1. Introduction

The field and applications of polymer micro- and nano-capsules have become a widespread research topic in biotechnology and nanomedicine. Drug delivery system (DDS) is typical and attractive example of the use of such capsules with the aim of maximizing therapeutic efficacies while minimizing adverse effects by controlling the behavior of medications spatially and temporally in the body. The controlled release of therapeutic molecules from a microcapsule at a targeted site in a desirable time is of growing importance for cutting-edge treatments including gene therapy, antibody therapy, and vaccine therapy [1,2]. There are different approaches to releasing an embedded drug at a specific target from a polymer microcapsule. In the last decade, hollow polymer microcapsules have received attention as a promising candidate for smart microcarriers that respond to external stimuli [3,4]. Several studies have reported on controlling the permeability of the shell of a microcapsule by using chemical stimuli [5], temperature [6], ultrasound [7] and lasers [8]. The controlled release using laser irradiation, among other methods, has advantages in spatial characteristics and applicability to catheter-based systems using optical fibers. The methods using lasers include ultraviolet light irradiation, which can be absorbed by polymers, for the destruction of capsules [8]. However, not only the limited penetration depth in tissue but also the considerable tissue damage associated with UV irradiation may cause problems in vivo. For in vivo applications of lasers, near-infrared wavelengths can penetrate deeply into tissue owing to low absorption, and they have the potential to be used for treatment of deeply located tissues. However, most of polymers show little absorption of near-infrared light, resulting in difficulties in the controlled-destruction of capsules followed by the release of embedded molecules. Near-infrared light irradiation of microcapsules doped with metal nanoparticles as an absorber is an alternative method for the controlled release due to a photothermal effect [9,10]. However, concerns with regards to the potential toxicity caused by long-lived residual metal nanoparticles and thermal damage to embedded molecules should be taken into consideration [11, 12].

Our group has been studying enhanced optical near fields and far fields generated by polymer spheres excited by a femtosecond (fs) laser pulse [13]. A transparent sphere, which has no hollow space inside, works as a microlens, and an enhanced optical field can be generated around polymer spheres. The highly- intense optical field produces a nonlinear interaction with transparent targets under the sphere such as cell membranes [14,15]. To date, however, hollow polymer microcapsules, which have diameters of submicron- to a few microns, have not been investigated as to whether they can be used for optical enhancement at a wavelength of 800 nm.

In the present study, we demonstrate the localized perforation of polymer microcapsules by irradiation of enhanced near-infrared fs laser pulses without any doping with metal nanoparticles. The highly-enhanced optical intensity can be generated at a spot on the shell of a microcapsule, which would in turn contribute to localized perforation. The optical intensity around a microcapsule is calculated to investigate the optical enhancement by a hollow microcapsule. On the basis of calculational results, we experimentally show the localized perforation of multiple polymer microcapsules using near-infrared fs laser pulses.

2. Simulation and experimental procedures

The optical field around a polymer microcapsule was calculated by the three-dimensional (3D) finite-difference time-domain (FDTD) method. The simulation system consisted of a polystyrene sphere (n = 1.577) in water (n = 1.326). A plane laser wave was incident on the microcapsule with the wave vector in the -z direction. The irradiated area was much larger than the diameter of a microcapsule, assuming unfocused laser irradiation. The incident wave of 800 nm wavelength was linearly polarized along the x axis. We simulated the enhanced optical field around microcapsules of 500, 750, 1000, 1250, 1500, and 1750 nm external diameter. The shell thickness was set to 200 nm for all the diameters calculated. The inside of the shell was filled with water. The minimal cell size in the calculation was 5 × 5 × 5 nm³.

We used polystyrene spherical microcapsules 1000 nm in diameter (Polysciences, Inc. Warrington, Pennsylvania) for the proof-of-concept experiments. The shell thickness of the microcapsules was measured to be approximately 200 nm by observation using scanning electron microscopy (SEM). Figure 1 shows a schematic of the experiments. The microcapsules, which were suspended in distilled water in a glass-bottomed dish 10 mm in diameter, were irradiated by a linear-polarized laser pulse from the top. The depth of the suspension was ~1 mm. A Ti:sapphire chirped pulse amplification laser system, which generates 80 fs laser pulses at 800 nm central wavelength at a repetition rate of 1 kHz, was used in the experiments. The laser beam was weakly focused using a plano-convex lens to a laser spot size of 300 μm, which was much larger than the diameter of the microcapsule, enabling the simultaneous treatment of multiple microcapsules. The irradiated laser intensity was 6.6 × 10¹² W/cm². The scanning area corresponds to the glass bottom area of 10 mm in diameter and the scanning velocity was 1 mm/s, corresponding to 300 shots per spot. The spherical microcapsules were highly dispersed in distilled water at 9.5 × 10⁴ capsules/ml. The irradiated microcapsules were collected and dispersed on a Si substrate. The alteration of the shape of the microcapsules was observed by SEM.

 

Fig. 1 Schematic of the proof-of-concept experiments. The laser spot was 300 μm, enabling the simultaneous irradiation of multiple microcapsules.

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3. Results and discussion

Figure 2 shows the optical intensity distribution for 800 nm incident wavelength around the microcapsule calculated by the 3D FDTD method. The optical near field in the vicinity of the microcapsules, which have shell thickness less than the incident wavelength, is governed by Mie scattering. The low enhancement factors obtained with external diameters smaller than 750 nm are attributed to the off-resonant Mie scattering regime. Highly-enhanced optical intensity can be generated on the boundary between the external surface of a microcapsule and water for external diameters of up to 1000 nm. The z-position of the peak intensity shifts downward with increasing diameter of a microcapsule, and the scattering is gradually dominated by far-field focusing. For a microcapsule 1750 nm in external diameter, two peaksare generated at the shell and 1300 nm under the microcapsule. Figure 3 shows dependence of optical intensity at the external bottom surface of a microcapsule (z = 0) on the external diameter of a microcapsule. The optical intensity at the shell is crucial for localized destruction and perforation of a microcapsule. The optical intensity reaches a peak at a diameter of 750-1000 nm, indicating the resonance of a scattering mode by a microcapsule. Then, the optical intensity decreases with increasing diameter, reaches a minimum at 1500 nm, and increases again as it approaches next resonance diameter. These results indicate that 1000 nm in diameter is a candidate to be used for the incident wavelength of 800 nm. Figure 4 shows dependence of the maximum intensity and its z-position on the external diameter of a microcapsule. The maximum intensity can be obtained in the vicinity of the microcapsule for external diameters of 500 nm and 750 nm, indicating that Mie scattering is dominant in this regime. The z-position of the maximum intensity increases with increasing diameter of a microcapsule which indicates the shifts from the Mie scattering near-field domain to far-field lens effect. The peak intensity reaches a peak at 1250 nm, and remains higher than 4 up to 1750 nm. The long-lasting focusing at 1000 nm in diameter [Fig. 2(C)] or far-field focusing for external diameters larger than 1250 nm [Figs. 2(D)-2(F)] may be used for advanced applications such as simultaneous treatments of controlled release of embedded molecules and cell membrane perforation.

 

Fig. 2 Optical intensity distributions on the x-z plane simulated by the 3D FDTD method for polymer microcapsules with different external diameters: (A) 500 nm, (B) 750 nm, (C) 1000 nm, (D) 1250 nm, (E) 1500 nm, and (F) 1750 nm. A plane wave was incident on the microcapsule with the wave vector in the -z direction. The shell thickness was set to 200 nm for all external diameters.

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Fig. 3 Dependence of the peak optical intensity at the external surface of a microcapsule on the external diameter of the microcapsule calculated by the 3D FDTD method. The vertical axis indicates optical intensity enhancement in relation to the incident optical intensity.

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Fig. 4 Dependence of the maximum optical intensity (blue line) and its z- position (red line) on the external diameter of a microcapsule. The increase in the z- position shows shifts from the Mie scattering near-field domain to far-field focusing.

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Figure 5 shows an SEM image of microcapsules before and after fs laser irradiation. Since the microcapsules were dispersed in water at the laser irradiation, the aggregation of microcapsules is due to the collection and dispersion on the Si substrate for the observation after the laser irradiation. A pore, which is smaller than the diffraction limit determined by the Abbe criterion for far-field optics, was fabricated in the shell of the microcapsules after fs laser irradiation. This is explainable on the basis of the nonlinear interaction between the fs laser pulse and the polymer at the localized spot on a shell. The irradiated laser intensities of 6.6 × 10¹² W/cm² was lower than the ablation thresholds for many transparent materials [16,17]. Figure 6 shows detailed optical intensity profiles for microcapsules 1000 nm in diameter. The full width at half maximum (FWHM) on the y-axis was 230 nm on the external surface on a shell, indicating that the interaction spot is localized at a space below the theoretical diffraction limit for far-field optics. The optical intensity increased by a factor of 3.0 on the surface of the microcapsule in relation to the incident optical intensity. The estimated intensities on the surface of a microcapsule are enhanced to 2.0 × 10¹³ W/cm². In this intensity domain, nonlinear optical interaction with transparent materials such as self-phase modulation, multiphoton absorption, and laser ablation are observed [17]. In our experiments, it is highly probable that an enhanced optical field with higher optical intensity was generated in a localized spot on the shell of the microcapsule, which resulted in a multiphoton ionization and other nonlinear interactions followed by the localized destruction of the microcapsule and fabrication of a pore in its shell. The femtosecond laser contributed to maintaining the spherical shape because the femtosecond laser can realize intense ablation on various kinds of materials with a limited heat-affected zone. In the experiment, both microcapsules with and without pores were observed in the experiment. This is due to the Brownian motion and the displacement of the microcapsules by laser-induced shock wave during laser irradiation [18]. The shape of the irradiated microcapsules was kept spherical except for the pore [arrows in Fig. 5(B)] in the shell for most of the microcapsules. The sizes of pores vary but are 150-400 nm in diameter which are smaller than the diffraction limit. By comparison with calculated optical intensity shown in Fig. 6, the experimental results indicate that the pore was fabricated by a localized-enhanced optical intensity by the microcapsule. Several microcapsules have two or more pores in the shell because we applied multiple pulses of fs laser. Reproducible results were obtained for 4 separate experiments under the same experimental conditions. The percentage of the microcapsules with a single or a few pores to the number of total microcapsules was 32% which was counted on the basis of the picture taken by SEM. However, only the top hemisphere of the microcapsules was observed by SEM and therefore more microcapsules should have a pore in their bottom hemisphere. Substantial disruption of a shell was also observed for some microcapsules shown as X in Fig. 5(B). The percentage of the microcapsules disrupted substantially was 16%. The difference in the morphology of microcapsules after laser irradiation is probably due to the dispersion of the diameter and the roughness of original microcapsules which affects the enhanced optical intensity around a microcapsule.

 

Fig. 5 SEM image of microcapsules: (A) before fs laser irradiation and (B) after fs laser irradiation at 6.6 × 10¹² W/cm². (C) Enlarged image of dashed square in (B). A nanopore [pointed out with white arrows in (B)] was fabricated in the shell of the microcapsules after fs laser irradiation. Substantial disruption of a shell was also observed indicated as X in (B). Scale bars indicate 1000 nm.

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Fig. 6 Optical intensity profiles along (A) the z- axis, (B) the x-axis and (C) the y-axis. (B) and (C) Optical intensity profiles on the external surface of a microcapsule (z = 0 nm) and that on the peak intensity (z = −375 nm). The FWHM on the y-axis at z = 0 nm was 230 nm, indicating that the interaction spot was localized to a submicron scale on a shell.

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4. Conclusion

In conclusion, we have demonstrated the nanoperforation of polymer microcapsules by using near-infrared fs laser pulses. To the best of our knowledge, this is the first demonstration of the localized destruction of transparent microcapsules by near-infrared laser, which can penetrate deeply in tissues, without doping with absorbing materials such as metal or metal oxide nanoparticles. The presented method is a promising approach for the controlled release of therapeutic drugs by using drugs-encapsulated in microcapsules without concerns for potential toxicity caused by nanoparticles. The method has potential to advance safe drug delivery because biodegradable polymers can be used.