The modified C-dots were observed under the blue-light excitation at a wavelength of 365 nm.

2.9 Quantum yield measurements

The quantum yield (QY) of the modified C-dots was determined by a comparative method (Mendes et al., 2017). Quinine sulfate in 0.1 M H₂SO₄ (literature ϕ = 54%) was selected as a standard to calculate the OY of the test samples (i.e. modified C-dots) was dissolved in ultrapure water at different concentration (Wu et al., 2013). All the absorbance values of the solutions at the excitation wavelength were measured with UV-vis spectrophotometer. Photoluminescence (PL) emission spectra of all the sample solutions were recorded by Perkin Elmer LS 55- Luminescence Spectrophotometer at an excitation wavelength of 350 nm (Quinine sulfate). The integrated fluorescence intensity is the area under the PL curve in the wavelength range from 380 to 600 nm. A graph, was then plotted using the integrated fluorescence intensity against the absorbance and a trend line was added for each curve with intercept at zero. Absolute values were calculated according to the following equation (Zhu et al., 2015):

2.10 Photothermal performance of C-dots

The photothermal effect of the C-dots and modified C-dots was determined by using methods as previously reported (Lee et al., 2016) with slightly modifications. C-dots and modified C-dots (C-dots-PEG_1500N and C-dots-BPEI) were dissolved in Phosphate buffered saline (PBS) at different concentrations (0 mg/mL to 20 mg/mL) separately. Each C-dots solution (1 mL) was introduced in a quartz cuvette and irradiated with UV-lamp (365 nm), visible light (LED spotlight) and 532 nm- laser light (Mendes et al., 2017) at a power density of 1 W/cm² for 5 min. To observe the photothermal characteristics of C-dots and modified C-dots solutions, a thermometer was inserted into the cuvette to contact the C-dots solutions, and the temperature was recorded every 1 min. PBS (0 µg/mL) was used as a control.

2.11 Fish embryo toxicity (FET) tests

E3 embryo media (5.03 m M NaCl, 0.17 m M KCl, 0.33 m M CaCl₂·2H₂O, 0.33 m M MgSO₄·2H₂O and 0.1% (w/v) methylene blue) were used to wash and maintain viable fertilised eggs at room temperature at all times. In a 96-well plate, the embryos were placed in single wells 1 hpf and were maintained in 180 μL of E3 embryo medium at room temperature. The toxicity effects of C-dots, C-dots-BPEI, and C-dots-PEG_1500N on the development of Danio rerio embryo were evaluated by exposing fertilised eggs to 0.5 mg/mL concentration of unmodified and modified C-dots respectively and three replicates, (n = 3) were set for each of the test. In this study, embryos from the three early embryonic development stages (24 hpf, 48 hpf, and 72 hpf) were sampled and analysed. These FET tests were carried out in accordance with the OECD draft guidelines (OECD, 2006, 2013).

3.1 Morphology and average particle size

The C-dots synthesis was performed by a chemical ablation method by dehydrating starch nanoparticles with concentrated H₂SO₄ followed by breaking the carbonaceous particles into individual C-dots with HNO₃ and finally, passivated with passivating agents such as PEG_1500N and BPEI. The morphological characteristics of synthesized and surface passivated C-dots were observed by TEM and HR-TEM. Fig. 1(a–c) shows the TEM and HR-TEM image of modified and unmodified C-dots. A congruent round shape was obtained for all C-dots samples and the C-dots ranged in size from 6 to 25 nm (C-dots), 3 to 12 nm (C-dots-PEG_1500N), and 4 to 20 nm (C-dots-BPEI). Further heating has reduced the size of particles without changing the shape. The size distribution of the unmodified and modified C-dots was further verified by dynamic light scattering (DLS) analysis and shown in Fig. S1 (Supporting Information). The average size of C-dots and modified C-dots was measured to about 24 ± 4 nm (C-dots), 4.7 ± 1.3 nm (C-dots-PEG_1500N), and 6 ± 0.8 nm (C-dots-BPEI). Crystalline structure of C-dots was confirmed by HR-TEM analysis (figure a-IV). C-dots exhibited lattice spacing of 0.25 nm attributed to (0 0 2) facet of graphite indicating that the synthesized C-dots are graphitic in nature.

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Fig. 1

Morphology image of (I) TEM, (II) HR-TEM, and (III) Lattice space of (a) carbon nanodots (C-dots) (b) surface modified C-dots with PEG_1500N (c) surface modified C-dots with BPEI.

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However, the synthesis of the C-dots resulted in particles with a variety of sizes. Therefore, for efficient applications of C-dots, complex separation methods including dialysis, chromatography, electrophoresis and sucrose density gradient centrifugation became mandatory to obtain uniformly sized C-dots (Li et al., 2012; Oza et al., 2015; Pandey et al., 2013). This step is necessary to determine the fluorescence properties of C-dots appropriately and for their use in the bioimaging and biomedical fields.

The shape and size of particles are concern in the biomedical applications. Most of the synthesized C-dots show spherical or round shape with size diameter below than 10 nm (Qin et al., 2013; Borse et al., 2017; Xu et al., 2013; Sha et al., 2013; Mehta et al., 2014; Li et al., 2014; Amjadi et al., 2014). Recently, a new and unique shape of C-dots called clathrates-like nanostructure was found and it was used as a nanocarrier. The clathrates-like morphology of C-dots was found to be extremely effective for the rapid release of drug and therefore it was labelled as a powerful drug vehicle (Pandey et al., 2017). The smallest size which is below 10 nm is preferable and it will easily penetrate through the cell membrane. In addition, the shapes of particles also affect the biological responses, and spherical, discoidal, rod-like, or filamentous shape allow better cell penetration (Barua and Mitragotri, 2014).

3.2 Absorption analysis of the modified C-dots

Fig. 2 shows the absorption peaks of unmodified and modified C-dots with PEG_1500N and BPEI aqueous solution. The unmodified C-dots exhibited an absorbance peak at 290 nm. The absorption of the C-dots is typically ascribed to the presence of n → π^∗ electron transitions associated with the C⚌O functional group. The absorption peak of the PEG_1500N -modified C-dots occurred at 290 nm, which is like that of the unmodified C-dots. This absorbance peak indicated that the C⚌O bond was not broken, but the PEG_1500N was bonded to the C-dots by amide bonds formed by condensation (Bartelmess and Giordani, 2014).

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Fig. 2

UV absorbance spectrum of surface modified C-dots with PEG_1500N and BPEI and aqueous solution of (a) starch (b) C-dots (c) C-dots- PEG_1500N (d) C-dots-BPEI under UV-lamp.

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The UV absorption spectrum of the C-dots with modified with BPEI contains absorption peaks at 360 nm. A similar observation was reported by Dong et al. (2012) who reported UV-vis absorption peaks at wavelengths of 360 and 240 nm, resulting from the π → π^∗ transitions of C⚌C electrons (Liu et al., 2012; Shen et al., 2013).

The aqueous solution of C-dots appears dark yellow colour, which emits green fluorescence under UV-lamp (365 nm) as displayed in the Fig. 2. Surface modified C-dots with PEG_1500N and BPEI shows blue and dark green luminescence under 365 nm UV-ray whereas, raw starch solution does not show fluorescence properties. Therefore, the luminescence shown by the modified C-dots is proposed to arise from both the C-dots and the modifying agents (PEG_1500N and BPEI).

3.3 Bonding configurations of the functional groups in the modified C-dots

FTIR spectroscopy was used to obtain information on the bonding in the C-dots and the location of the hydrogen atoms within the particles. The FTIR spectra obtained for the unmodified C-dots and those modified with PEG_1500N and BPEI (alongside the spectra of the modifying agents alone) are shown in Fig. 3a and b, respectively. Both modified C-dots spectra contain a broad absorption peak, followed by several overlapping peaks where most of the bands are due to the characteristic vibrations of the chromophores of different functional groups in the C-dots internal structure.

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Fig. 3

FTIR Spectrum of modified C-dots with (a) PEG_1500N (b) BPEI.

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Most of the bands in the spectrum of the unmodified C-dots can be associated with the C—H and C⚌C bonds that are characteristic of organic compounds, and some of these bonds arise from the dehydration step (Galvez et al., 2002). The main band can be divided to four bands. The peak at 3433 cm⁻¹ corresponds to the stretching vibration of the O—H bond (Ray et al., 2009; Yan et al., 2014). The second band between 1500–2000 cm⁻¹ contains a peak at 1682 cm⁻¹, arising from the stretching of the carbonyl group in the C-dots (Ray et al., 2009). The hydroxyl peak at 3433 cm⁻¹ reduces the intensity of the carbonyl absorption at 1682 cm⁻¹. An absorption peak at 829 cm⁻¹ indicates that the starting material used is a polysaccharide. Absorption peaks at 2930, 1130, 1104, 997, 925 and, in particular, 832 cm⁻¹ (study 829 cm⁻¹) in the FTIR spectrum are derived from the α-glucose polysaccharides in the starting material (Prasad et al., 2011). Finally, an additional peak can be seen at 2092 cm⁻¹, and this corresponds to C—H stretching, while that at 1456 cm⁻¹ is due to the bonds in the aliphatic CH₃ groups of the C-dots (Ray et al., 2009).

The main band from the PEG_1500N spectrum Fig. 3a) can be divided to four groups. The peak at 3431 cm⁻¹ indicates NH stretching vibration (in the range of 3500–3000 cm⁻¹, equivalent to stretching vibrations of OH groups or NH), whereas peaks at 2884, 2232, and 2157 cm⁻¹ suggest the presence of CH₂, CH₃, and CN groups (in the range of 3000–2000 cm⁻¹) (Sachdev et al., 2013). Peaks at 1643, and 1573 cm⁻¹ indicated that CH, and NH groups are presented (in the range of 2000–1500 cm⁻¹) (Sachdev et al., 2013), and vibrations at 1467, 1344, and 1279 cm⁻¹, similar to those in the 3000–2000 cm⁻¹ range, revealed the presence of CH₂, CH₃, and CN groups (in the range of 1500–1000 cm⁻¹) (Sachdev et al., 2013).

The absorption band at 3417 cm⁻¹ represents OH and NH groups, and the peaks arising from these vibrations overlapped each other. CH₂ and CH₃ stretching vibrations resulted in absorptions at 2920 and 2882 cm⁻¹. A stretching vibration at 1700 cm⁻¹ corresponded to the amide I (—HCONH—) stretch, whereas the absorption peak at 1639 cm⁻¹ is possibly due to the conjugated of CN bond, while the peak at 1575 cm⁻¹ corresponds to amide II (NH). The new absorption peak at 1700 cm⁻¹ confirmed that the surface of the C-dots had been successfully passivated with PEG_1500N. Absorption peaks at 1469 and 1344 cm⁻¹ corresponded to —CH₃. In addition, the sharp peak at 1280 cm⁻¹ is due to the amine I of CN bond. A peak at 1110 cm⁻¹ resulted from the symmetric vibrations of the —C—O—C— group (Sachdev et al., 2013).

Fig. 3b shows the BPEI spectrum, which is divided into two groups. The first group is in the range of 3500–2500 cm⁻¹, which is equivalent to the stretching vibration of OH or NH. The peak at 3440 cm⁻¹ indicates an NH stretching vibration, whereas the absorption peaks at 2942 and 2868 cm⁻¹ are due to the presence of CH₂ groups. The second band lies within the range of 1500–1000 cm⁻¹, where peaks at 1606, 1457, and 1307 cm⁻¹ indicate the presence of NH, CH₂ and CN (Liu et al., 2012).

As shown in Fig. 3b, the spectrum of the C-dots surface passivated with BPEI contains absorption bands that are characteristic of the NH groups in BPEI in the range of 3442 to 1304 cm⁻¹, i.e., CH₂ at 2942, 2836, and 2100 cm⁻¹, and CN at 1304 cm⁻¹. Peak at 1590 cm⁻¹ which is caused by C⚌C stretching. This suggested BPEI was stable during the passivation of the C-dots. Furthermore, the sharp peak at 1700 cm⁻¹, which is characteristic of an amide (—CONH—) group, suggested the BPEI was held on the surface of C-dots by amide bonds. The surface modification at a low temperature (<200 °C) allowed the complete carbonation without destroying the BPEI layers, as shown by FTIR. There are strong linkages (—CONH—) between the carbonyl groups of C-dots and NH₂ group of the BPEI, providing a stable coating (Liu et al., 2012).

Zeta potential analysis was conducted to determine the electrical charges of particles on the surface of an unmodified and a modified C-dots. Zeta potential of aqueous C-dots solution was found to be −32.2 ± 11 mV which shows the presence of negatively charged carbonyl functional group on the surface while the modified C-dots with PEG_1500N and BPEI was found to be 6.80 ± 2.93 mV and 7.77 ± 3.08 mV, respectively (as shown in Fig. S2). The positive surface of modified C-dots is due the presence of positively charged amide functional groups, NH₂⁺ on the surface from the passivating agents.

3.4 Fluorescence properties of modified C-dots

The florescence spectra of PEG_1500N, BPEI, starch, C-dots, C-dots-PEG_1500N, C-dots-BPEI was recorded by changing the excitation wavelength. Fig. 4 shows the emission spectra of unmodified and modified C-dots, while the emission spectra of control materials such as PEG_1500N, BPEI and starch were shown in Fig. S3 of Supporting Information. The unmodified C-dots, which were synthesized from Gadong starch, naturally showed strong fluorescence. Therefore, the fluorescence of the modified C-dots can be compared to that of the unmodified C-dots based on their emission spectra.

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Fig. 4

The emission spectra of (a) unmodified C-dots; (b) PEG_1500N-modified C-dots; and (c) BPEI-modified C-dots at different excitation wavelength.

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The excitation wavelength when changes from 340 nm to 480 nm, the emission peak position of C-dots, C-dots-PEG_1500N, C-dots-BPEI is red-shifted to longer wavelength from 350 nm to 500 nm and fluorescence intensity decreased remarkably, indicating a strong dependence on the excitation wavelengths. The fluorescence intensity of the modified C-dots is higher than the unmodified C-dots, indicating that the polycations had been successfully deposited on the surface of the C-dots.

Although until now the origins of fluorescence in C-dots are unclear, it is expected that this wavelength-dependent phenomenon comes from the different distribution of emissive energy traps on the surface of C-dots and the modified C-dots. As suggested in the literature, the functional groups of precursors can be affected to the origin of the fluorescence emitted from C-dots (Liu et al., 2011; Zhao et al., 2008). A combination of amine and carboxyl groups in the compound resulting to enhance the most fluorescence activity of the modified C-dots (Baker and Baker, 2010; Hsu and Chang, 2012).

The fluorescent properties can be described by using quantum confinement theory, where the modifying agent traps the emission energy, thus exhibiting strong fluorescence, which has been previously observed in silicon nanocrystals (Mao et al., 2010). The establishment of strong bonds between the PEG_1500N and BPEI with the C-dots trapped this energy, thus resulting in greater luminescence intensity compared to that of the unmodified C-dots. Functional groups such as carbonyl and amines were introduced to the surface of modified C-dots, and these also entrap the excitation energy, resulting in greater photoluminescence (Zhou et al., 2012).

Based on photoluminescence properties, the quantum yield (QY) of modified C-dots with PEG_1500N and BPEI was calculated by using quinine sulfate (excitation wavelength 350 nm) as a reference compound. Modified C-dots exhibited strong fluorescence with QY of C-dots-PEG_1500N and C-dots-BPEI was found to be 15% and 12.6% respectively (as shown in Fig. S3), which was comparable to previously reported modified C-dots (Huang et al., 2014; Wu et al., 2013; Liu et al., 2012; Zhu et al., 2015; Mehta et al., 2014).

3.5 Photothermal effects of C-dots

The photothermal properties of C-dots and modified C-dots was examined using UV-lamp, visible lamp and 532 nm laser light (1 W/cm²) for 5 min. The temperature changes of C-dots and modified C-dots (Fig. S5 Supporting Information) at different concentration (0.1–20 mg/mL) were measured and plotted in Fig. 5 (only certain significant concentrations of C-dots were shown while full result was reported in the Supporting Information section). In Photothermal study, the temperature increment was observed with increasing concentration of both C-dots. Modifying agents (PEG_1500N and BPEI) on C-dots shows a slight impact on the temperature increment. Table 1 summarized the temperature rose for each sample when irradiated for 5 min by using UV-lamp, laser and visible lamp. The initial temperature recorded 27 °C (for UV-lamp and Visible lamp). Based on the observation made for each instrument, the modified C-dot gave better photothermal stability performance compared to the unmodified.

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Fig. 5

Temperature increment of C-dots (0.1–20 mg/mL) after irradiation with UV-lamp (a), Visible lamp (b) and Laser (c) for 5 min.

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Using the similar method previously mentioned above, both samples (20 mg/mL) were further investigated to determine photo stability via their fluorescence properties (Figs. S6 and S7 Supporting Information). The intensity of the fluorescence increased upon exposure to the UV-lamp (365 nm) and laser (532 nm) for 15 min. There were no changes to the results although extension was made up to 30 min. This was in agreement with observation reported by Thakur et al. (2017). In contrast, the fluorescence intensity of both samples decreased with the increasing of time exposure (from 15 to 30 min) to visible light irradiation.

Consequently, to the elevated changes of temperature Table 1, the fluorescence intensity declined tremendous. Increasing in temperature is directly proportional to the energy transfer causes decreasing in collision time of the molecules, which is superior to an effectual collision quenching the fluorescence of the particle (Papadopoulou et al., 2005; Kuzkova et al., 2014). Kumawat et al. (2016) also reported the fluorescence intensity decreased over temperature increment. For further application, by using variation temperature in cells as model, his group has successfully justified that GQDs have the ability to detect the temperature changes occurred in the cellular environment. Researched by Menter (2006) were elegantly demonstrated that fluorescence was affected by proteins denaturation in cell due to the temperature changes. Due to that, the QDs can alternatively be used as temperature sensing probe. Likewise, C-dots also gave similar properties therefore, it was suggested to be used as a medium of thermal sensing in biomedical applications.

3.6 Fish embryo toxicity (FET) tests

C-dots were used to replace quantum dots (QD), which are composed of heavy metals. Li et al. (2012) reported that QDs are toxic both in vivo and in vitro. This makes QDs undesirable for biomedical and industrial applications because of the risk of health problems and aquatic and terrestrial pollution. C-dots, on the other hand, are viewed as a safer alternative to QDs (Li et al., 2012) because they can easily penetrate cells without disrupting the nuclei. Disruption of the nuclei would cause genetic instability, which is undesirable (Li et al., 2012; Jeong et al., 2009).

Fig. 6 shows the C-dots and modified C-dots were introduced into fish embryo to evaluate their toxicity capabilities using confocal microscopy test in vitro. Cell viability studies suggested C-dots exhibited cytotoxicity and posed no significant toxicity effect for the early 48 h. In the early stages, from 24 to 48 h, the fish embryos (70%) developed normally, whereas the embryos died after 72 h. The modified fluorescent C-dots with BPEI became toxic to the fish embryos after exposure for 24 h, as shown in Fig. 6 while the modified C-dots with PEG_1500N exhibited cytotoxicity and posed no significant toxicity effect. Within 72 h, fish embryos exposed with the C-dots-PEG_1500N were observed to develop normally (100%) without any sign of lethality.

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Fig. 6

Conditions of zebrafish embryos in different test solutions, (n = 3). (a) Representative images of zebrafish embryos treated with C-dots, C-dots-BPEI and C-dots PEG_1500N. The red indicated dead embryos, the blue indicated live embryos and the green indicated hatched embryos. (b) Percentage of survival embryos against C-dots, C-dots-BPEI and C-dots PEG_1500N.

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We selected zebrafish as model because it is a vertebrate organism and the embryos develop most of the major organ system present in mammals by less than a week (Rubinstein, 2006). Zebrafish embryos also an important for studying toxicity, in vivo system damage, the transport and biocompatibility of nanoparticles, their small size, transparency and it is easy to observe the transport and the effects of nanoparticles in real-time and ability to absorb compounds through the water. Besides that, the period of embryo development can be controlled by regulating the light-dark cycle. Importantly, zebrafish are very homologous with mammals and approximately 70% of human gene are orthologous to those in zebrafish, so modelling human disease with zebrafish as model becomes an important and fast strategy to use as a model organism (Howe et al., 2013; Kettleborough et al., 2013). Embryonic development is an important model for studying the toxicity and these possible risks posed to human health and the environment are proposed based on these developmental results. Therefore, zebrafish embryo is unique for these studies compared with other vertebrate model systems such as mice and rats (Wang et al., 2010; Kimmel et al., 1995).

The C-dots modified with BPEI were more toxic, suggesting that C-dots modified with BPEI are not safe for biological or biomedical applications, and this result agrees with previous findings (Moghimi and Andresen, 2009). The cytotoxicity of the BPEI is due to its positive charge, which causes serious damage to the cell membrane.

In contrast, the C-dots modified with PEG_1500N showed no significant toxic effects on the embryos. This is supported by a previous report that showed that PEG_1500N is not toxic to cells (Li et al., 2012). Modified C-dots contain two fundamental spatial components which is the core and the corona that interact with the environment or solvent. The reduction of toxicity of C-dots-PEG_1500N have been caused by reduced contact between the C-dots and embryos. PEGylation reduced the contact between the nanomaterials and cells (Chen et al., 2015).

The basic challenges of nanoparticles are include uptake by the reticuloendothelial system (RES), in which tiny particles are rapidly shuttled out of circulation to the liver, spleen or bone marrow and nonspecific binding of nanoparticles to non-targeted areas (Joker et al., 2011). Nanoparticles toxicity often arise due to this RES accumulation. Aggregation can lead to nanoparticles entrapment in the liver, lungs or elsewhere due to capillary occlusion (Knop et al., 2006). Therefore, addition of PEG to the C-dots surface can reduce many of these challenges. PEGylation has been widely used in nanomaterials because it can improve the biocompatibility and reduce the clearance by the reticuloendothelical (Gao et al., 2013; Gao et al., 2014). Addition of PEG to C-dots reduces RES uptakes and increases circulation time versus uncoated counterparts. PEG decreases the amount of attraction between C-dots by increasing the steric distance between them and increasing hydrophilicity via ether repeats forming hydrogen bonds with solvent. Other benefits of PEGylation include modifying the size of the particle. PEG modifies the C-dots flexibility and C-dots can become ‘softer’ after PEGylation than the underlying material, influencing extravasation (Joker et al., 2011).

Based on these results, it is suggested C-dots modified with PEG_1500N are suitable for bioimaging, biomedical, or other biological applications mainly due to their non-toxic characteristic to the zebrafish embryos.