Intracellular dynamic disentangling of Doxorubicin release from

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Biological and Medical Applications of Materials and Interfaces

Intracellular dynamic disentangling of Doxorubicin release from luminescent nanogold carriers by Fluorescence Lifetime Imaging Microscopy (FLIM) under two-photon excitation Sorina Suarasan, Ana-Maria Craciun, Emilia Licarete, Monica Focsan, Klara Magyari, and Simion Astilean ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21269 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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ACS Applied Materials & Interfaces

Intracellular Dynamic Disentangling of Doxorubicin Release from Luminescent Nanogold Carriers by Fluorescence Lifetime Imaging Microscopy (FLIM) under Two-Photon Excitation Sorina Suarasana, Ana-Maria Craciuna, Emilia Licareteb, Monica Focsana, Klara Magyaric, Simion Astileana,d,*

aNanobiophotonics

and Laser Microspectroscopy Center, Interdisciplinary Research Institute

in Bio-Nano-Sciences, Babes-Bolyai University, T. Laurian Str. 42, 400271, Cluj-Napoca, Romania bMolecular

Biology Center, Interdisciplinary Research Institute in Bio-Nano-Sciences, BabesBolyai University, T. Laurian Str. 42, 400271, Cluj-Napoca, Romania

cNanostructured

Materials and Bio-Nano-Interfaces Center, Interdisciplinary Research

Institute on Bio-Nano-Sciences, Babes-Bolyai University, T. Laurian Str. 42, 400271 ClujNapoca, Romania dBiomolecular

Physics Department, Faculty of Physics, Babes-Bolyai University, M. Kogalniceanu str. 1, 400084, Cluj-Napoca, Romania.

KEYWORDS: two-photon enhanced fluorescence, fluorescence lifetime imaging microscopy, Doxorubicin, gold nanoparticles, ovarian cancer

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ABSTRACT

There is still a lack of available technique to follow noninvasively the intracellular processes as well to track or disentangle various signals from the therapeutic agents at the site of action in the target cells. We present here the assessment of the intracellular kinetic of Doxorubicin (DOX) and gold nanoparticles carriers (AuNPs) by mapping simultaneously fluorescence and photoluminescence signals by Fluorescence Lifetime Imaging Microscopy under two-photon excitation (TPE-FLIM). The new nano-chemotherapeutic system AuNPs@gelatin-hyd-DOX has been fabricated by DOX loading onto the surface of gelatin biosynthesized AuNPs (AuNPs@gelatin) through a pH-sensitive hydrazone bond. The successful loading of DOX to the AuNPs was studied by spectroscopic methods and steady-state fluorescence and the nanosystem pH-responsive character was validated in simulated biological conditions at different pH buffers (i.e. pH 4.6, 5.3 and 7.4). Considering that the fluorescence lifetime of DOX molecules at a specific point in the cell is a reliable indicator for the discrimination of the different states of the drug in the internalization path i.e. released vs loaded, the kinetics of AuNPs@gelatin-hyd-DOX cellular uptake and DOX release was compared to free DOX, resulting two different drug internalization pathways. Finally, cell viability tests were conducted against NIH:OVCAR-3 cell line to prove the efficiency of our chemotherapeutic nanosystem. TPE-FLIM technique could be considered promising for non-invasive, highresolution imaging of cells with improved capabilities over current one-photon excited FLIM.


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1. Introduction Despite the recent progress in the design of smart cancer drug nanocarriers1–3, the kinetics of drug release and different pathways of internalization is not sufficiently understood currently. Fluorescence lifetime imaging microscopy (FLIM) is a non-invasive fluorescence imaging technique ideal to monitor fluorophores in cells or tissue by the spatial variation of the fluorescent lifetime, specifically the average time that a molecule remains in the excited state before emitting a fluorescence photon4. As the fluorescence lifetime is independent of variations in the dye concentration, photo-bleaching or excitation intensity but highly dependent on the local microenvironment of molecules, FLIM could become an efficient strategy to probe the transport and localization of fluorescent drugs inside the cell5,6. There are several chemo-drugs which exhibit good fluorescence characteristics but the most investigated was Doxorubicin (DOX), a well-known antitumoral drug. The optical spectra of free DOX as function of concentration were previously reported both by steady-state fluorescence and time-resolved fluorescence measurement7,8. Surprising, despite the fluorescence lifetime investigations recorded in simulated biological and aqueous solutions, FLIM has been slightly used to map the internalization of DOX in cells9,10 even it can also reveal its quantitative real-time release in live cells, as Adnan et al.11 recently proved by monitoring the time-dependent shift from longer to shorter fluorescent lifetime. On the other side, FLIM operating with excitation light in the visible range could be impeded by the use of so-called one-photon excitation (OPE) due to the background fluorescence, photon attenuation and scattering. Therefore, it is conceivable that by combining FLIM technique with two-photon excitation (TPE) by the use of near-infrared (NIR) excitation wavelengths pulse in the optical transparency window (700 – 1350 nm), the mapping of the internalization and kinetics of intracellular DOX release, will be achieved with light cells penetration at depth. Moreover, beyond the specific aim of DOX investigation, TPE-FLIM could in addition provide



visualization of luminescent nanocarriers with improved contrast, enhanced depth penetrability, reduced photodamage to surrounding living tissues and background free signal12,13, becoming thus nowadays a well-recognized tool in modern biomedicine research. Indeed, when used as drug carriers, plasmonic nanoparticles (NPs) not only act as efficient two-photon imaging contrast agents due to their own strong photoluminescence generated under TPE by near-infrared (NIR) pulse laser14,15 but also could amplify the TPE fluorescence of drugs due to coupling with localized surface plasmon resonance. There are only several reports that exploit the TPE of plasmonic NPs for cellular imaging. For example, Gao et al.14 have studied the ability of several shapes gold nanoparticles (AuNPs) to operate as twophoton imaging contrast agents in HepG2 cancer cells and Durret et al.16 demonstrated the strong signal, resistance to photobleaching and chemical stability of gold nanorods (AuNRs) as contrast agents for two-photon imaging of epithelial cancer. Zhao et al17 also demonstrated the ability of AuNRs to perform as dual photo-sensitizing and imaging platforms for TPE imageguided photodynamic therapy of cancer cells. Moreover, Qu et al.18 determined that AuNPs are a feasible alternative to fluorescent dyes for cancer diagnosis and cellular imaging using multiphoton FLIM. Our group has recently reported the potential of three different shaped AuNPs (i.e. nanospheres, nanorods and nanotriangles), biocompatibilized with gelatin, to act as reliable label free contrast agents to image by their intrinsic photoluminescence the signal from cancer cells by using FLIM technique under non-invasive NIR laser pulse15. We demonstrated also the capacity of TPE-FLIM technique to track and accurately localize the carboplatin loaded silver nanoparticles inside live NIH:OVCAR-3 cells13 and hollow Au−silver nanospheres grafted with antiCD19 monoclonal antibodies CD19(+) in cancer lymphoblasts12. Indeed, researchers concluded recently that TPE-FLIM is a promising noninvasive technique even for tracking skin penetration of nanoparticles19 and successfully compete with more invasive and time demanding cell imaging techniques. Surprisingly, to the best of our


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knowledge there is no study that investigates the TPE plasmonic induced metal enhanced fluorescence of DOX loaded onto AuNPs internalized in ovarian cancer cells. An efficient nano-chemotherapeutic system should improve the therapeutic effects of drugs to diseased tissues while reducing its side effects on healthy tissues and organs20,21. Considering the differences between normal and tumoral tissues in the lymphatic system, capillary walls of blood vessels and pH levels,22,23 body intrinsic stimuli responsive nanosystems are in the focus of recent studies. Amongst them, pH-responsive drug carriers have been mainly investigated24–26 since they are able to release the cargo drugs only at the site of the action, in the acidic microenvironment of the diseased tissue and in the acidic organelles of cells. In this context, the pH-cleavable hydrazone bond is an efficient linker between drugs – especially ones containing a ketone group such as DOX – and gold nanoparticles carriers27–29. Specifically, this linkage is fairly stable at neutral pH 7.4, with low or no DOX release expected at physiological conditions within the circulation, but – contrary, it is cleavable in the lower pH environments of cellular organelles28,30. Thus, by designing such delivery nanosystems will be possible not only to selectively deliver drugs by keeping them attached to the carrier during systemic circulation but also to provide a controlled release in acidic media of the tumoral cells31–34. However, is imperative to monitor the intracellular dynamic distribution and release of drugs11,35,36. Thus, TPE-FLIM imaging is the ideal tool for tracking the DOX intracellular dynamics since it can detect even slight changes in its distribution. In this study we prove the feasibility of fluorescence lifetime imagining microscopy (FLIM) in combination with two-photon excitation (TPE-FLIM) for the investigation of the DOX intracellular distribution from a new designed pH-responsive nano-chemotherapeutic system. Concretely, as nano-chemotherapeutic delivery system we propose AuNPs@gelatinhyd-DOX, specifically designed to trigger the drug release by its chemical responsivity at acidic pH. With this guidance, DOX was attached to the nanocarrier by the hydrazone bond realized



between the ketonic groups of DOX and hydrazine moiety37 fixed into gelatin layer around the AuNPs (see Scheme 1).

Scheme 1. Schematic illustration of the designed AuNPs@gelatin-hyd-DOX nanochemotherapeutic system.

First, the successful loading of DOX to the AuNPs was studied by UV-vis spectroscopic methods and steady-state fluorescence while the pH-responsive potential was validated in simulated biological conditions at different pH buffers (i.e. pH 4.6, 5.3 and 7.4). The kinetics of AuNPs@gelatin-hyd-DOX uptake by NIH:OVCAR-3 cell line and DOX release was monitored comparatively relative to free DOX by OPE- and TPE-FLIM. Cell viability tests were conducted to prove the potential of our new nano-system to perform as effective chemotherapeutic agent. Finally, we believe that it is for the first time when TPE and fluorescent lifetime is combined to generate a powerful imaging approach for DOX drug tracking, revealing consequently sensitive information about its intracellular distribution of the designed pH-responsive nano-chemotherapeutic systems.


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2. Materials and methods 2.1. Materials Hydrogen tetrachloroaurate-(III) trihydrate (HAuCl4 ∙ 3H2O, 99.99 %), gelatin type A, Doxorubicin hydrochloride (DOX), phosphate buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI), Penicillin/Streptomycin, L-glutamine, bovine insulin, and 3-(4,5dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution were purchased from Sigma-Aldrich. Fetal Calf Serum was purchased from HyClone and LysoTracker Deep Red from ThermoScientific. All chemicals were used as purchased and all solutions were prepared using ultrapure water (resistivity ~ 18 MΩ).

2.2. Nano-chemotherapeutic system preparation Spherical AuNPs were synthesized by the reduction of HAuCl4 with gelatin biopolymer solution using our previously reported method38. Briefly, a volume of the gelatin solution was mixed with an equal volume of 3 mM HAuCl4 solution and incubated for 6 h at 80 °C. The synthesized NPs were washed by centrifugation once for 15 min at 15000 RPM and then resuspended in water in the presence of NaOH. The obtained colloidal AuNPs@gelatin exhibits a plasmonic signature at 531 nm and an average diameter of 18±3.5 nm. The nano-chemotherapeutic system (denoted further as AuNPs@gelatin-hyd-DOX) was synthesized by loading the drug doxorubicin (DOX) to the gelatin from the surface of AuNPs through a hydrazone crosslinker. First, the colloidal AuNPs@gelatin was incubated with a hydrazine 1 mM solution for 24 h and then was allowed to react with a DOX solution (230 μM)



for another 24 h in dark at room temperature. Subsequently the complex was centrifuged once for 10 min at 8000 RPM to remove the unbound DOX and then re-dispersed in water. Finally, the DOX encapsulation efficiency (EE) and loading capability (LC) were quantified by already reported protocols, employing a calibration curve for DOX recorded at 485 nm maximum absorbance39.

2.3. DOX release study To trigger the release of DOX in the release experiments, 200 µl of AuNPs@gelatinhyd-DOX were added to 3.8 ml of buffer solutions previously thermo stated at 37 °C and lightly shaken. The DOX release trend from AuNPs@gelatin was investigated at pH 4.6, pH 5.3 and pH 7.4, respectively. The concentration of DOX released in the buffer medium at defined time intervals was quantified, as previously reported39, by measuring the fluorescence emission intensities of the release solutions at 593 nm against a standard calibration curve. The standard curve was obtained by recording the fluorescence emission intensity (λex 485 nm; λem 590 nm) of different concentrations of DOX solutions.

2.4. In vitro cellular studies 2.4.1. Cell culture NIH:OVCAR-3 human ovarian adenocarcinoma cells were grown in RPMI-1640 culture medium supplemented with 20% Fetal Calf Serum, 2 mM L-glutamine, 100 U/ml Penicillin/Streptomycin and 0.01mg/ml bovine insulin. Cells were maintained at 37 °C in a humidified incubator with 5% CO2.


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2.4.2. Cellular imaging assay For imaging analysis, the cells were seeded on Ibidi µ-Dish 50 mm in growth medium at a concentration of 30.000 cells/ml. After adhesion, the cells were treated for 24 and 48 h with AuNPs@gelatin-hyd-DOX. Untreated and treated cells with AuNPs@gelatin and free DOX at equivalent concentrations were used as control groups. Subsequently, the cells were fixed with 4% paraformaldehyde, as previously described15. For LysoTracker staining, treated, live cells were incubated for 30 min with Deep Red fluorescent dye to mark the acidic organelles and then washed several times with PBS prior to images acquisition.

2.4.3. In vitro AuNPs@gelatin-hyd-DOX cytotoxicity study The survival of NIH:OVCAR-3 cells after the treatment with AuNPs@gelatin-hydDOX, AuNPs@gelatin, and free drug was assessed by MTT protocol. Specifically, the cells were seeded in 100 µl RPMI-1640 medium, at a concentration of 10.000 cells/ml in 96-well plates maintained at 37 °C and 5% CO2 for 24 h. Then, the cells were treated with AuNPs@gelatin-hyd-DOX, AuNPs@gelatin and DOX at an equivalent drug concentration of 2.3 µg/ml and were incubated for another 24 and 48 h, respectively. Cells incubated without treatment were also prepared as control. After treatment, the medium from each well was replaced by 100 µl MTT solution and placed in the incubator for 1 h so that the live cells could convert the yellow tetrazolium dye MTT into purple formazan. Next, the solution was aspirated, and 150 µl DMSO was added to each well to solubilize the formazan crystals. The optical densities of each well were analyzed spectrophotometrically at 570 nm with a reference



wavelength of 670 nm. To confirm the results reproducibility, the experiments were repeated for 3 times in triplicate, and the mean value was reported.

2.5. Characterization The absorbance of free DOX solutions and the extinction signal of colloidal solutions were investigated by a Jasco V-670 UV–Vis-NIR single-monochromator spectrophotometer. The AuNPs@gelatin-hyd-DOX nano-chemotherapeutic system stability against physiological conditions (ionic solution of 0.9% NaCl at 37 °C) was evaluated with the same system equipped with a Peltier thermostatted single cell holder module. The hydrodynamic diameter and zeta potential of the colloidal solutions were analyzed by a Zetasizer Nano ZS90 from Malvern Instruments. The steady-state fluorescence spectra were collected employing a Jasco LP-6500 spectrofluorometer equipped with a xenon lamp as a light source. The fluorescence emission spectra were obtained at 485 nm excitation wavelength. The Fourier transform infrared (FT-IR) absorption spectra were recorded with a Jasco 6000 spectrometer, at room temperature, in the range 400–4000 cm-1, spectral resolution of 4 cm-1; using the well-known KBr pellet technique. Prior to analysis the samples were freeze dried using the Biobase BK-FD10S freeze dryer. TPE (two-photons) and OPE (one-photon) excited FLIM images were performed on cells treated with AuNPs@gelatin-hyd-DOX, AuNPs@gelatin and free DOX in 50 mm Ibidi µ-Dish using a MicroTime200 time-resolved confocal fluorescence microscope system (PicoQuant), described in detail in 15. The TPE images were obtained using 800 nm laser excitation


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while the OPE images were recorded using 485 nm diode laser excitation. The collection of the FLIM, bright and dark-field images, as well as the decay curves were previously described15.

3. Results and Discussion 3.1. DOX loading onto AuNPs@gelatin-hyd-DOX in solution The loading of DOX and stability of AuNPs@gelatin-hyd-DOX was investigated by UV-Vis and steady-state fluorescence spectroscopy in aqueous solution. Figure 1A shows the extinction spectra of AuNPs@gelatin recorded before and after loading of DOX molecules. The as-prepared colloidal AuNPs@gelatin exhibits the localized surface plasmon resonance (LSPR) band at 531 nm (Figure 1, spectrum a), which remains preserved after the loading of DOX molecules (Figure 1, spectrum c), excepting a 10 nm red shift (inset of Figure 1A) due to the local increase of refractive index around the nanoparticles induced by the addition of DOX molecules.

Figure 1. A. Extinction spectra of (a) biosynthesized AuNPs@gelatin, (b) AuNPs@gelatinhyd-DOX nano-chemotherapeutic system and (c) absorbance spectrum of free DOX in



solution. B. Fluorescence emission spectra of (a) free DOX in solution and (b) DOX loaded onto AuNPs@gelatin at the same concentration. The insets show the normalized spectra.

The chemotherapeutic system formation was also confirmed by steady-state fluorescence studies. The spectrum (a) presented in Figure 1B is definitory for the fluorescence emission of free DOX in solution prepared at 230 μM concentration upon excitation at 485 nm. However, after DOX loading onto AuNPs nanocarriers at the same concentration as free in solution, the fluorescence emission of DOX is highly quenched (Figure 1B, spectrum b). The modification of the fluorescence quantum yield together with the small bathochromic shift and a change in the ratio between the intensities of bands located at 560 nm and 635 nm (inset of Figure 1B) are consistent with energy transfer toward metallic surface of AuNPs and polarity modification after chemical attachment to gelatin layer. The loading of the DOX molecules onto AuNPs@gelatin and subsequent formation of AuNPs@gelatin-hyd-DOX nanosystem showed in TEM image from Figure 2A is also proved by the dynamic light scattering and zeta potential measurements. These investigations which provide data about the changes occurred in the hydrodynamic diameter and surface charge of NPs in a colloidal solution are ideal to evaluate the loading of DOX molecules. Specifically, the hydrodynamic diameter of AuNPs@gelatin slight increase in size from 70.63±5.55 to 86.46±4.82 nm (Figure 2B), while the negative –26±0.46 mV zeta potential value of AuNPs@gelatin shift to a positive value of +47.6±0.3 mV (Figure 2C) after the successful DOX loading. Therefore, the AuNPs@gelatin-hyd-DOX complex possess a positive charge that will allow later the binding with negatively charged cell’s membrane.


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Figure 2. Characterization of AuNPs@gelatin-hyd-DOX A. TEM image, B. DLS and C. zeta potential measurements (a) before and (b) after DOX loading

The FT-IR technique was also employed to prove the loading of the DOX molecules onto

AuNPs@gelatin

and

subsequent

formation

of

AuNPs@gelatin-hyd-DOX

chemotherapeutic system. The FT-IR spectrum of the gelatin presented in Figure S1 – spectra a, shows characteristic absorption bands as follows: amide A (3430 cm-1), asymmetric and symmetric CH2 stretching (2900 and 2800 cm-1), symmetric stretching of CH3 methyl groups (2960 cm-1), amide I (1650 cm-1), amide II (1550 cm-1), C-H2 bending (1450 cm-1), amide III (1335 cm-1) and skeletal stretching (1080 cm-1)

40,41.

These bands are clearly visible after

AuNPs@gelatin synthesis, proving the presence of gelatin at the surface of metallic NPs42 (Figure S1, sperctra b). The hydrazine bond to the gelatin from the AuNPs surface can be seen by the symmetric NH2 vibration at 840 cm-1 43. When the DOX is attached to the nanocarrier this vibration band disappears. This change in the spectrum of AuNPs@gelatin-hyd-DOX (Figure S1, spectra d) and band appearance at 1580 cm-1 assigned to C=N stretching vibration proves the hydrazone bond formed between the ketonic groups of DOX and hydrazine moiety of AuNPs@gelatin-hyd. The FT-IR spectrum of AuNPs@gelatin-hyd-DOX also shows specific vibrations at 1610, 1410 and 1071 cm−1 assigned to quinone and ketone carbonyl groups of DOX and characteristic peaks at 1285 and 984 cm−1 due to the stretching bands of the C–O–C groups 44–46, clearly demonstrating the loading of drug.



3.2. DOX release from AuNPs@gelatin-hyd-DOX in solution The AuNPs@gelatin-hyd-DOX nano-chemotherapeutic system developed in this study is based on the loading of DOX drug to the AuNPs surface through an acid labile hydrazone bond. We preferred here a hydrazone linkage considering that is a convenient method for conjugating drugs with AuNPs since these bonds are stable at physiological conditions (pH 7.4, 37 ºC) but are quickly cleaved under acidic pH of cellular organelles28,47. To further investigate the effectiveness of AuNPs@gelatin-hyd-DOX as a potential chemotherapeutic agent we evaluated the pH-dependent release of DOX at pH of 7.4, 5.3, and 4.6, respectively, chosen to mimic both physiological pH and the acidic medium of cellular organelles36. As discussed above, the fluorescence of DOX molecules loaded to AuNPs@gelatin is quenched due to the proximity to metallic surface. On the other hand, once the hydrazone bonds are cleaved, the fluorescence emission recovers with the DOX release from its carrier. To trigger the drug release, small volumes of DOX loaded AuNPs were incubated in different solution buffers at 37 ºC and kept under gentle shaking. At predefined time intervals, the fluorescence emission intensity of the release medium was recorded to estimate the amount of DOX released using a calibration curve39.Then, the obtained data were plotted as a function of time to generate the drug release profiles at the selected pH levels. Figure 3A show the highly pH-responsive character of DOX release from AuNPs@gelatin-hydDOX system. Specifically, it shows a minimal release of DOX at pH 7.4 in 24 h (Figure 3A) which demonstrates the stability of the chemotherapeutic system at physiological pH. Quite the opposite, at acidic pH, a burst release is observed in the first 2 h both for pH 5.3 and pH 4.6. Next, the DOX release trend from AuNPs system is slow and sustained, reaching 43.7 % and 65.18 % respectively at pH 5.3 and 4.6 after 24 h. However, when the chemotherapeutic system


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is incubated at pH 4.6 for 24 h, in the presence of trypsin, even a higher release rate is observed. Trypsin, notorious for degradation of the gelatin biopolymer was used to decompose the gelatin from the surface of AuNPs, which together with the highly acidic pH 4.6, favors a faster DOX release that reaches 71.34 % in 24 h (Figure 3A). Therefore, these results confirm the acidcleavable characteristic of the hydrazone linkage between DOX and AuNPs@gelatin, the pHdependent release of DOX as well as its more efficient release in the acidic media, specific for tumors and cancer cells. It is also important to note that after its release, DOX maintains its native fluorescence emission spectral shape and the initial ratio between the intensities of bands is restored, as it can be seen in Figure 3B and inset of Figure 3B, suggesting that the chemical structure of drug molecules remains unaffected.

Figure 3. A. Release curves of DOX as a function of medium pH B. Fluorescence emission of DOX (a) loaded onto AuNPs@gelatin-hyd-DOX and (b) recovered after drug release at pH 4.6 in the presence of trypsin. The inset show the normalized (a) and (b) spectra together with spectra of free DOX in solution (spectrum c)

In order to confirm this pH dependent DOX release profiles also at the cellular level, the cellular localization was investigated employing distinct organelle marker. Concretely, the



LysoTracker staining protocol was used to allow an accurate cellular observation of the AuNPs@gelatin-hyd-DOX internalization in the acidic organelles of the cells after staining them with Deep Red fluorophore highlighted in green, as presented in Figure S2B in the Supporting Information. The merged image from Figure S1C confirms the co-localization of DOX and chemotherapeutic system in the endosomes and lysosomes of the cells. Doubtlessly this means that the carried DOX molecules remain attached to the AuNPs during systemic circulation and the cargo is released only at the site of the action, in acidic pH of endosomes/lysosomes of the cell where starts the degradation of hydrazone bonds (see Figure S2 from Supporting Info).

3.3. AuNPs@gelatin-hyd-DOX internalization and DOX release in vitro

Next, to check and track the internalization of NPs in cells we performed dark-field microscopy and TPE-FLIM measurements on the same cells. These techniques are adequate to trace not only the nanoparticles in cells but also the whole chemotherapeutic system, thanks to the light-scattering properties of AuNPs coming from their surface plasmon resonances.


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Figure 4. Bright field, dark-field and TPE-FLIM images of NIH:OVCAR-3 control cells and cells treated with AuNPs@gelatin, AuNPs@gelatin-hyd-DOX and free DOX for 24 and 48 h, respectively. The scale bars represent 20 µm. Intensity and lifetime scale bars (0-7 ns) are the same for all TPE-FLIM images.

Figure 4, shows on the upper rows the bright field and dark-field images of untreated – control NIH:OVCAR3 cells and cells treated with AuNPs@gelatin, DOX loaded AuNPs@gelatin and free DOX, respectively. Compared with negative and positive control cells – untreated and treated with free DOX – which scatter light from biological organelles of the cells and have a slight intrinsic signal48,49, the AuNPs@gelatin treated cells present a little more intense and brighter signal. However, this signal is largely dependent on the size and aggregation state of the AuNPs. For instance, in the case of spherical AuNPs, large size and aggregation correspond to stronger signal. Judging by the low signal from cells treated with AuNPs@gelatin, we suppose that the negatively charged AuNPs are hardly internalized and remains rather isolated after internalization. On the other hand, a much stronger signal is



detected from the cells incubated with AuNPs@gelatin-hyd-DOX for 24 h. The positively charged surface of DOX loaded AuNPs@gelatin facilitates their interaction with negatively charged cell membrane and subsequent internalization in cells (Figure 4). After 48 h of treatment, the scattering signal is detected from a much larger area of cell’s cytoplasm which suggest a progressive internalization of drug loaded AuNPs in cells. Additionally, the signal is much brighter suggesting the aggregation of AuNPs after internalization. Supplementary images showing the same behavior are presented in Figure S2 from Supporting Info. For a more thorough localization, disentangle of various signals and better discrimination between incorporated and released DOX molecules in cells as determined by the sensitivity of fluorescence lifetime to local environment and lower scattering background, we have recourse to TPE-FLIM measurements. TPE-FLIM produces high intensity contrast images based upon the lifetime measurement of the AuNPs and DOX molecules after excitation in NIR with 800 nm pulse laser light. Under TPE, AuNPs exhibit photoluminescence due to the sequential recombination of the excited electrons in the sp conduction band with holes in the dband of metal surface, which makes them powerful contrast agents for NIR cell imaging12,13,15. Indeed, it can be seen from the intense bright spots, better than in the dark-field images, that AuNPs@gelatin are successfully internalized by the cells and locate in the whole cytoplasmic region (Figure 4). As regard the cells incubated with AuNPs@gelatin-hyd-DOX, TPE-FLIM provides valuable information about both AuNPs’s localization and drug distribution in cells. By comparing the cells treated with free DOX or DOX loaded AuNPs from Figure 4 and Figure 5, significant differences in the internalization behavior of free DOX and the released DOX are revealed, as a function of time. In particular, the fluorescence signal from cells incubated with free DOX for 24 and 48 h is almost exclusively nuclear, which was expected since the free DOX molecules are uptaken by the cells through their membranes via passive diffusion and are quickly accumulated in the cell nucleus39,50.


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On the other hand, after cell’s treatment with DOX loaded AuNPs for 24 h, the nanochemotherapeutic system is taken up by cells and internalized in cellular compartments, where the drug release activated by the acidic pH starts (as demonstrated in Figure S3 from Supporting Info). At this phase, from TPE-FLIM data we can assume that DOX can be found in two different states: released and still attached to AuNPs. The released DOX from cytoplasm migrates to the nucleus to bind with DNA exhibiting a fluorescence signal and, more important, a lifetime (see details later) similar with the one recorded from cells treated with free DOX. In contrast, DOX molecules attached to the AuNPs internalized in cell cytoplasm, gathers around the nuclear membrane and presents a strong signal. This signal could arise from a combined effect between intrinsic photoluminescence of aggregated AuNPs and fluorescence of DOX molecules. A fluorescence enhancement of DOX under two-photon excitation is measured despite of a quenched emission in solution, similar with the results obtained by Fischer et. al51. We hypothesize the enhancement of fluorescence emission of DOX molecules located at a favorable distance from the metallic surface and positioned in the hot-spots created between the AuNPs aggregated after the gelatin matrix from their surface is degraded by the metalloproteinases overexpressed in cancer cells52,53. Zhou et al54. noted previously that AuNPs that absorbs photons in the NIR region act as donors which interact with the excitation and emission properties of nearby acceptor, in our case DOX, and enhance fluorescence emission. After 48 h, we notice even a stronger signal both from the cell nucleus and cytoplasm due to the progressive AuNPs internalization, DOX release and subsequent accumulation in cell nucleus. TPE-FLIM might be also especially useful for deep cell imaging applications, as proved by images recorded at different depths z-levels (Figure S4).



Figure 5. OPE-FLIM and TPE-FLIM images of NIH:OVCAR-3 control cells and cells treated with AuNPs@gelatin, AuNPs@gelatin-hyd-DOX and free DOX for 24 and 48 h, respectively. The scale bars represent 20 µm. Lifetime scale bars (0-7 ns) are the same for all images.


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To further investigate the intracellular kinetics of DOX, we employed one- and twophoton excited FLIM imaging of cells. The different selection rules for OPE and TPE excitation can provide insight into the properties of excited DOX molecules. Moreover, the fluorescence lifetime of DOX molecules at a specific point in the cell is a reliable indicator for the discrimination of the different states of drug i.e. free or loaded onto AuNPs and is especially useful to monitor the intracellular DOX release in time5,55. In the case of 485 nm excitation in OPE-FLIM images (Figure 5, first row and Figure S5) of the control cells and cells incubated with AuNPs@gelatin, there was no signal detected from the cellular components or AuNPs. On the other hand, in the case of cells treated with free DOX for 24 and 48 h, the fluorescence lifetime distribution of DOX in cells shows that the signal is exclusively nuclear, with a lifetime value of ~1.2 ns (Figure 6A, curve a), attributed to intercalated DOX−DNA55,56. In the cells treated with chemotherapeutic complex AuNPs@gelatin-hyd-DOX, the fluorescence lifetime distribution displays two different regions corelated with nucleus and cytoplasm of the cell. In the cell cytoplasm it was recorded a signal corresponding to a biexponential decay with an average lifetime of 3.795±0.021 ns that consist of a slow and a fast lifetime component (Figure 6A, curve c). As we previously demonstrated39, the slow-longer lifetime component (4.105±0.027 ns) comes from DOX loaded to AuNPs while the fast lifetime component is similar with free DOX being attributed to released DOX (1.22±0.027 ns). After 48 h treatment, the fraction of the fast component increases in the lifetime recorded from cell cytoplasm suggesting the progressive release of DOX from its carrier AuNPs@gelatin after hydrazone linker cleavage in the acidic organelles of the cell10,20. Once DOX is released in cell cytoplasm, it migrates to nucleus and bind to DNA to initiate the apoptosis. Indeed, after 48 h OPE-FLIM revealed that the fluorescence signal of DOX is increased and almost entirely nuclear. Therefore, by investigating the lifetime of DOX in



different areas of the cell we were able to trace its release and assign its distribution thanks to the sensitivity of DOX to physico-chemical factors such as pH changes in the local environment of molecules and interactions with the drug carrier and DNA5.

Figure 6. Normalized fluorescence lifetime decays recorded from NIH:OVCAR-3 cells under A. OPE and B. TPE at 485 nm and 800 nm, respectively, from regions marked º in Figure 5. A. OPE (a) free DOX treated cell; AuNPs@gelatin-hyd-DOX treated cell (b) nucleus, (c) cytoplasm. B. TPE - cell treated with (a) AuNPs@gelatin, (b) free DOX and AuNPs@gelatinhyd-DOX – (c) cytoplasm, (d) cytoplasm from bright spots and (e) nucleus

The TPE-FLIM images of treated cells and the decay curves obtained at 800 nm excitation from different points of the cells treated with free DOX and DOX loaded AuNPs@gelatin for 24 h are presented in Figure 5 – second column and Figure 6B, respectively. As can be seen, there was not detected any signal from the cellular components in untreated cells. However, compared with OPE-FLIM image of cells incubated with AuNPs@gelatin there was recorded a slightly brighter signal coming from internalized AuNPs, which prove the


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effectiveness of two-photons excitation and validate the ability of metallic NPs to act as reliable contrast agents for NIR imaging assays. Moreover, TPE-FLIM images of cells treated with DOX and DOX loaded AuNPs@gelatin confirm the OPE-FLIM data on different pattern internalization of DOX. Indeed, the obtained fluorescence lifetime values of free DOX are close to the values found under OPE in cell nucleus being fitted with monoexponential decays (Figure 6B, curve b) with fluorescence lifetime of 1.249 ns and 1.186 ns at 24 and 48 h, respectively. AuNPs@gelatin-hyd-DOX treated cells also displayed different areas in cell defined by lifetime of DOX. In the nucleus of the cell, DOX released after 24 h treatment shows a lifetime of 1.41 ns (Figure 6B, curve c) that decrease to 1.22 ns after 48 h. In contrast, in the cytoplasm of the cell TPE-FLIM images reveals besides data recorded by OPE-FLIM, two different lifetimes. One of reduced intensity with a lifetime value similar to released DOX (Figure 6, curve c) and another recorded from the bright spots from Figure 5 with a lifetime of 1.3 ns which is composed from a slower lifetime corresponding to DOX and a faster lifetime of about 0.21 ns (Figure 6B, curve d). According to previous results12,13,15, we can hypothesize that this lifetime is the result of a combination between the lifetime of AuNPs which cannot be detected by excitation at 485 nm and the lifetime of DOX from AuNPs surface. A plausible origin for this signal can be associated with the formation of aggregated AuNPs that can significantly enhance the luminescence of AuNPs under 800 nm excitation by increasing the local-field intensity. Additionally, the coupled AuNPs are well known to have an LSPR band shifted toward the NIR region enabling the resonant excitation which significantly increases the TPE efficiency13. Moreover, the DOX molecules loaded onto AuNPs through gelatin layer and a hydrazone linker are affected by the high intensity of electromagnetic field from the hot-spots created between AuNPs which enhances their fluorescence emission and reduces the fluorescence lifetime due to the increase of radiative rate57,58. It is worth mentioning that after 48 h treatment, more bright spots can be distinguished in cell cytoplasm which prove the progressive internalization of



DOX loaded AuNPs in time. Also, its signal intensity diminished – indicating the DOX release – while the nucleus became brighter with the DOX accumulation (Figure 5).

Figure 7. Fluorescence spectra recorded from A. AuNPs@gelatin and B. DOX internalized in cell at 800 nm excitation from regions marked º in Figure 5

Figure 7 presents the normalized two-photon excited fluorescence spectra extracted from regions marked º in Figure 5 upon excitation at 800 nm. AuNPs@gelatin and AuNPs@gelatin-hyd-DOX exhibits a spectral profile that resembles to the extinction spectra of AuNPs but are quite broad and shows a red-shift which could indicate the intracellular aggregation process. The recorded TPE PL spectra are consistent with previous results and being assigned to interband transitions14,15,59. Withal, TPE PL of DOX recorded from cell treated with free DOX is identical with that recorded from cell incubated with DOX loaded AuNPs which not only supports the drug release in cell and subsequent accumulation in cell nucleus but TPE-FLIM results, as well.


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3.4. Cytotoxicity of AuNPs@gelatin-hyd-DOX nano-chemotherapeutic system

Figure 8. Cytotoxicity of free DOX, AuNPs@gelatin-hyd-DOX and AuNPs@gelatin against NIH:OVCAR-3 cells (incubation time 24 and 48 h)

To further confirm the intracellular release of DOX molecules, its accumulation in cell nucleus and interaction with DNA, we monitored the drug effects on cell viability by employing MTT assay. First, we incubated different concentrations of free DOX with cells for 24 h to determine the half maximal inhibitory concentration of drug (IC50) (data not shown). Then, the cytotoxicity of AuNPs@gelatin-hyd-DOX against NIH:OVCAR-3 cells was compared with that of equivalent doses of free DOX and AuNPs carrier, in a time-dependent manner. Considering that we used the equivalent concentration of free drug selected as to present a half maximal effect after 24 h treatment, the cell viability was monitored only in the first 48 h of



incubation. As observed in Figure 8, after the treatment with free DOX the viability of cells decreased to 50.65 % in first 24 h and reached 23.73 % in 48 h. However, the toxicity of AuNPs@gelatin-hyd-DOX was slightly lower compared to free DOX and showed a survival of cells treated of 25.74 % after 48 h. This result clearly evidences a minor retention in cytotoxic activity of DOX loaded AuNPs but is not surprising considering the sustained release of drug. It can just be explicated by the different internalization routes of free DOX and AuNPs@gelatin-hyd-DOX, as our OPE-TPE-FLIM data and previous result confirm39. Specifically, the higher cytotoxicity of free DOX is attributed to the rapid internalization of DOX in cells as free drug directly penetrates the cell membrane and diffuse to the nuclei. Contrary, AuNPs@gelatin-hyd-DOX are uptaken by the cells through the progressive endocytosis, reside inside cellular compartments where acidic media and intracellular enzymes cleaves the hydrazone bonds and DOX could be released slowly; only after a sustained release DOX diffuses to the cell nuclei to bind to DNA and exert its therapeutic effect. It is also worth pointing out that the cells treated with control AuNPs@gelatin carrier present excellent viability even after 48 h incubation, indicating that the cytotoxicity of AuNPs@gelatin-hyd-DOX is not due to the presence of AuNPs. Thus, by evidencing the cytotoxic effect of AuNPs@gelatinhyd-DOX we certainly proved the nuclear DOX internalization.

4. Conclusions

In this study, we successfully confirmed the applicability of TPE-FLIM microscopy to track the internalization of DOX loaded AuNPs@gelatin in NIH:OVCAR-3 cells and to disentangle various signals from the therapeutic agents in the target cells. After systematic investigation of DOX loading to the AuNPs@gelatin through a pH-responsive hydrazone bond


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and a thorough characterization of the AuNPs@gelatin-hyd-DOX obtained system, this was incubated with NIH:OVCAR-3cells. The kinetics of DOX molecules after intracellular release was monitored by TPE-FLIM thanks to the DOX molecules that act as reliable probes due to their sensitivity to environment changes. Finally, the efficient drug release and nuclear internalization was proved by the anticancer activity of our system against ovarian cancer cells. Thus, TPE-FLIM imaging is an ideal tool for tracking the AuNPs carriers and DOX intracellular kinetic by mapping simultaneously photoluminescence and fluorescence signals.

Supporting Information FT-IR characterization of AuNPs@gelatin-hyd-DOX system and its internalization in cells highlighted by LysoTracker Deep Red staining protocol. OPE- TPE-FLIM, bright and darkfield images of NIH:OVCAR-3 cells treated with AuNPs@gelatin-hyd-DOX.

AUTHOR INFORMATION Corresponding Author * Tel: +40 264 454554/116; e-mail: [email protected]

ACKNOWLEDGMENT The authors thank to Ministry of Research and Innovation, CNCS-UEFISCDI, project number PN-IIIP4-ID-PCCF-2016-0142.



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Intracellular dynamic disentangling of Doxorubicin release from

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