New Insight into a Cancer Theranostic Probe: Efficient Cell-Specific

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New Insight of Cancer Theranostic Probe: Efficient Cell Specific Delivery of SN-38 Guided by Biotinylated Poly (vinyl alcohol) Debabrata Dutta, Susan M. Alex, Kondapa Naidu Bobba, Kaustabh Kumar Maiti, and Sankarprasad Bhuniya ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10580 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016

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ACS Applied Materials & Interfaces

New Insight of Cancer Theranostic Probe: Efficient Cell Specific Delivery of SN-38 Guided by Biotinylated Poly (vinyl alcohol) †¶

¶

†

Debabrata Dutta, Susan M. Alex,‡ Kondapa Naidu Bobba, Kaustabh Kumar Maiti,*‡║ †

and Sankarprasad Bhuniya* ┴

†Amrita Centre for Industrial Research &Innovation, Amrita School of engineering, Coimbatore,

Amrita University, India 64112. ‡Chemical Sciences & Technology Division, CSIR-National Institute for Interdisciplinary Science & Technology (CSIR-NIIST), Industrial Estate, Pappanamcode, Thiruvananthapuram, Kerala, India 695019. ║

Academy of Scientific and Innovative Research, AcSIR, CSIR-NIIST, Thiruvananthapuram, Kerala, India 695019.

┴

Department of Chemical Engineering & Materials Science, Amrita School of Engineering, Coimbatore, Amrita Vishwa Vidyapeetham, Amrita University, India 641112.

KEYWORDS: Theranostic, Polyvinyl alcohol, Biocompatible, Blood serum, SN-38, Antiproliferative. 1

ACS Paragon Plus Environment


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ABSTRACT An optical modulated ‘turn-on’ theranostic prodrug TP1 has been explored and formulated with biotinylated polyvinyl alcohol (biotinPVA) to get desired pharmacokinetics. The TP1 consists of SN- 38 an antineoplastic camptothecin analogue and a fluorescent dye rhodol green, have been covalently conjugated through a disulfide bond. GSH triggered the release of drug and fluorophore has been well established by UV-vis measurement, the mass spectral analysis in physiological condition. The biocompatible biotinPVA formulated prodrug (PTP1) showed remarkably higher stability against blood serum and cell- specific activation in contrast to TP1. Significantly, PTP1 permits to monitor the delivery and release of well-known topoisomerase I inhibitor SN-38 by modulating fluorescence signal at λem 550 nm within intracellular milieus. Moreover, theranostic probe PTP1 exhibited dose-dependent antiproliferative activity against receptor positive HeLa cells; whereas it did not show such effect against receptor-negative NIH3T3 cells. Finally, the cell-specific antiproliferative activity of PTP1 via apoptotic pathway makes an efficient approach in cancer theranostic. Thus the futuristic PTP1 could be a promising agent where diagnostic and prognostic facts will be monitored synergistically.

INTRODUCTION Chemotherapy is a dominant tool in cancer treatment. However, its activity against cancer is hindered by undesired cytotoxicity to the normal cells/tissues.1 At the advent of genomics, proteomics and bioinformatics has directed to develop a new type of healthcare system to reduce the drug abuse, for precise treatment and safety. In search of an innovative health care product, the term, ‘theranostic’ gained interest in clinical science.2, 3 Cancer theranostic- a combination of chemotherapeutic and imaging modality in a tandem becomes attractive strategy in prognostic 2


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assessment. Currently, optical modulated theranostic system received more attention because of easy functionalization, purification and readily applicable to get spatial images.4-16 Typically it comprises ligated fluorophore in conjunction with chemotherapeutic via a self-immolative linker.17-19 The overexpressed chemical entities in cancer microenvironment, such as GSH,20-28 H2O2,29-31 pH32-33 and bio reductase enzymes34-36 lead to cleavage of the chemical bond and concomitantly release active drugs and fluorophores. This attractive strategy can push drug development research toward an era of precise and personalized treatment. To keep this promise, a theranostic should develop that compose of advanced imaging modalities, cancer biomarkers, and therapeutics. It is a fact that intracellular glutathione (GSH) level in tumor cells is relatively higher (~ 10-100 fold) than the normal cells and tissues.37 The varieties of GSH triggered theranostic systems have been developed for monitoring of therapeutic effect and tracking of the chemotherapeutics.19-27 Definitely, this strategy is more attractive than other strategies, 29-31, 34-36 as it works without any additional external agency. However, the presence of substantial amount of GSH (≥1.02 mM) and vicinal cysteine residue containing albumin protein in blood serum may cause a premature release of chemotherapeutics.38 Unfortunately, the stability of theranostic probe against in blood serum has less studied so far which turned out a serious concern to get an optimized therapeutic effect with a minimal perturbation. Thus, it needs to develop a superior strategy to overcome premature release of chemotherapeutic and to provide stability of GSHresponsive theranostic in blood serum. To keep this promise in mind, we have adopted a strategy, where the new theranostic prodrug (Scheme-1) was loaded into a nontoxic, slow degradable and biocompatible biotinylated poly (vinyl alcohol) (biotinPVA). We believe that the formulated system can overcome premature release of the drug; it can enhance aqueous solubility of the theranostic probe and targets towards selective tumor cells. Here we report the synthesis of new 3



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theranostic prodrug (TP1), its formulation, optical property, mechanism of GSH mediated drug release, tracking of chemotherapeutics, mechanism of cellular uptake and its anticancer activity in living cells. Biocompatible prodrug has been constructed by encapsulation of biotinylated PVA named as PTP1 which showed remarkably higher stability against blood serum and cellspecific activation compared to TP1. Significant release of well-known topoisomerase I inhibitor SN-38 has been observed in the pro-drug construct by modulating fluorescence signal at λem 550 nm in the intracellular milieus. Further, it exhibited dose-dependent antiproliferative activity against receptor positive HeLa cells; whereas no such effect pronounced in receptor-negative NIH3T3 cells. Finally, the cell-specific antiproliferative activity of PTP1 via apoptotic pathway makes it, to be a futuristic theranostic agent, which enables to monitor diagnostic and prognostic facts synergistically.

Experimental section General information and methods: Resorcinol (Loba chem., India), phthalic anhydride (Loba chem., India), 2-hydroxyethyldisulfide (Alfa Aesar), DMTr chloride (Alfa-Aesar), 4-nitrophenyl chloroformate (Alfa-Aesar), 4, 4ʹ - dimethoxytrityl chloride (Avra), SN-38 (Avra), N, Ndiisopropylethylamine (DIPEA) (Alfa-Aesar), GSH (Loba chem, India), TFA (Aldrich), TEA (Aldrich), DMF (Aldrich), and DCM (Loba chem. India) were purchased commercially and used without further purification. Analytical thin layer chromatography was performed using silica gel 60 (pre coated sheets with 0.25 mm thickness). Mass spectra were recorded on anion SpecHiRes ESI mass spectrometer. 1H and

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C NMR spectra were recorded in CDCl3 or DMSO-d6

(Cambridge Isotope Laboratories, Cambridge, MA) on a 400 MHz spectrometer (Bruker, Germany). 4


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Synthesis of 1 and A: Synthesis of 2-(4-diethylamino-2-hydroxybenyl) benzoic acid (1) and compound A were synthesized according to reported literature.39 Synthesis of B: To a solution of mono-O-dimethoxytrityl- 2-hydroxyethyl disulfide (472 mg, 1.034 mmol) in dichloromethane (10 mL) were added N, N-diisopropylethylamine (DIPEA) (0.751 mL, 4.136 mmol) and 4-nitrophenyl chloroformate (625 mg, 3.10 mmol) at 0 °C. The reaction mixture was then stirred at rt for 3 h. After completion of the reaction, the reaction mixture was then concentrated under reduced pressure. The resulting crude intermediate was dissolved in anhydrous Dimethylformamide (DMF) (10 mL) and cooled to 0°C. Then, compound A (400 mg, 1.034 mmol) was added followed by addition of triethylamine (TEA) (0.6 mL, 4.136 mmol) and the reaction mixture was allowed to stir at room temperature (rt) for 24 h. The reaction mixture was diluted with ice water and extracted into dichloromethane. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude compound was passed through a silica gel column using methanol (MeOH) (2.0 %) in dichloromethane as the eluent. The compound was dried in vacuo to afford Compound B (500 mg, 55.60%) as pale brown solid 1

H-NMR (400 MHz, CDCl3): δ 8.24 (q, 1H); 7.73 (d, 1H, j = 5.78 Hz); 7.50 (t, 2H, j = 7.01 Hz);

7.45 (d, 2H, j = 6.27 Hz); 7.36-7.29 (m, 9H); 7.03-6.98 (m, 3H); 6.87-6.84 (d, 1H, j = 8.98 Hz); 6.32 (d, 2H, j = 4.67 Hz).13C-NMR (100 MHz, DMSO-d6): 186.21, 164.62, 153.29, 149.89, 148.70, 144.42, 137.45, 137.36, 135.60, 135.19, 134.30, 134.11, 133.94, 133.76, 131.88, 131.58, 131.29, 129.60, 129.45,129.35, 129.29, 119.90, 110.31, 106.53. ESI- HRMS m/z (M+H): calcd. 869.27, found 870.27683. A base peak for dimethoxytrityl group (DMTr) (M-1) has appeared at 303.128 and another fragment for B - DMTr has appeared at 568.144 (M+1).

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Synthesis of TP1: To a solution of Compound B (500 mg, 0.57 mmol) in dichloromethane (8 mL), was added trifluoroaceticacid (TFA) (2 mL) drop wise at 0 °C. The reaction mixture was then stirred at rt for 3 h. Then, the reaction mixture was poured into ice water and extracted with dichloromethane. The organic layer was dried over sodium sulfate and concentrated in vacuo to afford a crude compound (250 mg, 78%). It was taken for next step without further purification. To a solution of above crude compound (100 mg, 0.176 mmol) in dichloromethane (10 mL) were added DIPEA (0.1 mL, 0.528 mmol) and 4-nitrophenyl chloroformate (107 mg, 0.528 mmol) at 0 °C. The reaction mixture was then stirred at rt for 3 h. Then the reaction mixture was concentrated under reduced pressure. The resulting crude intermediate was dissolved in anhydrous DMF (10 mL) and cooled to 0°C. Then, SN-38 (35 mg, 0.09 mmol) was added followed by addition of TEA (0.05 mL, 0.528 mmol) at the same temperature. Then, the reaction mixture was allowed to stir at rt for 24 h. The reaction mixture was diluted with ice water and extracted into dichloromethane. The organic layer was dried over sodium sulfate and concentrated in vacuo. The crude compound was passed through a silica gel column using MeOH (5.0 %) in dichloromethane as the eluent. The compound was dried in vacuo to afford TP1 (30 mg, 36.0 %) as pale brown solid. 1H-NMR (400 MHz, CDCl3): δ 8.26-8.20 (m, 2H); 8.14 (s, 1H); 8.01 (d, 1H, j = 6.21 Hz); 7.79-7.72 (m, 3H); 7.51 (t, 1H, j = 4.23 Hz); 7.32-7.30 (m, 3H); 6.98 (d, 1H, j = 4.91 Hz); 6.32 (t, 1H, j = 5.29 Hz); 6.53-6.42 (m, 4H); 5.43 (s, 2H), 5.32 (d, 2H, j = 5.91 Hz); 4.51 (q, 4H); 3.37 (t, 3H, j = 4.28 Hz); 3.19-3.13 (m, 6H); 1.86 (t, 2H, j = 7.01 Hz); 1.24 (t, 3H, j = 5.89 Hz); 1.07 (t, 6H, j = 3.21 Hz); 0.89 (t, 3H, j = 5.87 Hz).13CNMR (100 MHz, DMSO-d6): 172.50, 16.5, 158.29, 153.89, 152.70, 149.90, 137.20, 131.91, 130.60, 129.19, 127.60, 124.60, 122.91, 120.02, 118.31, 116.09, 110.92, 109.81, 104.30, 97.45,

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83.55, 73.02, 66.06, 64.31, 50.09, 42.00, 36.20, 30.91, 23.51. ESI- HRMS m/z (M+H): calcd. 957.22, found 986.265: HPLC purity: 98.106%. Synthesis of biotinPVA 500 mg of polyvinyl alcohol (PVA) was dissolved in dimethylsulfoxide (DMSO) (15 mL) and stirred for 30 min to obtain a homogeneous solution. Then, biotin (200 mg, 0.82 mmol), N-(3Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl) (236 mg, 1.24 mmol) and 4-dimethylaminopyridine (DMAP) (75 mg, 1.23 mmol) were added successively into the reaction flask and the resulting mixture was stirred at rt for 2 days. After completion of the reaction, the reaction mixture was diluted with ethyl acetate, centrifuged and dialyzed to afford biotinPVA as white color solid. The compound was confirmed by 1H-NMR. 1H-NMR (400 MHz, DMSO-d6): δ 6.41 (d, 1H); 6.02 (d, 1H); 4.70 (s, 2H); 4.52 (s, 2H); 4.52 (s, 2H); 4.30 (s, 2H); 3.81-3.72 (m, 4H); 3.60-3.01 (m, merged with DMSO moisture); 2.70-2.45 (m, merged with DMSO-d6); 1.89-2.01 (m, 4H); 1.51-1.31 (m, 6H). Preparation of PTP1 BiotinPVA (100.0 mg) was dissolve in ethanol-methanol (1:1) mixed solvent (50 mL) and then TP1 (20.0 mg) in

methanol (5.0 mL) was added into the polymer solution. The solution was

continuously stirred for another 2h. Then the solution mixture was dried concentrated and dried out vacuo to obtain biotinPVA formulated prodrug PTP1. The size of polymer encapsulated theranostic drug (PTP1) was confirmed by DLS and high resolution transmission electron microscopy (HR-TEM) studies.

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Particle Size Measurement The size of PTP1 was analyzed using Dynamic Light Scattering (DLS) performed in Zetasizer Nano ZSP (Malvern Instruments). Initially the polymer encapsulated theranostic probe was prepared in 1:5 ratio (w/w) (TP1: biotinPVA) and suspended in methanol-ethanol solvent mixture. The sample was kept for 30 min stirring till nanoparticles were formed and vacuum dried to remove the solvent. PTP1 was suspended in MilliQ for the following experiments. A drop of PTP1 in aqueous solution was casted onto a 230 mesh copper grid coated with carbon and air dried for HR-TEM analysis. Image was procured from a JEOL 2010 high-resolution transmission electron microscope with an accelerating voltage of 200 KV. In parallel to this PTP1 aqueous solution was subjected for DLS measurement. Sample was measured three times to calculate the average hydrodynamic diameter that corresponded to the particle size. Measurements were assessed in a 3 × 3 mm quartz cuvette and collected at a 90° scattering angle.

Absorption and Fluorescence Studies All fluorescence spectra were recorded in F-4500 FL spectrometer with a 1cm standard quartz cell and UV-vis absorption spectra were collected from UV-1800 spectrophotometer, respectively. Stock solutions (1 mM) of various analytes (GSH, cystine, alanine, histidine, arginine, tyrosine, Na+, K+, Ca2+, Cu2+, Fe2+, Fe3+, Zn2+, Ascorbic acid (AA), H2O2, NO, NO2-, NO3-, and S2O32-) were prepared in PBS buffer. The stock solution of probe, TP1 was prepared in PBS buffer (pH = 7.4) with 5% DMSO. Excitation was carried out at 510 nm with excitation and emission slit widths is 3 nm each. The fluorescence experiments (solution test) of

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prodrug, TP1 (5.0 µM) recorded in the presence of increasing concentrations of GSH (0-15 mM) in PBS buffer (pH = 7.4) with 5% DMSO.

Cell Culture and Fluorescence Imaging Two cell lines namely HeLa and NH3T3 cells were adopted for cellular studies. The cells were grown in DMEM medium supplemented with 10% FBS serum and maintained at 37ºC and 5% CO2 incubation. The next day, cells achieved 80% confluency and were trypsinised, centrifuged and seeded at a density of 105 cells per well into a 96-well cell culture plate. Each cell lines followed the similar procedure and were cultured in the 96-well cell culture plate accordingly for the following experiments. Cells were imaged using a Nikon Eclipse TS100 inverted fluorescence microscopy.

Cytotoxicity Study It is a matter of concern to know the nontoxic effect of polymeric probe nanoparticles towards cells and MTT assay was adopted to establish the cellular viability. The assay was carried out for TP1, PTP1 and SN-38 in two cell lines namely HeLa and NIH3T3 cells against concentration of 1µM, 5 µM, 10 µM and 20 µM for 24 h and 48 h incubation. The two cell lines were treated with four different concentrations of the samples suspended in serum free medium and incubated in the respective time periods. After the allotted time, cells were treated with MTT and followed by addition of DMSO to acquire the cell count reading at 570 nm. The percentage of cell viability was measured in terms of the ratio of absorbance of the three samples relative at different concentrations to the absorbance of control which is medium alone without any samples.

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Apoptosis Study The therapeutic application of theranostic probe PTP1 was estimated by identification of apoptotic cells using Acridine orange/ethidiun bromide assay (AO/EtBr). After 48h incubation, around 50 µL of AO/EtBr solution mixture was added to HeLa cells in each well separately treated with PTP1 (15 µM), treated with biotin (2.5 µg/mL) before adding PTP1 and untreated cells as control. Equivalent amount of acridine orange and ethidium bromide (0.1mg/l mL) was combined to make the solution mixture. Followed by 5 min incubation with AO/EtBr, the cells were observed under inverted fluorescent microscope in 20X magnification

RESULTS AND DISCUSSION The compound 1, A, and mono-O-dimethoxy trityl- 2-hydroxyethyl disulfide were synthesized as per reported method16, 39 and used for the synthesis of TP1. The theranostic prodrug TP1 was synthesized in three successive steps as shown in scheme 1. In the first step, mono-O-dimethoxy trityl- 2-hydroxyethyl disulfide was reacted with 4-nitrobezylchloroformate to form a reactive intermediate which was subsequently reacted with A to afford B with a yield of 55.60 %. Dimethoxytrityl group in B was deprotected by TFA and subsequently it was reacted with 4nitrophenyl chloroformate to provide activated intermediate, which was further reacted with SN38 to afford TP1. TP1 was characterized by 1H and 13C -NMR, and HRMS. The purity of TP1 was checked by HPLC analysis. The detailed experimental procedure and analytical data were provided in supporting information (Figure S1- S8). In this study we chose SN-38, a wellknown topoisomerase I inhibitor and a highly potent anti-cancer drug that can arrest over expressed topoisomerase I in various carcinoma cells.40

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Scheme 1. Synthesis of theranostic prodrug TP1

To justify whether the biologically relevant thiolate, e.g. reduced glutathione (GSH) enables to cleave –S-S- bond to release active SN-38 and fluorophores, we have recorded UV-vis absorption and fluorescence emission changes of TP1 in the presence of variable concentrations of GSH in an artificial physiological condition. As shown in Figure 1a, the UV- absorption, centered at 510 nm gradually increased with increasing concentrations of GSH and finally UVabsorption was ~20 fold enhanced in the presence of GSH (15 mM). The fluorescence emission intensity of TP1 was also 60-fold increased upon the following exposure to GSH (15.0 mM) (Figure 1b). The increment in UV-absorption and fluorescence emission for prodrug TP1 upon exposure with GSH is due to the formation of the free fluorophore (A) (Figure S9). The UVabsorption and emission data have well supported our expectation on 11



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Figure 1. (a) UV- absorption and (b) emission spectra of TP1 (5 µM) were recorded in the presence of variable concentrations of GSH (0- 15 mM) and absence of GSH in PBS buffer containing 5% (v/v) of DMSO. All the data were acquired 30 min after addition of GSH in the buffer solution at 37 oC. Excitation was effected at 510 nm with the excitation and emission slit widths both set at 3 nm.

GSH mediated release of active chemotherapeutic SN-38 and fluorophore (A). Considering the importance of the temporal release19-27 of SN-38 from TP1, we have monitored fluorescence emission intensity changes as a function of time at λem 550 nm upon exposure to GSH. The result depicted in Figure 2 indicates that the emission intensity reached a saturation point within 30 min upon exposure to 10 mM of GSH. It concurred with a conclusion that TP1 was completely dissociated by the action of GSH (10.0 mM) and consequently released free SN-38 and fluorophore. In contrast, in the absence of GSH, such enhancement in the fluorescence intensity at 550 nm as a function of time was not observed for prodrug TP1 (Figure 2). Thus, these findings lead to a suggestion that the active components (fluorophore and SN-38) will release shortly from TP1 in the cellular milieus. 12


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Figure 2. Time dependant fluorescence changes (λem 550 nm) of TP1 upon addition of various concentrations of GSH (0- 15 mM) in PBS buffer containing 5% (v/v) of DMSO at 37 oC. Excitation was effected at 510 nm with the excitation and emission slit widths both set at 3 nm.

Further to prove the GSH mediated release of fluorophore and active chemotherapeutic SN-38, the prodrug TP1 was treated with 10 mM GSH at 37 oC for 1 h. The aliquot was subjected to HR-MS analysis which reflected two major peaks associated with SN-38 ([M+H] = 393.144) and fluorophore ([M+H] = 487.146) (Figure S10). These result revealed that by the action of GSH, the self-immolative -S-S- linker cleaved and the resulting thiol undergoes an intra-molecular cyclization at the carbamate moiety, consequently releasing SN-38 in a pharmaceutically active form and free fluorophore ( Scheme 2).

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Scheme 2. Schematic presentation of GSH induced formation of active SN-38 and fluorophore (A) from TP1. Next, we have studied the reactivity of TP1 toward GSH at the biological relevant pH range (~4.5 – 8.0). As shown in Figure S11, the fluorescence intensity gradually was increased with pH in the presence of GSH (10 mM). The result concludes that the theranostic prodrug TP1 enables to read-out extent of drug release within cellular pH range. Before applying to in vitro cellular system, we examined whether the GSH mediated cleavage of self-immolative -S-S- linker can interfere by other biologically relevant analytes. Thus, changes of optical features of TP1 were recorded in the presence of nonthiol amino acids (cystine, alanine, histidine, arginine, and 14


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tyrosine), biologically relevant metal ions (Na+, K+, Ca2+, Fe 2+, Fe3+, Cu2+, Mg2+, and Zn2+) and reactive oxygen species (Ascorbic acid, NO-, NO3-, NO2-, H2O2, and S2O32-). There were no significant spectroscopic changes were observed in the presence of other analytes (Figure 3 and Figure S12). Therefore, we can conclude that self-immolative disulfide linker in TP1 undergoes GSH mediated cleavage without significant interference from other bio-analytes.

Figure 3. Fluorescence response of TP1 (5 µM) in the presence of various analytes (a: GSH, b: cystine, c: alanine, d: histidine, e: arginine, f: tyrosine, g: Na+, h: K+, i: Ca2+, j: Cu2+, k: Mg2+, l: Fe2+, m:Fe3+, n: Zn2+, o: ascorbic acid, p: NO-, q: NO2-, r: H2O2, s: S2O32-, t: NO3-). Bars represent comparative fluorescence changes at 550 nm. Each spectrum was acquired 30 min after addition of analytes at 37 °C in PBS containing 5% (v/v) of DMSO. Excitation was effected at 510 nm.

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The stability of TP1 against blood serum is very crucial to avoid premature release of active drug as blood serum contains a substantial amount of GSH. We observed that the fluorescence intensity of TP1 increased in the presence of blood serum as shown in Figure 4, which demonstrated the decomposition of TP1. To overcome premature drug release, prodrug TP1 was encapsulated in biotinylated polyvinyl alcohol (biotinPVA) (1:5 w/w). The biotinPVA encapsulated prodrug PTP1 is considerably stable in blood serum, as the emission intensity of prodrug PTP1 has not changed significantly even after 12h (Figure 4, Figure S13). The macromolecular nature of PTP1 permits it to survive in blood serum.41 The fluorescence intensity change of PTP1 in the presence of GSH (10 mM) indicates that the reactivity of polymer formulated PTP1 toward GSH, remains alike with TP1. The size and shape of PTP1 were further established by morphological examination with HR-TEM (Figure S14a-b). It showed uniform spherical particle size of around 270 nm; whereas the size uniformity was lost due to disassembly of PTP1 in the presence of GSH (15 mM) (Figure S14c-d). Similarly, DLS analysis (Figure S14e-f) of PTP1 solution also displayed size uniformity with average hydrodynamic diameter 450 nm; whereas upon glutathione (GSH) treatment, the particle size increased to above 1000 nm. It further supported the reactivity of PTP1 towards GSH causing the disassembly of PTP1.42

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Figure 4. Fluorescence changes of TP1 (5.0 µM) and PTP1 (5.0 µM) in the presence and absence of GSH (15 mM) in PBS buffer containing 5% (v/v) of DMSO and blood serum respectively. All the data were acquired 30 min after addition of GSH in the buffer solution at 37 oC. Excitation was effected at 510 nm with the excitation and emission slit widths both set at 3 nm. Altogether these provide information with an expectation that the biotinPVA formulated PTP1 can be a powerful strategy in cancer theranostic for targeted delivery of chemotherapeutic SN38. In order to demonstrate in vitro cell- specific recognition of PTP1, we have investigated the extent of fluorescent labeling in two different cell lines with different expression levels of the biotin-receptor. We observed in Figure 5 that the receptor positive human cervical cancer HeLa cells exhibited strong fluorescence within 1h of incubation with PTP1 (5.0 µM). In contrast, a weak fluorescence signal was noticed in receptor negative NIH3T3 cells under similar condition (Figure 5). Further, time- dependent fluorescence enhancement in receptor positive HeLa cells indicated that the extent of cellular uptake, the release of drug and fluorophore increased with time in the cellular milieus (Figure S15). In contrast, such strong fluorescence signal was not 17



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observed in TP1 pretreated HeLa cells (Figure S16a). When fluorescence intensities were compared (Figure S15b) using ImageJ software (National Institutes of Health, Bethesda, MD),43 PTP1 pretreated cells showed around five- fold higher fluorescence intensity compare to TP1. This is obviously due to lack of biotin residue in TP1. Altogether, these findings suggest that biotinPVA formulated PTP1 may be an efficient theranostic prodrug to get desired therapeutic effect in the target cells.

Figure 5. Fluorescence images of HeLa and NIH3T3 cells were recorded after pre-treatment with 5.0 µM of PTP1 (TP1: biotinPVA = 1:5 w/w) in PBS buffer for 2 h. Nuclear counterstaining using Hoechst (blue). Scale bar, 20 µm. Images were obtained using excitation wavelengths of 361 nm and 450 nm, with the emission being monitored over the 460–500 and 520–560 nm spectral regions for the blue and green signals, respectively.

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Further, to confirm the receptor-mediated uptake mechanism, the receptor-positive HeLa cell was pre-treated with an excess of biotin (2.5µg/mL) for 20 min prior to incubating with PTP1. The purpose of biotin pre-treatment of HeLa cells is that biotin may block the receptors to prevent or reduce cellular uptake of PTP1. The result presented in Figure 6 indicates that overall fluorescence intensity was significantly reduced or not available in the green channel region. This result of fluorescence imaging analysis revealed that lack of sufficient biotin receptor in biotin pre-treated HeLa cells disrupted the cellular uptake of PTP1. Thus, it leads to a conclusion that cellular uptake of PTP1 only occurs via a receptor-mediated endocytic pathway.

Figure 6. Cellular fluorescence images of HeLa cells. After pretreatment with biotin (2.5 µg/mL) for 20 min., cells were treated with PTP1 (5 µM) and incubated for 3 h. Nucleus was stained using Hoechst (blue). Scale bar, 20 µm. Images were obtained using excitation wavelengths of 361 nm with the emission being monitored over the 460–500 nm.

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The therapeutic efficacy of TP1, PTP1 and SN-38 were investigated by evaluating their cytotoxicity profile in HeLa cells and normal standard fibroblast cell NIH3T3 with a different expression level of biotin receptor.26As shown in Figure 7, PTP1 exhibited dose-dependent antiproliferative activity against receptor positive HeLa cells. The IC50 value for PTP1 against HeLa cell found to be 15±0.3 µM. As biotinylated polyvinyl alcohol (biotinPVA) did not show any cytotoxicity against HeLa cells (Figure S17); thus we can conclude that the cytotoxicity of PTP1 against HeLa cells is due to the active pharmaceutical ingredient SN-38 in PTP1. In contrast, PTP1 did not show such antiproliferative activity against receptor-negative NIH3T3 cells. In previous report, the small molecular theranostic showed considerable antiproliferative against receptor-negative NIH3T3 cells under similar condition.26 This result inferred that biotinPVA in PTP1 plays an important role on cell-specific recognition and targeted delivery of SN-38. The prodrug TP1 hardly showed antiproliferative activity against HeLa and NIH3T3 cells under similar condition. In contrast, SN-38 showed similar antiproliferative activity against HeLa and NIH3T3 cells irrespective of biotin receptor overexpression (Figure 7). Moreover, SN-38 showed undesired high toxicity to the normal NIH3T3 cells. The SN-38 release from PTP1 is an endogenous stimulus-responsive (GSH) slow release process; thus the antiproliferative activity of PTP1 is lower than SN-38 in a time-frame against receptor positive HeLa cell.26, 44 The slow release of SN-38 from PTP1 may reduce a probable cellular drug efflux; hence, it may avoid unexpected adverse effect to the normal cells.45

Altogether, these findings suggest that

theranostic prodrug PTP1 could be a potential candidate for cell-specific delivery of chemotherapeutic and its tracking by a fluorescence imaging without any adverse effect on normal cells.

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Figure 7. Cell viability of (a) HeLa and (b) NIH3T3 cells pretreated with various concentrations(1, 5, 10 and 20 µM) of TP1, PTP1 (TP1:biotinPVA = 1:5 w/w)and S (SN38)in PBS buffer solution. The cells were incubated for 48 h after treatment. Cell viability was assessed using a standard MTT assay.

Finally, the toxic impact of SN-38 drug is determined by assessing the apoptosis effect of PTP1 using acridine orange/ethidium bromide assay.46 Morphological imaging of apoptotic cells was observed under the inverted fluorescent microscope where acridine orange/ethidium bromide enabled double staining procedure that distinguished non-viable cells from viable ones by preferentially staining dead cells with ethidium bromide. In Figure 8a cells treated with PTP1 resulted in more number of cells with orange fluorescence than viable cells. It confirms the ability of PTP1 to release the drug for action within the cells effectively. On the other hand in Figure 9a, cells treated with biotin prior addition of PTP1 showed more viable cells with green fluorescence promoted by acridine orange dye. Hence, the study demonstrated that biotin 21



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targeted theranostic probe adopted biotin receptors in the cells for targeted delivery of the SN-38 drug into the cells. In contrast, as presented in Figure 8b and Figure 9b, the control cells without any treatment of PTP1 show clear cellular morphology indicating that apoptosis was completely absent.

Figure 8. Fluorescence images of apoptosis in HeLa cells. Cells were treated with a) PTP1 (15 µM) for 48 h and stained with Acridine orange/Ethidium bromide solution. b) Untreated control cells. Scale bar is 20 µm. Images were obtained using excitation wavelengths of 502 nm and 518 nm, with the emission being monitored over the 520–560 and 590-630 nm spectral regions for the green and red signals respectively to avail viable and non-viable cells.

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Figure 9. Acridine orange/EtBr assay for apoptosis study in HeLa cells. a) After pretreatment with biotin (2.5 µg/mL) for 20 min., cells were treated with PTP1 (15 µM), incubated for 48 h and observed under fluorescent microscope. b) Untreated control cells. Scale bar is 20 µm. Images were obtained using excitation wavelengths of 502 nm and 518 nm, with the emission being monitored over the 520–560 and 590-630 nm spectral regions for the green and red signals, respectively to avail viable and non-viable cells.

CONCLUSIONS In conclusion, we have described the synthesis and polymer formulation of stimuli-responsive theranostic prodrug TP1 and its characterization, anticancer activity as well as ferrying the chemotherapeutic monitored by intracellular fluorescence imaging. The cellular glutathione (GSH) triggered cleavage of –S-S- bond of TP1 allows intramolecular cyclization, which leads to the release of the antineoplastic camptothecin analogue SN-38 and rhodol-fluorophore. The enhanced fluorescence intensity of TP1 enables to quantify the extent of SN-38 release. The 23



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significant stability of biotinylated-PVA polymer formulated PTP1 in blood serum indicates its efficiency to overcome unexpected premature release of chemotherapeutic SN-38. The fluorescence microscopic images revealed that formulated prodrug PTP1 was preferably uptake by biotin receptor- positive HeLa cell via a receptor-mediated endocytic pathway. Further, cell specific antiproliferative activity via an apoptotic pathway of PTP1 against HeLa cell indicates that it is a unique promising strategy for the development of endogenous stimuli-responsive cancer theranostic for targeted therapy and trafficking of the chemotherapeutic at desired target site. The superiority of the theranostic potential of PTP1 warrants the need of detailed investigation of the molecular mechanism in cell lines and animal models.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 1

H NMR,

13

C NMR, and HR-MS spectra of all intermediates and final product, TEM images,

DLS data and microscopic fluorescence image of live cells. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (S. B.) *E-mail: [email protected] (K. K. M.) Author Contributions ¶

D. D. and S. M. A. equally contributed to this work

Notes The authors declare no competing financial interest 24


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ACKNOWLEDGEMENTS SB thanked to DST-SERB, India for a research grant (ECR/2015/00035). KKM wish to thank Department of Biotechnology, Govt. of India (DBT No: BT/ PR14698/NNT/28/832/2015) and Council of Scientific and Industrial Research (CSIR), Govt. of India, network project CSC-0134, BSC-0112 for research funding.

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Graphical Abstract

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New Insight into a Cancer Theranostic Probe: Efficient Cell-Specific

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