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Penetration of oxidized carbon nanospheres through lipid bilayer membrane: Comparison to graphene oxide and oxidized carbon nanotubes, and effects of pH and membrane composition Jiraporn Seemork, Titiporn Sansureerungsikul, Kamonluck Sathornsantikun, Tarit Sinthusake, Kazuki Shigyou, Thapakorn Tree-udom, Banphot Jiangchareon, Khajeelak Chiablaem, Kriengsak Lirdprapamongkol, Jisnuson Svasti, Tsutomu Hamada, and Supason Pattanaargson Wanichwecharungruang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07908 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 13, 2016
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Penetration of Oxidized Carbon Nanospheres through Lipid Bilayer Membrane: Comparison to Graphene Oxide and Oxidized Carbon Nanotubes, and Effects of pH and Membrane Composition Jiraporn Seemork,a Titiporn Sansureerungsikul,b Kamonluck Sathornsantikun,b Tarit Sinthusake,b Kazuki Shigyou,c Thapakorn Tree-Udom,d Banphot Jiangchareon,e Khajeelak Chiablaem,f Kriengsak Lirdprapamongkol,f Jisnuson Svasti,f Tsutomu Hamada,c,‡ and Supason Wanichwecharungruang*b,g,‡ a
Program in Petrochemistry, Faculty of Science, Chulalongkorn University, Thailand
b
c
Department of Chemistry, Faculty of Science, Chulalongkorn University, Thailand
School of Materials Science, Japan Advanced Institute of Science and Technology, Japan d
Nanoscience and Technology program, Graduate School, Chulalongkorn University
e
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Thailand f
Laboratory of Biochemistry, Chulabhorn Research Institute, Thailand
g
Nanotec-CU Center of Excellence on Food and Agriculture, Thailand
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KEYWORDS. Membrane leak, Membrane leakage, pH Responsive, Nuclear membrane penetration, Liquid ordered phase liposome, Liquid disordered phase membrane
ABSTRACT. Here we show that the ability of oxidized carbon particles to penetrate phospholipid bilayer membrane varies with the particle shapes, chemical functionalities on the particle surface, lipid compositions of the membrane and pH conditions. Among the similar surface charged oxidized carbon particles of spherical (Oxidized Carbon Nanosphere, OCS), tubular (Oxidized Carbon Nanotube, OCT), and sheet (Oxidized Graphene Sheet, OGSh) morphologies, OCS possesses the highest levels of adhesion to lipid bilayer membrane and penetration into the cell-sized liposome. OCS preferably binds better to the disordered lipid bilayer membrane (consisting of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) as compared to the ordered membrane (consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and cholesterol). The process of OCS-induced leak on the membrane is pH responsive and most pronounced under an acidic condition. Covalently decorating the OCS’s surface with poly(ethylene oxide) or (2-aminoethyl)trimethylammonium moieties decreases its ability to interact with the membrane. When used as carriers, OCSs can deliver curcumin into nucleus of A549 human lung cancer and human embryonic kidney cells, in contrast, curcumin molecules delivered by OCTs remain in the cytoplasm. OGShs cannot significantly enter cells and cannot induce noticeable cellular uptake of curcumin.
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Introduction Strategies to bring matters across cell membrane are needed in biological research and medicine. Small nonpolar gases such as oxygen,1 carbondioxide,2 and small polar molecules such as ethanol and water3 can diffuse across lipid bilayer membrane rapidly. Examples of other limited known natural molecules that can passively diffuse across cell membranes include glycerol4 and steroids.5 Cell penetrating peptides, 5–30 amino acid peptides that when linked to cargoes can bring the cargoes across cell membrane, is one of the popular strategies to bring nonpenetrate-able materials into cells.6-10 Positively charged lipid vesicles or the so-called positively charged liposomes are the most extensively used delivery system for gene delivery, although they have drawbacks in terms of their stability.11 Various other strategies to bring matter into cells and also to overcome endosome trapping have been demonstrated.12 The biologically derived vesicles such as red blood cell-derived vesicles,13 mesenchymal stem cell-derived vesicles,14,15 and biologically isolated exosomes,16 have shown improved results in biocompatibility and efficiency for in vivo gene delivery researches. These vesicular particles can fuse to cell membranes and thus can avoid the problem of being trapped in the endosome/lysosome. The fact that they are sophisticatedly derived from biological sources, however, elicits some safety concerns and availability in large scale. Delivering gene by viral vectors, although is highly effective, possesses a limitation on gene size and also raises concerns on safety.17 Loading of cargoes into all these delivery systems must be carried out prior to the application, and loading efficiency usually varies with size and polarity of materials to be loaded. Electroporation, a strategy that uses high electrical voltage to induce membrane leakage on cell membrane, although requires no loading step and thus allows unlimited materials in the vicinity to leak into cells, usually produces damages to cells.18 Since non-lipid vesicular particulate
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systems usually enter cells via protein mediated endocytosis, some features to overcome the endosome/lysosome trapping must be incorporated into these delivery systems.12,19-22 The use of carbon nanomaterials, especially graphene oxide and carbon nanotubes, in drug/gene delivery and cellular imaging, has been reported.23-26 We have reported that the oxidized carbon nanospheres (OCSs) with the size of 100-200 nm can induce leakages on the lipid bilayer membrane of cell-sized liposomes, and can deliver peptide nucleic acids into the nucleus of mammalian cells via endocytosis and endosome leakage.27 Although OCS shows greater ability to deliver cargo compared to a commercially available positively charged liposome system,27 its ability to bring matter into cells has never been compared with oxidized carbon particulates of different morphologies such as the frequently used oxidized graphene sheets (OGShs)24 and oxidized carbon nanotubes (OCTs).26 Here we compare the ability to interact with the phospholipid bilayer membrane of the OCSs, OCTs and OGShs, using cellsized liposomes, focusing on the effects of lipid composition of the membranes and functional groups on the surface of the oxidized carbon particles. We verify the ability of the OCSs to induce membrane leakages in the lipid bilayer membranes, and demonstrate the pH responsiveness of this membrane leak induction process. Finally, we show that by using oxidized carbon particulates of different shapes, curcumin can be delivered to different locations in cells.
Results and Discussion Synthesis and characterization of OCSs, OCTs, OGShs, mPEO-OCSs, mPEO-OCTs, mPEOOGShs, TMA-OCSs. Oxidized carbon particles with spherical and sheet morphologies were successfully prepared from graphite by sonication-assisted oxidation with sodium nitrate, sulfuric acid and KMnO4. After gradient centrifugation, OCSs and OGShs were obtained.
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Although OCTs were also observed in some centrifuged fractions, purification of OCTs from other shaped materials was unsuccessful. Therefore, we prepared OCTs by oxidizing SWCNTs. SEM and TEM images (Figure 1) clearly indicate that OCSs possess a spherical shape with the diameter of 100 nm whereas OCTs exhibit a tubular shape of a dimension of 1 µm × 100 nm, and OGShs exhibit a sheet shape with the dimension of 0.5 × 0.5 µm. Zeta potential values are 42.6 ± 0.07, -58.5 ± 1.27 and -71.4 ± 0.99 for OCSs, OCTs and OGShs, respectively. It should be noted here that the titration experiment gave the pKa value of approximately 4.8 for all three oxidized carbon materials. ATR-FTIR spectra indicate that OCSs, OCTs and OGShs exhibit similar functional groups, i.e., O-H stretching (3360-3367 cm-1), C=O stretching (1706-1715 cm1
), and C=C stretching (1612-1614 cm-1) (Figure S1, SI). C 1s and O 1s spectra obtained from
XPS analysis of OCSs, OCTs and OGShs reveal C-C, C=C, C-O, C=O and O-C=O functional groups on the surfaces of the three shaped particles (Figure S2, SI). Raman spectra of the three oxidized carbon particles (Figure S3, SI) show two strong broad absorption bands at 1300 cm-1 (D band, tetrahedral sp3 bonded carbons) and 1575 cm-1 (G band, sp2 bonded carbons), three small maxima at 2720, 2920, and 3200 cm-1 (2D or G’ band of disordered planar (sp2) planes), and insignificant absorption at 500 cm-1 (amorphous sp3 bonded carbons). These analyses indicate that we have obtained the oxidized carbon particles of three different shapes, all under a similar range of negative zeta potential values, similar chemical constituents, and similar functional groups on the surfaces. Next, OCSs, OCTs and OGShs were modified with methoxy-terminated polyethylene oxide or mPEO-COOH using standard coupling reaction and the obtained products were characterized by ATR-FTIR. All three mPEO-modified oxidized carbon particles show the characteristic peak of mPEO around 1095 cm-1, which corresponds to C-O stretching of PEO chain, and decreased
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3400 cm-1 peak (attributing to O-H stretching) (see a representative ATR-FTIR spectrum of OCSs in Figure S1, SI). Zeta potential values of the mPEO-OCSs, mPEO-OCT and mPEOOGSh in water are -41.4 ± 0.85, -56.3 ± 1.44 and -69.3 ± 0.19 mV, respectively. The values are quite similar to those of the unmodified particles. Surface functionalization of OCSs with (2-aminoethyl)trimethylammonium (TMA) moieties was carried out through general coupling reaction by EDCI and NHS, and the obtained TMAOCSs showed C=O stretching (amide I band) at 1640 cm-1 (Figure S1, SI) in their FTIR spectrum. The TMA-OCS particles possess zeta potential value of -34.0 ± 1.75 mV, which is significantly less than their original OCSs particles (- 42.6 ± 0.07 mV), agreeing well to the fact that the decorated TMA moieties are positively charged at pH of around 5.5 – 6.0. Both the mPEO-OCSs and the TMA-OCSs showed aggregation when kept in water, however, sonication could re-disperse them again. All seven particles, OCSs, OCTs, OGShs, mPEO-OCSs, mPEO-OCTs, mPEO-OGShs and TMA-OCSs were successfully labeled with rhodamine-B and fluorescein. The reaction was adjusted until the degree of dye substitution on the particles were in the same range (similar range of uncoupled fluorescence dyes obtained in the dialysates).
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Figure 1. SEM (top) and TEM (bottom) photographs of (column a) OCSs, (column b) OCTs and (column c) OGShs.
Particle-lipid bilayer membrane association. Fluorescein-labeled particles were mixed with both the liquid disordered or Ld liposomes (consisting of 1,2-dioleoyl-sn-glycero-3phosphatidylcholine) and the liquid ordered or Lo liposomes (consisting of 1,2-dipalmitoyl-snglycero-3-phosphatidylcholine and cholesterol), and the association between the particles and the liposomes were observed under CLFM for 30 min. Among the three shaped oxidized carbon particles, OCSFlu, OCTFlu and OGShFlu, the levels of fluorescence signals observed on the liposomes were highest for OCSFlu, followed with OCTFlu, whereas no signal was detected on liposomes which were mixed with OGShFlu (Figure 2 and Figure S4, SI). Similarly, among the three shaped mPEO-modified oxidized carbon particles, mPEO-OCSsFlu, mPEO-OCTsFlu and mPEO-OGShsFlu, the liposomes mixed with mPEO-OCSsFlu showed the highest fluorescence signal, followed with those mixed with mPEO-OCTsFlu, whereas those mixed with mPEO-
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OGShsFlu showed no detectable signal. The results indicate that spherical oxidized carbon particles possess higher membrane association level than the tubular and the sheet-like oxidized carbon particles. Comparing between the OCSFlu and the mPEO-OCSFlu, the former possesses significantly higher membrane association level (Figure 3 and Figure S5, SI), suggesting that the original oxidized carbon surface of the particles associates with phospholipids better than the mPEO-decorated oxidized carbon surface. Comparing between Ld and Lo liposomes, the former possesses better association with the particles (Figure 3 and Figure S5, SI). This result implies that membranes with higher fluidity bind more effectively to the oxidized carbon particles, thus suggesting a possible partitioning adhesion mechanism between the particles and the membrane.21 This result agrees well with the dose-dependent effects of polyunsaturated fatty acids over the function of immune cells reported previously, of which the authors have speculated that phagocytosis is more effective when the cell membrane contains more polyunsaturated fatty acids.28 This result also agrees with our previous report on the effective penetration of OCSs into the liposomes.27 As previously reported that the membrane association of particles is affected by particle shape.29 Free energy of membrane-particle wrapping and probability of favorable orientation of the particle toward the membrane, contribute greatly to the association level. The better membrane association of OCS over OCT can be explained through the fact that all orientations of OCS-membrane association are favorable (no sharp edge), whereas only the parallel OCTmembrane orientations are favorable. A little tilting from the parallel orientations will introduce sharp edge (tube end) to the membrane leading to unfavorable association due to the difficult packing of bilayer phospholipid molecules around the sharp edge (Figure S6, SI). The same reason can be used to explain the unfavorable membrane association of the OGSh.
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Interestingly, comparing to OCSFlu, TMA-OCSFlu possess lower ability to associate with the membrane, regardless of the fact that the zeta potential of the TMA-decorated particles in water is less negative than that of the unmodified particles (zeta potential of OCSs and TMA-OCSFlu in water are -42.6 ± 0.07 and -34.0 ± 1.75, respectively). Nevertheless, some penetration of the TMA-OCSs into the inside of the liposomes could be detected (Figure S7, SI). This indicates that electrostatic repulsion does not play a major role in the association between the negatively charged lipid bilayer membrane and the OCSs. It is likely that surface modification with TMA has decreased the surface energy of the particles, thus lowering their ability to associate with the membrane. It should be noted here that we have demonstrated earlier that OCSs possess higher ability to bring cargoes into cells as compared to the commercial positively charged liposomes.27 Therefore, we conclude that among OCSFlu, OCTFlu and OGShFlu, the OCSFlu possesses the highest membrane affinity. The Ld membrane is preferred over the Lo membrane. Surface functionalization the OCS with either mPEO or TMA decreases its membrane affinity.
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Figure 2. Association of OCSsFlu (top), OCTsFlu (middle) and OGShsFlu (bottom) with Ld liposomes at 30 min post mixing of the particles with liposomes. The graphs on the left show fluorescence intensity along the white dash line of each corresponding image shown on the right. All images were acquired using the same fluorescence microscope setting parameters.
Liposome leak. We speculate that the effective penetration of OCSFlu into the interior of the liposomes is a result of leakages at the membrane induced by the association of the particles to the membrane. To clarify this point, we performed the calcein fluorescence dye leak experiment by introducing the calcein solution to the liposome suspension (solution surronding liposomes), and observing the leak of calcein fluorescence signals into the liposome interior. And indeed, we observed calcein fluorescence signals at the interior of the Ld liposomes only when OCSs were
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introduced into the liquid outside the liposomes. With no OCS, no fluorescence signal was observed at the interior of the liposomes (Figure S8, SI). This result suggests that OCSs can induce leakages on the liposome membrane, and agrees well with our previous reports in which OCSs could induce anthocyanin leak from the liposomes.27 It should be noted here that OCTs and OGShs could not induce an obvious dye leak into the liposomes. To demonstrate that the ability to induce membrane leakage is quite unique and specific for the OCS material, we also investigated an ability of other nano-materials to induce transient leakages on the liposome membrane. In this experiment, fluorescence dye-filled liposomes were incubated with various tested nano-materials, and the dye-leakage from the inside of liposomes to the outside was monitored. We observed that the 100 nm polystyrene (PS) spheres, the 300 nm ethylcellulose (EC) nanospheres, or the 30 nm lanthanide-doped NaYF4 (NaYF4) quantum dots, could not induce any liposome leakage, i.e., no leakage of fluorescence dye from the dye-filled liposomes could be observed when each of these nano-materials was introduced into the liquid surrounding the dye-filled liposomes (Figure S9, SI). In contrast, when OCSs were introduced into the dye-filled liposome suspension (to the liquid surrounding the liposomes), obvious dye leak from the interior of the liposomes to the exterior could be observed. Therefore, we conclude that the ability to induce transient leakages on the lipid bilayer membrane is quite unique for the OCS material, and functionalization of the OCS surface with either mPEO or TMA decreases this ability.
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Figure 3. Association of OCSsFlu (A and B) and mPEO-OCSsFlu (C and D) with Ld (A and C) and Lo (B and D) membranes at 30 min post mixing of the particles with the liposome suspension. The plot showing fluorescence intensity (F.I.) along the white dotted line in each image is shown on the right of that image. All images were acquired using the same fluorescence microscope setting parameters. Effect of pH on liposome leakage. To quantitatively demonstrate the ability of OCSs to induce liposome leakage under different pH conditions, we monitored the liposome breakdown through the decrease in liposome numbers after being exposed to OCSs at pH 7.4 and pH 5.5. We can use this approach because severe liposome leakage will lead to liposome breakdown. The experiment was set up to compare the numbers of liposomes after being incubated with 0, 0.5 and 5 ppm of OCSs for 90 min. At pH 5.5, we observed lower number of liposomes at higher concentration of OCSs (Figure S10, SI). This result indicates severe instability of liposomes in the presence of OCSs, in a dose dependent manner under acidic condition. This is explainable probably through a higher surface energy of the OCSs particles under acidic pH, which leads to higher phospholipid adsorption to their surfaces.27 However, at pH 7.4, the numbers of liposomes in the
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suspension with no OCS and that with 0.5 ppm OCS were similar. This indicates that the ability of 0.5 ppm OCSs to induce the membrane leak under pH 7.4 was not very strong. However, at pH 7.4, when we increased OCS concentration to 5.0 ppm, higher numbers of liposomes were observed accompanied by smaller size of the liposomes compared to those observed at 0 and 0.5 ppm OCS under the same pH. The higher numbers of smaller liposomes at pH 7.4 at the higher OCS concentration can be explained through the breakdown of larger liposomes into smaller ones. Moderate instability of liposome can usually result in the disintegration of large liposomes into smaller ones. Therefore, we conclude that OCS can induce leakage of the lipid bilayer membrane at pH 7.4 and pH 5.5, but the induction is significantly more severe at pH 5.5. Curcumin-loaded OCS. The above results clearly reveal that OCS can destabilize phospholipid bilayer membrane, and this ability will be more pronounced under acidic condition. Nevertheless, it is interesting to see if the loading of some cargoes onto the OCS has any effect on this ability. Therefore, we investigated this question using curcumin as a cargo. The selection of curcumin was based on not only the ability of the compound to be loaded onto OCS effectively but also the strong auto-fluorescence of the material, which allowed easy detection via CLFM. Here the loading content of curcumin on OCSs was 33%. In this experiment, we used Ld liposomes, which OCSs show high adhesion level and high leakage induction ability. When curcumin-loaded OCSsRho were incubated with Ld liposomes, the curcumin signal could be seen on the surface of the liposomes shortly after mixing, and also at the inside of the liposomes after approximately 20-30 min post mixing (Figure 4 and Figure S11, SI). The result clearly indicates that even with cargo loading, OCSsRho can still bind and penetrate the lipid bilayer membrane.
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Figure 4. Interaction of OCSsRho (150 µg/mL) and curcumin-loaded OCSsRho (75 µg/mL curcumin, 150 µg/mL OCSsRho) with cell-sized liposomes (0.1 mM of the DOPC). Fluorescence images of the Ld liposomes at 30 min post addition of the OCSsRho (row 1) and curcumin-loaded OCSsRho (row 2): shown as the merged images of both crucumin fluorescence and OCSRho fluorescence (column a), only curcumin signal (green, column b), and OCSRho signal (red, column c). Delivering curcumin into cells. We further compared the abilities of OCSRho, OCTRho, OGShRho and the three PEO-modified oxidized carbon particles to deliver curcumin into A549 lung cancer cells. The results reveal that OCSRho can induce the highest level of curcumin uptake, whereas the OCTRho can induce moderate curcumin uptake level (Figure 5A). There was rarely any curcumin uptake when OGShRho was used. There was no signal from both curcumin and rhodamine-grafted particles from the cells when mPEO-OCSRho, mPEO-OCTRho or mPEOOGSRho were used to deliver curcumin, indicating no significant cellular uptake of these particles and their curcumin cargo into the cells (data not shown). These cellular penetration results agree well with the results of the above liposome experiments. We speculate that the presence of the
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mPEO moiety probably decreases surface activity of the oxidized carbon particles, and thus suppresses the ability of particles to interact with the lipid bilayer membrane. When delivered by OCSRho, fluorescence signals of curcumin and of particle were found in the nucleus of the cells. It is possible that the OCSRho penetrate the cell membrane via the transient membrane leakage mechanism. The OCSsRho can also penetrate the nuclear membrane, resulting in the accumulation of both OCSsRho and curcumin in the nucleus. When delivered by OCTRho, the particle and cargo signals were observed only in the cytoplasm. OCTsRho are particles with less surface energy compared to OCSsRho and probably cannot effectively induce leakage on the membrane (from the liposome experiment result). It was likely that the attached OCTsRho on the cell
membrane
were
endocytosed
into
cells.
Therefore,
they
remained
in
the
endosomes/lysosomes, located in the cytoplasm of cells. We further investigated the intracellular trafficking of the three shaped oxidized carbon particulates using HEK293T. Attention was paid over whether the materials would be trapped in lysosomes. We covalently labeled the particles with fluorescein so that the particles could be tracked under confocal fluorescence microscope. The fluorescence images (Figure 5B) taken at 4 h incubation show the signals of OCSsFlu in both cytosol and nucleus of the HEK293T cells. Their whereabouts were all over cells and were unrelated to the location of lysosomes (observed through the fluorescence signals of the lysosome trackers), suggesting that the OCSsFlu were not trapped in lysosomes. Highest amount of OCSsFlu could be observed around the outer nuclear membrane (Figure 5B). In fact, the spreading of the OCSsFlu particles all over cells could already be observed clearly at 2 h incubation of the particles with the cells (Figure S12, SI). Since the fluorescence signals indicated no organelle confinement of the OCSsFlu inside the cells, it was likely that OCSsFlu entered cells via the membrane leak mechanism. In contrast, the signals of
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OCTsFlu appeared to be confined in organelles inside the cells (not spreading all over cells, Figure 5B). The OCTFlu signals co-localized with signals from lysotrackers, indicating that OCTsFlu were trapped in lysosomes. This implied that OCTsFlu probably went through the endosome/lysosome trafficking pathway inside the cells. Our speculation is that the OCT particles bind moderately well to the membrane, but their ability to make membrane leakage is not effective enough, in stead; they were taken up into cells via endosome/lysosome pathway. We could slightly observe signals from OGShsFlu inside the cells, and their location co-localized with those of the lysosomes. Therefore, it was likely that the OGShsFlu could enter cells, although not very effectively, and they were trapped in the lysosomes. We conclude that OCSs can be taken up into HEK293T cells very effectively, and the particles do not get trapped in lysosomes but can diffuse all over cytosol and nucleus of cells. It is likely that OCSs enter cells via transient membrane leakages. In contrast, OCTs, although can be taken up into the HEK293T cells, they are likely to be trapped in lysosomes. Lastly, the OGShs can slightly be taken up into cells, but they end up being trapped in the lysosomes.
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Figure 5. Delivery of curcumin into cells by various shaped oxidized carbon particulates. A) Intracellular tracking of curcumin and rhodamine-labeled particles: Cells incubated with no particle (row 1), curcumin-loaded OCSsRho (row 2), curcumin-loaded OCTsRho (row 3), curcumin-loaded OGShsRho (row 4); images in fluorescence mode showing the signals from nucleus staining with Hoechst (blue, λex/λem = 405/450 nm, column a), curcumin (green, λex/λem = 488/525 nm, column b), and rhodamine-labeled oxidized carbon particulates (red, λex/λem = 561/595 nm, column c). The scale bar represents 20 µm. B) Intracellular tracking of fluoresceinlabeled oxidized carbon particulates and lysosomes: Cells incubated with no particles (row 1), OCSsFlu (row 2), OCTsFlu (row 3) and OGShsFlu (row 4); images in the fluorescence mode showing merged fluorescence signals (column a), nucleus staining with DAPI (blue, λex/λem = 359/461 nm, column b), fluorescein-labeled oxidized carbon particulates (green, λex/λem =
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495/519 nm, column c) and lysotracker (magenta, λex/λem = 635/660-710 nm, column d). The scale bar represents 20 µm.
Conclusion Here, through the use of cell-sized liposomes, we show that OCTs and OGShs with similar surface charges to OCSs, possess significantly lower ability to penetrate the membrane than OCSs. In addition, the OCSs associate better with Ld phospholipid bilayer membrane (membrane with high fluidity) than with Lo membrane (membrane with low fluidity). We also demonstrate here that OCSs can induce the membrane leakage on lipid bilayer membrane, and this ability to induce a membrane leakage is more pronounced under acidic pH conditions. Decorating the surface of OCSs with PEO or TMA diminishes their ability to penetrate the membrane. Therefore, OCS is an effective delivery vehicle for bringing cargoes across lipid bilayer membrane. Experiments using the A549 and HEK293T cell lines indicate that OCSs can effectively bring curcumin into the nucleus of the cells while OCTs cannot. OCS and its cargo are not trapped by lysosome. Although OCT and its curcumin cargo can enter the cells, they are trapped in lysosome and thus remain in the cytoplasm.
Methods Synthesis and characterization of OCSs, OCTs and OGShs. The carbon particles were prepared using the previously described method with some modifications.30 One gram of graphite and sodium nitrate were mixed together. After that, the mixture was dispersed in 50 mL of 18 M sulfuric acid, followed by sonication at 40 kHz at room temperature for 1 h. Next, KMnO4 (6.0 g) was slowly added into the mixture with stirring for 90 min. Then, 100 mL of
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water was added, allowing an increase of the temperature to 90 °C for 30 min. Three hundred milliliters of water was added and the mixture was stirred for 10 min. Next, the excess KMnO4 was removed by adding 5% (w/v) H2O2 (50 mL) and stirring at room temperature for 30 min, and dialyzing the mixture against water (CelluSep T4, MWCO of 12,000−14,000 Da, Membrane Filtration Products, USA), to the pH of 5−6. Then gradient centrifugation was used to stepwise separate OCSs, OCTs and OGShs. Firstly, the suspension was centrifuged at 9,400 g for 10 min to precipitate large sized carbon debrides, followed by centrifugation of the supernatant at 11,300 g for 15 min to precipitate small sized debrides. Next, OGShs was precipitated from the obtained supernatant by centrifuging at 21,100 g for 15 min. Finally, the supernatant was centrifuged at 37, 000 g for 30 min to remove the smaller OGShs and some OCTs. The supernatant from this step contains OCSs. Since it is very difficult to separate OCTs from small OGShs, we prepared OCTs by oxidizing single-walled carbon nanotubes (SWCNTs) using the same reaction steps as OCSs and OGShs but without any sonication and decreasing the reaction time to 30 min. All materials (OCSs, OCTs and OGShs) were subjected to attenuated total reflectance Fourier transform infrared spectrometry (ATR-FTIR, Nicolet 6700, Thermo Electron Corporation, Waltham, Massachusetts, USA), X-ray diffractometry (XRD, DMAX 2200/Ultima+, Rigaku, Akishima, Japan; using a Cu Kα radiation source and operating at 40 kV and 30 mA) and X-ray photoelectron spectroscopic analysis (XPS, Kratos AXIS Ultra DLD instrument, Kratos, Manchester, England; using a monochromatic Al Kα X-ray source at 1486.6 eV and operated at 150 W, 15 kV and 10 mA). Morphology was determined by scanning electron microscopy (SEM, JSM-6400, JEOL, Tokyo, Japan; the samples were coated with gold under vacuum at 15 kV for 90 s and observed at an accelerating voltage of 20 kV) and transmission electron microscopy (TEM, JEM-2100, JEOL, Tokyo, Japan; operated at 100–120 kV). Hydrodynamic
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diameter and zeta potential value were obtained from Zetasizer (nanoseries model S4700, Malvern Instruments, Worcestershire, UK). pKa of each oxidized carbon material was determined by acid-base titration. An aqueous suspension of the oxidized carbon particulate (10 mL) with pH ∼5.5 was titrated with 0.020 M NaOH until pH of the system reached 10.5. Then, the pH values (vertical axis) were plotted as a function of the corresponded volumes of NaOH used (horizontal axis). The equivalent point, which is a dramatic change of the slope, was obtained from the plot. After that, pKa was determined from the pH value at the intersection with the half way of the beginning to the equivalent point along the horizontal axis (Figure S13, SI). Decorating the oxidized carbon particulates with poly(ethylene oxide). Methoxy-terminated polyethylene oxide carboxylic acid (mPEO-COOH) was grafted onto the surface of the three shaped carbon materials using previous protocols31 (Figure S14, SI). Briefly, to prepare mPEOCOOH, polyethylene glycol methyl ether (mPEG, 10.00 g, Mn 5,000, Sigma-Aldrich, Missouri, USA) was reacted with an excess amount of succinic anhydride (Acros Organics, Geel, Belgium) in dimethyl formamide (20 mL) with a catalytic amount of pyridine (2 drops, Carlo Erba Reagents, Val de Reuil, France) at 60 °C for 8 h. The obtained product was purified by dialysis against water using benzoylate dialysis tubing (MWCO 2000; Sigma-Aldrich, Missouri, USA) before freeze-drying. Next, the mPEO-COOH was grafted onto the surface of our oxidized carbon particulates by coupling reaction. Firstly, mPEO-COOH (5.0 mg) was dissolved in water. Then, aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.30 mg) was slowly added into the reaction with stirring under N2 atmosphere at 0 °C for 30 min. After that, N-hydroxysuccinimide (NHS, 0.25 mg) was added into the reaction, followed by addition of five milligrams of the carbon materials. The reaction was further stirred for 8 h and then, purified by dialysis against water.
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Methoxy-terminated polyethylene oxide carboxylic acid (mPEO-COOH): Yield = 60%, 1H NMR (D2O, 400 MHz, ppm): 2.51 (-COCH2CH2COOH), 4.11 (-OCH2CH2OCOCH2CH2-COOH), 3.35–3.70 (-OCH2CH2OCH3) and 3.20 (-OCH2CH2OCH3).
Decorating of the oxidized carbon nanospheres with (2-aminoethyl)trimethylammonium (TMA). Surface functionalization of OCSs was performed with (2-aminoethyl) trimethylammonium by as follows (Figure S14, SI). Firstly, 10 mL of OCSs aqueous suspension (400 ppm) were chilled to 0 °C under N2 atmosphere. Then, EDCI (36 mg in 1 mL of water) was slowly dropped into the stirring suspension under N2 atmosphere. The mixture was stirred for 30 min. Next, NHS (21 mg in 1 mL of water) was added into the reaction, followed by the addition of 16 mg of (2-aminoethyl)trimethylammonium (TMA, Sigma-Aldrich, Missouri, USA) in water (1 mL). The reaction was further stirred overnight. After that, the excess amount of reagents was removed by dialysis against water using cellulose membrane. The obtained TMA-OCSs inside the dialysis bag were collected as aqueous suspension and were characterized by ATR-FTIR and zetasizer. Fluorescence-labeling. Rhodamine B sulfonyl chloride (Lissamine, Life Technologies, California, USA) was grafted onto the oxidized carbon particulates through the reaction between sulfonyl group of rhodamine and hydroxyl group of the oxidized carbon particulates (Figure S12, SI). Rhodamine B sulfonyl chloride solution (2 mg in 50 µL DMF) was stirred with aqueous suspension of the oxidized carbon particles for 8 h. Uncoupled dye molecules were eliminated by dialysis. The rhodamine B-labeled materials (in dialysis bag) were collected. 6-Aminofluorescein was grafted onto the oxidized carbon particulates through the coupling reaction using EDCI and NHS (Figure S12, SI). Firstly, 2.2 mg of EDCI in water (1.0 mL) was slowly added into aqueous
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suspension of the oxidized carbon particulates (400 ppm, 5 mL) at 0 °C, under N2 atmosphere. The mixed suspension was further stirred for 30 min. After that, 1.3 mg of NHS in water (1.0 mL) was dropped into the reaction, followed by an addition of 6-aminofluorescein solution in acetone (2000 ppm, 0.5 mL). The reaction was stirred for 8 h. All products were purified by dialysis against water using cellulose membrane. Dialysates were collected and subjected to UVvis spectrophotometry for the determination of dyes and dialysis was continued until there was no absorption peak of the dye. Total amounts of dye in the dialysates were used to estimate the amount of grafted dye moieties on the carbon particulates. The fluorescein-labeled oxidized carbon particulates, OCSFlu, OCTFlu and OGShFlu, were used to evaluate the interaction of the particles with lipid bilayer membrane (λex/λem = 488/525 nm). The rhodamine-labeled oxidized carbon particulates, OCSRho, OCTRho and OGShRho, were used in curcumin delivery experiments (λex/λem = 561/595 nm). Curcumin loading. Solution of curcumin in ethanol (0.2 mg curcumin, 0.2 mL) was slowly dropped into aqueous suspension of oxidized carbon particulates (400 ppm, 1.0 mL) under ultrasonication. The mixture was incubated at room temperature under light protection for 4 h. Then, it was purified by dialysis against water using cellulose membrane. The dialysate containing unloaded curcumin was collected and was subjected to curcumin quantification using absorption spectroscopy at the maximum wavelength of 425 nm with the aid of standard calibration curve. Loading of curcumin was finally calculated using the following equation:
% Curcumin loading = [(Winitial – Wunloaded) / Wtotal] x 100 where Winitial is the initial weight of curcumin, Wunloaded is the weight of unloaded curcumin and Wtotal is the weight of curcumin-loaded carbon particulates.
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Interaction with lipid bilayer membrane. Cell-size liposomes were prepared from 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids, Alabama, USA) for liquid-disordered phase liposome (Ld), and from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, Avanti Polar Lipids) with cholesterol (Avanti Polar Lipids) for liquid-ordered phase liposome (Lo).32 Twenty microlitres of the lipid solution (2 mM in chloroform) were added into glass vial, followed by an addition of 12 µL of glucose solution (10 mM in methanol). The mixed solution was dried under nitrogen gas flow to make a thin film. The dried film was kept under vacuum for 3 h. After that, water (200 µL) was added and the solution was kept at 37 °C for 2-3 h in order to hydrate the film and allow the formation of cell-size liposomes. To observe the liposome-carbon particulate interaction, the obtained liposome suspension in water was mixed with the tested carbon particulates (final concentrations of the lipid and the tested carbon particulates were controlled at 0.1 mM and 100 µg/mL, respectively), and the suspension was dropped on the glass slide with silicon chamber. The mixture was immediately observed with a confocal laser fluorescent microscope (CLFM, FV-1000, Olympus, Japan) with a diode laser (473 nm). Noted that the fluorescein-labeled carbon particulates were used. The curcumin-loaded OCSsRho were incubated with liposomes using similar procedure as described above. The final concentrations of the liposome suspension and the curcumin-loaded OCSsRho were controlled at 0.1 mM and 150 µg/mL (75 µg/mL curcumin, 150 µg/mL OCSs), respectively. The mixture was, then, dropped on the glass slide with silicon chamber and observed with the CLFM (FV-1000, Olympus, Japan) operated with diode lasers (473 nm for curcumin and 559 nm for OCSsRho).
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Liposomal leakage. The leakage of the liposomes was investigated using calcein fluorescence dye (Wako, Osaka, Japan). Cell-sized liposomes were prepared as described above. Then, the liposome suspension (1 mM, 20 µL) was mixed with calcein solution (68.5 µM, 2 µL) and the mixture was subjected to the CLFM (FV-1000, Olympus, Japan) with diode lasers (473 nm). After that, the OCSs suspension (1,260 ppm, 10 µL) was gently added into the liposome suspension (1 mM, 20 µL). The liposomes with and without fluorescence signals were then counted at 10, 20, 30, 40, 50 and 60 min after mixing. The experiments were set up to compare the membrane penetration ability among OCS, polystyrene (PS, 100 nm, obtained through the in-house preparation according to previous work,33 ethylcellulose (EC, 300 nm, obtained through the in-house preparation according to our previous work34 and lanthanide-doped NaYF4 (NaYF4, 30 nm, obtained through the in-house preparation according to our previous work29), by observing the dye leak from dye-filled liposome. Cell-sized liposomes were prepared using hydration method as described previously. However, aqueous solution of doxorubicin (1000 ppm, 2 mL, used as fluorescence dye) was used to hydrate the lipid film instead of pure water. The suspension of doxorubicin-filled liposomes was left at room temperature overnight to settle down the liposomes. The solution containing left over doxorubicin outside liposomes was, then, gently removed and the same volume of water was slowly added. Next, the oxidized carbon sample suspension (500 ppm, 200 µL) was added to the doxorubicin-filled liposomes suspension (200 µL). Final concentration of the tested sample and lipid were controlled at 250 ppm and 0.1 mM, respectively. Then the suspension was observed by CLFM (λex/λem = 561/595 nm) at 30 min incubation time. Liposome leak under different pH conditions. Cell-sized liposomes were prepared by electroformation method.35 Briefly, DOPC solution (10 mM in chloroform, 2 × 22.5 µL) was
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spread gently on the indium tin oxide (ITO) coated side of the ITO glass slide (Sigma-Aldrich, Missouri, USA) with the resistant of 30 – 40 Ω. After that, the solution was dried at 50 °C, to make a film. Then a silicone chamber (silicone wall with the thickness of around 1 mm) was placed on the lipid-coated ITO glass slide and the chamber was then covered with another ITO glass slide (ITO side down to the chamber). Then, sucrose solution (100 mM, 500 µL) was added into the chamber, and voltage (1 V, 10 Hz, 2 h) was applied to the system at 50 °C, to allow the formation of liposomes. To make a particle suspension in the medium of pH 5.5, 50 µL of the aqueous OCS suspensions (concentrations of 2 and 20 ppm) were mixed with 50 µL aqueous solution containing 2-(N-morpholino)ethanesulfonic acid (0.1 M Mes buffer, pH 5.5) and glucose (100 mM). To make OCS suspension in the medium of pH 7.4, 50 µL of the OCS suspensions (concentrations of 2 and 20 ppm) were mixed with 50 µL solution containing trisaminomethane (0.1 M Tris buffer, pH 7.4) and glucose (100 mM). Then, an equal volume of the liposome suspension (1 mM lipids) and the obtained OCSs suspensions were mixed together. The size and the numbers of liposomes were observed immediately after mixing and at 90 min after mixing. The counting was carried out under an optical microscope. Final concentrations of OCSs in the mixtures were 0.50 and 5.0 ppm with the final liposome concentration of 0.5 mM (expressed as lipid concentration). Cell culture. A549 human lung cancer cells were cultured in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL) and amphotericin B (125 ng/ mL) (fetal bovine serum and antibiotics were obtained from Gibco, Grand Island, New York, USA), in a humidified atmosphere of 5% (v/v) CO2 at 37 ºC. Human embryonic kidney (HEK293T) cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 1% (w/v) sodium pyruvate, 1% (w/v) N-2-
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hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 1% (w/v) penicillin/streptomycin (all reagents were purchased from HyClone, Utah, USA) in a humidified atmosphere of 5% (v/v) CO2 at 37 °C. Delivery of curcumin into cells. The intracellular uptakes of curcumin-loaded OCSsRho, curcumin-loaded OCTsRho and curcumin-loaded OGShsRho were investigated in A549 human lung cancer cells. The cells cultured in 6-well plates (1.0 mL/well) were treated by adding the sample (1.0 mL/well) to give final concentrations of the carbon particulates and curcumin of 10 and 0.33 µg/mL, respectively. In order to investigate intracellular trafficking, 120 nM lysotracker (120 µL/well, λex/λem = 650/668, Life Technologies, California, USA) was added to the cells together with the sample. Then the cells were incubated at 37 °C under 5% (v/v) CO2 for 4 h. Next, the media was removed and the cells were washed twice with culture media. For A549, nucleus was stained with Hoechst (0.1 mg/mL) in culture media. After that, the cells were washed with culture media, followed by fixing with 4% paraformaldehyde. The cells were washed with culture media again before observation under a confocal laser fluorescent microscope (CLFM, Nikon, Tokyo, Japan; Digital Eclipse C1-Si/C1Plus equipped with Plan Apochromat VC 100×, a 32-channel-PMT-spectral-detector and Nikon-EZ-C1 software). In the case of HEK293T, the cells were stained with DAPI (2 µg/mL) after fixing with 4% paraformaldehyde, prior to washing with PBS and then observing by mean of CLFM. Excitation was carried out using a Diode Laser (405 nm for Hoechst; 488 nm for curcumin; 561 nm for rhodamine, Melles Griot, Albuquerque, NM, USA), and fluorescence spectral signals at 450, 525 and 595 nm were collected for Hoechst, curcumin and rhodamine, respectively. To make sure that there was no fluorescence crosstalk among the three chromophores (rhodamine grafted to the carbon particles, Hoechst and curcumin), image of each fluorescence dye was obtained with
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only one excitation wavelength at a time, using laser excitation intensity that only the monitored dye showed emission.
ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge. Figure S1. ATR-FTIR spectra of OCSs, OCTs, OGShs, mPEO-OCSs and TMA-OCSs. Figure S2. C 1s and O 1s XPS spectra of OCSs, OCTs and OGShs. Figure S3. Raman spectra of OCSs, OCTs and OGShs. Figure S4. Additional images illustrating the association of OCSsFlu, OCTsFlu and OGShsFlu with Ld liposomes. Figure S5. Additional images illustrating the association of OCSsFlu with Ld liposomes and Lo liposomes, and mPEO-OCSsFlu with Ld liposomes and Lo liposomes. Figure S6. Illustration of particle-membrane association. Figure S7. Association of TMA-OCSsFlu with Ld liposomes at 30 min after mixing. Figure S8. Images and graph showing membrane leakage induction on liposome membrane by OCSs. Figure S9. Leakage of doxorubicin dye from Ld liposome with the presence of various types of particle, OCSs, EC, NaFY4 and PS. Figure S10. A graph showing numbers of liposomes observed after the liposome suspension was incubated with different concentrations of OCSs at pH 7.4 and 5.5 for 90 min. Figure S11. Additional images illustrating the interaction of curcumin-loaded OCSsRho with cell-sized liposomes. Figure S12. Intracellular trafficking of fluorescein-labeled oxidized carbon particulates in HEK293T cells at 2 h incubation time: Figure S13. Titration curves of OCSs, OCTs and OGShs with 0.02 M NaOH. Figure S14. Schematic illustration of the chemical derivatization for (a) mPEO-OCSs, (b) TMA-OCSs, (c) OCSsFlu and (d) OCSsRho.
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AUTHOR INFORMATION Corresponding Author *Supason Wanichwecharungruang, Department of Chemistry, faculty of Science, Chulalongkorn University,
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was financially supported by the Ratchadapiseksompot Fund (GCRS58032301) Chulalongkorn University (CU), the Nanotec-CU Center of Excellence on Food and Agriculture, Center of Excellence on Petrochemical and Materials Technology, CU, Thailand, and the MEXT KAKENHI (15H00807 and 26103516) grant, Japan. J. Seemork is funded by Chulalongkorn University Dussadiphiphat Scholarship. ACKNOWLEDGMENT Authors would like to thank Dr. Tatsuya Murakami at Nano-Platform, JAIST, Ms. Shino Mizuno and Ms. Akiko Nakade at JAIST for their technical supports.
ABBREVIATIONS OCS, oxidized carbon nanosphere; OCT, oxidized carbon nanotube; OGSh, oxidized graphene sheet. REFERENCES
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26. Varkouhi, A. K.; Foillard, S.; Lammers, T.; Schiffelers, R. M.; Doris, E.; Hennink, W. E.; Storm, G., Sirna Delivery with Functionalized Carbon Nanotubes. Int. J. Pharm. 2011, 416, 419425. 27. Arayachukiat, S.; Seemork, J.; Pan-In, P.; Amornwachirabodee, K.; Sangphech, N.; Sansureerungsikul, T.; Sathornsantikun, K.; Vilaivan, C.; Shigyou, K.; Pienpinijtham, P.; Vilaivan, T.; Palaga, T.; Banlunara, W.; Hamada, T.; Wanichwecharungruang, S., Bringing Macromolecules into Cells and Evading Endosomes by Oxidized Carbon Nanoparticles. Nano Lett. 2015, 15, 3370-3376. 28. Calder, P. C.; Bond, J. A.; Harvey, D. J.; Gordon, S.; Newsholme, E. A., Uptake and Incorporation of Saturated and Unsaturated Fatty Acids into Macrophage Lipids and Their Effect Upon Macrophage Adhesion and Phagocytosis. Biochem. J. 1990, 269, 807-814. 29. Tree-Udom, T.; Seemork, J.; Shigyou, K.; Hamada, T.; Sangphech, N.; Palaga, T.; Insin, N.; Pan-In, P.; Wanichwecharungruang, S., Shape Effect on Particle-Lipid Bilayer Membrane Association, Cellular Uptake, and Cytotoxicity. ACS Appl. Mater. Interfaces 2015, 7, 2399324000. 30. Arayachukeat, S.; Palaga, T.; Wanichwecharungruang, S. P., Clusters of Carbon Nanospheres Derived from Graphene Oxide. ACS Appl. Mater. Interfaces 2012, 4, 6808-6815. 31. Seemork, J.; Tree-Udom, T.; Wanichwecharungruang, S., A Refillable Fragrance Carrier with a Tuneable Thermal Switch. Flavour Frag. J. 2012, 27, 386-392. 32. Sankaram, M. B.; Thompson T. E., Cholesterol-induced fluid-phase immiscibility in membrane. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8686-8690.
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33. Ouyang, Y.; Shi, H.; Fu, R.; Wu, D., Highly Monodisperse Microporous Polymeric and Carbonaceous Nanospheres with Multifunctional Properties. Sci. Rep. 2013, 3, 1430. 34. Suwannateep, N.; Wanichwecharungruang, S.; Haag, S. F.; Devahastin, S.; Groth, N.; Fluhr, J. W.; Lademann, J.; Meinke, M. C., Encapsulated Curcumin Results in Prolonged Curcumin Activity in vitro and Radical Scavenging Activity ex vivo on Skin after UVBIrradiation. Eur. J. Pharm. Biopharm. 2012, 82, 485-490. 35. Hamada, T.; Morita, M.; Miyakawa, M.; Sugimoto, R.; Hatanaka, A.; Vestergaard, M. d. C.; Takagi, M., Size-Dependent Partitioning of Nano/Microparticles Mediated by Membrane Lateral Heterogeneity. J. Am. Chem. Soc. 2012, 134, 13990-13996.
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Figure 1. SEM (top) and TEM (bottom) photographs of (column a) OCSs, (column b) OCTs and (column c) OGShs. 78x51mm (300 x 300 DPI)
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Figure 2. Association of OCSsFlu (top), OCTsFlu (middle) and OGShsFlu (bottom) with Ld liposomes at 30 min post mixing of the particles with liposomes. The graphs on the left show fluorescence intensity along the white dash line of each corresponding image shown on the right. All images were acquired using the same fluorescence microscope setting parameters. 109x144mm (300 x 300 DPI)
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Figure 3. Association of OCSsFlu (A and B) and mPEO-OCSsFlu (C and D) with Ld (A and C) and Lo (B and D) membranes at 30 min post mixing of the particles with the liposome suspension. The plot showing fluorescence intensity (F.I.) along the white dotted line in each image is shown on the right of that image. All images were acquired using the same fluorescence microscope setting parameters. 77x34mm (300 x 300 DPI)
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Figure 4. Interaction of OCSsRho (150 µg/mL) and curcumin-loaded OCSsRho (75 µg/mL curcumin, 150 µg/mL OCSsRho) with cell-sized liposomes (0.1 mM of the DOPC). Fluorescence images of the Ld liposomes at 30 min post addition of the OCSsRho (row 1) and curcumin-loaded OCSsRho (row 2): shown as the merged images of both crucumin fluorescence and OCSRho fluorescence (column a), only curcumin signal (green, column b), and OCSRho signal (red, column c). 57x40mm (300 x 300 DPI)
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Figure 5. Delivery of curcumin into cells by various shaped oxidized carbon particulates. A) Intracellular tracking of curcumin and rhodamine-labeled particles: Cells incubated with no particle (row 1), curcuminloaded OCSsRho (row 2), curcumin-loaded OCTsRho (row 3), curcumin-loaded OGShsRho (row 4); images in fluorescence mode showing the signals from nucleus staining with Hoechst (blue, λex/λem = 405/450 nm, column a), curcumin (green, λex/λem = 488/525 nm, column b), and rhodamine-labeled oxidized carbon particulates (red, λex/λem = 561/595 nm, column c). The scale bar represents 20 µm. B) Intracellular tracking of fluorescein-labeled oxidized carbon particulates and lysosomes: Cells incubated with no particles (row 1), OCSsFlu (row 2), OCTsFlu (row 3) and OGShsFlu (row 4); images in the fluorescence mode showing merged fluorescence signals (column a), nucleus staining with DAPI (blue, λex/λem = 359/461 nm, column b), fluorescein-labeled oxidized carbon particulates (green, λex/λem = 495/519 nm, column c) and lysotracker (magenta, λex/λem = 635/660-710 nm, column d). The scale bar represents 20 µm. 109x68mm (300 x 300 DPI)
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