Polyelectrolyte coated BaTiO₃ nanoparticles for second harmonic generation imaging-guided photodynamic therapy with improved stability and enhanced cellular uptake

Abstract

Photodynamic therapy (PDT) based on novel nanomaterials has attracted considerable interest in recent years. In this work, we fabricate positively charged polyelectrolyte (PAH) coated barium titanate (BaTiO₃, BT) nanoparticles (BT-PAH) with improved colloidal stability and enhanced cellular uptake efficiency. The BT-PAH nanoparticles are demonstrated to be promising as second harmonic generation (SHG) imaging probes due to their coherent and non-bleaching signals under femtosecond laser excitation, which is useful for long-term monitoring of cell dynamics. Furthermore, Ce6, a type of photosensitizer for PDT, is successfully loaded on BT-PAH to form BT-Ce6-PAH nanocomposites. Enhanced PDT efficiency is achieved due to higher cellular uptake of Ce6 assisted by BT-PAH nanocomposite-loading, which is confirmed by SHG and fluorescence cell imaging. Our work illustrates that BT-PAH nanoparticles can serve as promising contrast agents for SHG imaging and highly efficient nanocarriers for SHG imaging-guided photodynamic therapy.

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1. Introduction

Photodynamic therapy (PDT) has attracted considerable attention as an exciting non-invasive strategy for selective cancer therapy, and it has been approved for the clinical treatment of many cancers, including lung cancer, superficial bladder cancer, head and neck cancer and skin cancer.¹ PDT is based on the activation of photosensitizers (PSs) under light irradiation to produce reactive oxygen species (ROS), which are cytotoxic and capable of destroying nearby cancer cells.^1,2 PS is the most critical component in PDT, and a great many types of PSs have been developed, including porphyrinoid compounds (like chlorins, phthalocyanines, bacteriochlorins) and non-porphyrinoid compounds (such as anthraquinones, xanthenes, and cyanines).^1,3 Some of these PSs have been approved and are being tested in clinical trials.^3,4 Unfortunately, most PSs are hydrophobic, and their poor-water solubility limits their applications in tumor therapy, because PSs have to be administered and transported to the target tissues (e.g. cancer cells and tumor tissues) where a certain amount of hydrophilicity is required.^1–4 With the development of nanotechnology, some alternative strategies, which use nanomaterials to carry and delivery PSs to overcome their disadvantage of hydrophobicity, have been proposed.^1,2,5 In recent years, various nanomaterials, including carbon nanotubes,⁶ gold nanomaterials such as gold nanorods and gold nanocages,^7,8 iron oxide (Fe₃O₄ nanoparticles),⁹ upconversion nanoparticles and graphene oxide,^10,11 have been developed as novel nanocarriers for PS delivery in PDT of cancer. The advantages of nanomaterial-assisted PDT include high payloads of PSs on the nanoparticles due to their large surface to volume ratios, improved stability of aqueous dispersion that bestows amphilicity to PSs, ease of surface modification for selective and targeting PDT of cancer cells and tumor tissues, and excellent biocompatibility. Furthermore, some of these nanomaterials exhibit linear or nonlinear optical properties, such as one-photon fluorescence, multi-photon luminescence and second harmonic generation (SHG) under continuous wave (CW) or femtosecond (fs) laser excitation, which can provide high contrast in bioimaging applications.^10,12–14 Nanomaterials serving as PS delivery vehicles and imaging probes will be very beneficial in imaging-guided PDT.

SHG is a nonlinear optical process that converts two photons into one photon with half the wavelength of the excitation laser. Usually, a scanning microscope equipped with a fs laser can be used for SHG microscopic imaging.¹⁵ Compared to one-/two-photon fluorescence, the SHG process is based on virtual electro energy transition without loss of nonradiative energy. Thus, SHG active materials are completely immune to bleaching and photothermal effects, and their signals are quite stable. These unique optical properties are beneficial for the observation and monitoring of dynamic processes in cells over a long period.¹⁶ In addition, the tunability of excitation and emission wavelength makes SHG microscopy suitable for deep-tissue imaging, as a laser with its wavelength in near infrared (NIR) range (known as an optical tissue window) can be adopted for SHG excitation.^14–17

Barium titanate (BaTiO₃) nanoparticles are a kind of SHG active nanomaterial due to their non-centrosymmetric structure, and the SHG property of BaTiO₃ (BT) nanoparticles has been reported recently. Periklis Pantazis et al. compared the SHG of BT nanoparticles with the fluorescence of quantum dots (QDs) and demonstrated the unique non-bleaching and non-blinking properties of BT nanoparticles.¹⁶ Davide Staedler et al. systematically investigated the SHG properties of five nonlinear nanomaterials, including BT nanoparticles, and demonstrated their great potential in cell imaging.¹⁷ In addition, BT nanoparticles have been studied for their potential applications in nanomedicine.¹⁵ Gianni Ciofani et al. functionalized BT nanoparticles with glycol–chitosan via a non-covalent binding and demonstrated their optimal cytocompatibility at high concentration.¹⁸ Furthermore, they loaded doxorubicin on the BT nanoparticles, and enhanced cytotoxicity was achieved. BT nanoparticles were also used as imaging probes for mRNA quantification by a second harmonic super-resolution microscopy.¹⁹ Chia-Lung Hsieh et al. prepared BT nanocrystals by covalently conjugating antibodies on their surface for specific labelling of membrane proteins of live cells.²⁰ Although progress has been made in biological imaging and nanomedicine applications based on BT nanoparticles, poor colloidal stability of aqueous dispersion and low cellular uptake efficiency of BT nanoparticles still limit their application as imaging probes or nanocarriers, especially for drug delivery applications where efficient cellular internalization is desired. Very recently, Davide Staedler et al. reported a diagnostic and therapeutic protocol based on a type of harmonic generating nanoparticles (bismuth ferrite) which is similar to BT nanoparticles and the deep ultraviolet light generated from bismuth ferrite nanoparticles can directly induce cell death without photosensitizing molecules.²¹ However, BT nanoparticles serving as nanocarriers for photosensitizer delivery and PDT have not yet been reported.

In this work, we prepared BT nanoparticles by coating a positively charged polyelectrolyte (PAH) on their surface, to improve the colloidal stability of aqueous dispersion and enhance their cellular uptake efficiency. In addition, in vitro SHG imaging of cells treated with BT-PAH nanoparticles was carried out to confirm their efficient cellular internalization and excellent photostability. Furthermore, Ce6 (photosensitizer for PDT) was loaded on the BT-PAH nanoparticles with high loading efficacy. Efficient cellular uptake of Ce6 assisted by BT-Ce6-PAH nanocomposites was confirmed by SHG and fluorescence imaging, and enhanced PDT efficiency of cancer cells was demonstrated (Fig. 1). Our results indicated that BT-PAH nanoparticles hold great promise as imaging probes and drug delivery vehicles for SHG imaging-guided PDT.

Fig. 1 Schematic illustration for the preparation of BT-Ce6-PAH nanocomposites and its applications for enhanced cellular internalization and second harmonic generation imaging-guided photodynamic therapy.

2. Experimental

2.1. Materials

Barium titanate (BT) nanoparticles, dimethyl sulfoxide (DMSO), 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) and poly(allylamine hydrochloride) (PAH, MW ∼ 15 000) were purchased from Sigma-Aldrich. Chlorin e6 (Ce6) was purchased from Frontier Scientific Inc. Deionized (DI) water was used in all experiments.

2.2. Preparation of BT-PAH nanoparticles

BT powder (5 mg) was dispersed in 18 mL DI water and sonicated for 1 h. 20 mg PAH (dissolved in 2 mL of 10 mM NaCl solution) was then injected into the aqueous dispersion of BT nanoparticles. After sonication for 2 h, the mixture was magnetically stirred for 24 h. The obtained BT-PAH nanoparticles were collected by centrifugation and washed twice with DI water.

2.3. Preparation of BT-Ce6-PAH nanocomposites

BT powder (5 mg) was dispersed in 18 mL DI water and sonicated for 1 h, then 2 mL of Ce6 (0.5 mg mL⁻¹, dissolved in DMSO) was added into the aqueous dispersion of BT nanoparticles. The mixture dispersion was sonicated for 2 h and stirred magnetically for 24 h. The BT-Ce6 nanoparticles were centrifuged and redispersed into 18 mL water, and 20 mg PAH (dissolved in 2 mL of 10 mM NaCl solution) was injected and stirred magnetically for 24 h. Finally, the obtained BT-Ce6-PAH nanocomposites were purified by centrifugation and washed twice with water. The loading efficiency of Ce6 on the BT nanoparticles was calculated by subtracting the amount of free Ce6 in the supernatant after centrifugation from the amount of Ce6 in the initial reaction solution.

2.4. Characterizations of the nanoparticles

The extinction spectra (including absorption and scattering) of the nanoparticles were measured with a Shimadzu 2550 UV-vis scanning spectrophotometer. Transmission electron microscope (TEM) images of the nanoparticles were captured by a JEOL JEM-1200EX microscope operating at 80 kV. Hydrodynamic size distribution and zeta potentials of the nanoparticles were measured on a Malvern Zetasizer Nano ZS-90. For the measurement of the SHG spectrum of the BT nanoparticles, a Ti–sapphire fs laser was used as the excitation source, and the operating wavelength was 800 nm. The SHG signals were collected by an objective (20×, NA = 0.75) and recorded with a spectrometer (PG 2000, Ideaoptics).

2.5. In vitro SHG imaging of BT-PAH nanoparticles treated cells

HeLa cells were used as the model for enhanced cellular uptake and imaging studies. HeLa cells were seeded in a 35 mm cell well at a density of 1 × 10⁵ cells per mL for 24 h and grew to 70–80% confluence. Then 2 mL of fresh culture medium containing 5 μg mL⁻¹ BT-PAH or BT nanoparticles was added to replace the original culture medium. After incubation for 24 h, the cells were washed three times with PBS to remove the nanoparticles that were not internalized by cells. A confocal scanning microscope (BX61W1 + FV1000, Olympus) equipped with a fs laser (operating wavelength: 800 nm) was used for SHG imaging of HeLa cells. The fs laser was focused on the cells by an objective (60×, NA = 1.0) with a power density of ∼2.8 × 10⁴ W cm⁻². The SHG signals from BT-PAH nanoparticles passed through a filter within the 380–420 nm range, and were collected by a photomultiple tube (PMT). To study the photostability of BT-PAH nanoparticles under fs laser excitation, 200 scans of the HeLa cells (incubated with 5 μg mL⁻¹ BT-PAH nanoparticles) were performed (2 μs per pixel, ∼2.1 s per scan).

2.6. Detection of ROS from BT-Ce6-PAH under laser irradiation

ABDA was used to evaluate the production of ROS from BT-Ce6-PAH nanocomposites. Typically, a 2 mL aqueous dispersion of BT-Ce6-PAH nanocomposites (100 μg mL⁻¹, equivalent concentration of Ce6: 16 μg mL⁻¹) containing 0.1 mg mL⁻¹ ABDA was irradiated with a CW laser at 660 nm (30 mW cm⁻²). The absorption spectra of ABDA were recorded after the laser irradiation at various points in time. A 2 mL aqueous dispersion of BT-PAH nanoparticles and 2 mL aqueous solution of free Ce6 were used as controls.

2.7. In vitro SHG and fluorescence imaging of BT-Ce6-PAH nanocomposites treated cells

HeLa cells were incubated with BT-Ce6-PAH nanocomposites (5 μg mL⁻¹, equivalent concentration of Ce6: 0.8 μg mL⁻¹) or free Ce6 (0.8 μg mL⁻¹) for 24 h, and washed with PBS three times. The aforementioned confocal scanning microscope (BX61W1 + FV1000, Olympus) equipped with the fs laser (operating wavelength: 800 nm) was used for SHG and fluorescence imaging of HeLa cells. The SHG signals from BT-Ce6-PAH nanoparticles passed through the filter within the 380–420 nm range, and were collected by the PMT. The fluorescence signals (from Ce6) were excited by a CW laser at 405 nm, and collected by a PMT after passing through a filter within the 620–720 nm range.

2.8. In vitro cytotoxicity and PDT-induced cell death

The cytotoxicities of BT, BT-PAH, BT-Ce6-PAH and free Ce6 toward HeLa cells were evaluated by using a CCK-8 assay. Typically, HeLa cells were seeded in a 96-well plate at a density of 5 × 10³ cells per well for 24 h and grew to 70–80% confluence. Then the cell culture medium was discarded, and fresh culture medium containing the above samples with various concentrations were added. After incubation for 48 h, the cells were washed three times with PBS, and a CCK-8 assay was performed to evaluate the cell survival rate according to the manufacturer's instructions.

The PDT effects of BT-Ce6-PAH nanocomposites against HeLa cells were studied. Typically, HeLa cells were seeded in a 48-well plate at a density of 1 × 10⁴ cells per well for 24 h and grew to 70–80% confluence. The cell culture medium was discarded and a fresh culture medium containing the BT-Ce6-PAH nanocomposites or free Ce6 with various concentrations were added. After incubation for 24 h, the cells were washed three times with PBS, and a fresh culture medium was added. The cells were then subjected to a CW laser irradiation (660 nm, 30 mW cm⁻²) with a spot diameter of 1 cm for PDT treatment. After laser irradiation, the cells were incubated for an additional 24 h, and the cell survival rate was determined by a CCK-8 assay. For direct observation of the morphological changes of the HeLa cells induced by PDT, HeLa cells treated with various concentrations of BT-Ce6-PAH nanocomposites or free Ce6 were subjected to PDT treatment (the same experimental condition with that in the CCK-8 assay). The bright field images of the HeLa cells were captured by an inverted microscope.

3. Results and discussion

3.1. Characterizations of BT and BT-PAH nanoparticles

Barium titanate (BT) nanoparticles were obtained from Sigma-Aldrich as the form of dry nanopowder. The nanopowder was dispersed in DI water and sonicated in order to be broken into individual nanoparticles. The transmission electron microscope (TEM) image of BT nanoparticles (Fig. 2a) showed that they are spherical or cubic in shape, with an average diameter of ∼63 nm. Poly(allylamine hydrochloride) (PAH) was mixed with BT nanoparticles via sonication, and BT-PAH nanoparticles were obtained after being stirred for 24 h. The TEM image of BT-PAH nanoparticles was very similar with that of BT nanoparticles (Fig. 2b). Fig. 2c showed the extinction spectra of BT and BT-PAH nanoparticles, which indicated that the PAH coating did not influence the extinction of the BT nanoparticles. The aqueous dispersion of BT nanoparticles was not stable, as they tend to form aggregates after standing for a period time, as shown in the inset of Fig. 2c. After PAH coating on the surface, the stability of BT nanoparticles was greatly improved. No obvious sediment of BT-PAH nanoparticles was found after standing in the aqueous dispersion for 24 h (inset of Fig. 2c). Hydrodynamic size distributions of BT and BT-PAH nanoparticles were measured by the dynamic light scattering (DLS) technique. As shown in Fig. 2d and e, the average hydrodynamic diameters of BT and BT-PAH nanoparticles were 91 nm and 80 nm, respectively. In addition, the polydispersity index (PDI) of the aqueous dispersion of BT-PAH nanoparticles was smaller than that of BT nanoparticles, indicating that the stability and monodispersity of the nanoparticles were improved after the PAH coating. As shown in Fig. 2f, the zeta potentials of BT nanoparticles increased from 21 mV (BT) to 45.4 mV (BT-PAH). PAH is a kind of positively charged polyelectrolyte and can be capped on the surface of BT nanoparticles, forming a stable dispersion without aggregation. It was attributed to the strong positive charge on the surface of BT, which allowed BT nanoparticles to keep repelling each other.²² These results demonstrated that a successful coating of positively charged polyelectrolyte (PAH) on the surface of BT nanoparticles was achieved. In addition, the stability and monodispersity of the aqueous dispersion of BT nanoparticles were greatly improved after the PAH coating.

Fig. 2 Characterizations of BT and BT-PAH nanoparticles. Representative TEM images of BT (a) and BT-PAH (b) nanoparticles. (c) Extinction spectra of BT and BT-PAH nanoparticles, and digital images of the aqueous dispersions of the two nanoparticles after different time periods of standing (inset). Hydrodynamic size distributions of BT (d) and BT-PAH (e) nanoparticles. (f) Zeta potentials of the two types of nanoparticles. Scale bars: 100 nm.

3.2. Enhanced cellular uptake of BT-PAH nanoparticles

BT nanoparticles exhibit a non-centrosymmetric crystal structure, which is essential prerequisite for efficient SHG. Here, SHG imaging was carried out to evaluate the internalization of BT-PAH nanoparticles, which has a peak emission at 400 nm upon 800 nm-fs laser excitation (Fig. 5d). After incubation with HeLa cells for 24 h, a great many BT-PAH nanoparticles were internalized by the cells, which was confirmed by the bright SHG signals emitted from the BT-PAH nanoparticles upon fs laser excitation (Fig. 3a–c). In comparison, some bright aggregates, rather than small dots, were observed in HeLa cells incubated with BT nanoparticles (Fig. 3d–f), indicating instability and insufficient cellular uptake of BT nanoparticles. These results demonstrated that the positively charged BT-PAH nanoparticles held the capability of enhancing cellular uptake for sufficient SHG imaging.

Fig. 3 SHG imaging of HeLa cells incubated with BT-PAH (a–c) and BT (d–f) nanoparticles. Excitation wavelength of the fs laser: 800 nm.

3.3. Photostability of BT-PAH nanoparticles upon fs laser excitation

Photobleaching is a common obstacle for commonly used fluorescent proteins and organic dyes for cell imaging.²³ Herein, the photostability of BT-PAH nanoparticles under fs laser irradiation was investigated. HeLa cells were incubated with BT-PAH nanoparticles for 24 h and subjected to the irradiation of a fs laser in the SHG microscope. The SHG images of BT-PAH nanoparticle-stained HeLa cells was obtained after each scan to calculate the mean SHG intensity. As shown in Fig. 4, the bright SHG signals from BT-PAH nanoparticles were quite stable after long-time laser irradiation (200 scans, 2.1 s per scan). The mean SHG intensity remained almost constant over 200 successive scans (Fig. 4f). The result indicated that BT-PAH nanoparticles have the advantage of photobleaching resistance, and their excellent photostability opens the way to long-term SHG imaging and monitoring of cellular dynamics.

Fig. 4 SHG imaging of HeLa cells incubated with BT-PAH nanoparticles with increasing numbers of scans. Bright field image (a) and SHG images after the 1st (b), 50th (c), 100th (d) and 200th (e) scan of the HeLa cells. (f) The normalized mean SHG intensity of BT-PAH nanoparticles in HeLa cells with increasing numbers of scans. Excitation wavelength of the fs laser: 800 nm. Excitation time: 2 μs per pixel, ∼2.1 s per scan.

3.4. Characterizations of BT-Ce6-PAH nanocomposites

Enhanced cellular uptake and sufficient SHG imaging of BT-PAH nanoparticles have been demonstrated above. Next, we designed BT-Ce6-PAH nanocomposites for SHG imaging-guided photodynamic therapy. Ce6, a widely used photosensitizer for PDT, was loaded on the surface of BT nanoparticles via electrostatic and/or hydrophobic interactions. PAH was then coated on the BT-Ce6 nanoparticles to form BT-Ce6-PAH nanocomposites. As shown in Fig. 5a, the extinction spectrum of BT-Ce6-PAH showed two characteristic peaks at ∼400 nm and ∼660 nm, which correspond to the absorption peaks of Ce6. After Ce6 loading, the aqueous dispersion of BT-Ce6-PAH nanocomposites exhibited a light green colour, which was quite different from the white colour of BT-PAH nanoparticles, indicating successful loading of Ce6 on the nanoparticles (Fig. 5b). In addition, the PAH coating greatly improved the colloidal stability of BT-Ce6-PAH nanocomposites, as shown in Fig. 5b and c. The TEM images of BT-Ce6 nanoparticles (Fig. 5e) and BT-Ce6-PAH nanocomposites (Fig. 5f) showed that a very thin layer covered the surface of BT nanoparticles, which may be the formation of Ce6 molecules. Zeta potential results (Fig. 5g) showed that the BT-Ce6 nanoparticles had a negative charge (−24.7 mV) while the BT-Ce6-PAH nanocomposites were positively charged (41 mV). The DLS analysis suggested that BT-Ce6 nanoparticles had an average hydrodynamic diameter of 165 nm (Fig. 5h), which was larger than that of BT nanoparticles (Fig. 2d). However, the average hydrodynamic diameter decreased to 86 nm after the PAH coating for the BT-Ce6-PAH nanocomposites (Fig. 5i), with a PDI of 0.105. These results demonstrated that the photosensitizer Ce6 had been successfully loaded onto the surface of the BT nanoparticles, and the PAH coating had greatly improved the colloidal stability and monodispersity of the BT-Ce6-PAH nanocomposites. The loading efficiency of Ce6 on BT was calculated to be ∼16%.

Fig. 5 Characterizations of BT-Ce6-PAH nanocomposites. (a) Extinction spectra of free Ce6, BT-PAH and BT-Ce6-PAH nanocomposites. Digital images of the aqueous dispersions of BT-PAH, BT-Ce6, BT-Ce6-PAH and free Ce6 after 0 h (b) and 24 h (c) standing period time. (d) SHG spectrum of BT-PAH nanoparticles excited by the fs laser at 800 nm. Representative TEM images of BT-Ce6 (e) and BT-Ce6-PAH (f). (g) Zeta potentials of BT, BT-Ce6 and BT-Ce6-PAH nanoparticles. Hydrodynamic size distributions of BT-Ce6 (h) and BT-Ce6-PAH (i).

3.5. ROS generation of BT-Ce6-PAH nanocomposites

ROS plays a significant role and is the main cause for cell death in PDT. The ROS producing ability of BT-Ce6-PAH nanocomposites upon laser irradiation at 660 nm was investigated. The generation of ROS was determined by ABDA, which has three absorption peaks at 360, 380 and 400 nm and will decrease due to the rapid reaction of ABDA with ROS.²⁴ As shown in Fig. 6a, the mixed dispersion containing BT-Ce6-PAH nanocomposites and ABDA exhibited a sustained decline in absorbance upon laser irradiation at 660 nm (30 mW cm⁻²), indicating rapid ROS generation from BT-Ce6-PAH nanocomposites. In addition, the ROS measurement of BT-PAH nanoparticles and free Ce6 (having the equivalent concentration of Ce6 with BT-Ce6-PAH nanocomposites) were also carried out as control experiments. As shown in Fig. 6b and d, free Ce6 could generate a significant amount of ROS, which was very similar to the case of BT-Ce6-PAH nanocomposites. After laser irradiation for 30 min, the mixed dispersion containing BT-PAH nanoparticles and ABDA showed a slight decrease in absorbance, which was consistent with the case of DI water (Fig. 6c and d). We attributed this to the photodegradation of ABDA induced by long-term laser irradiation. These results demonstrated that the BT-Ce6-PAH nanocomposites could produce ROS with high efficiency upon laser irradiation at 660 nm, and the ROS was generated from Ce6 molecules loaded onto the BT nanoparticles.

Fig. 6 Absorption spectra of ABDA after photo-decomposition by ROS generated from BT-Ce6-PAH (a), free Ce6 (b) and BT-PAH (c) upon laser irradiation (660 nm, 30 mW cm⁻²). (d) Normalized absorbance intensity of ABDA at 380 nm after photo-decomposition by ROS generated from BT-Ce6-PAH, free Ce6, BT-PAH and DI water upon laser irradiation (660 nm, 30 mW cm⁻²).

3.6. In vitro SHG and fluorescence imaging of BT-Ce6-PAH nanocomposites treated cells

The high ROS generation efficiency of BT-Ce6-PAH nanocomposites and sufficient SHG signals of BT nanoparticles encouraged us to apply these nanocomposites for SHG imaging-guided PDT of cancer cells. Firstly, we studied the co-localization of BT-Ce6-PAH nanocomposites that were internalized by cancer cells with SHG and fluorescence imaging. Bright SHG signals (Fig. 7b) excited by the 800 nm-fs laser and intense fluorescence signals (Fig. 7d) excited by the 405 nm-CW laser were observed in HeLa cells, which were incubated with BT-Ce6-PAH nanocomposites for 24 h. The result illustrated effective internalization of the BT-Ce6-PAH nanocomposites into the cancer cells. Furthermore, the uptake of the BT-Ce6-PAH nanocomposites with various incubation time was observed by fluorescence imaging. As shown in Fig. 8a, the red fluorescence signals from the internalized BT-Ce6-PAH nanocomposites in HeLa cells could be clearly observed, and the intensity of the fluorescence signals got higher with increasing incubation time, indicating a time-dependent internalization of BT-Ce6-PAH nanocomposites. However, the fluorescence signals exhibited a slow increase in HeLa cells incubated with the same concentration of free Ce6, and the final fluorescence intensity (after 24 h-incubation) was lower than that of HeLa cells incubated with BT-Ce6-PAH nanocomposites (Fig. 8b). The rapid uptake of BT-Ce6-PAH nanocomposites into HeLa cells may be attributed to the positively charged surface of the nanocomposites, which promoted the nanoparticles to bind onto the surface of the cell membrane and enhanced their internalization via endocytosis, as the cell membrane is negatively charged. However, the Ce6 molecule with low water solubility is negatively charged, which hindered its entry into cells through passive diffusion. These results demonstrated that the BT-PAH nanoparticles could be a highly efficient nanocarrier for Ce6 delivery, and the PDT effects towards cancer cells would be enhanced.

Fig. 7 Co-localization of BT-Ce6-PAH nanocomposites in HeLa cells by SHG and fluorescence imaging. Bright field (a and c), SHG (b) and fluorescence (d) images of HeLa cells incubated with BT-Ce6-PAH nanocomposites (5 μg mL⁻¹, equivalent concentration of Ce6: 0.8 μg mL⁻¹). (e) Merge of SHG and fluorescence signals from BT-Ce6-PAH nanocomposites in HeLa cells. Scale bars: 50 μm.

Fig. 8 Fluorescence images of HeLa cells incubated with BT-Ce6-PAH nanocomposites (5 μg mL⁻¹, equivalent concentration of Ce6: 0.8 μg mL⁻¹) and free Ce6 (0.8 μg mL⁻¹) for 3 h, 6 h, 12 h and 24 h. Excitation wavelength of the CW laser: 405 nm.

3.7. In vitro cytotoxicity and PDT-induced cell death

Before the in vitro PDT of cancer cells, the cytotoxicities of BT, BT-PAH, BT-Ce6-PAH nanoparticles and free Ce6 toward HeLa cells were evaluated by using a CCK-8 assay. As shown in Fig. 9a and b, all four samples showed good biocompatibility and no obvious cytotoxicity was observed in the analyzed concentration ranges. The results indicated that BT nanoparticles have excellent biocompatibility (Fig. 9a), which was in accordance with the previous report¹⁷ and benefited their applications in biology. In the absence of laser irradiation, both BT-Ce6-PAH and free Ce6 did not exhibit obvious cytotoxicity towards HeLa cells, indicating a negligible dark toxicity of Ce6 molecules (Fig. 9b).

Fig. 9 (a) Cell viabilities of HeLa cells incubated with various concentrations of BT and BT-PAH nanoparticles for 48 h. (b) Cell viabilities of HeLa cells incubated with various concentrations of BT-Ce6-PAH nanocomposites and free Ce6 for 48 h, but without laser irradiation. (c) Cell viabilities of HeLa cells incubated with various concentrations of BT-Ce6-PAH nanocomposites and free Ce6 under PDT treatment (660 nm, 30 mW cm⁻², 2 min). (d) Cell viabilities of HeLa cells incubated with 1.25 μg mL⁻¹ BT-Ce6-PAH nanocomposites (equivalent concentration of Ce6, 0.2 μg mL⁻¹) under PDT treatment (660 nm, 30 mW cm⁻²) with various laser-irradiation times.

Afterward, the PDT effects of BT-Ce6-PAH nanocomposites was assessed on HeLa cells. As shown in Fig. 9c, the cell viability showed a dramatic decrease in the presence of BT-Ce6-PAH nanocomposites, as well as laser irradiation at 660 nm (30 mW cm⁻², 2 min), and exhibited a dose-dependent PDT effect. The IC₅₀ (the half maximal inhibitory concentration) of the BT-Ce6-PAH nanocomposites was around 5 μg mL⁻¹ (the equivalent concentration of Ce6: 0.8 μg mL⁻¹). When the concentration of BT-Ce6-PAH reached 20 μg mL⁻¹ (the equivalent concentration of Ce6: 3.2 μg mL⁻¹), the cell viability decreased to ∼10%, indicating an efficient PDT activity for killing cancer cells. In comparison, the PDT effect of free Ce6 (with the same concentration of Ce6 as that in BT-Ce6-PAH nanocomposites) was much weaker than that of BT-Ce6-PAH nanocomposites. We attributed this enhanced PDT efficacy to higher cellular uptake of Ce6 assisted by BT-Ce6-PAH delivery, compared to free Ce6 (Fig. 8). Furthermore, the PDT effect of BT-Ce6-PAH nanocomposites under various laser-irradiation times was investigated. As shown in Fig. 9d, the cell viability showed a decrease with the increasing irradiation time, indicating a time-dependent PDT effect. It is worth nothing that the concentration of BT-Ce6-PAH was 1.25 μg mL⁻¹ (the equivalent concentration of Ce6: 0.2 μg mL⁻¹), which was a low concentration for PDT. The cell viabilities after 6 min and 9 min of laser irradiation showed no obvious changes for both BT-Ce6-PAH nanocomposites and free Ce6. The result illustrated that the amount of Ce6 delivered into the cells is critical for the PDT treatment, and only increasing the laser-irradiation time cannot improve the efficiency of PDT too much. Meanwhile, the bright field images of HeLa cells incubated with various concentrations of BT-Ce6-PAH nanocomposites and free Ce6 under laser irradiation at 660 nm (30 mW cm⁻², 2 min) were captured by an inverted microscope. As shown in Fig. 10, obvious morphological changes of HeLa cells were observed after the PDT treatment. The morphology of HeLa cells became round-like and their sizes decreased, indicating that they were under unhealthy conditions. With the increasing concentration of Ce6, the number of the survival cells exhibited a dramatic decline while the number of cells in apoptosis and dead conditions (round-like cells) increased, which was in accordance with the assessment results of cell viability after PDT (Fig. 9c). These results demonstrated that the BT-Ce6-PAH nanocomposites could serve as a promising drug delivery agent for enhanced PDT of cancer cells.

Fig. 10 Bright field images of HeLa cells incubated with 0, 2.5, 5 and 10 μg mL⁻¹ BT-Ce6-PAH nanocomposites and free Ce6 (equivalent concentration of Ce6, 0, 0.4, 0.8 and 1.6 μg mL⁻¹) after PDT treatment (660 nm, 30 mW cm⁻², 2 min).

4. Conclusions

In conclusion, we developed a novel BT nanoparticle-based platform for SHG imaging-guided PDT. A positively charged polyelectrolyte (PAH) was coated on its surface with BT nanoparticles, and improved colloidal stability and enhanced cellular uptake efficiency of the BT-PAH nanoparticles were demonstrated. In addition, the BT-PAH nanoparticles showed excellent photostability under fs laser excitation in SHG cell imaging. Furthermore, Ce6 was loaded on the BT-PAH nanoparticles with high loading efficacy (16%). Efficient cellular uptake of Ce6 assisted by BT-Ce6-PAH nanocomposites was confirmed by SHG and fluorescence imaging, and enhanced PDT efficiency towards cancer cells was demonstrated. Our work illustrated that the BT based nanocomposites could be used as promising contrast agents for SHG imaging and highly efficient nanocarriers for SHG imaging-guided photodynamic therapy.

Acknowledgements

This work was supported by the National Basic Research Program of China (973 Program, 2013CB834704), the National Natural Science Foundation of China (61178062, 61275190, and 91233208), the Program of Zhejiang Leading Team of Science and Technology Innovation (2010R50007), the Fundamental Research Funds for the Central Universities and the Swedish Research Council.

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