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In Vitro and in Vivo Demonstration of Photodynamic Activity and Cytoplasm Imaging through TPE Nanoparticles Dhanya T. Jayaram,† Sara Ramos-Romero,‡ Balaraman H. Shankar,† Cristina Garrido,‡ Nuria Rubio,‡ Lourdes Sanchez-Cid,‡ Salvador Borros Gómez,§ Jeronimo Blanco,‡ and Danaboyina Ramaiah*,†,∥ †

CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Thiruvananthapuram, 695019, India Institute of Advanced Chemistry of Catalonia (IQAC−CSIC), Barcelona and Biomedical Research Networking Center for Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Zaragoza, 08025, Spain § Grup d’Enginyeria de Materials (GEMAT), Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona 08017, Spain ∥ CSIR-North East Institute of Science and Technology (CSIR-NEIST), Jorhat, 785006, India ‡

S Supporting Information *

ABSTRACT: We synthesized novel tetraphenylethene (TPE) conjugates, which undergo unique self-assembly to form spherical nanoparticles that exhibited aggregation induced emission (AIE) in the near-infrared region. These nanoparticles showed significant singlet oxygen generation efficiency, negligible dark toxicity, rapid cellular uptake, efficient localization in cytoplasm, and high in vitro photocytotoxicity as well as in vivo photodynamic activity against a human prostate tumor animal model. This study demonstrates, for the first time, the power of the selfassembled AIE active tetraphenylethene conjugates in aqueous media as a nanoplatform for future therapeutic applications.

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AIE, where the compound become highly emissive in the aggregate state in which restriction of the intramolecular rotation (RIR) of phenyl substituent can suppress the nonradiative decay pathways.38 The fluorophores with AIE characteristics can serve as an alternative tool for the design of cancer diagnosis and therapeutics, since conventional photosensitizers show an ACQ effect. Recently, fluorescent nanoparticles based on AIE active chromophores have emerged as a new generation of bioimaging nanoprobes.39,41 They are found to have advantages over extensively studied variety of materials like inorganic quantum dots, organic dyes, and fluorescent proteins for the purpose of fluorescence imaging.40−48 In earlier reports, the AIE active nanoparticles have been fabricated by the use of bioconjugation with proteins or amphiphilic copolymers like DSPE−PEG2000 and DSPE−PEG5000− folate.39,47−49 These processes often require tedious synthetic efforts, and the reactions are normally found to be incomplete. To overcome the major hurdles of the existing therapeutic approaches like elaborate synthesis and sophisticated engineering control,13−15 we have utilized the combined action of aggregation induced emissive property and singlet oxygen producing efficiency of the tetraphenylethene (TPE) conjugates. We synthesized novel TPE-benzothiazole conjugates 4,

n recent years, there has been great interest in the development of drugs for cancer treatment which incorporate both imaging and therapeutic properties.1,2 The conventional therapeutics utilize a variety of physical encapsulation and chemical conjugation methods in order to accommodate different therapeutics and diagnostics.1,2 Photodynamic therapy (PDT) is an attractive alternative to the traditional cancer therapies due to the high selectivity in the destruction of tumor cells.3,4 The combined action of the photosensitizer, light, and molecular oxygen is the basic principle of PDT.5−16 A variety of photosensitizers (PSs) including porphyrins, chlorins, bacteriochlorins, phthalocyanines, cyanines, squaraines, and their derivatives have been examined for their potential in PDT.17−28 The search for efficient sensitizers with high specificity and efficacy has provided potential impetus because of the wide application of the PDT in both oncological and nononcological disease treatments.16−20 Moreover, in cancer therapy, a number of nanovehicles have been extensively utilized as drug delivery systems (DDSs) due to their enhanced permeation and retention (EPR) effect to deliver drugs to cancer.29−34 The development of an efficient nanoplatform for cancer treatment embracing both diagnostics and therapeutics holds great potential as a next generation nanocarrier systems. Tang et al. described the phenomenon of aggregation induced emission (AIE) to mitigate the aggregation caused quenching (ACQ).35−41 Tetraphenylethene (TPE), a propellershaped conjugated molecule, was reported to show excellent © 2015 American Chemical Society

Received: July 12, 2015 Accepted: October 22, 2015 Published: October 22, 2015 104

DOI: 10.1021/acschembio.5b00537 ACS Chem. Biol. 2016, 11, 104−112

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ACS Chemical Biology 5, and 6 in good yields and have investigated their photophysical, in vitro, and in vivo photobiological properties under different conditions. These systems having electron donor−acceptor (D−A) moieties exhibited favorable photophysical and intramolecular charge transfer (ICT) properties. Further, these novel TPE conjugates exhibited a unique selfassembly ability to yield spherical nanoparticles with the sizes of ca. 10 ± 5 nm, which showed aggregation induced near-infrared (NIR) emission, selectively localized in cytoplasm and generated cytotoxic singlet oxygen in significant yields. Herein, we report the nanoaggregates of TPE conjugates 4, 5, and 6 and their potential use as NIR fluorescent probes and photodynamic (PDT) agents. As far as we know, this is first report wherein the self-assembled NIR emitting AIE active tetraphenylethene derivatives have been utilized for therapeutic applications, such as for both bioimaging of cytoplasm and in vivo photodynamic activity against a human prostate tumor animal model.

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Figure 1. (A) Absorption and (B) emission spectra of the TPE derivatives 4−6 in acetonitrile. 4 (black, 10 μM), 5 (red, 10 μM), and 6 (blue, 10 μM). Changes in (C) absorption and (D) emission spectra of 4 (10 μM in various water/acetonitrile mixtures. Percentage of water (f w) (a) 0, (h) 70 and (j) 99%. Inset shows the visual observation of fluorescence changes of 4 in acetonitrile and in the water fraction ca. 99%. Excitation wavelength, 450 nm.

RESULTS AND DISCUSSION Synthesis of the alkyl substituted TPE derivatives 4 (hexyl, HTPE-BS), 5 (dodecyl, DTPE-BS), and nonalkyl substituted 6 (TPE-BS) were achieved in ca. 89%, 91%, and 92% yields, respectively, by refluxing their corresponding TPE precursors 3a, 3b, and 3c with benzothiazole salt in ethanol (15 mL) for 12 h (Scheme 1). The products thus obtained were

To understand the photophysical properties of the conjugates 4−6, we have carried out theoretical calculations using density functional theory (DFT) employing the B3LYP/ 6-31g* method. All these systems were found to exist in the twisted conformation. The calculated HOMO and LUMO energy levels and the band gap energies are found to be 2.06, 2.06, and 2.46 eV for the conjugates 4−6, respectively. From the optimized geometries, it can be concluded that the TPE chromophore orbitals predominantly contribute to the HOMO levels, while the LUMO levels have been dominated by the orbitals from the benzothiazolium core, confirming the existence of the intramolecular charge transfer (ICT) process in these cases. The calculated band gap of 2.46 eV for 6 (nonalkylated) is larger than that of 4 and 5, which is in agreement with the experimental observations. The fluorescence studies have indicated that the conjugates 4 and 5 show significantly reduced yields, when compared to the nonalkylated derivative 6, under similar conditions. These observations can be attributed to the existence of the twisted conformation and the restriction of the intramolecular rotation (RIR) of the phenyl groups of the conjugate 6, when compared to alkyl substituted systems 4 and 5. The optimized energy level diagram of the conjugates 4 (Figure S1) and 6 (Figure S2) has been shown in the Supporting Information, and the calculated HOMO−LUMO gaps (band gap) for these conjugates 4−6 were tabulated in Table S1. To investigate the aggregation induced emission properties, we have carried out the photophysical studies of these conjugates 4−6 in various water/acetonitrile solvent mixtures. The absorption bands of TPE-benzothiazole conjugate 4 in acetonitrile showed significant changes due to a gradual increase in the proportion of water in the acetonitrile solution as shown in Figure 1C. Initially, we observed negligible changes up to water fraction (f w) ≤ 60%, but when it was increased to ca. 70%, we observed a profound hypochromicity of ca. 49% at 450 nm along with a 27 nm bathochromic shift and broadening. The peak at 335 nm, on the other hand, showed red-shifted

Scheme 1. Synthesis of TPE-Benzothiazole Derivatives

unambiguously characterized by various analytical and spectroscopic techniques (Experimental Section, SI). The photophysical studies of the TPE-benzthiazole conjugates 4−6 were carried out in different organic solvents, and their absorption and fluorescence spectra in acetonitrile are shown in Figure 1 and data summarized in Table S1. The conjugate 4 showed two absorption bands, at 335 and 450 nm. The peak at 335 nm can be attributed to the characteristic absorption of the TPE chromophore, while the latter band at 450 nm corresponds to the intramolecular charge transfer (ICT) band from the TPE chromophore to the electron deficient benzothiazole moiety.38−40 Of these systems, the conjugates 4 and 5 showed significantly reduced quantum yields of fluorescence, whereas the derivative 6 exhibited emission maxima at 710 nm and a quantum yield of 0.034 (Figure 1B). 105

DOI: 10.1021/acschembio.5b00537 ACS Chem. Biol. 2016, 11, 104−112

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ACS Chemical Biology absorption to 345 nm along with the increase in the intensity under these conditions. Similar observations were made with the derivative 5 (Figure S3). In contrast, the nonalkylated derivative 6 showed a gradual decrease in the intensity of absorbance at 425 nm with the increase in water fraction (Figure S4). In this case, we observed a red shift of 7 nm in the absorption band from 325 to 332 nm. Meanwhile, in the emission spectrum, we observed significant changes with the increase in the water fraction. The conjugate 4 showed negligible fluorescence in acetonitrile. Upon increasing the water fraction to ca. 50%, we observed nonsignificant changes in the emission spectrum. Eventually, at an f w of ca. 70%, as shown in Figure 1D, we observed a significant enhancement in the emission intensity of 4 at 715 nm. From the absorption studies, we could get the indication of aggregation of the molecules at ca. 70% water/acetonitrile mixture. Since the tetraphenylethene (TPE) based systems are active as aggregation induced emissive (AIE) molecules, the observed profound enhancement in the emission can be due to the restricted intramolecular rotation (RIR) of the phenyl substituents. In the case of the dodecyl derivative 5, similar enhancement in the fluorescence intensity was observed at a water fraction (f w) of ca. 50% (Figure S3). However, at 99% water percentage, we observed a small decrease in the fluorescence intensity. In contrast, the nonalkylated derivative 6 exhibited an initial fluorescence at 710 nm, with a fluorescence quantum yield (ΦF) of ca. 3.4% (Figure S4). We observed an initial decrease in fluorescence intensity up to f w ≤ 60%. Further increase in the water fraction resulted in a prominent enhancement in the fluorescence intensity with a hypsochromic shift of ca. 60 nm and a fluorescence quantum yield (ΦF) of 13.2% at 95% water/ acetonitrile mixture (Table 1). On further increasing the water

Figure 2. (A) Plot of fluorescence intensity versus water fraction in the water/acetonitrile mixture of 6 [10 μM], (λem = 650 nm). (B) Fluorescence decay profiles of 6 [10 μM] in (a) acetonitrile, (b) 99% water/acetonitrile mixtures (λex, 440 nm).

time-resolved fluorescence techniques. For example, the fluorescence decay profile of the conjugate 6 in acetonitrile showed a monoexponential decay with a lifetime of 0.4 ns, which corresponds to the tetraphenylethene chromophore. Whereas in the water−acetonitrile mixture having 99% water, we observed a double-exponential decay with the major component (ca. 91%) exhibiting a lifetime of 5.7 ns, which can be attributed to the aggregated species, Figure 2B. Similar observations were made in fluorescence decay dynamics of the alkylated derivatives 4 and 5, wherein the aggregated state exhibited a double exponential decay (Table 1). This longer lifetime for the aggregate excited state has been in conjunction with the corresponding fluorescence enhancement. Motivated from the profound changes observed in the photophysical properties, we have further carried out their morphological analysis through dynamic light scattering (DLS), fluorescence microscopy, and transmission electron microscopy (TEM) studies. In the case of TPE conjugates 4−6 in acetonitrile solution, we observed negligible formation of selfassembled nanoparticles as analyzed through a DLS analyzer. However, when water was added to the acetonitrile solutions of the conjugates, we observed the homogeneous and transparent solutions, even at f w’s as high as 99% of water and formation of self-assembled nanosized structures. At ca. 99% of water/ acetonitrile mixture, the conjugates 4−6 showed formation of spherical nanoparticles (TPE-NPs) having sizes of ca. 10 ± 5 nm (Figure 3A and Figures S6 and S7). In addition, the nanoparticles thus formed from these conjugates have exhibited high yields of red emission (Figure 3B), indicating thereby the potential of the TPE nanoparticles (TPE-NPs) as fluorescent reporters for bioimaging applications. In the fluorescence sensory system, this AIE active organic nanoparticle is advantageous over the metal nanoparticles, wherein the latter will usually quench the fluorescence of the therapeutics due to the energy transfer between dye and nanoparticle, thus restricting their potential as imaging agents.50,51 Further, we have examined the photosensitized singlet oxygen generation efficiency of these conjugates 4−6, since singlet oxygen is known as the main cytotoxic agent in the photodynamic therapy. We have determined quantum yields [Φ(1O2)] of singlet oxygen by using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger. The sensitizer along with DPBF was irradiated with a light source >355 nm at different time intervals from 0 to 120 s. Yields for the generation of singlet oxygen were calculated using the standard, 1-acenaphthone,52 by plotting the ΔOD of DPBF against the irradiation time (Figure S8). These values are Φ(1O2) = 0.58 ± 0.03 for 4, 0.57 ± 0.02 for 5, and 0.60 ± 0.03 for 6 in methanol.

Table 1. Summary of Fluorescence Properties of TPE Derivativesa TPE

ΦF (%) CH3CN

ΦF (%) (H2O/ CH3CN)

τ (ns) acetonitrile

4x

a

7.6

a

5y

a

7.5

a

6z

3.4

13.2

T1 = 0.40 (100%)

τ (ns) (H2O/ CH3CN) T1 = 0.96 (10%), T2 = 3.87 (90%) T1 = 0.53 (21%), T2 = 2.15 (79%) T1 = 1.40 (9%), T2 = 5.66 (91%)

Φ(1O2) 0.57 ± 0.03 0.57 ± 0.02 0.60 ± 0.03

a

Average of more than three independent experiments. a, negligible fluorescence; ΦF, quantum yield of fluorescence; τ, fluorescence lifetime; x, y, and z indicate the percentage of water in water/ acetonitrile mixture. x = 70%, y = 50% and z = 95%, for 4, 5, and 6, respectively.

fraction to ca. 99%, we observed negligible changes in the emission intensity for 6. When the change in the emission peak intensity at 715 nm for 4 and 5 and 650 nm for 6 have been plotted versus the water fraction in the water/acetonitrile mixtures, it could be well understood that the TPEbenzothiazole conjugates 4−6 exhibit aggregation induced emission (Figure 2A and Figure S5). To have a better understanding of the fluorescence changes observed, we have analyzed the decay dynamics by varying the percentage of water (f w) in the acetonitrile solution of TPE derivatives through 106

DOI: 10.1021/acschembio.5b00537 ACS Chem. Biol. 2016, 11, 104−112

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ACS Chemical Biology

Figure 3. (A) TEM and (B) fluorescence microscopic images of the nanoparticles formed by 6 [10 μM] in water/acetonitrile mixtures with f w of 99%. Inset shows the DLS analysis of 6 [10 μM] under similar conditions.

emitting TPE-NPs of 4−6 were proportional to the nanoparticle concentration as shown by FACS analysis, Figures 5A and S10A and B. Confocal laser scanning microscopy (CLSM) imaging of green fluorescent PC3 cells incubated with TPENPs followed by Z-axis scanning of confocal microscope images further confirms that the NIR emitting nanoparticles were detected within the cell cytoplasm (Figure 5C,D,E). Further, the PC3 cells were incubated with TPE-NPs by counter staining with 4′,6-diamidino-2-phenylindole (DAPI). CLSM imaging with a 488 nm laser excitation followed by collecting the fluorescent signals above 650 nm for the TPE-NPs and 560 nm for DAPI, respectively, confirmed the internalization of the NPs in the cytoplasm of the cells (Figure S11). The potential singlet oxygen generation efficiency together with low toxicity and intrinsic capacity to effectively penetrate into the tumor cells suggest that TPE-NPs could be efficient photodynamic therapeutic agents. To investigate the in vitro photodynamic activity, PC3 cells were exposed to TPE-NPs of 4−6 and irradiated using a blue (450 nm, 7W) LED light as described in the Experimental Section. The MTT assay was used to determine cell survival. The IC50 values, a measure of cytotoxic activity, calculated for all three NPs and are found to be 5.6 μM, 27.4 μM, and >50 μM for 4, 5, and 6, respectively (Figure 6A and S12), showing that compound 4 is an effective photodynamic agent. This observation also suggests that the PDT activity of the NPs in cells depends not only on singlet oxygen yields but also on cellular uptake properties. These studies reveal that the TPE-NPs can act as therapeutic agents, wherein AIE active TPE-NPs can be utilized for both fluorescence imaging and as efficient photodynamic therapeutic agents under in vitro conditions. To establish the potential of the developed conjugates as PDT agents, we have compared with Photofrin, a U.S. Food and Drug Administration (FDA) approved and clinically used sensitizer as well as an aluminum phthalocyanine dye, which is currently in clinical trials. Our newly synthesized tetraphenylethene (TPE) conjugate 4 exhibited an IC50 value of 5.6 μM, which is around 2-fold better than Photofrin (8−12 μM) in all the tested cell lines.11 Moreover, the efficacy of tumor growth reduction by the newly synthesized conjugate 4 was also found to be similar to that of aluminum phthalocyanine dye. Gold nanoparticle (AuNP)-based photothermal therapy (hyperthermia) is also known to induce apoptotic cell death, which

The favorable photophysical properties and efficient singlet oxygen generation efficiency suggest that the TPE-NPs of the derivative 4−6 can act as an efficient bioimaging probes and potential candidates for PDT applications. To analyze cytotoxicity of TPE-NPs, human prostate cancer (PC3) cells were exposed to increasing concentrations of TPE-NPs of 4−6 in the dark. MTT assay of cell survival showed that more than ca. 97% cells were alive even after 48 h of incubation with the conjugates 4−6 (Figure S9) and NPs were cytocompatible up to 50 μM. Fluorescence activated cell sorting (FACS) analysis showed very effective internalization of nanoparticles by the cells following an overnight incubation with 5 μM TPE-NPs (Figure 4). In particular, after incubation for a period of