Squaric Acid-Coumarin-Chlorambucil: Photoresponsive Single

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Squaric Acid-Coumarin-Chlorambucil: Photoresponsive Single-Component Fluorescent Organic Nanoconjugates for Self-Monitored Therapeutics Amrita Chaudhuri, Yarra Venkatesh, Joyjyoti Das, Krishna Kalyani Behara, Smita Mandal, Tapas Kumar Maiti, and N. D. Pradeep Singh ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01533 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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ACS Applied Nano Materials

Squaric Acid-Coumarin-Chlorambucil: Photoresponsive Single-Component Fluorescent Organic Nanoconjugates for Self-Monitored Therapeutics Amrita Chaudhuri,† Yarra Venkatesh,† Joyjyoti Das,‡ Krishna Kalyani Behara,† Smita Mandal,† Tapas K. Maiti,‡ and N. D. Pradeep Singh*† †Department

of Chemistry, ‡Department of Biotechnology, Indian Institute of Technology

Kharagpur, 721302, West Bengal, India. KEYWORDS: Combination Therapy, Organic Nanoparticles, Phototrigger, Photodynamic Therapy, Drug delivery. ABSTRACT: In this paper, we have developed photoresponsive fluorescent organic nanoconjugates based on a single component system for effective cancer treatment by a synergistic combination of photodynamic therapy (PDT) and chemotherapy. Our single component system was synthesized by coupling the squaric acid and the coumarin-chlorambucil conjugate.

Next,

multifunctional

squaric

acid-coumarin-chlorambucil

(Sq-Cou-Cbl)

nanoconjugates were prepared by “simple reprecipitation technique”. The unique properties of the newly designed Sq-Cou-Cbl nanoconjugates are (i) a phototrigger for controlled anticancer drug (chlorambucil) release and a photosensitizer for PDT upon visible light irradiation (ii) cellular imaging and self-monitoring of the drug release by a non-invasive fluorescent technique and (iii) PDT activity of the released photoproduct thereby resulting in the improved therapeutic efficiency. Further, in vitro studies showed that Sq-Cou-Cbl nanoconjugates exhibited an enhanced anticancer activity by synergistic cytotoxic effect of PDT and chemotherapy on HeLa cells.

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INTRODUCTION: Photoresponsive multifunctional nanocarriers for combination therapy are desirable, as they are capable of on-demand delivery of therapeutic agents with effective spatial, temporal and dosage-control.1-3 To date, various types of photoresponsive multimodal nanocarriers for cancer treatment were developed utilizing different therapeutic mechanisms like PDT, photothermal therapy (PTT) and chemotherapy.4-5 Among them, combining PDT with chemotherapy is an intelligent approach since PDT and chemotherapy follows two different mechanistic pathways for the eradication of tumor cells.6 A number of photoresponsive nanocarriers were reported for the synergistic treatment of PDT and chemotherapy with an enhanced cytotoxicity on cancer cells.7-10 Kim and co-workers have developed doxorubicin loaded photoresponsive polymeric micelles based on methoxy-poly(ethylene) glycol, chlorin e6 and lipoic acid for both chemotherapy and photodynamic therapy.11 Khaliq et al. have synthesized heparin- pluronic nanoparticles (NPs) for combination therapy, where doxorubicin (prodrug) and methylene blue (photosensitizer) were assembled into a single structure.12 Wang and workers have fabricated core−shell−shell structured NPs composed of three layer- an upconversion (UC) core, a photosensitizer stacked silica middle shell, and, a βcyclodextrin assembled mesoporous silica outside shell for exhibiting triple role like-NIRtriggered drug delivery, PDT and, cell imaging.13 Chen group have made a drug nanosheet delivery system based on black phosphorous for synergistic photothermal, photodynamic and chemotherapy.14 Our group reported the folic acid attached TiO2 NPs tethered with a coumarin chlorambucil component for efficient combination therapy (PDT and chemotherapy).15 To date, photoresponsive nanocarriers for combination therapy have been developed based on liposomes, polymeric NPs, metallic NPs, dendrimers etc.16 The synthetic procedure for these


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hybrid (inorganic-organic) NPs for dual treatment involve four crucial steps: (i) synthesis of biocompatible nanocarrier (ii) surface decoration of nanocarrier by functional groups (iii) attachment of the phototrigger to the NPs for drug delivery and (iv) tethered with photosensitizer for PDT. To overcome the above-mentioned crucial steps, we need to develop a single component nanoconjugate using an organic chromophore17,18 which can perform dual functionality like PDT and chemotherapy.19 Hence, we developed single component photoresponsive organic nanoconjugates based on squaric acid-coumarin-chlorambucil conjugate for the synergistic treatment of PDT and chemotherapy (Scheme 1). We have chosen coumarin for caging anticancer drug because (i) coumarin is a well-known for its clean and efficient photorelease20 (ii) it has an inherent fluorescent property and (iii) it is biocompatible in nature. We have conjugated the coumarinchlorambucil with squaric acid because the squaraine dyes are widely known for their PDT activity.21,22 Our designed squaric acid-coumarin-chlorambucil (Sq-Cou-Cbl) nanoconjugates provided the following advantages like (i) the maximum absorption wavelength of our nanoconjugates extended to the visible wavelength region (ii) the fluorescent intensity of the nanoconjugates increases upon photolysis which helps in self-monitoring of the drug release and (iii) the released photoproduct (Sq-Cou-OH) exhibited PDT activity similar to Sq-Cou-Cbl nanoconjugates.


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Scheme 1. Squaric acid-Coumarin-Chlorambucil based single component multifunctional nanoconjugates for combination therapy (PDT and chemotherapy). 2. EXPERIMENTAL SECTION: Synthesis of the Squaric acid-Coumarin-Chlorambucil conjugate (3): Compounds 1 and 2 were synthesized using the procedure depicted in our previous work.23 Next, Compound 2 (0.21g, 0.44 mmol) and squaric acid (50 mg, 0.44 mmol) was taken in a R.B flask and was dissolved in hot methanol. The mixture was refluxed for 6 h until the deep yellow solid appeared. The obtained solid was filtered and then washed with several solvents likewater, hexane, ethyl acetate, and methanol to get the compound 3 as a deep yellow solid. Yield: 0.24g, 90 %. The resulting compound was characterized by 1H, 13C NMR, and HRMS analysis. (See Figure S1, S2 in Supporting Information (SI)). 1H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 7.96 (s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H), 7.01 (d, J = 7.8 Hz, 2H), 6.65 (d, J = 7.2 Hz, 2H), 6.26 (s, 1H), 5.27 (s, 2H), 4.12 (s, 1H), 3.67 (s, 8H), 2.49 (s, 2H), 2.22 (t, J = 5.8 Hz, 1H), 1.86 – 1.75 (m, 2H), 1.74 – 1.67 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 191.22, 173.23, 172.76, 167.94, 160.53, 160.06, 154.97, 154.37, 150.45, 145.50, 144.94, 129.85, 113.78, 112.28, 61.64, 52.64, 49.41, 41.58, 40.57, 40.36, 40.15, 39.94, 39.73, 39.52, 39.32, 33.14. HRMS (ESI+) calcd for C28H27Cl2N2O7 [M+H]+, 573.1190; found: 573.1168.


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Synthesis of the compound 4: Compound 1 (0.5 g, 1.96 momol) was refluxed in presence of sodium formate (0.15 g, 2.35 mmol) and ethanol (3 mL) to get the compound 4 as a yellow solid. Yield: 0.331g. 1H NMR (400 MHz, DMSO-d6) δ 6.73 (s, 1H), 6.24 (s, 1H), 6.77 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 4.62 (s, 2H).13C NMR (101 MHz, DMSO-d6) δ 165.97, 160.48, 155.56, 150.36, 130.83, 115.78, 113.58, 65.9. HRMS (ESI+) calcd for C14H9NO6 [M+H]+, 191.0582; found: 191.0591. Synthesis of the Sq-Cou-OH conjugate (5): Compound 4 (0.21g, 1.09 mmol) and squaric acid (12 mg, 1.09 mmol) were taken in a R.B flask and dissolved in hot methanol. The mixture was refluxed for 6 h until the deep yellow solid appeared. The obtained solid was filtered and washed with several solvents like- cold water, hexane, ethyl acetate, and methanol to get 5 as a deep yellow solid. Yield: 0.28 g, 95 %. The resulting compound was characterized by 1H, 13C NMR, and HRMS analysis. (See Figure S3, S4 in Supporting Information (SI)). 1H NMR (600 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.01 – 7.93 (m, 1H), 7.69 (d, J = 9.6 Hz, 2H), 6.35 (s, 1H), 4.71 (s, 2H), 4.64 (s, 1H).

13C

NMR (151 MHz,

DMSO-d6) δ 190.69, 178.69, 161.38, 160.59, 156.73, 154.24, 141.19, 125.89, 125.53, 114.91, 114.90, 114.23, 109.61, 59.48, 39.88. HRMS (ESI+) calcd for C14H10NO6 [M+H]+, 288.0503; found: 288.0493. Preparation of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates: Photoresponsive Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates were prepared by a reprecipitation technique.24,25 Millipore water (25.0 mL) was taken into a 25 mL vial and placed in a sonicator. A solution of Sq-Cou-Cbl conjugates (3 mM, 0.0016 g in 1 mL) in DMSO was prepared and kept in the dark. The 5 µL of the DMSO solution of a Sq-Cou-Cbl conjugates was slowly injected into the sonicated water kept in the vial, giving an effective concentration of Sq-


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Cou-Cbl nanoconjugates in water of 1 × 10-5 M. The resulting solution was then sonicated for a period of 12 min maintaining the temperature below 25 °C. Before starting the next sonication period, in every 10 min interval, a short break was given to cool down the solution to 10 °C. The sonication was continued up to one hour. Then the solution was filtered through a nitrocellulose disk filter (~0.8 µm) to remove the largest particles of nanoconjugates. For further study, the filtered solution was used. Using this procedure, the sizes of the particles were noted to be comparable between two independent preparations. Similarly, we have also prepared Sq-Cou-OH nanoconjugates. Photoinduced Anticancer Drug Release by Sq-Cou-Cbl nanoconjugates: Sq-Cou-Cbl nanoconjugates (0.0027 g) dispersed in 50 mL of distilled water was irradiated under visible light (≥ 410 nm) using 125 W medium pressure Hg vapour lamp with a filter 1 M NaNO2 solution (the incident light intensity (I0) is 2.886 × 1016 quanta S − 1) for a period of 60 min. During the photolysis, 0.2 mL aliquots were collected at regular intervals of irradiation time from the photolysate mixture, analyzed them by reverse phase (RP)-HPLC using ACN / water (9:1), a mobile phase at 1 mL/min flow rate (detection: UV 310 nm). From RP-HPLC, peak areas were determined, indicating a gradual decrease in concentration of the Sq-Cou-Cbl nanoconjugates with time (average of three runs). Based on the HPLC data, we have plotted a normalized [HPLC peak area] vs irradiation time which established an exponential correlation for the disappearance of the Sq-Cou-Cbl nanoconjugates with time. Hence, we can suggest that the photolysis of Sq-Cou-Cbl nanoconjugates is a first order reaction. Singlet oxygen generation from Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates: In order to confirm, the selective singlet oxygen generation by Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates, we have performed the conventional photodegradation study of 1,3-


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diphenylisobenzofuran (DPBF). 26 The degradation study was carried out individually with SqCou-Cbl and Sq-Cou-OH nanoconjugates. The singlet oxygen quantum yield (ΦΔ) was calculated using Rose Bengal as the reference (ΦR =0.76 in water). An equimolar solution of SqCou-Cbl nanoconjugates (34 μM) and DPBF (35 μM) was prepared in PBS buffer with 10 % DMSO. In the solution, the photosensitizer concentration was adjusted to maintain the same absorbance (typically 0.1) at 415 nm. The initial concentration of DPBF was 1.26 × 10−4 M. During the experiment, the solutions were stirred vigorously in the presence of O2 gas. The solutions were irradiated under visible light (λ≥ 410) nm. The course of DPBF degradation was monitored by UV-vis absorption spectroscopy. To measure the photostability of DPBF, the solution of only DPBF was irradiated (λ≥410 nm) and also studied by UV-vis absorption spectroscopy. The ΦΔ of each solution was calculated using the following equation 2. ФΔ= (KS/KR) × ΦR ……………….(2) Where K is the slope of photodegradation of DPBF vs time (m) plot, the subscripts S denote the sample and R denotes the reference, and ΦR is the singlet-oxygen generation quantum yield of the Rose Bengal (reference). In vitro cellular uptake studies of the Sq-Cou-Cbl nanoconjugates: The HeLa cells (1 × 104 cells / mL) were seeded on coverslips in MEM medium. After 24 h, one set of cells were treated with 10 µM of Sq-Cou-Cbl nanoconjugates and incubated them for 4 h and another set was used as control (no treatment). Cells were fixed using 3.7% paraformaldehyde. Then, the slides were washed thrice with PBS and prepared with D.P.X mountant. Next, imaging was carried out using a confocal microscope (Olympus FV 1000).


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Anticancer activity of the Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates on HeLa cell line: The cytotoxicity of the Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates in HeLa cells were determined using the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay on HeLa cells before and after irradiation. Anticancer activity before photolysis: The cells were seeded into a 96-well cell culture plate at 1 × 104 cells / mL. Afterward, different concentrations (3, 6, 9 and 12 µg / mL) of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates were added separately into the wells and an equal volume of PBS buffer was added in the control wells. The cells were then incubated in 5% CO2 at 37 °C for 24 h. After that, fresh MTT solution (0.20 mg/mL ) in PBS was added to the wells and incubated in the same cellular environment. After 4 h, solution containing MTT was removed and formed Formazan crystals were taken to dissolve in the DMSO. The absorbance was recorded at 595 nm using an ELISA plate reader Multiskan GO (Thermo Scientific, USA). Anticancer activity after photolysis: HeLa cells maintained in MEM medium (in a 96- well cell culture plate at a concentration of 1 × 104 cells / mL) were incubated independently with different concentrations (3, 6, 9 and 12 µg / mL) of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates for 4 h at 37 °C in 5% CO2. Thereafter, the cells were irradiated under visible light (≥410 nm) for 60 min, keeping the cell culture plate minimum 5 cm apart from the light source. After 60 min irradiation, the cells were again incubated for 24 h. Then the cell viability was measured using the MTT assay as described above.


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Lysosome specific cellular internalization studies of Sq-Cou-Cbl nanoconjugates in HeLa cell: To investigate cellular internalization pathway of Sq-Cou-Cbl nanoconjugates, we have done lysosome specific cellular internalization studies using lysosome-staining dye.27,28 For that, HeLa cells (105 cells per well) were incubated at 37°C with Sq-Cou-Cbl nanoconjugates in a humidified 5% CO2 atmosphere cell culture medium for 4 h. Followed by the treatment of 4% paraformaldehyde and washed with PBS buffer (1X, pH 7.4) for several times. Then the HeLa cells were treated with LysoTracker Red® DND-99 (200 nM) and incubated for another one hour. After that, the cells were washed with PBS buffer and imaging was done by confocal microscope with the appropriate filter (Olympus FV1000). Intracellular singlet oxygen generation by Sq-Cou-Cbl nanoconjugates: HeLa cells treated with Sq-Cou-Cbl nanoconjugates were washed several times with PBS buffer. Then the cells were incubated with 10 mM dichloro-dihydro fluorescein diacetate (DCFDA) in PBS buffer solution (4 mL) for 30 min. Then, cells were again washed and irradiated under visible light (λ≥ 410 nm) with a medium pressure mercury lamp for 3 min and 5 min respectively. After incubation of 24 h, the cells were washed with Dulbecco’s modified eagle medium (DMEM) and the fluorescence signal was determined with excitation/emission at 488/530 nm using the confocal microscope (Olympus FV1000). RESULT AND DISCUSSION: Synthesis of Squaraine-coumarin-chlorambucil (Sq-Cou-Cbl) and Squaraine-coumarinOH (Sq-Cou-OH) conjugates: Here, we synthesized two different conjugates: (i) Sq-Cou-Cbl which can show chemotherapy and PDT and (ii) Sq-Cou-OH (photoproduct) which can show only PDT, to show the advantage


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of the combinatorial therapy over the monotherapies. The synthesis of the Sq-Cou-Cbl conjugate (3) was carried out according to the described procedure in Scheme 2. First, compound 1 was synthesized using the procedure explained in our previous work.23 Next, coumarin-chlorambucil conjugate 2 was obtained by treating 1 with chlorambucil in the presence of K2CO3 and dry DMF. Finally, Sq-Cou-Cbl conjugate (3) was synthesized by refluxing 2 with squaric acid in methanol at 70°C for 4 h. Then, Sq-Cou-OH (5) was synthesized by refluxing compound 1 in the presence of sodium formate and ethanol for 8 h to give 4, followed by refluxing the compound 4 with squaric acid in methanol afforded 5. All the synthesized compounds were characterized by 1H,

13C

NMR, and mass spectral analysis (See Figures S1-S4 in Supporting

Information (SI)). Br

H2N

1

O

iii

O

O

O i

O H2N

2

O

3

Cl

N

O

Cl OH

OH

ii

O HN

HO

O

O 3

3

O

Cl

N Cl

O

iv H2N

O 4

HN

O HO

O

O 5

O

O

Reagents and Conditions: (i) chlorambucil, K2CO3, DMF, 60 C, 2 h (ii) squaric acid (1:1), methanol, reflux, 6 h (iii) sodium formate, ethanol, reflux (iv) squaric acid (1:1), methanol, reflux, 6 h

Scheme 2. Synthesis of Sq-Cou-Cbl and Sq-Cou-OH conjugates. Preparation and characterization of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates: The Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates were prepared using the “reprecipitation technique”. The shape and sizes of the nanoconjugates were determined by TEM and DLS studies respectively. TEM analysis revealed that Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates


10

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were globular in shape with an average diameter of ∼60 and ∼140 nm (Figure 1a and c), respectively. From the DLS data, we found that the average sizes of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates were ∼75 and ∼160 nm (Figure 1b and d), respectively.

Figure 1: TEM images and DLS presented in the intensity of Sq-Cou-Cbl (a,b) and Sq-Cou-OH (c,d) nanoconjugates, respectively. Photophysical properties of Sq-Cou-Cbl conjugate and (Sq-Cou-Cbl and Sq-Cou-OH) nanoconjugates: The photophysical properties of degassed solutions (1 × 10 − 5 M) of Sq-Cou-Cbl conjugate in acetonitrile and nanoconjugates (Sq-Cou-Cbl and Sq-Cou-OH) in water were recorded, individually (Figure 2a and b). From Figure 2a, we observed that the absorption maximum of Sq-Cou-Cbl conjugate was centered at ~450 nm. On the other hand, the absorption spectrum of Sq-Cou-Cbl nanoconjugates showed a broad absorbance band from 320 nm to 500 nm with the blue-shift of absorption maximum at around 410 nm. In case of emission spectrum, the emission maximum of Sq-Cou-Cbl conjugate was centered at ~ 480 nm in acetonitrile solvent, but in the


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case of Sq-Cou-Cbl nanoconjugates, it was blue shifted to 454 nm. Further, we also recorded the absorption and emission spectra of Sq-Cou-OH nanoconjugates. The absorption and emission maxima of Sq-Cou-OH nanoconjugates were 380 nm and 464 nm, respectively. The blue shift in absorption and emission maxima of Sq-Cou-Cbl nanoconjugates are believed to be a result of a "face-to-face" / sandwich type alignment of molecules in the aggregated state (H-type aggregation)29. Interestingly, we observed a large difference in fluorescence intensity between Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates. The difference is probably due to the “aggregation-caused quenching”30,31 fluorescence of the Sq-Cou-Cbl nanoconjugates. Initially, the Sq-Cou-Cbl nanoconjugates were hydrophobic in nature so that the interaction between the solvent (water) and the molecules of the nano-aggregates were very less which reduce the fluorescence intensity of Sq-Cou-Cbl nanoconjugates. In the case of Sq-Cou-OH nanoconjugates the presence of –OH, a polar functional group, the interaction with water molecule is comparatively greater which resulted in the enhanced fluorescence intensity of Sq-Cou-OH nanoconjugates. In addition, we have calculated the fluorescent quantum yields of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates and were found to be ~0.039 and ~0.15 respectively (quantum yield of 9,10-Diphenylanthracene is 0.95 in ethanol, as the standard).32

Figure 2. (a) UV-vis absorption and (b) Emission spectra of Sq-Cou-Cbl conjugate, (Sq-CouCbl and Sq-Cou-OH) nanoconjugates.


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Further, the hydrolytic stability of Sq-Cou-Cbl nanoconjugates was tested by keeping it under dark in PBS buffer solution (of pH=7.4) for a period of 15 days. From HPLC peak area, we found that less than 10% decomposition of our nanoconjugates (Table S1 in SI). Photoinduced anticancer drug release from Sq-Cou-Cbl nanoconjugates: To analyze the drug release process, we irradiated the PBS buffer solution of Sq-Cou-Cbl nanoconjugates (1 × 10–4 M , pH 7.4) in the visible wavelength region (λ≥410 nm). The release of anticancer drug (chlorambucil) from the Sq-Cou-Cbl nanoconjugates was monitored by RPHPLC (Figure 3). From HPLC data, we noticed that the peak at tR=3.98 min corresponding to Sq-Cou-Cbl nanoconjugates was decreasing with an increase in the irradiation time, indicating the photodecomposition of Sq-Cou-Cbl nanoconjugates with time. Additionally, two new peaks at tR=3.16 min and tR=4.18 min were appeared and gradually increased in intensity with increasing irradiation time, which shows the formation of photoproduct (Sq-Cou-OH) and anticancer drug chlorambucil, respectively. The confirmation of corresponding photoproducts was carried out by injecting the authentic samples and also isolated and characterized by mass spectrometry (MS) analysis (Figure S5 and S6 in SI). From the HPLC data, we noticed that almost 85% drug released after 60 min of visible light irradiation and less than 3% drug released from Sq-Cou-Cbl nanoconjugates without light irradiation (Figure S7a in SI). Next, we calculated the photochemical quantum yield (Φp) for chlorambucil release using potassium ferrioxalate as an actinometer and found to be 0.083.33,34


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Figure 3: HPLC overlay spectra for the photolysis of the Sq-Cou-Cbl nanoconjugates at different interval of irradiation time (0-60 min). Irradiation wavelength: λ≥410 nm. The y-axes are offset by 10 mAU and the x-axes are offset by 15 s to facilitate visualization. AU = arbitrary units. To demonstrate precise control over the anticancer drug release, we exposed our Sq-Cou-Cbl nanoconjugates to light and dark environment periodically. Figure S7b clearly showed that whenever the light source was off, drug release process was ceased, indicated that external stimulus light is entirely responsible for the anticancer drug release. The shape and sizes of the nanoconjugates after photolysis were determined by transmission electron microscopy (TEM) (Figure. 4b) and DLS study (Figure S8 in SI). The studies revealed that after photolysis the particle size of nanoconjugates increased, which can be attributed to the formation of Sq-Cou-OH nanoconjugates (photoproduct). Self-monitoring of drug release from Sq-Cou-Cbl nanoconjugates: The interesting property of Sq-Cou-Cbl nanoconjugates is that it shows fluorescence enhancement upon drug release (Figure S9). At 0 min, the excitation of Sq-Cou-Cbl nanoconjugates at 390 nm exhibited a blue-emission band at λ max=457 nm. The gradual increase


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in the irradiation time (0–60 min), resulted in a gradual increase in the fluorescence intensity of blue emission band. After 60 min of photoirradiation, we observed over 5-fold increase in the fluorescence intensity, which corresponds to Sq-Cou-OH nanoconjugates (photoproduct). To confirm, the fluorescence enhancement is due to the formation of Sq-Cou-OH nanoconjugates, we compared the emission spectra of Sq-Cou-OH nanoconjugates and photolysate recorded at 60 min (see Figure 2b and 4a). The results revealed that the emission spectrum of Sq-Cou-OH nanoconjugates was similar to the photolysate recorded at 60 min.

Figure 4: (a) Emission spectra of the Sq-Cou-Cbl nanoconjugates (1 x 10-4 M) during photolysis in PBS buffer at different time intervals (0-60 min). Irradiation wavelength: λ≥410 nm. (b) TEM image of Sq-Cou-Cbl nanoconjugates after photolysis. Singlet

oxygen

generation

by

Sq-Cou-Cbl

and

Sq-Cou-OH

(Photoproduct)

nanoconjugates: To examine the singlet oxygen generation ability of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates (photoproduct), we have performed the DPBF photodegradation study in the presence of our nanoconjugates under visible light (λ ≥ 410 nm) irradiation by absorption spectrometry. From Figure 5, we observed that the decrease in the absorption maximum of DPBF at 415 nm with increase in irradiation time clearly indicated the generation of singlet oxygen by our nanoconjugates. From the slope of the curve (degradation of DPBF vs time), the


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ΦΔ of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates were calculated to be 0.51 and 0.47, respectively (Rose Bengal was used as a reference with a ΦR of 0.76 in water)35. Here, we also observed that the Sq-Cou-OH nanoconjugates (photoproduct) generated singlet oxygen similar to the Sq-Cou-Cbl nanoconjugates. Further, in the absence of nanoconjugates, no significant degradation of DPBF was observed under visible light irradiation (Figure 5).

Figure. 5: Photodegradation of DPBF at 415 nm in presence of Sq-Cou-Cbl and Sq-Cou-OH nanoconjugates and Rose Bengal under visible light irradiation. Photolysis mechanism of Sq-Cou-Cbl nanoconjugates: Based on the literature,36,37 we have suggested a possible photorelease mechanism and singlet oxygen generation by Sq-Cou-Cbl nanoconjugates as shown in Scheme 3. After initial absorption of a photon by Sq-Cou-Cbl nanoconjugate, it gets excited to the singlet state (S1). In the singlet excited state, nanoconjugate undergoes heterolytic C-O bond cleavage to release the anticancer drug, chlorambucil. On the other hand, the Sq-Cou-Cbl nanoconjugate undergoes intersystem crossing to the triplet excited state and subsequently resulted in the generation of singlet oxygen.


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Scheme 3: Possible photolysis mechanism of Sq-Cou-Cbl nanoconjugates Cellular uptake study and Self-monitoring of drug release: To establish Sq-Cou-Cbl nanoconjugates as a versatile single component photoresponsive system for combination therapy, time-dependent cellular uptake studies of Sq-Cou-Cbl nanoconjugates by HeLa cells was monitored using confocal microscopy (Figure S10 in SI). Cells were incubated with 10 µM of Sq-Cou-Cbl nanoconjugates in cell culture medium for 4 h. After incubation for 4 h, the cells emitted blue fluorescence on excitation at 405 nm (Figure 6b), indicating that Sq-Cou-Cbl nanoconjugates were readily internalized into the cells. To demonstrate the self-monitoring ability of the Sq-Cou-Cbl nanoconjugates, the in vitro time-dependent drug release studies upon irradiation on HeLa cells were performed. Figure 6b clearly showed that before irradiation (at 0 min) the Sq-Cou-Cbl nanoconjugates in the HeLa cells showed less intense blue fluorescence, whereas after 60 min of irradiation under visible light (λ≥410 nm) showed intense blue fluorescence (Figure 6c, at 60 min), suggesting that the decomposition of Sq-Cou-Cbl nanoconjugates and concurrent release of anticancer drug (Cbl) inside the cells.


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Figure 6. Confocal images of internalization of Sq-Cou-Cbl nanoconjugates in HeLa cells (a) DIC (differential interference contrast) (b) before photolysis (0 min) (c) after photolysis (60 min). Scale bar = 20 µm. To understand whether Sq-Cou-Cbl nanoconjugates are internalized via lysosomal pathway, we have carried out lysosome specific cellular internalization studies using lysosome-staining dye LysoTracker Red® DND 99. From the confocal images, we observed co-localisation of blue and red fluorescence of Sq-Cou-Cbl nanoconjugates and lysotracker dye, respectively (Figure. S11 in SI). The above observation indicates that the internalization of nanoconjugates might occur via lysosome. Intracellular singlet oxygen generation by Sq-Cou-Cbl nanoconjugates: The qualitative estimation of intracellular singlet oxygen generation by Sq-Cou-Cbl nanoconjugates was done using DCFDA staining on the HeLa cell line .38 Initially, DCFDA is a non-fluorescent in nature, upon oxidation by the reactive oxygen species (ROS) it forms dichloro fluorescein (DCF). The DCF shows strong green fluorescence upon excitation at 488 nm. To understand the cell death pathway by PDT, the HeLa cells were co-incubated with DCFDA and Sq-Cou-Cbl nanoconjugates (λex = 390 nm, λem = 460 nm). Before irradiation, DCF exhibits less intense green fluorescence (Figure 7 b1), indicating negligible singlet oxygen generation by the nanoconjugates. After 5 min of irradiation, strong green fluorescence of DCF was observed


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inside the HeLa cell (Figure 7b3), indicating our Sq-Cou-Cbl nanoconjugates can generate singlet oxygen inside the cells.

Figure 7: Intracellular generation of singlet oxygen by Sq-Cou-Cbl nanoconjugates using DCFDA staining experiment (λex = 488 nm, λem = 530 nm). (a) DIC, (b) fluorescent and (c) merged images of DIC and fluorescent images of (1) control cells with Sq-Cou-Cbl nanoconjugates in dark, (2) after 3 min and (3) 5 min of irradiation with light of ≥410 nm. Scale bar = 10 µm. Cytotoxicity studies of Sq-Cou-Cbl, Sq-Cou-OH nanoconjugates and chlorambucil before and after photolysis: In vitro cytotoxicity of the Sq-Cou-Cbl, Sq-Cou-OH nanoconjugates and cbl were evaluated by MTT assay (before and after photolysis) on HeLa cells. Cells were incubated with different concentrations of Sq-Cou-Cbl, Sq-Cou-OH nanoconjugates and chlorambucil (anticancer drug, cbl) independently for 4 h. It was observed that cell viability of Sq-Cou-Cbl and Sq-Cou-OH


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nanoconjugates remains more than 80% at a studied concentration (Figure 8a). In case of cbl the cell viability is around 48% (at 12 µg/mL). After 60 min of irradiation (at λ≥ 410 nm), we observed 18% (at 12 µg/mL) of the cancer cells survived in the case of the Sq-Cou-Cbl nanoconjugates however ~55% (at 12 µg/mL) cell viability was observed in the case of the SqCou-OH nanoconjugates (Figure 8b). The cbl has not shown any change after irradiation. This enhanced cell cytotoxicity of the Sq-Cou-Cbl nanoconjugates compared to the Sq-Cou-OH nanoconjugates can be ascribed to the combined effect of PDT and chemotherapy whereas, in the case of Sq-Cou-OH nanoconjugates, cytotoxicity was observed only from PDT. Further, we calculated the combination index value of our nanoconjugates (for the combination of PDT and chemotherapy) and found to be ~ 0.7.39

Figure 8. Cell viability assay of Sq-Cou-Cbl, Sq-Cou-OH nanoconjugates and chlorambucil in HeLa cell line: (a) before photolysis (b) after photolysis. Values are presented as mean ± SD. CONCLUSIONS: We have successfully developed single component photoresponsive organic nanoconjugates based on squaric acid-coumarin-chlorambucil for effective cancer treatment by a combination therapy (chemotherapy and PDT). On exposure to visible light, the prepared Sq-Cou-Cbl nanoconjugates simultaneously released chlorambucil and generated singlet oxygen.


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Interestingly, the photoproduct formed during photolysis also generated singlet oxygen similar to the Sq-Cou-Cbl nanoconjugates. Finally, the Sq-Cou-Cbl nanoconjugates have been demonstrated for the in vitro cellular imaging, singlet oxygen generation and self-reporting of drug release ability. The cytotoxicity assay revealed that the enhanced anticancer activity of SqCou-Cbl nanoconjugates compared to the Sq-Cou-OH nanoconjugates is due to the synergistic effect of released anticancer drug chlorambucil and PDT. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes. The authors declare no competing financial interests. ASSOCIATED CONTENT Supporting Information Synthesis details, characterization data, and other experimental details. The Supporting Information is available free of charge on the ACS Publications website. ACKNOWLEDGEMENTS We thank DST SERB (Grant No. EMR/2016/005885) for financial support and DST (SR/FST/CSII-026/2013) for 500 MHz NMR. Amrita Chaudhuri is thankful to UGC-New Delhi for fellowship and Yarra Venkatesh is thankful to the Indian Institute of Technology Kharagpur for the fellowship. AUTHOR CONTRIBUTIONS The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.


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Squaric Acid-Coumarin-Chlorambucil: Photoresponsive Single

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