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In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse.

Terakawa M, Tsunoi Y, Mitsuhashi T - Int J Nanomedicine (2012)

Bottom Line: A polylactic acid (PLA) sphere, a biodegradable polymer, was used.Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres.Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Electronics and Electrical Engineering, Keio University, Yokohama, Kanagawa, Japan. terakawa@elec.keio.ac.jp

ABSTRACT
Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.

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Dependence of perforation efficiency evaluated by using fluorescein isothiocyanate-dextran (closed squares) and survival rate (open squares) on the laser fluence.Notes: A single shot of 80-fs laser pulse was irradiated. The corresponding peak focused intensity under the polylactic acid sphere which was derived from Figure 2 is also shown on the top horizontal axis.Abbreviation: fs, femtosecond.
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f5-ijn-7-2653: Dependence of perforation efficiency evaluated by using fluorescein isothiocyanate-dextran (closed squares) and survival rate (open squares) on the laser fluence.Notes: A single shot of 80-fs laser pulse was irradiated. The corresponding peak focused intensity under the polylactic acid sphere which was derived from Figure 2 is also shown on the top horizontal axis.Abbreviation: fs, femtosecond.

Mentions: Figure 5 shows the dependence of perforation efficiency and the survival rate on the laser fluence. The estimated peak intensity under the PLA sphere which was derived from Figure 2 is also shown in the figure. At the laser fluences lower than 0.8 J/cm2, the perforation efficiency was lower than 10%. The perforation efficiency was steeply increased at the laser fluences exceeding 0.88 J/cm2, in which the corresponding peak laser intensity under the sphere is higher than 1.08 × 1014 W/cm2. In a previous study23 using PS sphere of 1000 nm in diameter, the steep increase in perforation efficiency was observed at 1.11 × 1014 W/cm2. These results suggest that the perforation is governed by the optical intensity under the sphere. At laser intensities of between 1013 and 1014 W/cm2, nonlinear optical interactions with liquid and solid media, such as self phase modulation, self-focusing, multiphoton absorption, white-light continuum generation, laser-induced breakdown, laser ablation, are observed.27–29 The utilization of a fs laser at such intensities realizes dissection of axon,30 ablation of corneal stroma,31 and protein crystallization.32 It was reported that the peak pressure produced by fs laser-induced breakdown reaches several hundreds of MPa.33 The peak pressure generated by the femtosecond laser-induced breakdown is much higher than that used for transfection by using a nanosecond laser-induced stress-wave.34,35 Although the membrane permeabilization depends not only on the peak pressure but also on the rise time and the impulse (temporal integration of pressure),36,37 it is highly probable that the femtosecond laser-induced cavitation bubble and the shock wave, as well as ablation of cell membrane, contributed to the cell membrane perforation with the peak intensity at 1014 W/cm2, which is higher than the experimental breakdown threshold in water (5.6 × 1013 W/cm2).38 As can be seen in Figure 5, at laser fluences lower than 0.8 J/cm2 a slope of 0.61 is obtained, while at laser fluences higher than 0.88 J/cm2 the slope is 1.69. At laser fluences lower than 0.8 J/cm2 with the slope of 0.61, liquid water absorbs laser photons mainly as a linear absorption process. The absorption coefficient of liquid water at 800 nm is as small as 0.01 cm−1. In this low fluence domain, the laser heating process may govern the perforation process. While at the higher laser fluence domain, the laser absorption process is mainly due to the two-photon nonlinear optical process. Actually, the cornea ablation by fs laser is reported at approximately 1 J/cm2.39 In the present study, the high perforation rate is achieved by the two-photon absorption process. The survival rate was defined by the fraction of the cells in the illuminated area that stained with trypan blue. The survival rate slightly decreased with the increase of laser fluence, but it showed still higher than 95% at the highest laser fluence of 1.24 J/cm2 (1.50 × 1014 W/cm2 under the sphere). A slight decrease in the survival rate may be due to the excessive number of pores on the cell membrane for the cells which have too many spheres on the membrane.


In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse.

Terakawa M, Tsunoi Y, Mitsuhashi T - Int J Nanomedicine (2012)

Dependence of perforation efficiency evaluated by using fluorescein isothiocyanate-dextran (closed squares) and survival rate (open squares) on the laser fluence.Notes: A single shot of 80-fs laser pulse was irradiated. The corresponding peak focused intensity under the polylactic acid sphere which was derived from Figure 2 is also shown on the top horizontal axis.Abbreviation: fs, femtosecond.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368514&req=5

f5-ijn-7-2653: Dependence of perforation efficiency evaluated by using fluorescein isothiocyanate-dextran (closed squares) and survival rate (open squares) on the laser fluence.Notes: A single shot of 80-fs laser pulse was irradiated. The corresponding peak focused intensity under the polylactic acid sphere which was derived from Figure 2 is also shown on the top horizontal axis.Abbreviation: fs, femtosecond.
Mentions: Figure 5 shows the dependence of perforation efficiency and the survival rate on the laser fluence. The estimated peak intensity under the PLA sphere which was derived from Figure 2 is also shown in the figure. At the laser fluences lower than 0.8 J/cm2, the perforation efficiency was lower than 10%. The perforation efficiency was steeply increased at the laser fluences exceeding 0.88 J/cm2, in which the corresponding peak laser intensity under the sphere is higher than 1.08 × 1014 W/cm2. In a previous study23 using PS sphere of 1000 nm in diameter, the steep increase in perforation efficiency was observed at 1.11 × 1014 W/cm2. These results suggest that the perforation is governed by the optical intensity under the sphere. At laser intensities of between 1013 and 1014 W/cm2, nonlinear optical interactions with liquid and solid media, such as self phase modulation, self-focusing, multiphoton absorption, white-light continuum generation, laser-induced breakdown, laser ablation, are observed.27–29 The utilization of a fs laser at such intensities realizes dissection of axon,30 ablation of corneal stroma,31 and protein crystallization.32 It was reported that the peak pressure produced by fs laser-induced breakdown reaches several hundreds of MPa.33 The peak pressure generated by the femtosecond laser-induced breakdown is much higher than that used for transfection by using a nanosecond laser-induced stress-wave.34,35 Although the membrane permeabilization depends not only on the peak pressure but also on the rise time and the impulse (temporal integration of pressure),36,37 it is highly probable that the femtosecond laser-induced cavitation bubble and the shock wave, as well as ablation of cell membrane, contributed to the cell membrane perforation with the peak intensity at 1014 W/cm2, which is higher than the experimental breakdown threshold in water (5.6 × 1013 W/cm2).38 As can be seen in Figure 5, at laser fluences lower than 0.8 J/cm2 a slope of 0.61 is obtained, while at laser fluences higher than 0.88 J/cm2 the slope is 1.69. At laser fluences lower than 0.8 J/cm2 with the slope of 0.61, liquid water absorbs laser photons mainly as a linear absorption process. The absorption coefficient of liquid water at 800 nm is as small as 0.01 cm−1. In this low fluence domain, the laser heating process may govern the perforation process. While at the higher laser fluence domain, the laser absorption process is mainly due to the two-photon nonlinear optical process. Actually, the cornea ablation by fs laser is reported at approximately 1 J/cm2.39 In the present study, the high perforation rate is achieved by the two-photon absorption process. The survival rate was defined by the fraction of the cells in the illuminated area that stained with trypan blue. The survival rate slightly decreased with the increase of laser fluence, but it showed still higher than 95% at the highest laser fluence of 1.24 J/cm2 (1.50 × 1014 W/cm2 under the sphere). A slight decrease in the survival rate may be due to the excessive number of pores on the cell membrane for the cells which have too many spheres on the membrane.

Bottom Line: A polylactic acid (PLA) sphere, a biodegradable polymer, was used.Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres.Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Electronics and Electrical Engineering, Keio University, Yokohama, Kanagawa, Japan. terakawa@elec.keio.ac.jp

ABSTRACT
Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.

Show MeSH
Related in: MedlinePlus