Limits...
Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide.

Rubio N, Mei KC, Klippstein R, Costa PM, Hodgins N, Wang JT, Festy F, Abbate V, Hider RC, Chan KL, Al-Jamal KT - ACS Appl Mater Interfaces (2015)

Bottom Line: Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol-ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM).Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, King's College London , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.

ABSTRACT
Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol-ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.

No MeSH data available.


Confocallaser scanning microscopy (CLSM) and multiphoton (MP)fluorescence microscopic images of MUC1+ cells incubated with anti-MUC1IgG, n-GO-PEG or n-GO-PEG-MUC1. MUC1+ cells were incubated with individualtreatments for 3 h then stained with Cy3 labeled antihuman IgG (H+L)(red) and DAPI (nuclei, blue) and imaged with CLSM (anti-MUC1 IgG,red) or MP microscopy (GO, green). Objective used was 40×/1.25NA. Scale bars = 15 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4559840&req=5

fig2: Confocallaser scanning microscopy (CLSM) and multiphoton (MP)fluorescence microscopic images of MUC1+ cells incubated with anti-MUC1IgG, n-GO-PEG or n-GO-PEG-MUC1. MUC1+ cells were incubated with individualtreatments for 3 h then stained with Cy3 labeled antihuman IgG (H+L)(red) and DAPI (nuclei, blue) and imaged with CLSM (anti-MUC1 IgG,red) or MP microscopy (GO, green). Objective used was 40×/1.25NA. Scale bars = 15 μm.

Mentions: The n-GO-PEG-MUC1 sample was further tested in the two MDA-MB-231breast cancer cell lines in order to confirm the intracellular deliveryand the possible targeting effect. Cells were imaged using confocallaser scanning microscopy (CLSM) and MP fluorescence microscopy inorder to detect the anti-MUC1 IgG antibody and GO, respectively (Figure 2). The multiphotonemission properties of GO have been recently described;15 these studies demonstrated the broad emissionspectra of GO in the visible range, when excited using a femtosecondlaser. We have relied on these optical properties for the detectionof GO in cells. MUC1+ cells were incubated with anti-MUC1 IgG, n-GO-PEGor n-GO-PEG-MUC1, at concentrations equivalent to 2.5 μg/mLanti-MUC1 IgG for 3 h. This was equivalent to 10 μg/mL n-GOs,a concentration that has proven to be nontoxic to cells under theseconditions. Cells were then fixed, permeabilized and immunostainedwith cyanine dye (Cy3)-labeled antihuman IgG, to track anti-MUC1 antibody.4′-6-Diamidino-2-phenylindole (DAPI) was used to visualizethe nucleus (CLSM). Direct imaging of GO was carried out using MP(green channel). The free antibody was internalized in cells within3 h of incubation. Stronger green signals were detected in cells incubatedwith n-GO-PEG-MUC1 than n-GO-PEG (Figure 2), suggesting higher specificity of the former.CLSM confirmed the presence of anti-MUC1 IgG within the cells (redchannel), red signals corresponding with anti-MUC1 IgG colocalizedwith the green signals of n-GO-PEG-MUC1, corroborating that anti-MUC1IgG remained conjugated to n-GO-PEG after cellular internalization.As expected, no red signals were observed for n-GO-PEG (lacking theantibody). Images showing uptake in MUC1– cells are shown in Figure S7. No uptake of anti-MUC1 IgG (red channel)was observed in this instance because of the absence of MUC1 receptors.The same cells showed reduced uptake of n-GO-PEG-MUC1 than in MUC1+cells adding further confirmation of the targeting effect of n-GO-PEG-MUC1.


Solvent-Free Click-Mechanochemistry for the Preparation of Cancer Cell Targeting Graphene Oxide.

Rubio N, Mei KC, Klippstein R, Costa PM, Hodgins N, Wang JT, Festy F, Abbate V, Hider RC, Chan KL, Al-Jamal KT - ACS Appl Mater Interfaces (2015)

Confocallaser scanning microscopy (CLSM) and multiphoton (MP)fluorescence microscopic images of MUC1+ cells incubated with anti-MUC1IgG, n-GO-PEG or n-GO-PEG-MUC1. MUC1+ cells were incubated with individualtreatments for 3 h then stained with Cy3 labeled antihuman IgG (H+L)(red) and DAPI (nuclei, blue) and imaged with CLSM (anti-MUC1 IgG,red) or MP microscopy (GO, green). Objective used was 40×/1.25NA. Scale bars = 15 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559840&req=5

fig2: Confocallaser scanning microscopy (CLSM) and multiphoton (MP)fluorescence microscopic images of MUC1+ cells incubated with anti-MUC1IgG, n-GO-PEG or n-GO-PEG-MUC1. MUC1+ cells were incubated with individualtreatments for 3 h then stained with Cy3 labeled antihuman IgG (H+L)(red) and DAPI (nuclei, blue) and imaged with CLSM (anti-MUC1 IgG,red) or MP microscopy (GO, green). Objective used was 40×/1.25NA. Scale bars = 15 μm.
Mentions: The n-GO-PEG-MUC1 sample was further tested in the two MDA-MB-231breast cancer cell lines in order to confirm the intracellular deliveryand the possible targeting effect. Cells were imaged using confocallaser scanning microscopy (CLSM) and MP fluorescence microscopy inorder to detect the anti-MUC1 IgG antibody and GO, respectively (Figure 2). The multiphotonemission properties of GO have been recently described;15 these studies demonstrated the broad emissionspectra of GO in the visible range, when excited using a femtosecondlaser. We have relied on these optical properties for the detectionof GO in cells. MUC1+ cells were incubated with anti-MUC1 IgG, n-GO-PEGor n-GO-PEG-MUC1, at concentrations equivalent to 2.5 μg/mLanti-MUC1 IgG for 3 h. This was equivalent to 10 μg/mL n-GOs,a concentration that has proven to be nontoxic to cells under theseconditions. Cells were then fixed, permeabilized and immunostainedwith cyanine dye (Cy3)-labeled antihuman IgG, to track anti-MUC1 antibody.4′-6-Diamidino-2-phenylindole (DAPI) was used to visualizethe nucleus (CLSM). Direct imaging of GO was carried out using MP(green channel). The free antibody was internalized in cells within3 h of incubation. Stronger green signals were detected in cells incubatedwith n-GO-PEG-MUC1 than n-GO-PEG (Figure 2), suggesting higher specificity of the former.CLSM confirmed the presence of anti-MUC1 IgG within the cells (redchannel), red signals corresponding with anti-MUC1 IgG colocalizedwith the green signals of n-GO-PEG-MUC1, corroborating that anti-MUC1IgG remained conjugated to n-GO-PEG after cellular internalization.As expected, no red signals were observed for n-GO-PEG (lacking theantibody). Images showing uptake in MUC1– cells are shown in Figure S7. No uptake of anti-MUC1 IgG (red channel)was observed in this instance because of the absence of MUC1 receptors.The same cells showed reduced uptake of n-GO-PEG-MUC1 than in MUC1+cells adding further confirmation of the targeting effect of n-GO-PEG-MUC1.

Bottom Line: Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol-ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM).Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Science, King's College London , Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.

ABSTRACT
Polyethylene glycol-functionalized nanographene oxide (PEGylated n-GO) was synthesized from alkyne-modified n-GO, using solvent-free click-mechanochemistry, i.e., copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The modified n-GO was subsequently conjugated to a mucin 1 receptor immunoglobulin G antibody (anti-MUC1 IgG) via thiol-ene coupling reaction. n-GO derivatives were characterized with Fourier-transformed infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), Bradford assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and atomic force microscopy (AFM). Cell targeting was confirmed in vitro in MDA-MB-231 cells, either expressing or lacking MUC1 receptors, using flow cytometry, confocal laser scanning microscopy (CLSM) and multiphoton (MP) fluorescence microscopy. Biocompatibility was assessed using the modified lactate dehydrongenase (mLDH) assay.

No MeSH data available.