Understanding the Capsanthin Tails in Regulating the Hydrophilic

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Understanding the Capsanthin Tails in Regulating the HydrophilicLipophilic Balance of Carbon Dots for Rapid Crossing Cell Membrane Jing Chen, Xiang Zhang, Ye Zhang, Wei Wang, Shuya Li, Yucai Wang, Mengyue Hu, Li Liu, and Hong Bi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01992 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Understanding the Capsanthin Tails in Regulating the Hydrophilic-Lipophilic Balance of Carbon Dots for Rapid Crossing Cell Membrane Jing Chen,†,

‡

Xiang Zhang,† Ye Zhang,† Wei Wang,‡ Shuya Li,§ Yucai Wang,§

Mengyue Hu,† Li Liu, † and Hong Bi†,* †

College of Chemistry and Chemical Engineering, Anhui University, Hefei 230601,

China ‡

School of Life Sciences, Hefei Normal University, Hefei 230601, China

§

The Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic

Diseases, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China

KEYWORDS: carbon dots, hydrophilic-lipophilic Balance (HLB), polarity, cell membrane, capsanthin

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ABSTRACT

Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dots (A-Dots) that exhibit bright, multicolored fluorescence and contain hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared with highly hydrophilic carbon dots (H-Dots), a control sample prepared from microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be uptaken by CHO, HeLa and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic-lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with a HLB value around 6.4 are easier and faster for crossing cell membrane. As we regulate HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces in alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step towards understanding the importance of regulating the HLB values as well as surface polarity of the C-Dots for their practical usage in bio-imaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging or not.

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INTRODUCTION

In recent years, carbon dots (C-Dots) have been extensively studied in biomedical applications such as bio-imaging, labeling and sensors,1-4 due to their low cytotoxicity, good biocompatiblity, excellent photostability and superior optical properties.5 Generally, a variety of C-Dots with various kinds of surface groups have been synthesized by “top-down” or “bottom-up” methods.6 Compared with the former, the bottom-up method that usually employs natural products (e.g., bee pollens, eggs, orange juice, gelatin, silkworm chrysalis and so on) as starting materials7-19 proves to be cheaper and more environmental-friendly. In general, the C-Dots synthesized from natural precursors can be classified into two categories: one with abundant surface hydrophilic groups such as -OH, -COOH or -NH2, the other mainly containing lipophilic groups like alkyl or ester groups and etc. Unfortunately, both the hydrophilic and the lipophilic C-Dots have no excellent effect to expectation for bio-imaging, which restricts their practical applications. In our previous work, N-doped C-Dots have been prepared from konjac flour, which own more lipophilic surface groups (i.e. acetyl group) than hydrophilic groups resulting in a bright photoluminescence in cytoplasm but a moderate water solubility.20 On the contrary, the hydrophilic C-Dots synthesized from green tea have an excellent water solubility but show a poor cell imaging effect.21

As well-known, the cell membrane is generally composed of a phospholipid bilayer membrane while the intracellular matrix up to 95% is comprised of water.22 Previous reports have revealed several cell internalization pathways of C-Dots, 3


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including energy-dependent via caveolae-mediated or clathrin-mediated pathway,23,24 receptor-mediated endocytosis,25,26 direct non-endocytic pathway,27 and the multiple internalization pathways of hydrophobic C-Dots.28 Anyway, the highly hydrophilic C-Dots (H-Dots) are incompatible to the cell membrane and thus cannot cross the cell plasma membrane barrier. Other than H-Dots, the lipophilic C-Dots are prone to penetrate the cell membrane more easily according to Meyer-Overton rule,29 but they can only disperse in organic solvents such as dimethyl sulfoxide (DMSO) or ethanol, etc., and tend to aggregate in aqueous solution.30 Therefore, an ideal C-Dot for cell imaging is expected to have balanced hydrophilicity and lipophilicity besides good biocompatibility.

For

practical

bio-imaging

applications,

understanding

the

importance of regulating the hydrophilic-lipophilic balance (HLB) values of C-Dots as well as improving their biocompatibility is fundamental and most important. Certainly, amphiphilic C-Dots have been prepared via surface engineering, i.e. surface

modification

with

ionic-liquid,31,32

coating

amphiphilic

synthetic

polymers,33,34 or capping red blood membrane lipids directly.35 However, ionic-liquid-modified amphiphilic C-Dots are highly charged with potential toxicity which is not conductive to bio-imaging application.31 Meanwhile, high-cost, complex preparation process and reduced quantum yield (QY) are inevitable drawbacks of surface coating amphiphilic synthetic polymers.

In this work, we select an everyday vegetable – paprika as a raw material to prepare a new kind of amphiphilic carbon dots (A-Dots). Chinese paprika is comprised mainly of cellulose, ascorbic acid, capsanthin and inorganic salts such as 4


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Na2SO4 and NaCl. Among these components, capsanthin is a lipophilic compound derives from carotenoid (its molecular structure is shown in Figure 1a) that has a similar structure to the phospholipid inside cell membranes,36 Carotenoids is believed having played an essential role in early evolution and appear in most of the cellular membranes. Previous studies have demonstrated that β-carotene-decorated carbon nanoparticles could promote the intracellular delivery for robust detection at cellular and tissue level, but β-carotene is known to exert significant toxicity, hence phospholipid was used to coat carbon nanoparticles with improved function and biocompatibility for in vivo studies.37 However capsanthin is much less toxic and more biocompatible than β-carotene. In addition, both cellulose and ascorbic acid are hydrophilic, which makes paprika an ideal candidate to be selected for preparation of A-Dots that are expected to be used both in vitro and in vivo eventually. The generated A-Dots are composed of carbon core and organic shell containing hydrophilic/lipophilic

surface

groups,

especially

with

the

phospholipid-like

capsanthin tails. For comparison of the A-Dots, the H-Dots as a control sample are prepared from citric acid according to the procedure in literature.38 An objective method to calibrate HLB value of the A-Dots is developed and the cell internalization pathway aided by the capsanthin tail has also been investigated for further understanding the mechanism of C-Dots crossing cell membrane.

RESULTS AND DISCUSSION

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Figure 1. (a) A scheme of the synthesis and purification process of A-Dots. (b) A typical transmission electron microscopy (TEM) image of the A-Dots dispersed in ethanol. (c) A high-resolution TEM image of an individual A-Dots in b. (d) The lateral size distribution of the A-Dots.

Preparation and characterization of A-Dots. As shown in Figure 1a, A-Dots were synthesized by pyrolysis of paprika at 400 oC (determined by thermogravimetric (TG) analysis, Figure S1) for 1.5 h and then extracted with ethanol and purified via silica column using a mixture of dichloromethane and methanol as the eluent. At so high temperature, ester bond and other weak bonds of capsanthin broke up but the long alky chain group would not decompose and then remained on the surface of the generated A-Dots. In order to remove inorganic salts such as NaCl and Na2SO4 in the sample, the aqueous solution containing A-Dots was dialyzed (MWCO 1000, Spectrum) against distilled water for several days, and then centrifuged, frozen and lyophilized. Figure 1b shows a typical TEM image of the quasi-spherical A-Dots mono-dispersed in ethanol, and the corresponding high-resolution TEM (HRTEM) 6


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image of an individual A-Dot, as shown in Figure 1c, reveals the lattice fringes of 0.219 nm that is in accordance with the in-plane lattice spacing of graphite, suggesting the formation of graphitic crystalline core in the A-dot. Meanwhile, amorphous shell can be observed owing to the presence of abundant organic surface groups. Figure 1d shows the lateral size distribution of the as-prepared A-Dots, in which the solid red-line going through the histogram is the best fitting of the data using a lognormal distribution function, indicating the average diameter of the A-Dots is about 3.05 nm. Moreover, no other crystalline phases but graphitic carbon can be observed in X-ray diffractionmeter (XRD) pattern of the A-Dots (Figure S2), indicating that inorganic salts have been removed successfully from the purified sample. Further, Figure S3 shows Raman spectrum of the A-Dots excited by a 325 nm laser. The peaks centered at 1381 and 1613 cm-1 are assigned to D and G bands, which correspond to the sp3- and sp2-hybridized carbon atoms in the A-Dots, respectively. In general, Raman spectroscopy is a powerful tool to identify the chemical state of carbon in C-Dots, and the intensity ratio of “disordered” D band to crystalline G band (ID/IG) is often used to compare the structural order between amorphous and crystalline graphitic systems. Our prepared A-Dots present an ID/IG ratio of 0.56 that is higher than that of both electrochemically synthesized graphene quantum dots (0.5)39 and the konjac flour-derived C-Dots (0.45),20 which reveals the increased ratio of amorphous shell in the A-Dots due to the existence of abundant surface groups.

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Figure 2. (a) The general XPS survey, and High-resolution (b) C1s and (c) N1s spectra of the A-Dots. (d) FT-IR spectrum of the A-Dots. (e) 1H-NMR spectra of the A-Dots and H-Dots. (f) The HLB values of the A-Dots and H-Dots determined on the basis of standard curves of Span 80 and Tween 80.

In order to investigate the nature of the abundant surface groups on the A-Dots, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) and 1H nuclear magnetic resonance (1H-NMR) spectra have been employed. As shown in Figure 2a, three typical peaks at 284.9, 399.8 and 531.8 eV appearing in the XPS full survey spectrum are assigned to C1s, N1s and O1s binding energies, respectively. Further, the C1s core level spectrum (Figure 2b) can be resolved into four Gaussian-fitting peaks at 284.7, 285.9, 287.3 and 288.6 eV, corresponding to sp2-carbon (C–C/C=C), sp3-carbon (C–O/C–N), carbonyl (C=O) and carboxylic (O=C–OH) groups, respectively.20,40 Besides, the N1s core level spectrum (Figure 2c) can be deconvoluted into three different peaks at 398.6, 399.4 and 400.6 eV, which 8


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are attributed to pyridinic-N, pyrrolic-N and quaternary-N, respectively.41 Moreover, the FT-IR spectrum as shown in Figure 2d reveals the co-existence of both hydrophilic and lipophilic groups on the surface of A-Dots. Table S1 summarizes the major hydrophilic groups including hydroxyl (O–H, 3398 cm−1), amine (N–H, 3305 cm−1) and carboxyl groups (C=O at 1648 cm −1 plus O–H at 1268 cm-1). Besides amine, carbonyl, hydroxyl and ether bond (C–O–C, 1018 cm−1), the FT-IR spectrum also shows the presence of hydrocarbon such as 2926, 2860 cm-1(CH2) and 1407 cm-1 (CH3), which provides a clear evidence of remnant capsanthin present on the surface of A-Dots since capsanthin molecule is a di-hydroxyl, keto-carotenoid composed of hydroxyl, amine, carbonyl and ether bond as well as long-chain hydrocarbon, as illustrated in Figure 1a. All of the results confirm that the yielded A-Dots possess abundant hydrophilic and lipophilic groups on their surface simultaneously, which endows the A-Dots with prominent amphiphilic property. As a control, Figure S4 shows the H-Dots prepared from citric acid and ethylenediamine with an average diameter of 3.50 nm that are mono-dispersed in aqueous solution. Far different from that of the A-Dots, the FT-IR spectrum of the H-Dots (Figure S5) shows only the sign of various kinds of hydrophilic surface groups (i.e. O–H, N–H and O=C–OH), as listed in Table S2. The hydrophilic-lipophilic balance (HLB) value of A-Dots. It is known that some traditional chemotherapeutic drugs are highly lipophilic which have lower pharmacokinetics and bioavailability.42,43 Previous studies have demonstrated that the amphiphilic nature provides the drugs with remarkable properties and great potential 9


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for use in biomedical and pharmaceutical contexts.44 Therefore, emulsifiers and amphiphilic block copolymers are conventionally employed as self-assembled carriers/devices to help the delivery of drugs as well as to enhance their absorption in vivo and improve the bioavailability. Typical examples are Doxil, Genexol-PM which are

stealthy

liposomal

glycol)-block-polylactide

formulation loaded

of

with

doxorubicin45 paclitaxol,46

and

poly(ethylene

respectively.

Besides,

hydrophility/lipophicity of various emulsifiers can be evaluated by self-assembled their HLB values ranging from extremely hydrophobic oleic acid of 1.0 to extremely hydrophilic sodium dodecyl sulfate of 40. Certainly, HLB value of a drug delivery system is one of the most important parameters for clinical applications.47 Inspired by this, we attempt to evaluate the hydrophilicity/lipophilicity of different kinds of C-Dots based on the concept of their HLB values. Generally, there are several methods (e.g., emulsification, critical micelle concentration (CMC), turbidimetric method and etc.) to determine HLB values of different emulsifiers. Here we employ 1

H-NMR spectroscopy to assess the HLB value of C-Dots utilizing the empirical

equation as follows: HLB = 18.24R + 1.8,48 where R = Ʃ H(W) /( Ʃ H(W) + Ʃ H(O)), H(W) represents for the intensity of integral curve of hydrophilic group and H(O) represents for the intensity of integral curve of lipophilic group. According to the 1H-NMR spectra data (Figure 2e), R values of the A-Dots and the H-Dots are 0.25 and 0.91, thus the calculated HLB values of the A-Dots, the H-Dots and the hydrophilic C-Dots derived from paprika are 6.4 and 18.4, respectively (Table S3). Meanwhile, we also estimate HLB values of the C-Dots in a traditional emulsification method that is based 10


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on standard curves of one typical lipophilic and the other hydrophilic emulsifiers.49,50 Thereby we select a classical lipophilic emulsifier sorbitan oleate (Span 80) as well as a common commercial hydrophilic emulsifier polyoxyethylenesorbitan mono-oleate ether (Tween 80) as standards. The HLB values of the purchased Span 80 and Tween 80 have been determined to be 4.3 and 15.0, respectively. According to the definition formula of HLB value: HLB0 = (WAHLBA +WBHLBB) / (WA+WB), where HLB0 represents the HLB value of the compound to be determined, WA, HLBA and WB, HLBB are masses and the HLB values of two different emulsifiers A and B those are employed as one lipophilic and the other hydrophilic standards.49,50 As determined from the standard curves of the Span 80 and Tween 80 (Figure 2f), the estimated HLB values of A-Dots and H-Dots are 6.5 and 18.3, respectively. It is noteworthy that the HLB value results of A-Dots and H-Dots obtained from these two different methods (1H-NMR and emulsification method) are almost identical. Compared with the HLB value of the control hydrophilic H-Dots, the distinctly lower HLB value of the as-prepared A-Dots confirms the more lipophilic than hydrophilic character of the A-Dots due to remnant capsanthin on their surface. Moreover, we have estimated HLB values of other kinds of C-Dots using the same 1H-NMR method, i.e., that of C-Dots prepared from corn cob and green tea is 9.2 and 15.0, respectively (see also Table S3). Although the issue of hydrophilicity or hydrophobicity of the prepared C-Dots has always been emphasized,32,51 and several approaches to regulating hydrophilicity/hydrophobicity of C-Dots have been previously reported,31-35,52,53 to the best of our knowledge, it is the first time to calibrate the HLB values of different 11


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kinds of C-Dots. It has demonstrated that the HLB-calibration method is a versatile and convenient way to rank the amphiphilic property of various kinds of C-Dots, which could be extended to other semiconducting (i.e., II-VI or III-V group) quantum dots and even to all of inorganic nanoparticles. The solubility, polarity and photoluminescence property of A-Dots. Generally, amphiphilic compounds have a good solubility in either an organic solvent or water. We observe that the A-Dots can keep stable dispersions in ethanol, DMSO and water for one month. However, the H-Dots can dissolve very well in water at a high concentration of 500 µg/mL but will precipitate very quickly in ethanol even at a much lower concentration of 50 µg/mL. As shown in Figures 3a and 3b, the average hydrodynamic diameter of H-Dots measured by dynamic light scattering (DLS) is ca. 12 nm in 50 µg/mL aqueous solution while that of the A-Dots becomes as large as 175 nm in aqueous solution at the same concentration. The corresponding TEM images (as shown in Figures 3c and 3d) reveal that each H-Dot is mono-dispersed in water whereas the A-Dots have self-assembled into bigger-sized (170 ~ 180 nm) aggregates in aqueous solution, which agrees well with the DLS result and provides a direct evidence of aggregated state of the A-Dots that possibly arising from lipophilic capsanthin tails on the adjacent A-Dots entangled with each other in the aqueous solution. In sequence, the DLS diameters of the A-Dots in EtOH, DMSO and H2O are 6 < 12 < 175 nm (see also Figure 3a and Figure S6), while the DLS diameters of the H-Dots in EtOH, DMSO and H2O are 35, 7 and 12 nm, respectively (see also Figure 3b and Figure S7). 12


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Figure 3. (a) Hydrodynamic diameters of A-Dots in EtOH and H2O, respectively (inset: the illustration of A-Dots self-assembly in aqueous phase). (b) Hydrodynamic diameters of H-Dots in EtOH and H2O, respectively. (c) Representative TEM image of the self-assembled A-Dots in aqueous solution (inset: the magnified image of self-assembled A-Dots). (d) A typical TEM image of the H-Dots dispersed in aqueous solution. (inset: the magnified image of an individual H-Dots). (e) The UV absorbance and PL emission spectra (λex = 330 nm) of the A-Dots dispersed in aqueous solution. (f) The UV absorbance and PL emission spectra (λex = 350 nm) of the H-Dots dispersed in aqueous solution (the insets in (e and f) show photographs of

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the aqueous solutions containing A-Dots and H-Dots under visible light and an UV lamp of 365 nm, respectively.).

These informative values can be interpreted by "like dissolves like" principle. The diverse sizes of the A-Dots (6 < 12 < 175 nm) in three solvents, for example, just match with the increasing sequence of polarity parameters of three solvents: EtOH (4.3) < DMSO (7.2) < H2O (10.2). The mono-dispersed state of the A-Dots in EtOH reveals that the surface polarity of the A-Dots is approximate to 4.3. Such a weak polarity sounds reasonable because the yielded A-Dots possess a larger ratio of amorphous shell on their surfaces, just as above-mentioned, they contains not only hydrophilic groups (i.e. –OH) but also remnant capsanthin tails with a long hydrocarbon chain (see also Figure 1a). In contrast, the H-Dots contain a plenty of highly polar and hydrophilic groups such as –OH, N–H and O=C–OH on their surface (see also Table S3), hence they have a very good solubility in a strong polar solvent (i.e. H2O) but a very poor solubility in a weak polar solvent (i.e. EtOH). Nonetheless, the A-Dots still show a bright blue fluorescence in water (λex = 330 nm/λem = 385 nm) with a relatively high QY of 45 % (Table S4) and a slight and broad UV-Vis absorption center at ca. 280 nm arising from the remnant capsanthin tail on the shell54 (Figure 3e). Far differently, the H-Dots show one typical UV-Vis absorption peak at ca. 240 nm assigned to the π-π* transition of C=C55 and another broad absorption in the range of 300-400 nm attributed to the n-π* transition of O=C– OH surface groups (Figure 3f).53,56,57 In addition, it could be found that the photoluminescence (PL) emission bandwidth of A-Dots in H2O is wider than that of 14


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the H-Dots. This behavior may result from the broad size distribution, inhomogeneous surface chemical structure and possible diverse PL centers of A-Dots.58,59 Besides, it can be understood that the PL intensity of H-Dots exhibit a peculiar pH-dependent behavior due to their surface groups. As shown in Figure 4a, the PL decreases to the lowest extent in a strong acidic solution (pH = 1-2) as well as in an extremely strong basic solution (pH = 12-14). The chemical states of O=C–OH and NH2 groups are strongly influenced by the pH values of the solution, where the PL intensity of H-Dots may decrease owing to protonation or deprotonation of the surface functional groups, as often reported previously.53,60 In contrast, the PL intensity of A-Dots aqueous solution rises slowly with pH vary ranging from 1 to 11 and then decreases slightly at pH = 13, confirming that the A-Dots contain more lipophilic and non-protonated groups

such

as

capsanthin

tails

than

those

hydrophilic

and

easily

protonated/deprotonated surface groups such as O=C–OH and NH2. As shown in Figure S8, the hydrodynamic diameters of A-Dots in aqueous solution continuously rise with the increase of concentration. Simultaneously, PL intensity of the A-Dots increases monotonously with concentration increasing from 20 µg/mL to 1 mg/mL, as shown in Figure 4b. This is not an aggregation-induced emission (AIE) phenomenon,61 but it has already been reported many times in C-Dots related literatures.2,62 This result demonstrates that the aggregated state of A-Dots does not induce fluorescence quenching, but can increase PL intensity. This remarkable advantage of A-Dots can ensure its application in bio-imaging under various conditions. The DLS and PL analysis results suggest that surface polarity of C-Dots 15


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must play a more important role in modulating their solubility in different solvents than their HLB value. However, strong surface acidity/basicity of the C-Dots may have a negative effect on their PL intensity in aqueous solution.

Figure 4. (a) The pH-fluorescence intensity curves of the A-Dots and H-Dots dispersed in aqueous solution, respectively. (b) PL emission spectra (λex = 330 nm) of the A-Dots in aqueous solution with different concentrations (inset: PL intensity histogram of varying A-Dots concentration).

Cytotoxicity test. The cytotoxicity of the C-Dots is evaluated with HeLa cells by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 5a, cell viabilities still keep as high as 90% and 85% after the HeLa cells co-incubated with 300 µg/mL H-Dots and A-Dots for 24 h, respectively. In fact, both H-Dots and A-Dots exhibit very low cytotoxicities at various concentrations of 50, 100, 150, 200, 250 and 300 µg/mL, but A-Dots show a slightly higher cytotoxicity to HeLa cells compared with H-Dots. Further, we employ lactate dehydrogenase (LDH) assay to investigate if the A-Dots will cause damage while crossing cell membranes, and thus induce the slight cytotoxicity to HeLa cells. As is well known, the amount of LDH released is proportional to the number of cells 16


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membrane being damaged. Figure 5b shows that the HeLa cells exposed to A-Dots for 24 h have a bit higher LDH leakage than those exposed to H-Dots, although both have presented a concentration-dependent increasing behavior within the same range of 50 - 300 µg/mL. This result demonstrates that the A-Dots can cause a bit more damage to cell membranes than H-Dots, since the A-Dots shell contains phospholipid-like capsanthin tails, as illustrated in Figures 1 and 2, which have a strong affinity to the fatty acyl chains in the lipid bilayer and then disrupt cell membranes, resulting in a slightly more cytotoxicity while penetrating into cells.

Cell imaging effect. Generally, fluorescent cell imaging effect is influenced by C-Dots concentration and co-incubation time with cells, besides cellular uptake efficiency that is mainly dependent on the HLB value of C-Dots. As shown in Figure 5c, CHO, HeLa and HFF cells are co-incubated with 50 µg/mL A-Dots and H-Dots at 37 oC for 2 h, respectively. A confocal laser scanning microscopy (CLSM) was used to observe cell imaging effect with an excitation at 408 nm. It could be found that all of the three cell lines co-incubated with A-Dots exhibit bright and blue emissions in their cytoplasmic regions. In sharp contrast, extremely weak fluorescence can only be seen inside those cells co-incubated with H-Dots in 2 h. We further quantify the fluorescence intensity inside the cells by flow cytometric analysis (Figure 5d), the results confirm that a larger number of A-Dots are localized inside the cells than that of H-Dots. In addition, Figure S9 shows that the A-Dots localized in HeLa cells display multi-colored (blue, green, and red) imaging under excitations of 408, 488 and 549 nm, respectively. 17


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Figure 5. (a) MTT results of HeLa cells viabilities after co-incubation with the A-Dots and H-Dots for 24 h at the different concentrations. Data are represented as mean ± SD (n = 5). (b) LDH release from HeLa cells treated with the A-Dots and H-Dots for 24 h at the different concentrations, respectively. Data are represented as mean ± SD (n = 5). (c) DIC and CLSM images of the CHO, HeLa, HFF cell lines treated with 50 µg/mL of the A-Dots or H-Dots for 2 h, respectively. The blue emission was excited by 408 laser pulse. Scale bar: 50 µm. (d) The fluorescence

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intensity were quantified by a flow cytometer. H-Dots: blue curves, A-Dots: red curves. The black curves correspond to cells incubated in the absence of C-Dots.

Notably, as the co-incubation time increase to 4 h, as shown in Figure S10, a weak fluorescence of HeLa cells stained by the H-Dots for 4 h, but those H-Dots are found only aggregated and stranded on the cell membrane but not internalized in cytoplasm. The results possibly due to the low affinity and the resistance of hydrophilic H-Dots to cell membranes, probably originating from the strong repulsion between the hydrophilic surface of H-Dots and the hydrophobic inner layer of the bilayer, which is the possible reason that prevents them from going through the phospholipid bilayer into the cytoplasm.

Figure 6. (a) FT-IR spectra of (І) the A-Dots, (II) the refluxed A-Dots. (b) DIC and CLSM images of the HeLa cells treated with 50 µg/mL of the refluxed A-Dots. The blue emission was excited by 408 laser pulse.

Regulating the HLB of A-Dots. Furthermore, we regulate the HLB value of A-Dots from 6.4 to 15.3 by refluxing the A-Dots in alkali solution so as to remove the capsanthin tails from their surfaces. As shown in Figure 6a, the FT-IR spectrum of A-Dots shows clearly the presence of capsanthin (2926, 2860, 1648 and 1407 cm-1 as 19


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marked in blue, green and orange colors, respectively), however, all of these peaks disappear in the FT-IR spectrum of the refluxed A-Dots while the majority of hydrophilic groups such as hydroxyl and amine groups still remain on their surfaces, which indicates that capsanthin tails have been almost removed from the surface of A-Dots. Correspondingly, the HLB value of the refluxed A-Dots has changed to be 15.3 (see also Table S2) which is determined from the 1H-NMR spectrum (Figure S11). More interestingly, as above-mentioned the DLS diameters of the A-Dots in EtOH and H2O are 6 and 175 nm, respectively (Figure 3a), but the DLS diameters of the refluxed A-Dots in EtOH and H2O change inversely to 295 and 9 nm (Figure S12). All of the results demonstrate that the refluxed A-Dots have turned from amphiphilic surface to hydrophilic surface in accordance with the HLB value varying from 6.4 to 15.3. Further, we find that the refluxed A-Dots can hardly cross HeLa cell membranes, and thus no obvious fluorescence can be observed inside HeLa cells as shown in Figure 6b. Similarly, it can be seen from the context that those highly hydrophilic carbon dots such as the C-Dots prepared from green tea (HLB = 15.0) and the H-Dots (HLB = 18.3) show poor cell imaging effect inside HeLa cells, too.

Cellular uptake fashion studies. C-Dots are considered to be one of the promising candidates as drug and gene delivery carriers, intracellular biomarkers and bioimaging probes, depending on that the C-Dots can be delivered into cells rapidly and high efficiently.63-65 Generally, endocytosis (including phagocytosis, pinocytosis and receptor-mediated endocytosis) has been long argued as an effective delivery pathway of nanoparticles,66-68 in which the nanoparticles would be wrapped by the cell 20


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membrane and then pinch off to cell interior, for instance, the internalized nanoparticles are often trapped in certain sites such as endosomes and lysosomes along the endocytic pathways.69-71 Otherwise passive transmembrane penetration provides an alternative pathway for cellular delivery of the nanoparticles.72 Notably, the surface chemical properties of nanoparticles can strongly affect the interactions between the nanoparticles and the cell membranes.73 In particular, previous studies have reported several cell internalization pathways of C-Dots, e.g., energy-dependent endocytosis of hydrophilic C-Dots,24 hyaluronic acid modified C-Dots25 or PEI passivated C-Dots26 and the multiple internalization pathways of hydrophobic C-Dots.28 In our case, the quasi-spherical A-Dots and H-Dots own homogeneous sizes about 3.05 nm and 3.50 nm as well as approximate zeta potential values of -9.74 mV and -5.41 mV in the same cell culture, as listed in Table S5. Although both A-Dots and H-Dots are negatively-charged, the A-Dots can be uptaken by CHO, HeLa and HFF cells more easily and rapidly than the H-Dots (Figures 5c and 5d). We speculate that the amphiphilic A-Dots might penetrate through lipid bilayers of cell membrane into cell cytoplasm. In further experiments, HeLa cells were co-incubated with 50 µg/mL A-Dots for 2 h under different temperatures of 4 oC and 37 oC, respectively. Determination of the optimum co-incubation time of 2 h is based on the experimental result that accumulation of A-Dots in cytoplasm becomes almost saturated within 2 h and will not increase in the prolonged co-incubation time, as demonstrated in Figure S13. Additionally, we co-incubated HeLa cells with four inhibitors including 25 µM Nystatin, 4 µM Cyto D, 20 µM Nocodazole and 0.05 wt % NaN3 for 45 min, 21


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respectively. Among the four inhibitors, Cyto D is a cell-permeable and potent inhibitor of actin polymerization, which can inhibit macropinocytosis endocytosis. NaN3 is known to inhibit ATP-dependent endocytosis. Nystatin is an inhibitor of the lipid raft and caveolae endocytic

Figure 7. (a) CLSM images of HeLa cells co-incubated with 50 µg/mL A-Dots for 2 h at 37 oC and 4 oC, and treated with different inhibitors (λex = 408 nm). (b) Quantitative analyses of intracellular fluorescence intensities in (a) by using flow cytometry. (c) A quantitative fluorescence intensity histogram corresponding to (b). Data are represented as mean ± SD (n = 3).

pathway. Nocodazole disrupts microtubules, and inhibits trafﬁcking of endosomes. The resulting cellular distributions and fluorescence intensities detected by confocal 22


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microscopy and flow cytometry are shown in Figures 7a and 7b, where all of the HeLa cells co-incubated with A-Dots exhibit blue fluorescence with a similar brightness in their cytoplasm regions not only at different temperatures of 4 oC and 37 o

C, but also under treated with four inhibitors respectively. The quantitative analysis

results as shown in Figure 7c confirm that neither different temperatures nor various inhibitors have influence on cell internalization of the A-Dots, which proves that our prepared A-Dots have an extraordinary cell-penetrating ability, with crossing cell membranes in a temperature and energy-independent fashion.

As a proof-of-concept, we use giant plasma membrane vesicles (GPMVs) composed of natural cell membranes as an intermediate model of endocytosis-free membrane system. As illustrated in Figure 8a, the GPMVs were mixed with 50 µg/mL A-Dots and H-Dots for 2 h, respectively, and then visualized in bright field by using a fluorescent inverted microscope. Figure 8b shows distribution of A-Dots and H-Dots in the GPMVs monitored under 408 and 488 nm channels independently. In comparison with the H-Dots, the A-Dots exhibit a diffused distribution with a stronger fluorescence in GPMVs. The quantitative analysis result of fluorescence intensity in GPMVs by using ImageJ is demonstrated in Figure 8c, which shows the great difference in cellular uptake number of A-Dots and H-Dots within the same co-incubation time. Further, the flow cytometric data (Figures 8d and 8e) confirm that A-Dots can penetrate into the GPMVs much more than H-Dots. Next, we perform a membrane co-staining-based fluorescence co-localization experiment to elucidate whether the A-Dots are infiltrated transport through the phospholipid bilayer. As 23


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shown in Figure 8f, green fluorescence of the GPMVs stained with the A-Dots (left panel) can overlap completely with red fluorescence of the GPMVs stained with a membrane labelling dye, DilC (middle panel) in the merged image (right panel). The result demonstrates that capsanthin tails on the surface of A-Dots can facilitate ability of crossing cell membranes due to their similar structure to phospholipid bilayer.

Figure 8. (a) Illustration of formation process of GPMVs and staining with A-Dots and H-Dots, respectively. (b) Fluorescence microscopy images of the GPMVs stained with 50 µg/mL A-Dots and H-Dots for 2 h. (c) Quantitative analysis of fluorescence 24


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intensity of (b) by ImageJ (n = 3, data are mean ± SD, *** p < 0.001). (d) Flow cytometric profiles of the GPMVs stained with 50 µg/mL A-Dots and H-Dots for 2 h, respectively. (e) A quantitative fluorescence intensity histogram of (d) (n = 3, data are mean ± SD,* p < 0.05 and *** p < 0.001). (f) Fluorescence microscopy images of the GPMVs stained with A-Dots (left panel), DilC (middle panel), and the merged image of them (right panel).

CONCLUSION

In summary, a novel kind of amphiphilic carbon dots (A-Dots) has been prepared by a facile one-step pyrolysis of Chinese paprika. The obtained A-Dots show bright fluorescence, high quantum yield, low cytotoxicity and good biocompatibility. In particular, the A-Dots contain both hydrophilic groups and lipophilic capsanthin tails on their surfaces. The phospholipid-like capsanthin tails can promote cell internalization of the A-Dots by means of crossing cell membranes rapidly in an energy-independent fashion. On the other hand, the abundant hydrophilic groups on the surface can keep the A-Dots water-soluble and prevent them from aggregation in cell culture. In comparison with control H-Dots, the as-prepared A-Dots can be uptaken by CHO, HeLa and HFF cells more easily due to their appropriate HLB value of 6.4 as well as the capsanthin tails on their surfaces. When regulating the HLB value of the A-Dots from 6.4 to 15.3 near to that of H-Dots (18.4), it is found that they can hardly cross HeLa cell membranes. Besides, the permeability test performed by using 25


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GPMVs as a model has demonstrated that the capsanthin tails can facilitate ability of crossing cell membranes due to their similar structure to phospholipid bilayer, whereas the H-Dots are only accumulated outside cell membrane showing a very poor imaging effect. Our work proves that regulation of surface HLB values of carbon dots (C-Dots) indeed take effect in improving cellular uptake as well as bio-imaging efficiency. Further understanding the role of surface chemistry (especially HLB values) of C-Dots is essential and beneficial for putting their potential applications such as live imaging, biosensing and etc. into real practice.

MATERIALS AND METHODS Materials. The paprika was purchased from the market (Hefei, China). Aitric

acid, ethylenediamine, dimethyl sulfoxide (DMSO), ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Dithiothreitol was purchased from Energy Chemical Reagent Co., Ltd. (Shanghai, China), 4% paraformaldehyde was purchased from Solarbio Chemical Reagent Co., Ltd. (Beijing, China), 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, USA). Dulbecco’s Modified Eagle’s medium (DMEM), penicillin/streptomycin, trypsin−EDTA solution (0.25%) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT). Cytochalasin D, Nystatin, Nocodazole, NaN3 were purchased from Aladdin (Shanghai, China). DilC was purchased from Invitrogen (Shanghai, China). All chemicals were used as received without 26


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further purification. Ultra-purified water was obtained from a Milli-Q Synthesis System (Millipore, Bedford, MA).

Characterization. The morphology of the C-Dots was observed on a transmission electron microscope (TEM) (JEM-2100, JEOL, Tokyo, Japan). The powder X-ray diffraction (XRD) patterns were collected on a XD-3 X-ray diffractometer equipped (Beijing Purkinje General Instrument Co., Ltd. Beijing, China) with Cu Kα radiation (λ = 1.5406 Å) over the 2θ range of 5 ~ 60 degree. Raman spectra were recorded using a Laser Confocal Micro-Raman Spectroscopy

(InVia-Reflex,

ultraviolet-visible

(UV-Vis)

Renishaw, spectra

were

London,

Britain).

recorded

on

a

The UV759

spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd. Shanghai, China). X-ray photoelectron spectroscopy (XPS) studies were carried out with ESCALAB250 XPS (Thermo Fisher Scientific, Waltham, MA). Fourier transform infrared (FT-IR) spectroscopy was performed on a NEXUS 870 FTIR (Thermo Nicolet, Waltham, MA) spectrometer using the KBr pellet method. 1H-NMR measurements were performed on a Bruker Avance 500 NMR

spectrometer.

The

photoluminescence

(PL)

measurements

were

performed using F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Cytotoxicity assay was quantified by a microplate reader (318C, Shanghai INESA Scientific Instrument Co., Ltd. shanghai, China). The zeta potentials and hydrodynamic diameters of C-Dots were measured by a Zetasizer analyzer (Nano ZS90, Malvern Instruments Ltd, Worcestershire, UK). 27


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Fluorescence microscopy images were taken by Olympus IX-51 inverted fluorescence microscope (Olympus, Tokyo, Japan). Confocal laser scanning microscopy images of cell uptake C-Dots were taken by Zeiss LSM 710 (Jena, Germany).

Preparation and purification of A-Dots. Paprika (1.0 g) was pyrolyzed under air atmosphere at 400 oC for 1.5 h with a heating rate of 5 oC/min in muffle furnace (KJ Group, Hefei, China). The black carbonized solid was then ground into fine powder by agate mortar. The powder was mixed with 20 mL distilled water under stirring overnight to extract the A-Dots, and then filtrated with a 0.22 µm filter membrane (Millipore). The obtained solution was purified via silica column chromatography using a mixture of dichloromethane and methanol as the eluent. Thereafter, the solution was dialyzed (MWCO 1000, Spectrum) against distilled water for 3 days, frozen and lyophilized, and then stored at 4 °C before use.

Synthesis of H-Dots. H-Dots was prepared as reported previously with minor modifications as follows38: 5 mL of ethylenediamine was mixed thoroughly in 50 mL of aqueous citric acid solution (0.50 mol/L) by 20 minutes ultrasonication, and then this mixture has been heated for 5 minutes using a domestic microwave oven (700 W, Galanz, China) until the solution turned brown yellow. After being cooled to room temperature naturally, the brown-yellow solution was filtrated through a 0.22 µm filter membrane for thrice and then dialyzed (MWCO 1000) for 72 hours in

28


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distilled water to remove the residual reactants, frozen and lyophilized, and then stored at 4 °C before use.

Synthesis of hydrophilic paprika derived C-Dots by alkali solution refluxing. To get rid of the residual capsanthin group of the surface on the A-Dots, 0.5 mg A-Dots dispersed in 100 mL 1M NaOH aqueous solution, heated and refluxed at 120 oC for 24 h. After the reaction completed, adjust pH to the neutral using 1M HCl, and then filtrated with a 0.22 µm filter membrane. Finally, through a dialysis membrane (MWCO 500), residual inorganic salt will be detached. frozen and lyophilized, and then stored at 4 °C before use.

Preparation of giant plasma membrane vesicles (GPMV). CHO (Chinese Hamster Ovary) cells were cultured in DMEM medium with 10% Fetal Calf Serum up to 70-80% of confluence. GPMVs were isolated by chemically inducing cell blebbing with 25 mM paraforlmaldehyde and 2 mM dithiothreitol in GPMV buffer (10 mM Hepes, 150 mM NaCl, 2 mM CaCl2, pH 7.4) for 1 h at 37 oC as previously described.34 The mixtures were incubated at 4 oC.

Determination of Fluorescence Quantum Yield (QY) of C-dots. Quinine sulfate dissolved in 0.1 M H2SO4 (QY = 0.54),32 was selected as a control standard. The QY of C-dots was estimated according to the following equation:

Φ = Φr ×

I Ar n 2 × × A I r nr2

29


(1)

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Where the Φ is the QY, I is the integrated PL emission intensity (excited at 340 nm for C-dots and quinine sulfate), n is the refractive index (1.334 for distilled water), and A is the absorbance value (less than 0.1) of UV-Vis absorbance at 340 nm (distilled water). The subscript “r” refers to the standard. Quantitative estimation of the hydrophilic-lipophilic (HLB) value of different types of C-Dots. For nonionic amphiphilic the degree of lipophilicity is commonly quantified by the hydrophilic-lipophilic balance (HLB). In the typical process,49,50 the mixing of Span 80 and Tween 80 emulsifiers with different ratio. The different standard samples 0.5 g of surfactant agents were in the 100 mL conical flask. Isopropyl alcohol and toluene mixture solutions (volume ratio 100:15) 5.8 mL, configured into mass fraction about 10% of the surface active agent organic solvent. The white paper with a type 3 font was put on the bottom of the conical flask by constantly shaking, and the same time distilled water was dropped into the flask slowly. To the bottom 3 font fuzzy for the ending, with the HLB value as the abscissa and the consumption volume of distilled water. The rise of the number of vertical coordinates, the production of standard curve. The HLB value was estimated according to the following equation:

HLB0 = (WAHLBA + WBHLBB) / (WA + WB)

(2)

Where is HLB0 for compound with HLB value of sample, WA is one of the quality of the surfactant component, HLBA for the other surfactant component HLB value, WB for another kind of surfactant component quality HLBB value.

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Meanwhile, the HLB values have been further determined by 1H-NMR spectra data. Nonionic surfactant of NMR mapping on the hydrophilic group and lipophilic group of chemical shift position, mostly throughout between 0 ~ 6. The hydrophilic group δ chemical shift was in low field area, and the lipophilic group δ chemical shift was in high field area. According to the measured structure, δ < 2.5 areas for lipophilic group. δ > 2. 5 areas for the hydrophilic group. The HLB value was estimated according to the following equation:

HLB = 18.24R + 1.8

(3)

Where R =ƩH(W)/(ƩH(W)+ƩH(O)), H(W) for The intensity of integral curve of hydrophilic group, H(O) for The intensity of integral curve of lipophilic group. Cells culture. CHO (Chinese Hamster Ovary cell), HeLa (Human cervical cancer cell) and HFF (Human foreskin fibroblast cell) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. The cells were maintained in a humidified 5% CO2 incubator (Thermo scientific, Waltham, MA) at 37 °C, and then routinely harvested by treatment with a trypsin−EDTA solution (0.25%).

Cytotoxicity study of C-Dots. The MTT assay is a colorimetric assay for assessing cytotoxicity. HeLa cells were initially seeded in a 96-well cell culture plate at a density of 1×104 cells per well for 12 h before containing various concentrations of the C-dots (50-300 µg/mL) were added into each well further incubation for 24 h, and 31


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the viability of the cells was measured. Controls were cultivated under the same conditions without the addition of the C-dots. Then, 10 µL of MTT (5 mg/mL in PBS buffer solution) was added into the wells and further incubated for an additional 4 h. Subsequently, culture supernatants were aspirated and purple formazan crystals were dissolved into 150 µL of DMSO for an additional incubation of 15 min in the shaker incubator with gentle shakes. Then the concentration of the reduced MTT in each well was measured at a reference wavelength of 630 nm while using a test wavelength of 570 nm employing a microplate reader (318C, INESA Instrument, China). The cell viability was estimated according to the following equation:

Cell Viability (%) =ODtreated / ODcontrol × 100%

(4)

Where ODcontrol was obtained in the absence of C-dots, and ODtreated was obtained in the presence of C-dots. Each measurement was performed in quintuplicate.

Membrane permeability assessment of C-Dots. The level of extracellular lactate dehydrogenase (LDH) release was assessed as an indicator of membrane permeability and cytotoxicity. In the LDH assay, cells seeded in a 96-well cell-culture plate at a density of 1×104 cells per well were cultured for 24 h. Then, different concentrations of the C-Dots (50-300 µg/mL) were added into different wells, and co-incubated with for 24 h. The release of the LDH in each sample was determined by measuring the absorbance at a reference wavelength of 630 nm while using a test wavelength of 490 nm with the micro-plate reader. The results of the LDH assays were estimated according to the following equation: 32


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LDH release rate (%) = (Atreated-Acontrol) / (Amaximum release-Acontrol) × 100%

(5)

Where Acontrol was obtained in the absence of C-dots, and Atreated was obtained in the presence of C-Dots. Amaximum release was obtained from cell disruption. The experiments were performed in quintuplicate.

Analysis of cellular uptake fashion of C-Dots. To analyze the cellular uptake of C-Dots, CHO, HeLa and HFF cells were seeded at a density of 1×106 cells/well in a 35 mm polystyrene dish containing a bottom glass and allowed to adhere for 24 h in a humidified atmosphere of 5% CO2 at 37 oC, and then the cells were precooled at low temperature (e.g., 4 oC) incubation and cellular uptake inhibitors, such as 0.05 wt% NaN3, 10 µM Nystatin, 3 µM Cytochalasin D (Cyto D) and 10 µM Nocodazole treatments for 45 min prior to replacement of the medium with fresh temperature-equilibrated complete medium containing C-Dots (50 µg/mL), and then continue incubation for 2 h, washed thrice with fresh temperature-equilibrated medium and cells were observed by using a confocal microscope (Zeiss LSM 710) with a 40 × objective and a 408 nm laser.

Flow cytometry analysis. The CHO, HeLa, and HFF cells were seeded in a 12-well cell culture plate at a density of 1 × 106 cells per well and further co-incubated with 50 µg/mL A-Dots and H-Dots for 2 h, respectively. After being washed three times with PBS, the cells were harvested and suspended in PBS buffer again and analyzed with a flow cytometer (C6, Becton-Dickinson, USA). Cells incubated without the C-Dots were used as a control. 33


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Data and Statistical analysis. All experiments were performed at least in three independent batches (n = 3). Data were represented as the mean ± standard deviation (SD). Statistical evaluation was conducted by Student’s t-test (two-tailed) using GraphPad Prism 6 (GraphPad Software, Inc., USA). Statistical differences between samples were performed with a level of significance as * p < 0.05, ** p < 0.01, *** p < 0.001.

ASSOCIATED CONTENT

Supporting Information

Supporting Information is available from the ACS Publications website or from the author. Supplementary figures including: (1) The thermogravimetric analysis. (TG) and differential thermogravimetric analysis (DTG) curves of paprika in air atmosphere. (2) XRD pattern of the A-Dots. (3) Raman spectrum of the A-Dots. (4) A typical TEM image of H-Dots in distilled water. (5) FT-IR spectrum of the H-Dots. (6) Hydrodynamic diameters of A-Dots in DMSO solvent. (7) Hydrodynamic diameters of H-Dots in DMSO solvent. (8) Hydrodynamic diameters of A-Dots in aqueous solution with different concentrations. (9) DIC and CLSM images of the HeLa cells treated with 50 µg/mL of the A-Dots for 2 h. The blue, green, and red emissions were excited by 408, 488, and 549 nm laser pulses, respectively. (10) DIC and CLSM images of the HeLa cells treated with 50 µg/mL of the H-Dots for 4 h. The blue emission was excited by 408 laser pulse. (11) 1H-NMR spectra of the different C-Dots. (12) Hydrodynamic diameters of the refluxed A-Dots in H2O and EtOH solution, respectively. (13) Intracellular fluorescence intensity of HeLa cells co-incubated with 50 µg/mL A-Dots and H-Dots for different incubation time of 15 ~ 34


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240 min, respectively. Supplementary tables including: 1. Peak assignments in FT-IR spectrum of A-Dots. 2. Peak assignments in FT-IR spectrum of H-Dots. 3. HLB values of various kinds of C-Dots prepared from different precursors. 4. QY of the A-Dots dispersed in distilled water. 5. Zeta potentials of A-Dots and H-Dots. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Author Contributions H. B. and J. C. designed research; J. C., X. Z., Y. Z., W. W., H. M. Y. and L. L. performed research; J. C., Y. C. W. and H. B. analyzed data and wrote the paper.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financed by National Natural Science Foundation of China (Grant No. 51272002). All of co-authors thank the Collaborative Innovation Center of Modern Bio-manufacture, Anhui University.

ABBREVIATIONS

C-Dots, Carbon dots; A-Dots, amphiphilihc carbon dots; H-Dots, hydrophilic carbon dots; HLB, hydrophilic-lipophilic balance; DMSO, dimethyl sulfoxide; CMC, critical micelle concentration; Span 80, sorbitan oleate; Tween 80, polyoxyethylenesorbitan 35


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mono-oleate

ether;

TG,

thermogravimetric

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analysis;

DTG,

differential

thermogravimetric analysis; TEM, transmission electron microscopy; XPS, X-ray

photoelectron spectroscopy; HRTEM, high-resolution transmission electron microscopy; XRD, X-ray diffractionmeter; FT-IR, Fourier transform infrared; DLS, dynamic light scattering; PL, photoluminescence; CLSM, confocal laser scanning microscopy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; GPMVs, giant plasma membrane vesicles.

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Figure 1. (a) A scheme of the synthesis and purification process of A-Dots. (b) A typical transmission electron microscopy (TEM) image of the A-Dots dispersed in ethanol. (c) A high-resolution TEM image of an individual A-Dots in b. (d) The lateral size distribution of the A-Dots. 82x47mm (300 x 300 DPI)


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Figure 2. (a) The general XPS survey, and High-resolution (b) C1s and (c) N1s spectra of the A-Dots. (d) FT-IR spectrum of the A-Dots. (e) 1H-NMR spectra of the A-Dots and H-Dots. (f) The HLB values of the ADots and H-Dots determined on the basis of standard curves of Span 80 and Tween 80. 177x97mm (300 x 300 DPI)


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Figure 3. (a) Hydrodynamic diameters of A-Dots in EtOH and H2O, respectively (inset: the illustration of ADots self-assembly in aqueous phase). (b) Hydrodynamic diameters of H-Dots in EtOH and H2O, respectively. (c) Representative TEM image of the self-assembled A-Dots in aqueous solution (inset: the magnified image of self-assembled A-Dots). (d) A typical TEM image of the H-Dots dispersed in aqueous solution. (inset: the magnified image of an individual H-Dots). (e) The UV absorbance and PL emission spectra (λex = 330 nm) of the A-Dots dispersed in aqueous solution. (f) The UV absorbance and PL emission spectra (λex = 350 nm) of the H-Dots dispersed in in aqueous solution (the insets in (e and f) show photographs of the aqueous solutions containing A-Dots and H-Dots under visible light and an UV lamp of 365 nm, respectively.). 177x185mm (300 x 300 DPI)


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Figure 4. (a) The pH-fluorescence intensity curves of the A-Dots and H-Dots dispersed in aqueous solution, respectively. (b) PL emission spectra (λex = 330 nm) of the A-Dots in aqueous solution with different concentrations (inset: PL intensity histogram of varying A-Dots concentration). 85x30mm (300 x 300 DPI)


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Figure 5. (a) MTT results of HeLa cells viabilities after co-incubation with the A-Dots and H-Dots for 24 h at the different concentrations. Data are represented as mean ± SD (n = 5). (b) LDH release from HeLa cells treated with the A-Dots and H-Dots for 24 h at the different concentrations, respectively. Data are represented as mean ± SD (n = 5). (c) DIC and CLSM images of the CHO, HeLa, HFF cell lines treated with 50 µg/mL of the A-Dots or H-Dots for 2 h, respectively. The blue emission was excited by 408 laser pulse. Scale bar: 50 µm. (d) The fluorescence intensity were quantified by a flow cytometer. H-Dots: blue curves, A-Dots: red curves. The black curves correspond to cells incubated in the absence of C-Dots. 177x181mm (300 x 300 DPI)


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Figure 6. (a) FT-IR spectra of (І) the A-Dots, (II) the refluxed A-Dots. (b) DIC and CLSM images of the HeLa cells treated with 50 µg/mL of the refluxed A-Dots. The blue emission was excited by 408 laser pulse. 177x47mm (300 x 300 DPI)


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Figure 7. (a) CLSM images of HeLa cells co-incubated with 50 µg/mL A-Dots for 2 h at 37 oC and 4 oC, and treated with different inhibitors (λex = 408 nm). (b) Quantitative analyses of intracellular fluorescence intensities in (a) by using flow cytometry. (c) A quantitative fluorescence intensity histogram corresponding to (b). Data are represented as mean ± SD (n = 3). 85x96mm (300 x 300 DPI)


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Figure 8. (a) Illustration of formation process of GPMVs and staining with A-Dots and H-Dots, respectively. (b) Fluorescence microscopy images of the GPMVs stained with 50 µg/mL A-Dots and H-Dots for 2 h. (c) Quantitative analysis of fluorescence intensity of (b) by ImageJ (n = 3, data are mean ± SD, *** p < 0.001). (d) Flow cytometric profiles of the GPMVs stained with 50 µg/mL A-Dots and H-Dots for 2 h, respectively. (e) A quantitative fluorescence intensity histogram of (d) (n = 3, data are mean ± SD,* p < 0.05 and *** p < 0.001). (f) Fluorescence microscopy images of the GPMVs stained with A-Dots (left panel), DilC (middle panel), and the merged image of them (right panel). 177x259mm (300 x 300 DPI)


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TOC 80x42mm (300 x 300 DPI)


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Understanding the Capsanthin Tails in Regulating the Hydrophilic

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