We demonstrated that SWNTs can amplify electro-stimulation by LV pulses to
achieve cell EP, by which various sizes of molecules can be delivered to the cells without
harmful effects. Although the HV pulses could induce pore formation in both the cell
membrane and cell organelles, thereby enhancing the transfection efficiency to a greater
degree than that by LV/SWNT stimulation, the cell mortality will increase significantly.
Moreover, in animal studies, we demonstrated the combination of SWNT and LV pulses
could enhance the EPR effect, and thus increase the accumulation of nanoparticles in the
tumor. We also confirmed that SWNT pre-injection, followed by exposure to LV pulses,
could enhance ECT in various nanoparticle drugs. In clinical research, several ECT
studies have confirmed the valid antitumor effect on patients with malignant melanoma,
basal cell carcinoma, and other superficial tumors [19]. Besides, bipolar electrodes
combined with our new modality could also be applied in conventional endoscopy to open
through the working channel of the endoscope, ensuring that the tip–tissue distance
remains constant as the device performs EP [61,62]. This new platform has the potential
to be used for both superficial and deep-seated cancer treatment precisely and safely in
the future.
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Scheme 1. Synthetic scheme for the fabrication of SWNT/PEG-VNc.
Fig 1. Elemental analysis of SWNT and oxidized SWNT.
Fig. 2. (A) Difference in fluorescence emission after mixing THA stock solution with
raw SWNT or oxidized SWNT. (B) Fluorescent spectra of THA in the presence of raw
SWNT and oxidized SWNT
Fig. 3. Zeta potential distribution of SWNT-COOH and SWNT-PEG were analyzed by DLS.
Fig. 4. 1H-NMR spectrum of SWNT-PEG. The arrow indicates the characteristic peak of PEG at approximately 3.5 ppm.
Fig. 5. (A) VNc suspended in water, DMF and DMSO. (B) SWNT-PEG/VNc suspended in water.
Fig. 6. TEM image of SWNT-PEG/VNc. The length of SWNT-PEG/VNc deduced from TEM image was approximately 188.64 nm.
Fig. 7. HRTEM of SWNT-PEG and SWNT-PEG/VNc.
Fig. 8. Representative EDX spectra of SWNT-PEG/VNc.
Fig. 9. X-ray diffraction analysis of SWNT, SWNT-PEG/VNc and VNc.
Fig. 10. UV-visible absorbance spectra of VNc (blue), SWNT-PEG/VNc (red), and SWNT-PEG (black).
Fig. 11. Singlet oxygen generation of VNc in DMSO by Singlet Oxygen Sensor Green reagent. The sample was exposed to irradiation (808 nm) for 1O2 generation
Fig. 12. The thermal curves and of PBS and VNc (1, 2, 5 µg/ml in DMSO) under 808 nm laser irradiation at 1.3 W/cm2 for 5 min.
Fig. 13. The thermal curves and of PBS and SWNT-PEG/VNc under 808 nm laser irradiation at 1.3 W/cm2 for 3 min.
Fig. 14. PI signals of HT-29 cells detected by flow cytometry. We used pulsing buffer with (PI/SWNT/LV and PI/SWNT/HV group) or without SWNT (PI/HV and PI/LV group)
during the pulsation.
Fig. 15. The data of histogram correspond to (Fig. 14).
Fig. 16. The cellular uptake of SWNT-PEG/VNc treated to HT-29 cells. (A) No EP group.
(B) EP group. HV (1600 V, 10 ms, 3 pulses); LV (50 V, 40 ms, 100 pulses).
Fig. 17. Cell viability of HT-29 cells treated with SWNT-PEG and SWNT-PEG/VNc for 24 h.
Fig. 18. Apoptosis and necrosis assay of the cells detected by fluorescence microscopy after incubation with SWNT-PEG and SWNT-PEG/VNc for 24 h with laser irradiation.
(scale bar = 100 µm)
Fig. 19. Cell viability of HT-29 cells after incubation with PEG and SWNT-PEG/VNc for 24 h with or without laser irradiation.
Fig. 20. The comparison of cell viability of HT-29 cells under different treatment.
Fig. 21. (A) Pulsing buffer with (LV/SWNT group) or without (HV and LV group) SWNT during the pulsation. The fluorescence of the PI signal expression of HT-29 cells
at 5, 15, and 60 min after transfecting with PI dye under HV (1600 V, 10 ms, 3 pulses)
and LV (50 V, 40 ms, 100 pulses) electro-pulses (scale bar = 100 µm). (B) The integrated
optical density (IOD) of the PI signal expression from the fluorescence image (A)
Fig. 22. Fluorescence micrographs captured from movie at 40 min of HV and LV/SWNT groups after being transfected with the PI dye (Gray scale) (scale bar = 25 µm).
Fig. 23. Concept of electric stimulation enhancement by CNTs. After giving the LV pulses, the electric stimulation is amplified by CNTs at their tips so that the cell
electropermeabilization could occur, and the delivery efficiency of biomolecules could
be enhanced
Fig. 24. High magnification of field emission electron micrographs in the near-membrane
region following in vitro cellular exposure to no-treatment, LV, and LV combined with
SWNT pulsing buffer. Green arrows demonstrated SWNT-like structures around the pore
in the surface of the cell membrane. The red star indicated the SWNT-like structures
appearing proximal in the pore.
Fig. 25. (A) TEM image of SWNT-PEG6k. High magnification of the TEM images
showed that the diameter of SWNT-PEG6k was approximately 20 nm. (B) Size
distribution of SWNT-PEG6k was deduced from (B). The length of SWNT-PEG6k was
approximately 187.36 nm.
Fig. 26. Cell viability after treatment with different intensities of electrical stimulations.
The HT-29 cells were transfected with the PI dye under HV (1600 V, 10 ms, 3 pulses)
and LV (50 V, 40 ms, 100 pulses) pulses. At 24 h after EP, the cell viability was assessed
by staining with calcein AM. (SWNT pulsing buffer = LV/SWNT group, pulsing buffer
without SWNT = HV and LV group). Live cells appeared to be green (calcein AM),
cellular uptake with PI appeared to be red, and the merged images appeared to be yellow
(scale bar = 50 µm).
Fig. 27. Apoptosis and necrosis assay of the cells detected by flow cytometry after
different EP conditions (Control; 50 V, 40 ms, 100 pulses; 100 V, 40 ms, 50 pulses; 50
V, 40 ms, 10 pulses; 700 V, 20 ms, 3 pulses; 1300 V, 20 ms, 3 pulses; 1600 V, 10 ms, 3
pulses).
Fig. 28. The data of histogram correspond to (B). The results showed the obvious escalation of necrotic cells when the applied voltage was increased.
Fig. 29. Cell viability of HT-29 cells exposed to LV (50 V, 40 ms, 100 pulses) and HV (1600 V, 10 ms, 3 pulses) pulses by using SWNT pulsing buffer and commercial T-buffer®.
Fig. 30. Investigation of cell morphology. (A) The SEM images showed the cells
following fixation immediately after receiving LV (50 V, 40 ms, 100 pulses) and HV
(1600 V, 10 ms, 3 pulses) electric stimulation (SWNT pulsing buffer = LV/SWNT group,
pulsing buffer without SWNT = HV and LV group).
Fig. 31. Dot plot representations of flow cytometry data showed the cell size and granularity after the same EP condition from Fig. 30.
Fig. 32. Dot plot data from flow cytometry showing the morphology change of cells after
different EP conditions. Compared to the control (no EP) group, the cells treated by HV
(1300V * 20 ms * 3 pulses and 1600 V * 10 ms * 3 pulses) stimulation revealed that the
cell size (FSC-H) decreased while the cell granularity (SSC-H) increased.
Fig. 33. The SEM images showed the cells following fixation at 0, 5, 15, and 60 min after receiving HV (1600 V, 10 ms, 3 pulses) electric stimulation.
Fig. 34. Scanning electron microscope images of the cells. Cell membrane appearances
at 1 and 60 min after exposing to LV/SWNT electrical stimulation. Pores were easily
identified within the surface of cells at 1 min after EP. However, the pore numbers were
decreased at 60 min after EP. Green arrows showed the pores distributed numerously on
the cell membrane.
Fig. 35. Investigation of the transmembrane potential of HT-29 cells by the membrane
potential probe. (A) Fluorescence images showed DIOC6-highlighted changes in the
membrane potential immediately after EP. White arrows showed the decrease in
fluorescence intensity (scale bar = 50 µm).
Fig. 36. Flow cytometry of HT-29 cells. Conditions were the same as in Fig. 35.
Fig. 37. Membrane potential monitored by JC-1 dye at 5, 30, 60, and 120 min after EP.
The results showed the same fields of view of cells, before and after electro-pulsation.
The loss of orange J-aggregate fluorescence and cytoplasmic diffusion of green monomer
fluorescence occurred after the exposure of electro-stimulation in the HV and LV/SWNT
groups (scale bar = 50 µm).
Fig. 38. HT-29 cells transfected with minicircle DNA encoding GFP by using transfection
reagent or exposing to LV (50 V, 40 ms, 100 pulses) and HV (1600 V, 10 ms, 3 pulses)
pulses. Histogram data analysis by flow cytometry showed the fluorescence intensity of
GFP expressed in HT-29 cells at 48 h after transfection (SWNT pulsing buffer =
LV/SWNT group, pulsing buffer without SWNT = HV and LV group).
Fig. 39. PI dye transferred into cells at 3 h after EP. Fluorescence images: Lysotracker (green), PI (red), and Merge (yellow). High magnification showing the different modes
of entrance of PI for HV compared with the LV/SWNT group (Scale bar = 100 µm).
Fig. 40. EPR effect enhancement following low-intensity electric stimulation enlarged
due to CNTs. The accumulation of nanoparticles in the tumor tissue occurred due to not
only the original intercellular space between the endothelial cells but also the tumor
vascular permeability increased by the electric stimulation effect.
Fig. 41. (A) In vivo IVIS images of the mice bearing HT-29 tumors at 1, 24, and 96 h
after injection with nanoparticles, respectively. Accumulation of nanoparticles in the
tumors was different between the different EP conditions. The treatment methods
demonstrated are from (A) (HV = 700 V, 20 ms, 3 pulses, LV = 50 V, 40 ms, 10 pulses).
(B) Ex vivo imaging of nanoparticles in the heart, liver, spleen, lung, kidney, and tumor
of mice at 96 h after the same treatments as in (A).
Fig. 42. Comparison of the tumor surface (right side tumor) after treating with different
EP conditions. The mice with the SWNT post-injection treated with LV pulse stimulation
(LV/SWNT) showed no harmful effect of the tumor. However, an obvious ablation was
observed on the tumor treated with HV pulses (HV). (The red arrow indicated the tumor
treated by HV after 1 h with severe blood stasis).
Fig. 43. Different regions of tumors were imaged using fluorescence microscopy for 10 min post-injection. Nanoparticles (red), tumor cells (Hoechst, blue). (Scale bar = 100 µm).
Fig. 44. (A) Typical intravital micrograph of tumors treated without electro-stimulation
after injecting nanoparticles. The nanoparticles were completely filled inside the tumor
vessels at 10 min after injection. At 60 min, the nanoparticles aggregated along the tumor
vessels without obvious appearance in the tumor tissue. Tumor cells (blue), blood vessels
(green), and nanoparticles (red) (scale bar = 100 µm). (B) Tumor treated with LV pulses
after SWNT injection, followed by intravenous administration of nanoparticles. White
arrows indicated the leakage of nanoparticles into the tumor interstitial tissue at 60 min
after EP. Tumor cells (blue), blood vessels (green), and nanoparticles (red) (scale bar =
100 µm).
Fig. 45. Comparison of LV/SWNT with LV groups by enhancement of nanoparticle accumulation. Tumor sections were extracted at 3 h after administering nanoparticle
injection. (A) The tumor treated with SWNT post-injection showed an apparent
aggregation of nanoparticles in the tumor region. Nanoparticles (red), tumor cell (Hoechst,
blue). (Scale bar = 50 µm). (B) The tumor treated without SWNT showed less aggregation
of nanoparticles compared to (A). Nanoparticles (red), tumor cell (Hoechst, blue). (Scale
bar = 50 µm).
Fig. 46. SWNT-PEG was loaded with doxorubicin non-covalently by π-π stacking.
Fig. 47. Particle size and zeta potential analyses of SWNT-COOH, SWNT-PEG, and
SWNT/DOX by DLS. SWNT with much more modification showed a larger size. Zeta
potentials of the SWNT showed that SWNT-PEG had negative charges. Loading DOX
increased the zeta potentials of SWNT-PEG.
Fig. 48. UV-visible absorbance spectra of SWNT/DOX (blue), DOX (red), and SWNT-PEG (black). The drug loading efficiency was 56.4%.
Fig. 49. SWNT combined with LV pulses for improving the tumor therapy efficacy by nanomedicine. Schematic representation of ECT in a HT-29 human colon cancer
xenograft model.
Fig. 50. Body weights of all the groups showed no notable changes.
Fig. 51. Tumor volume ratio of HT-29-bearing mice among the various groups. Animals
that received a combination of pulses and nanomedicine showed significant tumor growth
suppression (SWNT/DOX + EP vs SWNT/DOX, *p < 0.05; SWNT + EP + LIPO-DOX
vs LIPO-DOX, *p < 0.05).
Fig. 52. Photographs of dissected tumors. Measurement of tumor weight for each group
after 36 days. The effective EP combined with nanomedicine revealed a high reduction
in the tumor weights (SWNT/DOX + EP vs SWNT/DOX, **p < 0.01; SWNT+ EP +
LIPO-DOX vs LIPO-DOX, *p < 0.05).
Fig. 53. Hematoxylin and eosin (H&E) staining of organs of HT-29 tumor xenograft–
bearing mice treated with PBS (control), LIPO-DOX, SWNT/DOX, and SWNT + EP +
LIPO-DOX. Scale bar = 50 μm.
Fig. 54. H&E-stained images revealed severe damage in the tumor tissues of SWNT/DOX + EP and SWNT + EP + LIPO-DOX groups. No notable damage was
observed in the PBS and SWNT + EP groups. Scale bar = 50 μm and 25 μm.
Fig. 55. Photograph of SWNT-PEG and SWNT/DOX suspended in PBS lasting for a
Fig. 55. Photograph of SWNT-PEG and SWNT/DOX suspended in PBS lasting for a