Imaging Laser-Triggered Drug Release from Gold Nanocages with

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Imaging laser triggered drug release from gold nanocages with transient absorption lifetime microscopy (TALM) Yongkui Xu, Qi Liu, Ruoyu He, Xianchong Miao, and Minbiao Ji ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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ACS Applied Materials & Interfaces

Imaging laser triggered drug release from gold nanocages with transient absorption lifetime microscopy (TALM) Yongkui Xu, Qi Liu, Ruoyu He, Xianchong Miao, Minbiao Ji* State Key Laboratory of Surface Physics and Department of Physics, Key Laboratory of Micro and Nano Photonic Structures (Ministry of Education), Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai 200433, China E-mail: [email protected] Keywords: drug delivery, nanomedicine, transient absorption microscopy, photodynamic therapy, nanobomb. Abstract: Nanoparticles have shown promise in loading and delivering drugs for targeted therapy. Many progresses have been made in the design, synthesis and modification of nanoparticles to fulfill such goals. However, realizing targeted intracellular delivery and controlled release of drugs remain challenging, partly due to the lack of reliable tools to detect the drug releasing process. In this paper, we applied femtosecond laser pulses to trigger the explosion of gold nanocages (AuNCs) and control the intracellular release of loaded AlPcS molecules for photodynamic therapy (PDT). AuNCs were found to enhance the encapsulation efficiency and suppress the PDT effect of AlPcS molecules until they were released. More importantly, we discovered that the excited-state lifetimes of the AlPcS-AuNCs conjugate (~3 ps) and free AlPcS (~11 ps) differ significantly, which was utilized to image the released drug molecules by transient absorption lifetime microscopy (TALM) with the same laser source. This technique extracts similar information as fluorescence life time imaging microscopy (FLIM), but is superior in imaging molecules that hardly fluoresce or prone to photo-bleaching. We further combined a dual-phase lock-in detection technique to show the potential of real-time imaging based on the change of transient optical behaviors. Our method may provide a new tool for investigating

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nanoparticle assisted drug delivery and release.

1. Introduction Controlled drug delivery has become a new strategy for targeted therapy by using carriers to load drugs and release them only in the disease sites in a steerable manner,1-3 thus reduce side-effects caused by drugs invading into normal tissues. However, the current targeting effects of drugs remain to be improved due to the complexity of living systems.4 With the rapid development of nanotechnology, nanoparticles have been suggested as efficient carriers for drug delivery.5-6 Gold nanoparticles have many unique properties that allow them to be used extensively in biophysics and biochemistry researches,7-10 these properties include chemical inertness, biocompatibility, wide and tunable absorption spectra, plasma resonance and high extinction coefficient. For instance, gold nanoparticles exhibit giant one- and twophoton absorption cross sections in the visible and near-infrared (NIR) region due to localized surface plasmon resonances (SPR),

11-14

enabling their applications for photo-thermal therapy

and photo-acoustic imaging.11, 14-16 Ever since the early report of synthesized gold nanocages (AuNCs), many efforts have been made on their preparations and applications.7,

10, 17-18

Although several studies have reported photo-thermal induced drug release from AuNCs, the low release efficiency and lack of quantifying the amount of drug release have hindered their applications.19-21 On the other hand, SPR was found to induce explosion of nanoparticles under the irradiation of ultrashort laser pulses,

22-23

which suggests that AuNCs may become

an effective means for drug delivery and efficient release with the proper use of pulsed lasers. In addition to the chemical effects of released drugs, the explosion of AuNCs may also induce photothermal and mechanical damage to the host cells, further strengthening the killing effects. ACS Paragon Plus Environment

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Although conceptually feasible, practically controlling drug release via laser triggered explosion remains challenging. Besides the controlled release, being able to detect the released drugs in living systems would be critical in understanding the dynamical process of how the molecules escape from their carriers. Although fluorescence based methods have been developed to probe such processes,

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they suffer from photo bleaching and rely heavily on the strong fluorescent

signal of drug molecules, which is often not the case. For example, photodynamic drugs tend to have weak fluorescence because of the energy transfer to triplet state oxygen. We have previously applied fluorescence-lifetime imaging microscopy (FLIM) to detect drug release based on the change of fluorescence lifetime during the releasing process.19 However, FLIM is difficult to provide the high speed for real-time imaging. Therefore, developing novel imaging techniques to measure drug release in living cells is highly desired. In this work, we studied the effect of femtosecond (fs) laser irradiation on AuNCs loaded with AlPcS, a common photodynamic therapy (PDT) drug. Unlike the continuous wave (CW) laser irradiation, where the morphology of AuNCs changed insignificantly; pulsed laser generates heat in a very short amount of time, resulting in the explosion of nanostructures to become formless debris.25 We further developed an imaging technique to detect released drugs after the triggered explosion. The technique is based on time-resolved transient absorption (TA) spectroscopy, which measures the differential absorption of the probe beam induced by the excitation of the pump beam, and has been widely used in studying photo- carrier dynamics, hot electron and phonon relaxation, etc.26-27 TA based microscopy has also been applied to image various metallic and semiconductor nanostructures.28-34 We have discovered a significant difference between the excited-state ACS Paragon Plus Environment


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lifetimes of AlPcS-AuNCs conjugate (~3 ps) and free AlPcS (~11 ps). Therefore, TA imaging taken at multiple delay times would allow us to extract the lifetime information and visualize the released AlPcS molecules. Moreover, a dual-phase lock-in detection scheme was applied to simultaneously image at two delay times, showing the potential of real-time imaging. Our study provides a way of controlling drug release using pulsed lasers and imaging the released drug with the same laser source.

2. Results and discussions Characterizations of synthesized AgNCs, AuNCs and AlPcS-AuNCs conjugates are shown in Figure 1. TEM images show a size of ~35 nm for both AgNCs and AuNCs (Figures 1A & B), which is suited for passive targeting because of the enhanced permeability and retention (EPR) effect of tumor tissues.35 UV-Vis absorption spectra indicate that AgNCs and AuNCs have SPR bands at 410 nm and 780 nm respectively (Figure 1D). We covered the surface of the nanocages with positively charged PVP to ensure their well dispersion in water and decrease toxicity. The absorption and fluorescence spectra of AlPcS are shown in Figure 1E, along with its molecular structure. AlPcS has two absorption bands at 360 nm (B band) and 665 nm (Q band) and a fluorescence peak at 675 nm. Notice that AlPcS is negatively charged because of the presentence of 4 sulfonic groups. Thus, the AlPcS-AuNCs composites are easy to form due to the electrostatic interactions between positively charged nanoparticles and negatively charged AlPcS molecules. The loading experiment was carried out by mixing 1 mL 20 pM AuNCs, with 10 µL 3 mM AlPcS in dark and shaking on a vortex for over 24 hours. These composite samples were centrifuged to separate the precipitated composites and free AlPcS in the supernatant. The ACS Paragon Plus Environment

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decreased zeta potential (from +33±4 mV to +6±3 mV) indicates that AlPcS molecules have successfully conjugated with AuNCs (Fig. S1). The encapsulation efficiency was estimated to be ~80% (Fig. S2) by comparing the fluorescence intensity of free AlPcS in the supernatant with that of AlPcS at the initial concentration (10 µM). Since the SPR band of AuNCs appears in the near infrared (NIR) region, we applied 780 nm laser pulses (100 fs, 80 MHz) to induce rapid explosion of AuNCs and subsequent release of loaded AlPcS molecules. After irradiation with unfocused 780 nm pulsed laser for 20 s at high power density (50W/cm2), the shape of nanocages became irregular with both larger chunks and smaller debris, as shown in Figure 1C. In contrast, irradiation under 780 nm CW laser with the same power density and duration of time did not induce severe explosion (Fig. S3). In vitro laser induced explosion was also performed and verified by in-situ transmission electron microscope (TEM), as shown in Fig. S4. To investigate the release efficiency, we used the same pulsed laser to irradiate the AlPcS composite in aqueous solution for 30 s, and collected the supernatant AlPcS solution after centrifugation. Fluorescence intensity measurements were used to determine the released amount of AlPcS in the supernatant, and the releasing efficiency was calculated to be ~70% (Fig. S5). The setup of our transient absorption microscopy is shown in Figure 2A. Briefly, a femtosecond laser optical parametric oscillator (OPO, Insight DS+, Spectra-Physics, CA) with dual-outputs was used as the light source. The tunable OPO output (690 – 1300 nm, ~120 fs) was tuned to 780 nm to serve as the probe beam, and the fundamental beam of 1040 nm was frequency doubled to 520 nm to serve as the pump beam. These two beams were combined with a 550 nm long pass dichroic mirror (DMLP550, Thorlabs), overlapped in space and time, guided into a laser scanning microscope (FV1200, Olympus) and focused at the sample with ACS Paragon Plus Environment


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an objective lens (UplanSApo, 60×, NA 1.2). The pump beam was intensity modulated at 20 MHz by modulating the 1040 nm beam using an electro-optical modulator (EOM, EO-AM-NR-C2, Thorlabs). The probe beam was filtered out by an 890 nm band pass filter (CARS890/220 M) and detected by the Si photodiode. The weak TA signal in the probe beam was then demodulated by a lock-in amplifier (HF2LI, Zurich instruments). Time-resolved experiments were realized by measuring the TA signals while changing the delay time between pump and probe pulses, by scanning the optical delay line (DL) with a motorized stage (MFA-CC, Newport). Notice that the pump and probe wavelengths were set away from the resonance bands of AlPcS to minimize the photodamage, thus the transitions most likely came from two-photon excitations of AlPcS. The 780 nm beam was chosen to meet the SPR band of AuNCs so that it could trigger their explosion and detect the transient signals from AuNCs, free and conjugated AlPcS. The measured time-resolved TA signals of these three samples are shown in Figure 2B, which could be fitted with bi-exponential decay functions:

∆ = exp − + exp −

(1).

For AuNCs, the results show a fast decay time of ~2.7 ps, which originates from the cooling of hot electrons via electron-phonon interations.36 And a slower time constant of ~50 ps may come from interband relaxation processes. Whereas AlPcS shows a major decay of ~11.4 ps and a much slower radiative decay that could be treated as a constant offset. Interestingly, the AlPcS-AuNCs conjugates appear to decay mono-exponentially with a time constant of ~2.9 ps, close to that of AuNCs. The distinguished differences between the excited-state lifetimes of free AlPcS and AlPcS-AuNCs conjugates allow us to detect AlPcS released from AlPcS-AuNCs conjugates based on the change of TA decay time constant, in a similar way as FLIM. ACS Paragon Plus Environment

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Next we imaged HeLa cells incubated with free AlPcS, AuNCs and AlPcS-AuNCs conjugates using bright field, two-photon excited fluorescence (TPEF) and transient absorption lifetime microscopies (TALM), as shown in Figure 3. TPEF images show much enhanced signal from the AlPcS conjugates compared with free AlPcS treated cells, indicating that AuNCs could efficiently adsorb AlPcS molecules and deliver them into cells (Figure 3, column 2). TA microscopy was measured at four time delays (τ= 0.5 ps, 1.5 ps, 2.5 ps, and 3.5 ps) in a sequential imaging mode (~1 s/frame), and the decay dynamics was fitted with single exponential for each pixel. The TA lifetime images are color coded with the decay time shown in the color bar (Figure 3A-C, column 3 and 4). We took measurements after different amounts of irradiation time by continuously scanning the 780nm laser beam across the samples. The 520 nm beam was unblocked only to acquire TA images with duration of 4 s for each data set. As seen in Figure 3A and B, cells treated with free AlPcS and AuNCs maintained their lifetimes of ~10 ps and ~2.1 ps respectively during the 5 min of 780 nm irradiation, indicating that such irradiation does not alter the lifetimes of AlPcS and Au nanoparticles, even though the AuNCs have already undergone morphological changes as shown in Figure 1. In contrast, the intracellular AlPcS conjugates experienced gradual changes of excited-state lifetime, from initial ~2.2 ps to ~6.5 ps after 5 min of 780 nm irradiation under the same laser intensity (Figure 3C and Fig. S7). Statistical data of the lifetimes analyzed on each pixel of the three cell groups is shown in Figure 3D. And comparison of the decay data at 0 and 5 min of irradiation times is plotted in Figure 3E, which represents the averaged signal from the rectangular area in Figure 3C. These results indicate that AlPcS molecules with longer lifetimes were indeed released from AlPcS-AuNCs conjugates, and subsequently slowed down the overall TA decay. Control experiments with ACS Paragon Plus Environment


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bare HeLa cells were shown in Fig. S6. The above experiments with sequential frame by frame imaging method have a few drawbacks. First, it could not accurately measure the dynamical processes shorter than 4 seconds. Second, potential drifting of intracellular organelles and nanoparticles during the image acquisition processes will cause “motion artifacts” that disturb final images and generate more noises. Therefore, parallel imaging methods will be much more suited for real-time measurements. As a proof of principle demonstration, we applied a simultaneous two-color imaging method with a dual-phase modulation and detection scheme (Figure 4), as reported earlier by our group.37-38 The 520 nm pump beam was turned into a Mach-Zehnder interferometer geometry, where the time delays of the two arms (Pu1 and Pu2) were precisely controlled so that their modulation phases differ by π/2. The modulation frequency of EOM was synchronized to a quarter of the laser repetition rate (f0=80 MHz), thus each modulation period contains exactly four pulse intervals. Then adding a delay of one pulse interval (1/f0) between Pu1 and Pu2 will shift the phase difference to π/2. By fine adjustment of the two delay lines, we could generate TA signals at two different time delays (τ1 and τ2) modulated in quadrature phase. The total signal could be written as: = τ sin

+ τ cos

(2).

Therefore, a phase sensitive lock-in amplifier could be used to detect τ and τ simultaneously through the in-phase (X) and quadrature (Y) output channels. Figure 5 shows the dual-phase, parallel TA images of AlPcS-AuNCs conjugates in Hela cells taken at two delay times (0.5 ps and 2.5 ps) within 1 second. The imaging speed could in principle reach video rate if a resonant scanner was applied. The square area in Figure 5D was irradiated with 780 nm pulsed laser for 5 min, and demonstrated a clear lifetime contrast with ACS Paragon Plus Environment

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the surrounding areas, indicating that the dual-phase TA technique is feasible in rapid detection of the drug release from drug-nanoparticle conjugates. This result also demonstrates the selective release of AlPcS molecules from AlPcS-AuNCs conjugates with controlled laser targeting. Although increasing parallel channels will produce more accurate lifetime information, our two-channel experiment has demonstrated the feasibility of utilizing this concept for future studies of drug release in real time. Laser triggered drug release was further verified by measuring the PDT effects of different forms of AlPcS on incubated HeLa cells (Figure 6). AlPcS is a well-known photosensitizer which produces 1O2 under red light illumination. The efficiency of PDT effect of a drug depends on its efficiency of 1O2 production. We used 1,3-diphenylisobenzofuran (DPBF) to probe the concentration of 1O2 generated by AlPcS in aqueous solution under the irradiation of 660 nm CW light, which corresponds to the absorption Q band of AlPcS. We observed the fluorescence intensity of DPBF decayed with increasing photo-activation time for AlPcS, due to the quenching effect of generated 1O2 on DPBF fluorescence (Figure 6A). Thus, we can quantify the rate of 1O2 production by measuring the decay rate of DPBF fluorescence intensity. As shown in Figure 6B, 1O2 producing rate of free AlPcS is about 11 times faster than that of intact AlPcS-AuNCs conjugates. In great contrast, when the AlPcS-AuNCs conjugates were irradiated by 780 nm fs light, their 1O2 producing rate increased by 8 times, close to that of free AlPcS. These results indicate that the AlPcS molecules inside AlPcS-AuNCs conjugates could not efficiently produce 1O2, whereas the released AlPcS after laser triggered explosion could recover the high efficient 1O2 production. The reduced 1O2 producing rate of AlPcS-AuNCs conjugates might result from multiple factors, including the spatial isolation of AlPcS from solvated O2, energy transfer from excited ACS Paragon Plus Environment


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AlPcS to AuNCs. Such a “shielding” effect of AuNCs plays the key role in controlled PDT with laser triggered AlPcS release. In vitro studies in HeLa cells to evaluate the PDT effect of AlPcS-AuNCs are shown in Figure 6C. Results are presented as the mean ± standard deviation (SD) from four groups: the control group with cells only (cyan), cells with AuNCs (green), cells with AlPcS (red), and cells with AlPcS-AuNCs conjugates (yellow). Each group contained six wells with different experimental parameters: no light illumination (blank), 660 nm red light irradiation to activate AlPcS (vertical lines), 780 nm NIR femtosecond light irradiation to trigger AuNCs explosion (horizontal lines), and NIR fs light irradiation followed by red light irradiation (grids). These results could be summarized as follows. (1) For the control group, neither the red light nor the NIR fs light could cause severe damage to the cells due to their weak light absorption in the red-NIR region. (2) For cells incubated with AuNCs, the survival rates were found to decrease with increasing AuNCs concentration, indicating measurable toxicity which might come from the release of residual silver ions,39 though it is considerably lower than CTAB mediated AuNRs.40-41 We also observed fs laser induced damage due to photo-thermal and nano-explosion effects. (3) For cells incubated with AlPcS, normal PDT effects were seen under red light illumination. (4) And for cells incubated with AlPcS-AuNCs conjugates, the PDT effects induced by red light illumination were much weaker than with free AlPcS. However, if the composites were irradiated with NIR fs light (30 s) prior to red light illumination, the PDT effects were found to increase dramatically. Under the same amount of red light illumination time (30 min), the survival rate has dropped efficiently from ~70% to ~10% due to the pre-irradiation of NIR fs light. Combining all these observations, we could conclude that NIR fs laser could indeed trigger the release of AlPcS, and thus provide much ACS Paragon Plus Environment

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higher PDT effect, which is otherwise suppressed by AuNCs. The key factor to realize our laser controlled drug delivery and imaging experiments is the “shielding” effect of AuNCs. These AuNCs not only suppress the PDT effect of encapsulated AlPcS molecules, but also minimize their influence on the transient lifetime of AlPcS conjugates. The lifetime of AlPcS has changed from ~11 ps in the free form to ~ 3 ps in conjugates, which is close to that of bare AuNCs. These may result from the gold surface plasmon assisted relaxation that quenches the excited-state of AlPcS,42 thus reduces the PDT effect as well as the transient lifetime of AlPcS. Notice our approach does not quantitatively measure the concentrations of AlPcS in the free or conjugate forms, partly due to the lack of standard methods to precisely calibrate the local concentration of AlPcS in AlPcS-AuNCs conjugates. The single exponential fitting method of the TA imaging data could only provide semi-quantitative assessment of the lifetimes. In principle, TA signal is linearly proportional to chemical concentration, and it is possible to calculate the concentration of each chemical species in a mixture given the pre-knowledge of their “spectral” or temporal behaviors.43-45 However, more precise controls need to be accomplished in both sample purification (of AlPcS) and TA measurements. The current work provides a new perspective in resolving chemical species – utilizing the ultrafast transient lifetimes to probe the change of molecular states, which has potential applications in drug release and other chemical reactions that result in significant change of lifetimes.

3. Conclusion In conclusion, we have demonstrated that AuNCs could efficiently adsorb AlPcS molecules and enter live cells, as well as release the drugs in a controllable manner by NIR femtosecond ACS Paragon Plus Environment


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laser irradiation. We verified that multiple factors have enable AuNCs to be a promising candidate for targeted therapy. First, AuNCs could assist cellular uptake

of AlPcS, due to

their high encapsulation efficiency. Second. AlPcS conjugate has ~10 fold suppressed PDT effect, due to the limited contact of AlPcS with oxygen, making it relatively safe to deliver drugs. Third, fs laser triggered explosion of AuNCs could effectively set free the loaded AlPcS, resulting in high PDT effect. Furthermore, we have applied transient absorption lifetime microscopy (TALM) to image the laser triggered release of drug molecules by taking advantage of the different transient lifetimes between AlPcS and free AlPcS, and demonstrated that parallel TA microscopy could potentially offer real-time imaging capability to study the dynamics of drug release. We believe our strategy of femtosecond laser controlled drug release and imaging technique would provide a new route for researches in the fields of drug delivery and nanomedicine.

4. Materials and methods 4.1. Materials HAuCl4, AgNO3 were obtained from Sigma-Aldrich. AlPcS was provided by Frontier Scientific, Logan, UT, USA. NaOH, Na2S, diethylene glycol (DEG) and polyvinyl pyrrolidone (PVP, MW=55 000) was purchased from Aladdin Industrial Inc. The D.I. water was self-produced with ions resin. 3-Diphenylisobenzofuran (DPBF) was bought from J&K Chemical. Hela cells (human epithelial cervical cancer cell line) and KB cells (a sub-line of the keratin-forming tumor cell line) were obtained from the cell bank of Shanghai Science Academy. Dulbecco's modified eagle medium (DMEM), fetal bovine serum, 0.25% ACS Paragon Plus Environment

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trypsin-EDTA (1x), Phosphate-Buffered Saline (PBS) and penicillin streptomycin were purchased from Gibco. WST-1 (a new kind of Cell Proliferation and Cytotoxicity Assay Kit), was

obtained

from

Beyotime

and

applied

as

a

replacement

for

MTT

(3-

（4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. 4.2. Synthesis of AuNCs We synthesized AuNCs using the galvanic replacement reaction between HAuCl4 and silver nanocubes (AgNCs), which was used as the template.10, 46 Synthesis method can be separated into two steps. First, synthesis of AgNCs using the sulfide-mediated polyol process as described in previous publications.10, 46 Typically, 20 mL of DEG was preheated to 150 °C for 1 h under magnetic stirring in a 250 mL round-bottomed flask. Then a flow of nitrogen was introduced at a rate of 1 L/min for 10 min. DEG solutions containing Na2S (3 mM, 180 µL) and PVP (20 mg/mL, 3.75 mL) were then quickly injected into the flask, followed by the injection of AgNO3 (282 mM, 1.2 mL). Then the reaction solution would go through four distinct stages of color change, from golden yellow to deep red, reddish gray, and then green ocher within about 3 hours. The reaction solution was then quenched by placing the reaction flask in an ice-water bath. The resultant product was washed 3 times with acetone at a volume ratio of 1:3 and centrifuged (8000g, 10 min) to remove excess DEG and PVP. Finally, the AgNCs were redispersed in 20 mL of DI water for further characterization and usage. Second, the AgNCs were converted to AuNCs. Briefly, 500 µL of the AgNCs was added to 5 mL of deionized water containing PVP (1 mg/mL) hosted in a 50 mL flask under magnetic stirring and then heated to boil for 10 min. The HAuCl4 (0.5 mM) was then added to the flask via syringe pump at a rate of 20 mL/h. Every 5 min, the solution was extracted to test ACS Paragon Plus Environment


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the absorption spectrum with UV-VIS spectroscopy (Agilent 8453). When the solution had an optical extinction peak at 780 nm, it was refluxed for another 20 min until the color of the reaction became stable. Once cooled to room temperature, the sample was centrifuged and washed with saturated NaCl solution to remove AgCl and with water several times to remove PVP and NaCl before further characterization and usage. 4.3. Characterization of nanoparticles We measured the absorption spectra of AlPcS and composites using Agilent 8453 with quartz cuvettes of 10 mm optical path length. The TEM images were taken by placing samples on carbon coated copper grids with a JEM-2100 transmission electron microscope operated at an accelerating voltage of 200 kV. Zeta potential was measured in the Malvern Zetasizer Nano S90 with a standard 633 nm laser. Fluorescence spectra were measured in a spectrometer (Hitachi, F-2500) with a 10 mm optical path quartz cuvette. 4.4. Cell culture Cells were maintained in DMEM medium with 10% calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin and 100 µg/mL neomycin in a humidified standard incubator with a 5% CO2 atmosphere at 37 ºC. When the cells reached 80% confluence with normal morphology, the tested compound was added and the cells were incubated for 4 hours for both AuNCs and AlPcS conjugates, and 24 hours for free AlPcS. The cells were washed three times with PBS before fluorescence and TA imaging measurements. 4.5. Cell imaging The fluorescence imaging of AlPcS in cells was acquired in a laser scanning confocal microscope (FV1200, Olympus) equipped with a band-pass filter of 660±15 nm in front of the PMT. White field images were recorded simultaneously in a transmission channel to show the ACS Paragon Plus Environment

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cell morphology. 4.6. Cytotoxicity of AlPcS, and AlPcS-AuNCs composites WST-1 was used to measure the dark toxicity of AlPcS, AuNCs, and AlPcS-AuNCs composites on cells. 200 µL cell suspensions with a consistency of 103 cells per mL was seeded in each well of a 96-well flat bottom tissue culture plat and allowed to attach to the plat and proliferate. When the cells reached 80% confluence with normal morphology, the AlPcS and AlPcS composites were added into corresponding wells, respectively, and incubated for the desired time. Then, the cells were incubated in 100 µL DMEM with 10 µL MTT solution (5 mg/mL) for another 3 h. Finally, the optical densities (OD) at 450 nm and 630 nm of each well were measured in an iEMS Analyzer (Lab-system). The cell viability in each well was determined by comparing the OD value with that of untreated control cells in other wells of the same plate. All results were presented as the mean ± SE from three independent experiments with 6 wells in each. 4.7. Singlet oxygen (1O2) measurements DPBF, a sensitive 1O2 probe, was used to measure the 1O2 photo-produced by AlPcS in different situations. Upon oxidative degradation by 1O2, the fluorescence of DPBF was quenched, so that the reducing rate of DPBF fluorescence in the sample solution is proportional to the 1O2 production. In the experiment the DPBF (10 mM) was added into the sample solution. The 10 mW 660±15 nm light was used to irradiate the sample solutions at different time interval. Then the fluorescence of DPBF was measured accordingly to quantify the relative 1O2 production. 4.8. PDT effect of released AlPcS from AlPcS on cells



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A culture plate with 96 wells, previously seeded in each well with 200 µL cells with concentration of 3×103 cells/ mL, was used in this experiment. When the cells reached 80% confluence with normal morphology, the AlPcS was added into each well and incubated for 3 h. After incubation, the cells were washed with PBS three times and replenished with fresh medium. Then these cell wells were irradiated by a 780 nm fs laser with power density of 500 mW/mm2 for desired times for different wells to make the AlPcS released from cellular AlPcS composites. After that, the cell wells were further irradiated by a CW light beam (660±15 nm) with the power density of 10 mW/cm2 to carry out PDT. After the irradiation, the cells were incubated for another 24 h. Then, the cells were incubated in 100 µL DMEM with 10 µL MTT solution (5 mg/mL) for 2 h. Finally, the optical densities (O.D) at 450 nm and 630 nm of each well were measured in an iEMS Analyzer (Lab-system). The cell viability in each well was determined by comparing the O.D value with that of untreated control cells in some wells of the same plate. All results were presented as the mean ± SE from three independent experiments with 6 wells in each.

Supporting Information: zeta potential; measurement of encapsulation and releasing efficiency; control experiments of cell imaging; TEM images; and TALM images at more time delays.

Acknowledgments We are grateful for financial support from the National Key Research and Development Program of China (2016YFC0102100, 2016YFA0301000); National Natural Science Foundation of China


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(81671725); Shanghai Rising Star program (15QA1400500); and Shanghai Action Plan for Scientific and Technological Innovation program (16441909200).

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Figure 1. Characterization of silver nanocubes (AgNCs), gold nanocages (AuNCs), AlPcS and AlPcS-AuNCs conjugates. TEM images of (A) AgNCs, (B) AuNCs and (C) AuNCs irradiated by 50 W/cm2 780 nm fs laser for 20 s. (D) UV-Vis absorption spectra of AgNCs and AuNCs. (E) Absorption and fluorescence spectra of AlPcS aqueous solution. Scale bar: 200 nm.



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Figure 2. Experimental setup of transient absorption microscope. (A) Optical layouts. EOM: electro-optical modulator; PD: photo-diode; PBS: polarizing beam splitter; DL: delay line; LIA: lock-in amplifier. (B) Measured time-resolved transient absorption curves of AuNCs, free AlPcS and AlPcS-AuNCs conjugates in aqueous solutions.


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Figure 3. Imaging nanoparticles and drug molecules in HeLa cells. Cells were incubated with (A) AlPcS (10 µM) for 24 hours, (B) AuNCs (10 pM) for 4 hours, and (C) AlPcS-AuNCs conjugates (12 µM to 10 pM) for 4 hours. Images were taken with bright field microscopy (column 1), two-photon excited fluorescence (TPEF) microscopy (column 2), and transient absorption lifetime microscopy (TALM) under different irradiation times of 780 nm femtosecond laser (0 and 5 min for column 3 and 4, respectively). TALM images are color coded with the fitted lifetime, as shown in the color bar. (D) Statistical data of TALM images showing the percentage of pixels with lifetime shorter (red) or longer (green) than 4 ps. (E) Mean transient signal within the rectangular area in (C). Scale bar: 50 µm



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Figure 4. Illustration of the dual-phase transient absorption microscope for parallel imaging. (A) Optical setup. (B) Modulated pulse trains of the two pump beams with a π/2 phase difference. (C) Detailed profiles of the pump and probe pulses.


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Figure 5. Images of AlPcS-AuNCs cultured HeLa cells taken with dual-phase transient absorption microscopy. (A) White field image and (B) two-photon excited fluorescence image of cells after 780 nm laser irradiation for 5 min in the green square region. (C) and (D) are TALM images before and after irradiation. The scale bar is 50 µm.



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Figure 6. Photodynamic effects of AlPcS, AuNCs and AlPcS–AuNCs after femtosecond laser triggered release. (A) Fluorescence intensity decay of DPBF (10µM) in AlPcS –AuNCs (10 µM-10 pM) aqueous solution. AlPcS was irradiated by an unfocused 660 nm light beam (10 mW) to generate 1O2, and DPBF was excited by 405 nm CW laser to generate fluorescence spectra. (B) Decay rates of DPBF fluorescence intensity under the irradiation of 660 nm reveal 1O2 concentration. (C) Cell viability (%) measured by MTT assay. HeLa cells were incubated with AuNCs (green), AlPcS (red) or AlPcS-AuNCs conjugates (yellow), and irradiated with 50 W/cm2 780 nm unfocused fs laser to trigger AuNCs explosion (horizontal lines) or 10 mW/cm2 660 nm red light (R, vertical lines) to induce PDT effect.


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Table of Contents (TOC):


Imaging Laser-Triggered Drug Release from Gold Nanocages with

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