Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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A Supramolecular Approach for Modulated Photoprotection, Lysosomal Delivery, and Photodynamic Activity of a Photosensitizer Indranil Roy,† Sharan Bobbala,‡ Ryan M. Young,†,∥ Yassine Beldjoudi,† Minh T. Nguyen,† M. Mustafa Cetin,† James A. Cooper,† Sean Allen,‡ Ommid Anamimoghadam,† Evan A. Scott,† Michael R. Wasielewski, and J. Fraser Stoddart*,†,#,§
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Department of Chemistry, ‡Department of Biomedical Engineering, and ∥Institute for Sustainability and Energy at Northwestern, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States # Institute of Molecular Design and Synthesis, Tianjin University, 92 Weijin Road, Nankai District, Tianjin, 300072, China § School of Chemistry, University of New South Wales, Sydney, New South Wales 2052, Australia S Supporting Information *
ABSTRACT: Prompted by a knowledge of the photoprotective mechanism operating in photosystem supercomplexes and bacterial antenna complexes by pigment binding proteins, we have appealed to a boxlike synthetic receptor (ExBox·4Cl) that binds a photosensitizer, 5,15diphenylporphyrin (DPP), to provide photoprotection by regulating light energy. The hydrophilic ExBox4+ renders DPP soluble in water and modulates the phototoxicity of DPP by trapping it in its cavity and releasing it when required. While trapping removes access to the DPP triplet state, a pH-dependent release of diprotonated DPP (DPPH22+) restores the triplet deactivation pathway, thereby activating its ability to generate reactive oxygen species. We have employed the ExBox4+-bound DPP complex (ExBox4+⊃DPP) for the safe delivery of DPP into the lysosomes of cancer cells, imaging the cells by utilizing the fluorescence of the released DPPH22+ and regulating photodynamic therapy to kill cancer cells with high efficiency.
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INTRODUCTION In oxygenic photosynthetic organisms, light absorption by pigment molecules such as chlorophyll can cause severe oxidative damage, leading to cell death.1 Harmful long-lived chlorophyll triplet states, which form during the so-called “light” reactions, can readily react with molecular oxygen to yield reactive oxygen species (ROS). Various protective mechanisms2 operate on the time scale of seconds to minutes in the photosynthetic apparatus to regulate the amount of ROS generation and control photooxidative damage. Pigmentbinding proteins3 in higher plants and algae play a key role in protecting photosystem (PS) supercomplexes by regulation of the excess energy through nonphotochemical quenching4−6 (NPQ) and/or by a photosynthetic energy-harvesting process7 involving fast electron and/or energy transfer along an electron transport chain together with ROS scavenging.8 Similar features are seen in photosynthetic bacterial antenna complexes,3,9 which consist of light-absorbing pigments associated noncovalently with integral membrane proteins. Inspired by the photoprotection by pigment-binding proteins operating in the photosynthetic apparatus, we decided to call upon a synthetic receptor that can bind a photosensitizer of interest and provide photoprotection. Receptors, such as cyclodextrins10 and cucurbiturils (Figure 1),11 exhibit diverse host−guest chemistry;12 however, they cannot13 provide photoprotection to a photosensitizer because of the lack of active chemical components in their constitutions that © XXXX American Chemical Society
can regulate the light energy on an ultrafast time scale. Designing a synthetic receptor having (i) a geometrically defined, (ii) structurally rigid, yet (iii) locally flexible cavity made of light-energy-regulating components that can accommodate a large-sized π-electron-rich photosensitizer and form a 1:1 host−guest complex is challenging and, therefore, has been hardly explored14 as a means of modulating the photoprotection of a photosensitizer. In particular, the underlying mechanism of the photoprotection of the photosensitizer inside a host molecule has not been investigated14 in great detail. Here, we aim to use the principle of photoprotection of photosensitizers to ensure their safe delivery into the required cellular destinations, where they can be activated upon release by means of a stimuli-responsive mechanism. Such a photosensitizer-bound host can be used in regulated photodynamic therapy15−17 (PDT) depending on spatially controlled release, while the side effects of overall photosensitization of the entire body during the treatment are reduced. PDT, a noninvasive treatment for killing microorganisms16 and cancer cells,17 requires light, a photosensitizer, and molecular oxygen. Under oxygenic conditions, the triplet excited state of the photosensitizer reacts with molecular oxygen by electron transfer and/or an energy transfer process to generate ROS, which kill cancer cells by apoptosis or Received: April 13, 2019 Published: June 30, 2019 A
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
Figure 1. Light-energy regulation and photoprotection inside the cavity of neutral and charged receptors.
Moreover, since host−guest complexation does not require additional covalent functionalization of porphyrins to render them water-soluble, tedious synthetic procedures can be avoided. Porphyrins also undergo protonation (imino nitrogen, pKa ∼ 4−5)23 in an acidic environment, leading to the formation of mono- and dicationic species,24 subjecting the porphyrins to nonplanar conformational distorsions.25 Thus, diprotonated DPP (DPPH22+) can be released in acidic environments on account of the electrostatic repulsions between the tetracationic ExBox4+ and DPPH22+. We anticipated that, in the complex, photoinduced ultrafast electron transfer from the lowest excited singlet state of DPP (1*DPP) to ExBox4+ would outcompete kinetically the formation of the DPP triplet state (3*DPP) by spin−orbitinduced intersystem crossing, leading to the formation of a charge-separated species. At low pH, DPPH22+ may be released, restoring access to the triplet state and activating its ability to generate ROS. Thus, ExBox4+ may ensure the safe delivery of DPPH22+ into the low-pH compartments (pH 4− 5)26 of living cells such as lysosomes and modulate the anticancer activity of DPP by means of regulated PDT. Furthermore, the fluorescence of the released DPPH22+ may be utilized to track the cellular internalization process. Herein, we report the synthesis and characterization of the water-soluble supramolecular complex ExBox4+⊃DPP. We demonstrate that the synthetic receptor ExBox4+ photoprotects DPP in the host−guest complex through ultrafast photoinduced electron transfer from DPP to ExBox4+, leading to the quenching of 1*DPP and elimination of the triplet product state. We show that when ExBox4+⊃DPP is internalized into the lysosome, DPPH22+ is released due to protonation, thereby activating its ability to generate ROS to kill cancer cells.
necrosis. We anticipated that a suitable photosensitizer-bound receptor might offer (i) solubility to the hydrophobic photosensitizer in aqueous media; (ii) a chemical environment for fast electron transfer from the photosensitizer to the receptor, providing photoprotection; (iii) the stimuli-responsive release of the photosensitizer from the receptor cavity, leading to its activation; (iv) straightforward synthetic routes for making the next-generation drugs for regulated PDT; and (v) a model platform for understanding both the photoprotective mechanism and the fate of the host−guest complex in cellular environments. A tetracationic, boxlike synthetic receptor, ExBox4+, incorporating two extended viologen units (ExBIPY2+) linked end-to-end by two p-xylylene linkers, can complex, as its tetrachloride, with hydrophobic π-electron-rich guests and render them soluble in aqueous media18 (except for the singlecrystal X-ray crystallography studies, throughout this paper, the counterions to ExBox4+ are chloride, i.e., 4Cl−). Furthermore, ExBox4+ has an overall rigid yet locally flexible cavity, which may potentially shield active substrates from the outside environment by embracing them. Our previous investigations19,20 have shown that ExBox4+ can function as an electron acceptor in both surface-bound19 and host−guest complexes.20 The host−guest properties of ExBox4+ and its ability to accept photoinduced electrons on a picosecond time scale make it an ideal candidate to photoprotect a photosensitizer by quenching its excited state, much like a pigment-binding protein in PS supercomplexes. Importantly, ExBox4+ is noncytotoxic21 and stable under physiological conditions and so is also a suitable choice as a cellular delivery vehicle. In previous work, we have shown21 that a glowing cyclophane, ExTzBox4+, readily internalizes into the lysosomes of living cells. Structurally and spatially, ExBox4+ is similar to ExTzBox4+ and, therefore, in principle, may also be internalized into the lysosomes to deliver a loaded photosensitizer. Porphyrins22 are employed commonly as photosensitizers in PDT on account of their ability to generate ROS efficiently. ExBox4+ has an ideal cavity size for binding a porphyrinsuch as the commercially available 5,15-diphenylporphyrin (DPP)to form a water-soluble 1:1 host−guest complex.
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RESULTS AND DISCUSSION The synthesis of ExBox·4Cl was carried out following a previously reported procedure.18 Solid DPP (0.46 mg, 1 mmol) was dissolved in Me2CO (1 mM, 1 mL) and added to a solution of ExBox·4Cl in H2O (1.0 mM, 1 mL). The mixture was sonicated for 15 min, Me2CO was removed under vacuum, and the aqueous portion was passed through a 0.45-μm filter to B
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Figure 2. Host−guest inclusion complex (ExBox4+⊃DPP) formation and binding affinity. (a) Scheme of 1:1 host−guest inclusion complex (ExBox4+⊃DPP) formation. (b) 1H NMR titration experiment with DPP in (CD3)2CO. (c) Measurement of the binding constant (Ka) for the in/ out equilibrium process conducted from a 1:1 binding model using the resonances for the Hε protons (indicated with asterisk). All experiments were performed at 298 K.
Figure 3. Solid-state (super)structures of ExBox4+⊃DPP deduced from single-crystal X-ray crystallography. (a) Tubular representation showing the distances between the DPP plane and the ExBIPY2+ sides and the torsional angles associated with the boxlike geometry. (b) Space-filling representation of ExBox4+⊃DPP complex. (c) Space-filling representations showing intermolecular π−π interactions between the two ExBIPY2+ sides of the neighboring ExBox4+⊃DPP complexes. (d) Space-filling representations showing an alternate offset arrangement of the neighboring ExBox4+⊃DPP complexes.
yield the 1:1 ExBox4+⊃DPP complex (Figure 2a) dissolved in water. Upon complex formation, the colorless, aqueous ExBox· 4Cl solution changed to light orange-pink or tangerine. The 1H NMR spectrum (Figure 2b, and Supplementary Figure 2) of ExBox4+⊃DPP in (CD3)2CO displays significant upfield chemical shifts of the resonances for the β and γ protons on ExBox4+ and all the signals for the DPP protons. Small
downfield shifts were observed for the signals from the bridged phenylene protons of ExBox4+. The resonances for the α and ε protons of ExBox4+ were affected only slightly by the shielding effect of DPP, as they are located at the corners and display only small changes in their chemical shifts. These observations are in excellent agreement with the inclusion of DPP inside ExBox4+ in aqueous solution. Importantly, only one set of C
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society proton signals was observed for both ExBox4+ and DPP, most likely as a result of the various species that exist in equilibrium undergoing fast exchange on the 1H NMR time scale. The association constant (Ka) obtained from a 1:1 binding model was calculated to be 12 600 ± 300 M−1 from the chemical shifts of 1H NMR titrations experiments (Figure 2b,c) carried out between ExBox4+ and DPP in (CD3)2CO. Further evidence of host−guest complex formation between ExBox4+ and DPP came from the X-ray diffraction (XRD) analysis (Figure 3 and Supplementary Figure 3) of single crystals of ExBox4+⊃DPP, grown by slow vapor diffusion of i Pr2O into a solution of ExBox·PF6− and DPP in Me2CO/ MeCN (1:1) over the course of 1 week. The cavity of ExBox4+ is 14.6 Å long and 6.3 and 7.1 Å wide at the periphery and center, respectively. The two torsional angles between the pyridinium and phenylene rings18 associated with the ExBIPY2+ unit in ExBox4+ are 32° and 27°, which are reduced to 23° and 17° upon complexation with DPP in order to facilitate its inclusion inside the ExBox4+ cavity (Figure 3a) while good π−π overlap between the π-electron-rich DPP and π-electron-poor ExBIPY2+ units of ExBox4+ is achieved. DPP is accommodated perfectly in the middle of ExBox4+ with a π−π distance of 3.3 Å from the ExBIPY2+ unit on both sides. C− H···π interactions were observed between the m-hydrogen atoms and p-xylylene rings (C−H··· π distance 3.8 Å). In order to minimize the steric strain, the two meso-phenyl groups of DPP remain outside of the ExBox4+. Intermolecular π···π interactions were observed (Figure 3c,d) between the two phenyl rings of adjacent DPP guests and the two phenylene rings of the ExBIPY2+ units of adjacent ExBox4+ hosts facing each other with a 25° tilt and short intermolecular π···π distances of 3.0 and 3.3 Å, respectively. ExBox4+ showed (Figure 4a) a strong absorbance in H2O in the ultraviolet region at 318 nm, while ExBox4+⊃DPP exhibited absorbance peaks at 320, 414, 513, 547, 585, and 635 nm. Of these, the peak at 320 nm originates from ExBox4+, while the peak at 414 nm could be the Soret band, and the remaining four peaks are the Q bands of DPP. The peak at 547 nm was not prominent and only appeared as a shoulder to the 513 nm peak. The presence of four Q bands in ExBox4+⊃DPP indicates no change in symmetry of DPP after complexation; i.e., the 1:1 complex exhibits the same vibronic features as those of free DPP. A low intensity and broad charge transfer (CT) band (650−1150 nm) appeared in the visible and NIR regions of the spectrum (Figure 4a), suggesting the existence of an inclusion complex in H2O and a strong electronic interaction between the π-electron-rich DPP and the πaccepting ExBox4+. Steady-state fluorescence spectroscopy of ExBox4+⊃DPP confirmed the quenching of DPP-based emission, suggesting the involvement of electron transfer between DPP and ExBox4+. Importantly, ExBox4+⊃DPP did not generate any singlet oxygen in H2O upon photoexcitation, indicating the complete deactivation of the triplet pathway of DPP inside the ExBox4+ cavity. In order to elucidate the fluorescence quenching of DPP in the 1:1 complex, we performed femtosecond transient absorption (TA) experiments. Analysis of the TA data for ExBox4+⊃DPP in H2O by exciting the Soret band showed (Figure 4b), on the basis of the appearance of the characteristic ExBIPY+• bands19,20,27 at 527, 993, and 1150 nm, along with the porphyrin cation absorption at 384 nm, an ultrafast electron transfer to the extended viologen units (ExBIPY2+) from 1*DPP within the first few picoseconds. Since these
Figure 4. Absorption and femtosecond transient absorption spectroscopy. (a) Normalized UV−vis spectra of ExBox4+⊃DPP and ExBox4+ (inset: closeup spectra showing the CT band). (b) Femtosecond TA spectra of ExBox4+⊃DPP in H2O excited at λex = 414 nm. (c) Evolution-associated spectra (EAS) generated by fitting to an A → B → ground state kinetic model. State A represents the locally excited 1*DPP state with some of the charge-separated state formed within the instrument response, and state B is the fully chargeseparated state.
bands were present immediately following the instrument response, we conclude that electron transfer may also compete with rapid internal conversion to the DPP S1 state. Similar electron transfer was also observed when exciting the Q band of DPP at λex = 575 nm (Supplementary Figures 6 and 7), suggesting that the ∼2 ps rise in ExBIPY+• absorptions corresponds to electron transfer from the lowest singlet excited state of DPP. The multiple, rapid electron transfer time scales observed most likely owe their existence to different binding orientations in solution. According to the evolution-associated spectra from the kinetic analysis, the charge-separated state lives for 80 ps, regardless of excitation wavelength. More importantly, the long-lived triplet state of DPP was not observed following excitation (Figure 4c), consistent with the observation (Supplementary Figure 11) of no singlet oxygen generation by ExBox4+⊃DPP. Free DPP does not produce singlet oxygen in H2O, as it is insoluble in H2O. Therefore, the D
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society singlet oxygen quantum yield of free DPP is measured (Supplementary Figure 13) in Me2CO: 0.43. The measured (Supplementary Figure 12) singlet oxygen quantum yield of DPPH22+ is 0.38 in H2O. The charge separation observed in the TA experiments was corroborated by density functional theory (DFT). Timedependent DFT calculations were carried out at the RB3LYP/ 6-31G*+ level of theory on the optimized gas-phase geometries (Supplementary Figure 14) of DPP and ExBox4+⊃DPP. The energy levels of the orbitals for ExBox4+⊃DPP are shown in Supplementary Figure 17. While the HOMO is localized on DPP, the LUMO is localized on ExBox4+. The calculated HOMO−LUMO gap is small (ΔEHOMO−LUMO = 1.6 eV) and consistent with the observed CT band in the absorption spectrum of the ExBox4+⊃DPP complex. The first DPP-based unoccupied molecular orbital is LUMO+2, which has a small contribution from the host ExBox4+, a situation that may facilitate the excited-state electron transfer from DPP to ExBIPY2+. In fact, the calculated HOMO−LUMO+2 transition (2.57 eV) of ExBox4+⊃DPP is slightly smaller than the HOMO−LUMO gap (2.72 eV) of free DPP (Supplementary Table 7). At low pH, DPP can be protonated. Therefore, the electrostatic repulsion between the dicationic DPP (DPPH22+) and the tetracationic host ExBox4+ can trigger release of DPPH22+ in H2O. Indeed, when an aqueous solution of ExBox4+⊃DPP is acidified with HCl (pH 4.5), DPPH22+ was released immediately in H2O, leading to a 17-fold increase in fluorescence signals (Figure 5a) when compared to the ExBox4+⊃DPP complex. Furthermore, femtosecond TA spectra of ExBox4+⊃DPP in H2O at pH 4.5, while exciting the Soret band at λex = 414 nm (Figure 5c), revealed the identical charge-separated state living for 80 ps alongside a new, slower component with a lifetime of ∼11 ns in addition to the characteristic excited-state absorption (473 and 1170 nm) and stimulated emission (615 nm) features of 1*DPP. The residual population survived for several microseconds and had a transient absorption spectrum of 3*DPP: the 2.4 μs time constant (Supplementary Figure 10) approximates the decay of the long-lived triplet state in aerated, room-temperature aqueous solution. These results support the protonation and release of DPPH22+ in an acidic environment (Figure 6), turning off the electron transfer in a fraction of the population of the 1:1 complex, and offers access to the triplet state of DPP. We estimate this fraction to be approximately 30% at the ∼20 μM concentration of the transient absorption experiments. ExBox4+ is not cytotoxic21 in the dark as well as under the light irradiation (Supplementary Figure 18). We tested the in vitro cytotoxicity of ExBox4+⊃DPP in the dark on A2780 (ovarian) and MCF-7 (breast) human cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Supplementary Figure 21). Incubation of physiologically relevant phosphate-buffered saline (PBS) solutions of ExBox4+⊃DPP (0.625−100 μM) with A2780 and MCF-7 cells showed >95% viability, indicating the noncytotoxic nature of ExBox4+⊃DPP. In order to understand the fate of ExBox4+⊃DPP in cellular environments and the ability of ExBox4+ to deliver DPPH22+ into cancer cells, we performed (Figure 7) confocal microscopic analysis. Concentration-dependent studies showed that the cells glow red and DPPH22+-based fluorescence are traceable (Supplementary Figure 23) as punctated areas inside A2780 cells at
Figure 5. Fluorescence and femtosecond transient absorption spectroscopy. (a) Fluorescence spectra (λex = 414 nm) of ExBox4+⊃DPP and DPP at pH 7 and 4.5 in H2O. (b) Femtosecond TA spectra (λex = 414 nm) of ExBox4+⊃DPP in H2O at pH 4.5. (c) Evolution-associated spectra (EAS) generated by fitting to a kinetic model for parallel populations decaying via A → B → ground state and C → D processes to account for the released protonated DPP (DPPH22+). State A represents the locally excited 1*DPP state with some amount of charge-separated state formed within the instrument response, and state B is the fully charge-separated state; the other states C and D represent the 1*DPPH22+ (wavelengths denoted in blue) and 3*DPPH22+ populations, respectively, of the free DPPH22+ in solution. All experiments were performed at 298 K.
concentrations as low as 0.1 μM with red laser excitation, indicating the release of DPPH22+ inside cellular compartments having an acidic environment. Next, colocalization studies were performed for DPPH22+ with both LysoTracker and Mitospy stains, known to localize in the lysosomes and mitochrondia, respectively. After 1 h incubation of the A2780 cells with ExBox4+⊃DPP, confocal microscopy revealed only very little DPPH22+-based red fluorescence and no lysosomal colocalization. On increasing the incubation time up to 12 h, colocalization of DPPH22+-based red fluorescence with the green fluorescence of LysoTracker was detected, with significantly higher fluorescence signals (Supplementary Figure 24, Figure 7h), indicating an endolysosomal26 uptake of ExBox4+⊃DPP and release of DPPH22+. The fast appearance E
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Figure 6. Decomplexation of ExBox4+⊃DPP at low pH.
Figure 7. Live-cell confocal microscopy images of A2780 cells showing cellular uptake and colocalization of ExBox4+⊃DPP. Cells were incubated with (a) ExBox4+⊃DPP (10 μM in PBS solution) and (b) NucBlue. (c) Bright-field image of the cells and (d) merged images with red and blue channels. Cells were incubated with (e) ExBox4+⊃DPP (10 μM in PBS solution) and (f) LysoTracker green. (g) Bright-field image and (h) merged images with red and green channel showing the colocalization of ExBox4+⊃DPP and LysoTracker green, evidencing the presence of ExBox4+⊃DPP in the lysosome of the living cells. Z-Stacks of images were deconvolved using the ImageJ package.
of DPPH22+-based fluorescence indicates that a diffusionmediated releasing mechanism is not involved. Independently, flow cytometry revealed a time-dependent increase in ExBox4+⊃DPP uptake and the DPPH22+-based fluorescence signal in A2780 cells. The uptake after 12 h was significantly higher than that at shorter incubation time (Supplementary Figure 24c), consistent with the confocal microscopy results. In contrast, after a 12 h incubation, we observed separate DPPH22+-based red and Mitospy (green) signals inside A2780 cells, demonstrating no mitochondrial internalization (Supplementary Figure 25). Free DPP is insoluble in water and PBS buffer and can only be delivered into the cells when DPP is inside the cavity of the ExBox4+. DPPH22+ can be taken up, however, by the cells into the lysosomes. Although the lysosomal conditions for protonation of DPP and the release of DPPH22+ from the ExBox4+ cavity are different from this control experiment, the lysosomal uptake (Supplementary Figure 20) of DPPH22+ is consistent with the uptake of ExBox4+⊃DPP. The cellular uptake, however, is less efficient in the case of DPPH22+ in comparison to the uptake of the complex ExBox4+⊃DPP, as evidenced from the lower DPPH22+-based fluorescence intensity inside the cells. As a control experiment we evaluated the PDT efficacy of DPPH22+ on A2780 cells as free DPP is insoluble in water and PBS buffer and cannot be delivered into the cells. We found that ∼90% cells are killed with a 10 μM PBS solution of DPPH22+ and a light dose of 0.078 J cm−2. This result confirms that DPPH22+ can kill cancer cells when ExBox4+⊃DPP is taken up by them into the lysosomes and DPP is delivered as
DPPH22+. Next, we determined the in vitro PDT efficacy of ExBox4+⊃DPP on A2780 cells. Initially, we tested a range of concentrations (0.01−10 μM) of ExBox4+⊃DPP in PBS on A2780 cells to determine the working concentrations for PDT. After 10 min of visible light irradiation (385−740 nm) with different doses (0.078 and 0.156 J cm−2), 10 μM of ExBox4+⊃DPP showed >95% killing of A2780 cells, while other concentrations (0.1, 0.05, and 0.01 μM) were less efficient. In the MTT assay, upon exposure to 0.078 and 0.156 J cm−2 light doses, 2.5, 5, and 10 μM concentrations of ExBox4+⊃DPP showed >90% A2780 cell death, while lower concentrations (1.25 and 0.625 μM) had (Figure 8b) an insignificant effect on cell viability. A light dose of 0.032 J cm−2 was efficacious only at 5 and 10 μM concentrations of ExBox4+⊃DPP, while a light dose of 0.016 J cm−2 had no effect on cell viability under the same ExBox4+⊃DPP concentrations. We note that this photoinduced toxicity may be attributed to the combined effect of the singlet oxygen production from the released DPPH22+ as well as the formation of radical cations as a result of the electron transfer process in the residual undissociated complex ExBox4+⊃DPP in lysosomes. PDT efficacy of ExBox4+⊃DPP on A2780 cells was supported using a calcein-AM/ethidium homodimer-1 assay where live and dead cells interact with calcein-AM and ethidium homodimer1, respectively. Under dark conditions, A2780 cells, treated with ExBox4+⊃DPP, interacted mainly with calcein-AM, resulting in (Figure 8c) bright green fluorescence. In contrast, after irradiation with 0.156 J cm−2 light dose for 10 min, A2780 cells showed red fluorescence, indicating an interaction with F
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Figure 30). At 10 μM ExBox4+⊃DPP and 0.156 J cm−2 light dose treatment, a greater number of necrotic cells were apparent, indicating necrotic cell death. The 10 μM ExBox4+⊃DPP and 0.078 J cm−2 light dose treatment and 5 μM ExBox4+⊃DPP, at both light dose treatments (0.156 and 0.078 J cm−2), show a greater number of apoptotic cells than necrotic cells. In contrast, A2780 cells, treated with 5 or 10 μM ExBox4+⊃DPP under dark conditions, showed 79 and 72% intact cells, respectively. These results suggest that ExBox4+⊃DPP irradiation treatment may initiate apoptotic or necrotic cell death, depending on the concentration and light dose. We also analyzed for any signs of cell injury under irradiation using real-time confocal microscopy. After 5 min of continuous irradiation (laser intensities were adjusted to 25 mW), A2780 cells, treated with ExBox4+⊃DPP, showed blebs on their surface, indicative (Movie 1 and Figure 8a) of cell necrosis or apoptosis. Normal imaging conditions, however, showed no signs of cell injury or blebbing. Intracellular ROS generation induced by photosensitizer-irradiation has been reported28 to be the major cause of oxidative stress and tumor cell death. Therefore, we measured the intracellular ROS generation, post-irradiation in A2780 cells. Significantly, a 20fold increase in intracellular ROS was evident after ExBox4+⊃DPP irradiation (10 μM/0.156 J cm−2 light dose), compared (Supplementary Figure 29) with that in dark or untreated cells. In addition, intracellular ROS generation after the 0.156 J cm−2 light dose was found to be greater than that after 0.078 J cm−2 light dose, demonstrating a direct relation of ROS generation and light dose. The amount of ROS generation also depended (Supplementary Figure 29) on the irradiation time.
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Figure 8. Photodynamic therapy efficacy of ExBox4+⊃DPP. (a) Livecell confocal images of A2780 cells following incubation with 10 μM PBS solution of ExBox4+⊃DPP (red) for 12 h. Images were obtained after 5 min exposure to 630 nm confocal laser with 20% gain (25 mW). White arrows on the merged confocal image identify blebs on the cell surface. Scale bar is 15 μm. (b) A2780 cell viability measured using an MTT assay after treatment with ExBox4+⊃DPP (10, 5, 2.5, 1.25, and 0.625 μM) and light irradiation (0.156, 0.078, 0.032, 0.016 J cm−2) for 10 min. Error bars represent SD, n = 4. (c) Confocal microscopy images of A2780 cells stained with calcein-AM and ethidium homodimer-1 after treatment with a 10 μM PBS solution of ExBox4+⊃DPP. Top row and bottom row images represent without (in dark) and with light exposure (after irradiation), respectively. Scale bar is 50 μm.
CONCLUSIONS Surveying the big picture, we identified a biomimetic photoprotective mechanism in the supramolecular complex ExBox4+⊃DPP, synthesized by the trapping of a hydrophobic photosensitizer, DPP, inside the cavity of a tetracationic receptor, ExBox4+. The hydrophilic ExBox4+ renders DPP soluble in H2O and modulates the phototoxicity of DPP. While trapping leads to quenching of the DPP singlet state to provide photoprotection by inhibiting the formation of 3*DPP by picosecond electron transfer, a pH-dependent release of DPPH22+ recovers its triplet state decay pathway and thereby activates its ability to generate ROS in oxygenic conditions. The mechanism of photoprotection was investigated by transient absorption spectroscopy and DFT analysis. Furthermore, the ExBox4+⊃DPP treatment of cells did not show cytotoxicity in the dark, even with a loading concentration as high as 100 μM. Confocal microscopy revealed that ExBox4+⊃DPP is taken up by the lysosomes in cancer cells, where DPP becomes protonated in the acidic environment, leading to the release of DPPH22+. This cellular internalization process was monitored by utilizing the fluorescence of the released DPPH22+ over a broad range of concentrations (0.1− 10 μM). A time-dependent study indicated that the cellular uptake process could be governed by endocytosis. In vitro investigations revealed that, upon irradiation with visible light, DPPH22+ generates 20 times more ROS inside cancer cells, as compared to in the dark, and kills them efficiently in a single treatment within 10 min, even with a dose as low as 0.078 J cm−2 and a loading concentration of 10 μM. The ability of the multifunctional synthetic receptor ExBox4+ to preside over (i) photoprotection, (ii) efficient lysosomal delivery, and (iii) pH-
only ethidium homodimer-1, which was consistent with the MTT assay (Figure 8b). The irradiation time required was found to be dependent on the ExBox4+⊃DPP concentration and light dose (Supplementary Figure 26). With 0.156 J cm−2 light dose and 10 μM ExBox4+⊃DPP treatment, 2 min of irradiation was sufficient to kill >90% of cells, while 0.078 J cm −2 light dose and 5 μM ExBox 4+ ⊃DPP required (Supplementary Figure 26) 10 min to kill >90% A2780 cells. A similar trend was observed (Supplementary Figure 27) with the MCF-7 cancer cell line treated with ExBox4+⊃DPP under light irradiation. We performed an Annexin V FITC/7-AAD assay after 3 h irradiation on ExBox4+⊃DPP-treated A2780 cells to understand the cellular behavior and measure apoptotic and dead cells. Annexin V binds to phosphatidylserine in the plasma membrane in early apoptotic cells or live cells and 7-AAD (7amino-actinomycin D) binds strongly to DNA but excludes intact or live cells during flow cytometry (Supplementary G
DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
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triggered release of a photosensitizer facilitates the use of ExBox4+⊃DPP in anticancer therapy through regulated PDT. This approach could be useful in supramolecular medicine29 for (i) developing new pharmaceutical formulations of photosensitive drugs, (ii) enhancement of drug photostability, and (iii) regulating their therapeutic activity.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b03990. Experimental and synthetic procedures, NMR spectrocopy, crystallographic characterization, transient absorption spectroscopy, detection of singlet oxygen, DFT analysis, and cell experiments (PDF) Jmol image of ExBox4+⊃DPP (CIF) Video of A2780 cells, treated with ExBox4+⊃DPP, after 5 min of continuous irradiation (AVI)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Ryan M. Young: 0000-0002-5108-0261 Yassine Beldjoudi: 0000-0002-9500-4308 Minh T. Nguyen: 0000-0002-9043-9580 M. Mustafa Cetin: 0000-0002-6443-0232 Evan A. Scott: 0000-0002-8945-2892 Michael R. Wasielewski: 0000-0003-2920-5440 J. Fraser Stoddart: 0000-0003-3161-3697 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research is part of the Joint Center of Excellence in Integrated Nano-Systems (JCIN) at King Abdulaziz City for Science and Technology (KACST) and Northwestern University (NU). The authors would like to thank both KACST and NU for their continued support of this research. This work was also supported by the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, U.S. Department of Energy (DOE) under grant no. DE-FG02-99ER14999 (M.R.W., photophysical studies)
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REFERENCES
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DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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DOI: 10.1021/jacs.9b03990 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
