Glutathione-degradable polydopamine nanoparticles as a versatile platform for fabrication of advanced photosensitisers for anticancer therapy

Abstract

A series of glutathione (GSH)-responsive polydopamine (PDA) nanoparticles (NPs) were prepared using a disulfide-linked dopamine dimer as starting material, of which the size could be tuned systematically by adjusting the amount of ammonia solution used. Molecules of a phthalocyanine (Pc)-based photosensitiser and an epidermal growth factor receptor (EGFR)-targeting peptide were then sequentially immobilised on the surface of the NPs through coupling with the surface functionalities of PDA. The immobilised Pc molecules in the resulting nanosystem were photodynamically inactive due to the strong self-quenching effect and the quenching by the PDA core. Upon exposure to GSH in phosphate-buffered saline or EGFR-positive cancer cells, namely A549 and A431 cells, the NPs were disassembled through cleavage of the disulfide linkages to release the Pc molecules, thereby restoring their fluorescence emission and singlet oxygen generation. The NPs with the smallest size (ca. 200 nm in diameter) exhibited the highest cellular uptake and high photocytotoxicity with IC₅₀ values as low as 0.05 μM based on Pc. These NPs could also accumulate and be activated in the tumour of A431 tumour-bearing nude mice, lighting up the tumour with fluorescence over a period of 72 h and completely eradicating the tumour through laser irradiation for 10 min (675 nm, 20 J cm⁻²). The results suggest that these biodegradable and versatile PDA-based NPs can serve as a promising nanoplatform for fabrication of advanced photosensitisers for targeted photodynamic therapy.

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1. Introduction

Biodegradable nanomaterials have received tremendous attention for the application in drug delivery, particularly in the field of cancer therapy.¹ Because of their nanoscale nature, they can prolong the circulation and accumulate at the tumour site by the enhanced permeability and retention (EPR) effect, yet they can be removed from the body via the urinary system after degradation.^2–4 To date, a broad range of biodegradable nanocarriers have been developed, such as the carbon-silica nanocomposite,⁵ Mn₃O₄ nanoparticles (NPs),⁶ poly(β-amino esters) NPs,⁷ DNA nanohydrogels⁸ and so on. Stimuli-responsive properties have also been imparted to these nanostructures to enable them to be activated in response to specific stimuli in the tumour microenvironment for controlled drug release that can minimise the toxicity to healthy cells.⁹ Owing to the various potential advantages of these biocompatible and smart nanocarriers, further development of new biodegradable nanomaterials with advanced pharmaceutical features remains as a contemporary research focus.

Polydopamine (PDA) is a highly versatile material that can be prepared readily through self-polymerisation of dopamine.¹⁰ With reactive functionalities such as amine, imine and catechol on the surface, this polymeric material can facilitate the loading of various noble metals, biomolecules and theranostic agents, as well as the crosslinking with thiol- and amine-containing molecules.^11–13 In addition, PDA possesses an excellent biocompatibility, adhesion property and fluorescence quenching ability. Owing to these advantageous characteristics, PDA-based nanomaterials have been widely used for various biomedical applications, such as biosensing, bioimaging, antimicrobials and anticancer therapy.^14–17 However, most of these materials reported so far have limited biodegradability that has greatly obstructed their clinical translation. To the best of our knowledge, biodegradable PDA-based NPs have only been reported sporadically.^18,19 We have recently developed a novel thioketal-linked PDA-based nanosystem that is responsive towards the endogenous reactive oxygen species (ROS).²⁰ We report herein an extension of this study, using disulfide linkages to render the PDA-based NPs susceptible to intracellular thiols, particularly glutathione (GSH), which is the most abundant intracellular thiol and is often over-produced in cancer cells.²¹ There are several examples of GSH-responsive PDA-based nanomaterials,²² including PDA-coated nanoparticles^23,24 and disulfide-linked PDA-anticancer drug NPs.²⁵ Compared with these nanosystems, our nanoplatform involves only a single component that would facilitate the fabrication process and possesses a degradable nature that can promote renal clearance after the treatment.⁴ The use of these NPs as a versatile nanoplatform of photosensitisers for targeted photodynamic therapy (PDT) was also studied in detail.

PDT is a clinically approved anticancer modality. It involves the interactions of a photosensitiser, light with an appropriate wavelength and endogenous oxygen to generate ROS for eradicating the cancer cells and tissues.^26,27 Compared with the traditional anticancer modalities, PDT is relatively non-invasive and can be applied repeatedly without the likelihood of resistance due to its unique mechanism.²⁸ The clinical translation of PDT, however, is still limited by the low initial selectivity of the currently used photosensitisers (e.g. photofrin) towards the tumour and the prolonged skin photosensitisation that results in a severe adverse effect to the patients. Therefore, there is a great demand of advanced photosensitisers that can exhibit enhanced tumour localisation and controlled ROS generation to minimise the side effect and improve the therapeutic outcome.²⁹ We believed that the aforementioned GSH-responsive PDA-based nanoplatform could facilitate the incorporation of various functional components to circumvent part of these problems.

Fig. 1 illustrates the design and working principle of this nanophotosensitising system. These biodegradable PDA-based NPs were prepared by base-promoted self-polymerisation of a disulfide-linked dopamine dimer. Their surface was then incorporated with molecules of a zinc(II) phthalocyanine-based photosensitiser (labelled as Pc) and the tumour-targeting peptide sequence QRHKPREGGGSC (labelled as QRH) via coupling with the surface functionalities. Owing to the various desirable characteristics, zinc(II) phthalocyanines have been well documented as superior photosensitisers for PDT.³⁰ The heptapeptide QRHKPRE in QRH has also been shown to exhibit high affinity towards the epidermal growth factor receptor (EGFR) overexpressed on the membrane of a variety of human cancer cells.³¹ This sequence can also bind specifically to the target cells within a few minutes with a high binding affinity (K_d = 50 nM).³² It was expected that the Pc molecules were stacked and quenched on the resulting NPs (labelled as PDA-Pc-QRH) by the PDA core and the self-quenching effect. Therefore, this nanosystem was photodynamically inactive in its native state. Upon selective binding to the EGFR-positive cancer cells followed by receptor-mediated endocytosis, the NPs were internalised and then degraded by the intracellular GSH to release the immobilised Pc molecules, thereby restoring their fluorescence and photosensitising properties in a controllable manner. Hence, this new class of biodegradable PDA-based NPs could serve as a tumour-targeting and activatable photosensitiser that can overcome the drawback of non-specific phototoxicity in PDT.

Fig. 1 Schematic illustration of the design and working principle of the GSH-responsive PDA-based nanosystem.

2. Results and discussion

2.1. Preparation and characterisation of PDA-based NPs

The GSH-cleavable dopamine dimer (see its structure in Fig. 1) was prepared by connecting two 3,4-dihydroxy-L-phenylalanine units via a disulfide linker according to the previously described procedure.³³ It underwent self-polymerisation in a mixture of deionised water and ethanol (4 : 1, v/v) with different amounts of ammonia solution (NH₄OH) added to tune the pH of the medium and with oxygen as an oxidant.³⁴ The solution turned brown almost immediately, showing that the self-polymerisation proceeded readily. By using 0.7%, 0.6% and 0.5% (v/v) of NH₄OH, three batches of PDA-based NPs with different size were prepared, namely PDA-1, PDA-2 and PDA-3 respectively. After stirring the mixture at room temperature for 36 h, the resulting NPs were collected by centrifugation and washed with deionised water, followed by lyophilisation.

These three batches of NPs were then treated with Pc (see its structure in Fig. 1), which was prepared according to Scheme S1 (ESI†) as reported by us previously,³⁵ in water containing 0.5% Tween 20 at 60 °C for 14 h. With a nucleophilic amino functionality, Pc could be immobilised readily on the surface of these NPs through Schiff base reaction or Michael addition.¹¹ The Pc-modified NPs were obtained by centrifugation followed by washing with water for three times. Based on the Pc's Q-band absorbance at ca. 700 nm of the supernatant, the amount of free Pc could be determined, from which the Pc loading was estimated to be 20%, 19% and 24% (by weight) for PDA-Pc-1, PDA-Pc-2 and PDA-Pc-3 respectively.

To achieve active tumour targeting, these NPs were further modified with the QRH peptide, which contains the EGFR-targeting heptapeptide QRHKPRE sequence and a non-functional GGGS spacer.²⁰ By simply mixing the NPs with the peptide in deionised water at 4 °C overnight, the resulting conjugates (labelled as PDA-Pc-QRH-1, PDA-Pc-QRH-2 and PDA-Pc-QRH-3) were formed via coupling between the thiol group of the cysteine residue of QRH and the reactive functionalities (e.g. quinone via Michael addition) on the PDA surface.¹¹

The size and morphology of all these NPs were studied using transmission electron microscopy (TEM) and dynamic light scattering (DLS). As shown in Fig. 2a, these PDA-based NPs were generally homogeneous and spherical in shape with a dimension depending on the amount of NH₄OH used. The size was increased in the order PDA-1 < PDA-2 < PDA-3 ranging from 136 to 323 nm in diameter, showing that a reduced amount of NH₄OH used promoted the formation of larger NPs. After loading with Pc, a darkened shell appeared surrounding the PDA core, which indicated the immobilisation occurring on the surface of the NPs, and the size was increased to 182–355 nm. After further conjugation with QRH, the size was increased further to 194–368 nm (Table 1). The average diameters of these NPs determined by TEM were generally in accordance with the hydrodynamic diameters determined by DLS (Fig. 2b and Table 1). The small polydispersity index (PDI) (0.04–0.07) indicated that these NPs were well dispersed in water without significant aggregation.

Fig. 2 (a) TEM images of the PDA-based NPs. Scale bar: 500 nm. (b) Normalised hydrodynamic diameter distribution of these NPs in water measured by DLS. (c) Electronic absorption and (d) fluorescence (λ_ex = 610 nm) spectra of PDA-Pc-QRH NPs and free Pc ([Pc] = 2 μM) in PBS with 0.5% Tween 20. (e) Comparison of the rates of decay of DPBF (initial concentration = 90 μM) sensitised by PDA-Pc-QRH NPs and free Pc ([Pc] = 2 μM) in PBS with 0.5% Tween 20.

Table 1 Characterisation data for the PDA-based NPs

Nanosystem	Physical size^a (nm)	Hydrodynamic diameter^b (nm)	Zeta potential^b (mV)	PDI^b
a The values were obtained from the analysis of 100 particles in the TEM images using ImageJ. b The values are reported as the mean ± standard deviation (SD) of three independent measurements.
PDA-1	136.3 ± 6.4	166.2 ± 0.5	−29.4 ± 0.4	0.04 ± 0.01
PDA-Pc-1	182.4 ± 8.5	215.3 ± 0.6	−24.8 ± 0.6	0.06 ± 0.02
PDA-Pc-QRH-1	194.2 ± 9.4	224.3 ± 1.1	−16.2 ± 1.4	0.05 ± 0.02
PDA-2	245.1 ± 8.2	287.0 ± 3.3	−28.2 ± 0.4	0.05 ± 0.01
PDA-Pc-2	301.2 ± 11.3	326.1 ± 4.9	−23.2 ± 0.6	0.05 ± 0.03
PDA-Pc-QRH-2	311.2 ± 14.5	341.6 ± 4.8	−15.4 ± 0.4	0.07 ± 0.02
PDA-3	323.3 ± 12.8	347.9 ± 2.4	−28.9 ± 0.6	0.03 ± 0.02
PDA-Pc-3	355.5 ± 13.1	375.7 ± 4.8	−24.1 ± 1.2	0.06 ± 0.01
PDA-Pc-QRH-3	368.4 ± 17.5	382.4 ± 4.5	−16.2 ± 0.3	0.05 ± 0.04

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The zeta potentials of these NPs were also determined by DLS (Table 1). It was found that conjugation of QRH led to a less negative value (−16.2 mV for PDA-Pc-QRH-1, −15.4 mV for PDA-Pc-QRH-2 and −16.2 mV for PDA-Pc-QRH-3) compared with the non-peptide-conjugated NPs (−24.8 mV for PDA-Pc-1, −23.2 mV for PDA-Pc-2 and −24.1 mV for PDA-Pc-3), which could be attributed to the positive charges of the peptide. The raw data of the hydrodynamic diameters and zeta potentials of these NPs are given in Fig. S1 and S2 (ESI†).

Fig. 2c shows the electronic absorption spectra of the three batches of PDA-Pc-QRH NPs in phosphate-buffered saline (PBS) with 0.5% Tween 20, which was added to increase the solubility of Pc in this aqueous medium. The spectrum of free Pc is also included for comparison. All these NPs showed the Q-band absorption at ca. 700 nm, which was significantly broadened and weaker compared with that of free Pc. Upon excitation at 610 nm, a very weak fluorescence band due to Pc was observed at 705 nm for all the three batches of NPs (Fig. 2d). The intensity was much weaker than that of free Pc. In addition, the singlet oxygen generation of these NPs was also evaluated using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger.³⁶ As shown in Fig. 2e, the absorbance of the DPBF's absorption at 415 nm was virtually unchanged upon irradiation (λ > 610 nm), showing that they could not generate singlet oxygen. In contrast, free Pc could efficiently consume DPBF through sensitising the formation of singlet oxygen. All these spectral results strongly indicated that the Pc molecules were significantly stacked and self-quenched in the NPs.

2.2. GSH-responsive properties of PDA-Pc-QRH

To examine the effect of GSH on the release of Pc from the NPs of PDA-Pc-QRH, we first monitored the change in fluorescence spectrum of PDA-Pc-QRH-1 in PBS with 0.5% Tween 20 in the presence of different concentrations of GSH (0, 0.005, 0.5 and 5 mM) at 37 °C over a period of 24 h. As shown in Fig. S3 (ESI†), the intensity of the fluorescence emission of Pc at 707 nm increased gradually with time and with concentration of GSH added. While the percentage of fluorescence recovery was less than 10% in the absence of GSH after 24 h, the value reached ca. 70% when 5 mM of GSH was used, which could mimic the intracellular environment in which the concentration of GSH is in the millimolar range (1–10 mM)³⁷ (Fig. 3a). We also compared the Pc release profile of PDA-Pc-QRH-1 with that of PDA-Pc-QRH-2 and PDA-Pc-QRH-3 in the presence of 5 mM GSH. Both the rate and extent of increase in fluorescence emission were similar [Fig. S4 (ESI†) and Fig. 3a]. The results suggested that the disulfide linkers in all the three batches of NPs were cleaved by GSH, triggering the degradation of the NPs and the release of the Pc molecules. The size of the NPs did not have a significant influence on the release profile of Pc.

Fig. 3 (a) Recovery of Pc fluorescence (λ_ex = 610 nm, λ_em = 707 nm) for PDA-Pc-QRH ([Pc] = 2 μM) in PBS with 0.5% Tween 20 upon exposure to different concentrations of GSH at 37 °C over a period of 24 h. (b) Comparison of the rates of decay of DPBF (initial concentration = 90 μM) sensitised by PDA-Pc-QRH ([Pc] = 2 μM) in PBS with 0.5% Tween 20 after treatment with different concentrations of GSH at 37 °C for 24 h. (c) Change in hydrodynamic diameter distribution of PDA-Pc-QRH-1 ([Pc] = 2 μM) in PBS with 0.5% Tween 20 before and after the treatment with 5 mM GSH at 37 °C for 24 h. The inset shows the corresponding TEM images. Scale bar: 200 nm. (d) Fluorescence intensities of PDA-Pc-QRH-1 ([Pc] = 2 μM) in PBS with 0.5% Tween 20, DMEM or FBS measured after 24 h at 37 °C and after treatment with different species at a concentration of 1 mM in PBS with 0.5% Tween 20 at 37 °C for 24 h. Data are reported as the mean ± SD of three independent experiments.

The released Pc also promoted the formation of singlet oxygen. As shown in Fig. 3b, when PDA-Pc-QRH-1 was used as the photosensitiser, the rate of decay of DPBF increased with the concentration of GSH, showing that the singlet oxygen generation efficiency of these NPs was also higher at a higher concentration of GSH. Upon exposure to 5 mM GSH, the efficiency was comparable with that of PDA-Pc-QRH-2 and PDA-Pc-QRH-3. The trend was consistent with that observed for fluorescence recovery as shown in Fig. 3a. Hence, GSH could also activate the singlet oxygen generation of the immobilised Pc and the efficiency was not dependent on the size of the NPs.

The GSH-induced degradation of PDA-Pc-QRH-1 was also studied using DLS. As shown in Fig. 3c, the hydrodynamic diameter distribution was significantly changed after the treatment with 5 mM GSH for 24 h. The NPs disassembled forming several forms of aggregates with different hydrodynamic diameters. TEM also showed the breakdown of the core–shell structure of these NPs upon the action of GSH (see the inset of Fig. 3c).

To examine the stimuli selectivity of PDA-Pc-QRH-1, we recorded the fluorescence spectra of these NPs in the presence of various potential interferential species, including Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Mg²⁺, Ca²⁺, L-ascorbic acid (Vit. C), GSH and dithiothreitol (DTT), and compared with the spectrum in neat PBS with 0.5% Tween 20 (Fig. S5, ESI†). Fig. 3d summarises the corresponding fluorescence intensities. It can be seen that only GSH and DTT could largely increase the fluorescence intensity of Pc through cleavage of the disulfide linkages in the NPs. For all the other conditions, the change in Pc fluorescence was negligible.

2.3. Cellular uptake and intracellular degradation of PDA-Pc-QRH

The cellular uptake of the three batches of PDA-Pc-QRH was first investigated and compared using the EGFR-positive A549 human lung carcinoma cells and A431 human epidermoid carcinoma cells³⁸ to reveal the effect of the size of the NPs. After incubation with these NPs ([Pc] = 0.5 μM) for 6 h, the fluorescence images of the cells were captured using a confocal microscope (Fig. 4a). As the photoactivities of Pc should be quenched when attached to the NPs inside the cells, the fluorescence observed could be attributed to the released Pc upon degradation of the NPs. Considering the similar Pc loading and zeta potential of these three batches of NPs, their intracellular fluorescence intensity could be directly compared, which should be correlated with the extent of cellular uptake. As shown in Fig. 4a, the fluorescence intensity decreased along the series PDA-Pc-QRH-1 > PDA-Pc-QRH-2 > PDA-Pc-QRH-3. The same trend was observed in flow cytometric study (Fig. 4b). The strongest intracellular fluorescence intensity of PDA-Pc-QRH-1 suggested that a smaller size of these NPs could promote the cellular uptake and/or release of Pc. Hence, PDA-Pc-QRH-1 was selected for further biological studies.

Fig. 4 (a) Confocal images of A549 and A431 cells after incubation with different PDA-Pc-QRH ([Pc] = 0.5 μM) for 6 h. (b) Flow cytometric measurements for the two cell lines after the above treatment. (c) Confocal images of HepG2, HEK293, A549 and A431 cells after incubation with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by incubation in a NP-free medium for 12 h. (d) Flow cytometric measurements for the four cell lines after the above treatment. (e) Confocal images of A549 and A431 cells being pretreated with the culture medium in the absence or presence of free QRH (50 or 500 μM) for 1 h, and then with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by incubation in a NP-free medium for 12 h. (f) Flow cytometric measurements for the two cell lines after the treatment conditions described above. (g) Confocal images of A549 and A431 cells upon incubation with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by incubation with different concentrations of GSH-OEt (0, 1 and 10 mM) for 4 h. (h) Flow cytometric measurements for the two cell lines after the treatment conditions described above. All scale bars: 25 μm. Data are reported as the mean ± SD of three independent measurements. To obtain the confocal images, the excitation and emission wavelengths used were 638 and 650–750 nm respectively. For the flow cytometric studies, the excitation and emission wavelengths for Pc were 640 and 720–840 nm respectively.

The selective uptake of PDA-Pc-QRH-1 was demonstrated using a range of cancer cell lines with different expression levels of EGFR. Apart from the two EGFR-positive A549 and A431 cell lines, the HepG2 human hepatocarcinoma cells and HEK293 human embryonic kidney normal cells were also used, which are known to have low expression of EGFR.³¹ These four cell lines were incubated with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by incubation in a NP-free medium for 12 h to provide sufficient time for the internalised NPs to degrade inside the cells. As shown in Fig. 4c, the fluorescence was extremely weak for HepG2 and HEK293 cells, which could be attributed to the low cellular uptake of PDA-Pc-QRH-1 by these EGFR-negative cells. In contrast, for the EGFR-positive A549 and A431 cells, the intracellular fluorescence was much stronger, which indicated that these NPs were preferentially taken up by these cells followed by degradation to release Pc. The intracellular fluorescence intensities for all these four cell lines were quantified by flow cytometry (Fig. 4d). The results were in good agreement with those obtained by confocal fluorescence microscopy.

It was believed that the preferential uptake of these NPs was due to the EGFR-binding property of the QRH peptide. To verify this hypothesis, a competition assay with free QRH peptide was performed. A549 and A431 cells were preincubated with different concentrations of free QRH peptide (0, 50 and 500 μM) for 1 h. They were then washed with PBS and then incubated further with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by incubation in a NP-free medium for 12 h. The confocal images clearly showed that the intracellular fluorescence intensity was generally weaker as the concentration of free QRH increased for both cell lines (Fig. 4e). Similar results were obtained by flow cytometry (Fig. 4f). The average fluorescence intensity decreased by ca. 50% for both cell lines after preincubation with 500 μM free QRH, showing that part of the surface EGFR was occupied by free QRH peptide.

To demonstrate the GSH-responsive fluorescence emission of this nanosystem, A549 and A431 cells were incubated with PDA-Pc-QRH-1 ([Pc] = 0.5 μM) for 1 h, followed by treatment of different concentrations of GSH-OEt (0, 1 and 10 mM) for 4 h after washing away the NPs. The fluorescence images of the cells were then captured with a confocal microscope. As shown in Fig. 4g, the fluorescence intensity increased upon treatment with a higher concentration of GSH-OEt. The same trend was observed by flow cytometry (Fig. 4h). The results indicated that the externally added GSH (produced through hydrolysis of GSH-OEt) could promote the degradation of the NPs and release of Pc.

2.4. In vitro PDT

The cytotoxic effect of PDA-Pc-QRH-1 was examined against the four cell lines, both in the dark and upon light irradiation (λ > 610 nm, 40 mW cm⁻², 48 J cm⁻²). A live/dead double staining protocol based on calcein acetoxymethyl ester (calcein AM) and ethidium homodimer-1 (EthD-1)³⁹ was first used to qualitatively assess the effect. As shown in Fig. 5a, for the EGFR-negative HepG2 and HEK293 cells, after the treatment with PDA-Pc-QRH-1 ([Pc] = 0.4 μM), the cells remained largely viable as evidenced by the intense green intracellular calcein fluorescence regardless of whether light irradiation was applied or not. Red fluorescence of EthD-1, which reflects the number of dead cells, remained extremely weak. This was in accordance with the low uptake of these NPs to these cells. In contrast, for the EGFR-positive A549 and A431 cells, the fluorescence of calcein was significantly reduced upon light irradiation, while notable fluorescence of EthD-1 was observed, indicating the presence of less viable cells and more dead cells under these conditions. These results could be explained by the selective uptake of these QRH-modified NPs and the PDT effect of the activated Pc.

Fig. 5 (a) Fluorescence images of HepG2, HEK293, A549 and A431 cells upon incubation with PDA-Pc-QRH-1 ([Pc] = 0.4 μM) for 1 h, and then with a NP-free medium for 12 h with or without subsequent light irradiation (λ > 610 nm, 40 mW cm⁻², 48 J cm⁻²). At 14 h post-irradiation, the cells were stained with calcein AM (for viable cells in green) and EthD-1 (for dead cells in red) in the dark for 30 min. Scale bar: 100 μm. (b) Cell viability of HepG2, HEK293, A549 and A431 cells upon treatment with different concentrations of PDA-Pc-QRH-1 with or without light irradiation (λ > 610 nm, 40 mW cm⁻², 48 J cm⁻²). Data are reported as the mean ± standard error of the mean of three independent experiments, each performed in quadruplicate.

The cytotoxicities of PDA-Pc-QRH-1 under all these conditions were then quantified by an MTT assay [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].⁴⁰Fig. 5b shows the corresponding dose-dependent survival curves. In the absence of light irradiation, PDA-Pc-QRH-1 was essentially noncytotoxic against all the four cell lines (up to 0.4 μM). Upon light irradiation (λ > 610 nm, 40 mW cm⁻², 48 J cm⁻²), the nanosystem was slightly photocytotoxic towards the EGFR-negative HepG2 and HEK293 cells. The percentage cell viability was decreased to ca. 80% and 70% respectively. For the EGFR-positive A549 and A431 cells, the photocytotoxicity of the nanosystem was significantly increased. The IC₅₀ values were found to be 0.05 μM for A549 cells and 0.06 μM for A431 cells (with respect to the concentration of Pc). These results were in good agreement with those obtained by the live/dead double staining method as described above.

It has been reported that both PDA¹¹ and phthalocyanines⁴¹ could exhibit a photothermal effect. To reveal whether it would contribute to the photocytotoxicity of PDA-Pc-QRH-1, we prepared the reference nanosystem PDA-QRH-1 from PDA-1 and the QRH peptide according to the procedure described above for comparing their photothermal property. As shown in Fig. S6 (ESI†), these NPs were also spherical in shape with an average diameter of 140.2 ± 10.4 nm as measured by TEM, which was in accordance with the hydrodynamic diameter (174.0 ± 0.5 nm) determined by DLS. As expected, the size of these NPs lay between those of PDA-1 and PDA-Pc-QRH-1. The small PDI (0.07 ± 0.03) indicated that the NPs were well dispersed in water without significant aggregation. The zeta potential of PDA-QRH-1 (−20.3 ± 0.5 mV) was less negative than that of PDA-1 (−29.4 ± 0.4 mV) as a result of the positive charges of the peptide. After the characterisation, we examined and compared the photothermal property of PDA-QRH-1 and PDA-Pc-QRH-1 in water upon laser irradiation at 675 nm. It was the laser used in the subsequent in vivo study (see below), and the fluence rate was adjusted to 40 mW cm⁻² (at 0.1 W) to mimic the light condition for the in vitro photocytotoxicity study. As shown in Fig. S7 (ESI†), the temperature was increased from the ambient temperature (21 °C) to 31 and 33 °C respectively over 15 min. When the nanosystems had been pre-treated with GSH (5 mM) for 12 h, the photothermal effect was significantly weaker. For PDA-QRH-1, the increase in temperature was the same as that for water, showing that the photothermal effect of the degraded PDA-based NPs was negligible. Hence, despite the PDA and Pc components in PDA-Pc-QRH-1 could generate heat upon laser irradiation, the temperature increase under these conditions was significantly lower than that for related nanosystems used for photothermal therapy,^42,43 and it was not sufficient to cause cell death. Therefore, the photocytotoxicity of PDA-Pc-QRH-1 should be mainly due to the photodynamic effect of the released Pc.

2.5. In vivo fluorescence imaging

It has been well-documented that NPs with a size greater than 200 nm will be quickly excreted from the blood stream and accumulate in the liver and spleen.^44,45 Hence, among the three batches of PDA-Pc-QRH being studied, PDA-Pc-QRH-1 has a suitable size for in vivo studies. In addition, these NPs also exhibited preferential uptake towards the EGFR-positive A549 and A431 cells. Therefore, this nanosystem was selected for detailed in vivo studies. We first investigated the biodistribution and tumour-localisation property of PDA-Pc-QRH-1 in tumour-bearing nude mice using whole-body fluorescence imaging. Nude mice bearing an A431 tumour were treated with an intravenous dose of PDA-Pc-QRH-1 in PBS ([Pc] = 10 μM, 200 μL). The fluorescence due to Pc at the tumour site was monitored continuously over a period of 72 h (Fig. 6a, upper panel). The mice showed negligible fluorescence at the tumour in the first 8 h as the NPs required a longer time to accumulate and disassemble in the tumour. The intratumoural fluorescence intensity was then increased steadily and continuously over 72 h, showing that the NPs were accumulated in the tumour gradually and the immobilised Pc molecules were also released therein. To reveal whether the immobilised QRH peptide contributed to the tumour localisation, the mice were also injected with free QRH peptide (200 μM) as a competitive ligand of EGFR together with the NPs ([Pc] = 10 μM) in PBS (200 μL). It was found that the fluorescence intensity at the tumour was significantly reduced over the whole period of time (Fig. 6a, lower panel). After 72 h, the intensity was reduced by ca. 40% in the presence of the competitive free peptide (Fig. 6b). The results suggested that apart from the potential EPR effect arising from the nanoscale nature of the NPs, PDA-Pc-QR-1 also exhibited an active EGFR-targeting effect due to the QRH peptide chains on the surface.

Fig. 6 (a) Whole-body fluorescence images of A431 tumour-bearing nude mice before and after intravenous injection with PDA-Pc-QRH-1 in PBS ([Pc] = 10 μM, 200 μL) (upper panel) and PDA-Pc-QRH-1 ([Pc] = 10 μM) with free QRH peptide (200 μM) in PBS (200 μL) (lower panel). The tumours are indicated by black circles. The excitation and emission wavelengths used were 680 and ≥700 nm respectively. (b) The corresponding fluorescence intensities (per unit area) at the tumour sites at different time points. (c) Ex vivo fluorescence images of the tumours and major organs harvested at 72 h post-administration. (d) Quantified fluorescence intensities (per unit area) at the tumour and major organs harvested at 72 h post-administration. (e) Tumour growth curves of tumour-bearing nude mice after different treatments. The mice were intravenously injected with PDA-Pc-QRH-1 or PBS as control and the tumour region was irradiated with laser (675 nm, 0.9 W) for 10 min (20 J cm⁻²) to trigger the PDT effect or without irradiation. (f) The corresponding tumour weight at day 14 after different treatments. The inset shows the corresponding tumour images collected at day 14 after different treatments. (g) Body weights of the mice in different treatment groups. (h) Hematoxylin and eosin (H&E) staining of the tumour sections collected from the mice in different treatment groups. Scale bar: 50 μm. For figure (b) and (d) to (g), data are reported as the mean ± SD (n = 4). n.s., not significant; **p < 0.01; ***p < 0.001.

In addition, we also examined the organ-level distribution of PDA-Pc-QRH-1 by sacrificing the mice and harvesting the tumour and major internal organs, including kidney, liver, spleen, lung and heart at 72 h post-injection for ex vivo fluorescence imaging (Fig. 6c, left part). It was found that the fluorescence intensity of the tumour was remarkably high and comparable with those of the kidney and liver, while the intensities of spleen, lung and heart were significantly lower (Fig. 6d). The high accumulation of NPs in kidney and liver can be explained by the excretion mechanisms of the renal and hepatobiliary systems for the clearance of NPs.⁴⁶ Co-injection of PDA-Pc-QRH-1 with free QRH peptide led to a significant reduction (by ca. 40%) in tumour accumulation, while the effect on all the organs was not significant (Fig. 6c, right part, and Fig. 6d). This observation further supported the in vivo EGFR-targeting effect of the immobilised QRH peptide.

2.6. In vivo PDT

The in vivo therapeutic effect of PDA-Pc-QRH-1 was further investigated in A431 tumour-bearing nude mice. After intravenous injection of PDA-Pc-QRH-1 in PBS ([Pc] = 10 μM, 200 μL) for 72 h, the tumour was irradiated with a diode laser at 675 nm for 10 min (20 J cm⁻²). The tumour size was then monitored continuously for 14 days. As the negative controls, the mice were just injected with PDA-Pc-QRH-1 without the light treatment or injected with PBS with the same light treatment. The corresponding tumour growth curves are shown in Fig. 6e. The images of the tumour-bearing mice before and after the different treatments are given in Fig. S8 (ESI†). It can be seen that PDA-Pc-QRH-1 could greatly suppress the tumour growth upon laser irradiation. In contrast, PDA-Pc-QRH-1 without laser irradiation basically exerted no effect on the tumour growth, similar to the other negative control group for which the mice were simply injected with PBS with laser irradiation. The weight and image of the tumour for each group were also determined. As shown in Fig. 6f, the results were in good agreement with the tumour growth curves shown in Fig. 6e. The body weights of the mice for each group were also monitored over a period of 14 days. It was found that they were essentially unchanged (Fig. 6g), suggesting that the side effect of all these treatments was not notable.

After 14 days of all these treatments, all the mice were sacrificed to harvest the tumour and major organs, including heart, liver, spleen, lung and kidney for histological examination. For the tumour tissues for the treatment group of PDA-Pc-QRH-1 with laser irradiation, severe tissue damage was observed, but not on those for the other two control groups (Fig. 6h). In contrast, there was no major abnormality in the stained organ slides for all the treatment groups (Fig. S9, ESI†), showing that the nanosystem did not induce significant toxicity in the absence of direct laser irradiation.

3. Conclusion

A series of GSH-responsive PDA-based NPs were prepared by a solution oxidation method using a disulfide-linked L-DOPA dimer as starting material. The size of these NPs could be tuned systematically through adjusting the amount of NH₄OH added. These NPs were then modified readily on the surface with molecules of a Pc-based photosensitiser and an EGFR-targeting QRH peptide to give the cell-selective and activatable nanophotosensitising systems PDA-Pc-QRH. The smallest nanosystem PDA-Pc-QRH-1 with an average diameter of 194 nm exhibited the highest cellular uptake towards the EGFR-positive A549 and A431 cells and high photocytotoxicity with IC₅₀ values as low as 0.05 μM based on Pc. It could also effectively eradicate the tumour in A431 tumour-bearing nude mice upon laser treatment without causing any significant side effect. The results show that these tailor-made NPs can serve as a smart and potent photosensitiser for targeted anticancer therapy. It is also envisaged that this new biodegradable and biocompatible nanomaterial can be used to fabricate other functional nanomedicines for various theranostic applications.

4. Experimental section

4.1. General

All the reactions were performed under an atmosphere of nitrogen. Chromatographic purification was performed on silica gel (Macherey Nagel, 230-400 mesh) with the indicated eluents. All solvents and reagents were of reagent grade and used as received. Electronic absorption and steady-state fluorescence spectra were recorded on a Cary 5G UV-Vis-NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer respectively. TEM images were obtained on a FEI Tecnai G2 Spirit transmission electron microscope operated at 120 keV acceleration voltage. Samples dispersed in Milli-Q water (10 μL) were deposited on carbon film-coated Cu grids (200 mesh) and dried under air before the analysis. Hydrodynamic diameters and zeta potentials were measured using a DelsaMax Pro analyser.

4.2. Preparation of PDA

A typical solution oxidation method was used to prepare the NPs of PDA. Ammonia solution (NH₄OH) (0.5%, 0.6% or 0.7%, v/v) was mixed with ethanol (6 mL) and deionised water (24 mL) under mild stirring at room temperature for 30 min. L-DOPA dimer (40 mg, 80 μmol) was dissolved in deionised water (2 mL). The solution was then injected into the above mixtures. The colour of the mixtures turned to pale brown immediately and gradually changed to dark brown. The reaction was allowed to proceed for 30 h. The resulting NPs were collected by centrifugation and washed with water for three times followed by freeze drying. Three batches of NPs with different size, namely PDA-1, PDA-2, PDA-3 were prepared.

4.3. Preparation of PDA-Pc

PDA-1, PDA-2 or PDA-3 (10 mg) was mixed with Pc (2.6 mg, 3 μmol) in water (50 mL) with 0.5% Tween 20. After stirring at 60 °C for 14 h, the mixture was centrifuged. The absorbance of the supernatant and initial solution was determined. The amount of Pc conjugated to the PDA NPs was calculated by subtracting the amount of Pc in the supernatant from the amount of Pc in the initial solution. The loading of Pc was estimated by dividing the weight of Pc by the weight of PDA. The precipitated NPs were washed with water twice and then resuspended in deionised water (50 mL) for further conjugation.

4.4. Preparation of PDA-Pc-QRH

A solution of QRH peptide in deionised water (0.1 M, 100 μL) was added to PDA-Pc-1, PDA-Pc-2 and PDA-Pc-3 prepared as described above. The mixtures were then stirred at 4 °C for 12 h, and then subject to centrifugation. The products PDA-Pc-QRH-1, PDA-Pc-QRH-2 and PDA-Pc-QRH-3 were suspended in deionised water (10 mL) for further use.

4.5. GSH-responsive fluorescence emission

The NPs of PDA-Pc-QRH ([Pc] = 2 μM) were mixed with different concentrations of GSH (0, 0.005, 0.5 and 5 mM) in PBS with 0.5% Tween 20 at 37 °C over a period of 24 h. At different time intervals, the mixture was centrifuged and the supernatant was extracted to obtain the release profile by fluorescence spectroscopy.

4.6. GSH-responsive singlet oxygen generation

The NPs of PDA-Pc-QRH ([Pc] = 2 μM) were treated with different concentrations of GSH (0, 0.005, 0.5 and 5 mM) in PBS with 0.5% Tween 20 at 37 °C for 24 h. The mixture was centrifuged and the supernatant was collected, to which (396 μL) an aqueous solution of DPBF (9 mM, 4 μL) was added. Before each measurement of the absorbance at 415 nm, the solution was illuminated with red light from a 100 W halogen lamp after passing through a water tank for cooling and a colour glass filter (Newport) cut on at λ = 610 nm for 5 s. The decay of DPBF was monitored for a total irradiation time of 180 s.

4.7. Cell lines and culture conditions

A549 (ATCC, no. CCL-185), A431 (ATCC, no. CRL-1555), HepG2 (ATCC, no. HB-8065) and HEK293 (ATCC, no. CRL-1573) cells were maintained in DMEM supplemented with FBS (10%) and penicillin–streptomycin (100 units per mL and 100 μg mL⁻¹ respectively). All the cells were grown in a humidified incubator with 5% CO₂ at 37 °C.

4.8. Confocal laser scanning microscopy

Approximately 4 × 10⁵ cells in DMEM (2 mL) were seeded on a confocal dish and incubated overnight at 37 °C with 5% CO₂. After different treatments, the medium was removed. The cells were rinsed with PBS and examined with a Leica TCS SP8 high speed confocal microscope. The excitation and emission wavelengths of Pc were 638 and 650–750 nm respectively.

4.9. Flow cytometry

Approximately 2 × 10⁵ cells were inoculated into each of the wells in a 12-well plate and incubated in DMEM overnight at 37 °C with 5% CO₂. After different treatments, the cells were rinsed with PBS and harvested by adding 0.5 mL of 0.25% trypsin–ethylenediaminetetraacetic acid (Invitrogen). After adding 0.5 mL of the medium to quench the activity of trypsin, the cell mixture was centrifuged at 1500 rpm for 3 min at room temperature. The cell pellet was washed with 1 mL of PBS and subject to centrifugation for three times. After resuspending the cells in 1 mL of PBS, their intracellular fluorescence intensities were measured by using a BD FACSVerse flow cytometer (Becton Dickinson) with 10⁴ cells counted in each sample. For Pc, the excitation wavelength was 640 nm and the emission wavelength was 720–840 nm. Data collected were analysed by using the BD FAC-Suite. All experiments were performed in triplicate.

4.10. Two-colour fluorescence cell viability assay

Cells were seeded onto a confocal dish at a density of 2 × 10⁵ cells per well and incubated for 24 h. The culture medium was replaced by 1 mL of fresh medium containing PDA-Pc-QRH-1 ([Pc] = 0.4 μM). After incubating for 1 h, the cells were incubated in a NP-free medium for 12 h. The cells were rinsed with 100 μL of PBS twice, replenished with 1 mL of fresh medium, and then irradiated. The light was emitted from a 300 W halogen lamp and passed through a water tank for cooling and a colour glass filter (Newport) cut on at λ = 610 nm. The fluence rate (λ > 610 nm) was 40 mW cm⁻². Illumination for 20 min led to a total fluence of 48 J cm⁻². After incubation for further 14 h, the cells were stained with calcein AM and EthD-1 (Invitrogen) in the dark for 30 min to distinguish the live (green) from the dead (red) cells. The cells were imaged under a confocal microscope (Leica TCS SP8 MP) to evaluate the drug efficacy.

4.11. Photocytotoxicity

Approximately 2 × 10⁴ cells were inoculated into each of the wells in a 96-well plate and incubated in DMEM overnight at 37 °C with 5% CO₂. The cells were incubated with different concentrations of PDA-Pc-QRH-1, followed by incubating in a NP-free medium for 12 h. The cells were rinsed with 100 μL of PBS twice and replenished with 100 μL of fresh medium. For the dark cytotoxicity assay, the plate was directly incubated at 37 °C overnight. For the photocytotoxicity test, the cells were illuminated with the aforementioned light source at room temperature for 20 min and then incubated overnight. A solution of MTT (Sigma) in PBS (3 mg mL⁻¹, 50 μL) was added to each well. After incubation for 4 h under the same condition, 100 μL of dimethyl sulfoxide was added to each well and the plates were placed on a Bio-Rad microplate reader to detect the absorbance at 490 nm. The average absorbance of the blank wells (not seeded with cells) was subtracted from the measured absorbance values of wells of various treatment groups. Cell viability was determined by the equation: %viability = [∑(A_i/A_control × 100)]/n, where A_i is the absorbance of the i^th datum (i = 1, 2, …, n), A_control is the average absorbance of control wells in which the nanosystem was absent. The size of treatment group (n) is 4.

4.12. Animals

All experiments involving live animals were performed in strict accordance with the animal experimentation guidelines and were approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (CUHK) (ref. no. 20-028-GRF). Licence to conduct animal experiments was obtained from the Department of Health, Government of the Hong Kong Special Administrative Region. Female Balb/c nude mice (20–25 g), obtained from the Laboratory Animal Services Centre at CUHK, were kept under a pathogen-free condition with free access to food and water. A431 cells (10⁷ cells in 200 μL) were inoculated subcutaneously on the back of the mice. Once the tumours had grown to a size range of 60–100 mm³, the mice were fed on a low-fluorescence diet (TestDiet, AIN-93M) for 4 days.

4.13. In vivo imaging

PDA-Pc-QRH-1 in PBS ([Pc] = 10 μM, 200 μL), with or without free QRH peptide (200 μM), was injected intravenously to tumour-bearing mice via the tail vein. In vivo fluorescence imaging was performed before and after the injection at different time points with an Odyssey infrared imaging system (excitation wavelength: 680 nm, emission wavelength: ≥700 nm). The images were digitised and analysed by the Odyssey imaging system software (no. 9201-500). The animals were sacrificed after the last scan. The tumour and various internal organs were harvested, and their fluorescence intensities were measured with the Odyssey infrared imaging system ex vivo. Four mice were used for each treatment group.

4.14. In vivo PDT

A431 tumour-bearing mice were randomly divided into three groups: (i) injection of PBS with laser irradiation, (ii) injection of PDA-Pc-QRH-1 without laser irradiation and (iii) injection of PDA-Dox-QRH-1 with laser irradiation. PDA-Pc-QRH-1 in PBS ([Pc] = 10 μM, 200 μL) was intravenously injected to the tumour-bearing mice via the tail vein. At 72 h of post-injection, the tumour was irradiated with a diode laser (Biolitec Ceralas) at 675 nm operated at 0.9 W. Illumination on a spot size of 26 cm² for 10 min led to a total fluence of 20 J cm⁻². The tumour size of the nude mice was monitored periodically for the next 14 days. Tumour volume as calculated using the equation: volume (mm³) = (length × width²)/2 was compared among all the treatment groups. At day 14 of post-treatment, the mice were sacrificed, and the tumour and internal organs were harvested. Tissues were fixed with 4% neutral buffered formalin, dehydrated with an increasing gradient of ethanol and mixed with a solution of xylene and paraffin (1 : 1, v/v). Finally, the tissues were embedded in paraffin for sectioning and stained by H&E. The stained tissue sections were examined using a Ti-E motorised inverted fluorescence microscope (Nikon).

4.15. Statistical analysis

Data shown on figures are presented as the mean ± SD. The data were analysed using Student's t-test with p values <0.05 considered as significant; *p < 0.05; **p < 0.01; ***p < 0.001. Statistical calculations were performed using a Microsoft Excel spreadsheet (Microsoft Corporation, Redmond, WA, USA).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by a General Research Fund from the Research Grants Council of the Hong Kong Special Administrative Region, China (project no. 14306320).

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Footnote

† Electronic supplementary information (ESI) available: Synthetic route for Pc, hydrodynamic diameters and zeta potentials of the PDA-based nanosystems, GSH-dependent fluorescence spectra of PDA-Pc-QRH in PBS, effects of various interferential species on the fluorescence spectra of PDA-Pc-QRH-1 in PBS, characterisation data for PDA-QRH-1, photothermal effect of PDA-QRH-1 and PDA-Pc-QRH-1, and photographic images of the mice and histological images of their major organs after various treatments. See DOI: 10.1039/d1bm01482j

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