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Acridine Orange Conjugated Chitosan Reduced Gold Nanoparticles: A Multifunctional Probe for Combined Photodynamic and Photothermal Therapy Kalpana Hari and Kumpati Premkumar Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu, India ABSTRACT Plasmonic metal nanoparticles can be used in photomedicine owing to their unique photophysical properties like surface plasmon resonance, high optical extinction, and extensive thermal stability, which makes the metal nanoparticles more suitable for the application of cancer phototherapy. Here, we exploited the appropriateness of chitosan reduced Gold nanoparticles (AuNPs) as a photothermal converter, photodynamic carrier and contrast agent to exhibit bioimaging, photothermal (PTT) and photodynamic therapy (PDT) concurrently to destroy the malignant cells. Also, synthesized AuNPs tested for cellular uptake and phototoxicity potential in breast cancer cells. AuNPs were successfully conjugated by non-covalent interaction with a hydrophilic photosensitizer, acridine orange (AO) through Glutathione (GSH) to achieve an improved PDT and PTT therapy. Results of this study demonstrated that AO tethered AuNPs (AO@GSHAuNPs) conjugate exhibited high loading efficiency and excellent photothermal stability with enhanced cellular uptake. The anticancer activity of AO@GSH-AuNPs in combination with blue light irradiation (492 nm) was studied in in vitro using human MCF-7 breast cancer cells. Furthermore, the enhanced antitumor effect of AO@GSH-AuNPs with irradiation may be due to both the cytotoxic photodynamic effect of AO coupled nanoconjugate and photothermal ablation intervened by AuNPs. From the results we conclude that AO@GSH-AuNPs in the combined PDT and PTT based approach, could be employed as a multifunctional single nanocarrier for enhanced anticancer therapy.

Nanoparticles

AuNPs

GSH-AuNPs

AO@GSH-AuNPs

Hydrodynamic diameter (d.nm)

24.09 ± 0.349

71.78 ± 0.99

85.06 ± 1.79

PDI

0.254 ± 0.01

0.27 ± 0.01

0.271 ± 0.041

Zeta Potential (mV)

50.77 ± 2.15

46.4 ± 0.78

33.3 ± 1.27

OBJECTIVE Synthesis, characterization and evaluation of multifunctional properties of chitosan reduced gold nanoparticles for combined photodynamic and photothermal therapy against cancer (b)

BACKGROUND Ø Nanomaterials with multifunctional property have become an essential tool for enhancing the efficacy of chemotherapeutics in recent years. Ø Among the various metal nanoparticles, gold nanoparticles have distinctive attributes due to their unique physico-chemical and custom designing properties like SPR, strong photothermal conductivity, high optical extinction and enhanced permeation retention. Ø Furthermore AuNPs can be readily functionalized with multiple targeting molecules and also shown potential application in various biomedical approaches including targeted therapy, photodynamic and photothermal therapy to effectively eradicate the cancer cells

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Fig. 4. In vitro cytotoxicity studies of MCF-7 cells treated with different concentrations of free AO & AO@GSH-AuNPs with and without exposure (3 min) of blue light of 60 W cm-2 power density at 490 nm followed by 24 h incubation. All experiments were repeated thrice.

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Fig. 5. (a) Fluorescent changes showing time-dependent cellular uptake of MCF-7 cells treated with fixed concentration (1.35 µM) of free and conjugated AO at different time intervals. (b) Comparison of fluorescence enhancement between free and conjugated AO at fixed concentration. (inset) Emission spectra of naked AuNPs. (c) Absorption spectra of AO@GSH-AuNPs upon addition of CT-DNA in the absence (top line) and presence of CT DNA upon increasing the concentrations (100-500 µl of DNA -6 addition). [AO@GSH-AuNPs] = 36.5µM; [DNA] = (0-18x10 M). The arrow showing the change in absorption upon increasing DNA concentration that shows the hypochromicity with CT DNA. (Insert) plots of [DNA] / (Ɛa –Ɛf) vs. [DNA] for the titration of DNA with AO@GSH-AuNPs.

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Fig. 1. (a) UV-visible spectrum of naked AuNPs (λ max 520 nm). (b) SEM images of AuNPs. (c) HR-TEM image of AuNPs (d) Size distribution of AuNPs and the mean particle size was found to be 14±3.69 d.nm. (e) Diffraction (SAED) pattern of AuNPs, (f) AFM image of AuNPs, (g) EDX spectrum of AuNPs confirms the presence of Au at (2.02eV). (h) Zeta potential of naked AuNPs.

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CONCLUSION In this study, we examined the appropriateness of chitosan reduced AuNPs as an effective photosensitizer carrier system as well as photothermal agent by investigating their controlled synthesis, photostability and consistency of drug loading, serves a multifunctional nanoparticle system for enhanced PDT and PTT mediated anticancer therapy. (b)

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Fig. 6. Comparison effect of DNA damage at different concentration of free AO and -2 AO@GSH-AuNPs exposed by blue light irradiation using MCF-7 cells at (13W cm ) power density. (b) Photodestruction of MCF-7 cells shown by both bright (20X) and fluorescence field (40X) with free and conjugated nanoparticles at the fixed concentration (1.35 µM) irradiated by 490 nm at (13 W/cm2) power density for 3 min. (c) Variations of NBT reduction among 5 different concentrations under blue light irradiation.

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Fig. 3. (a) Temperature measurements for free and conjugated AuNPs at fixed concentration (36.35 µM) Fig. 2. (a) Extinction Spectra of naked and conjugated AuNPs. (b) FT-IR spectra of free AO (b) Photostability of free AO and AO@GSH-AuNPs at 490 and AO@GSH-AuNPs. (c) Emission overlapping spectra for AO and AO@GSH-AuNPs. nm of blue light emission. (d) Effect of GSH-AuNPs on emission spectra of free AO.

REFERENCES 1. Jeongmin Oh, Hwanjun Yoon and Ji-Ho Park, (2013) Biomed Eng Lett 3:67-73 2. Byamnajav Lkhagvadulam et al, (2013), BioMed Research International, http://dx.doi.org/10.1155/2013/720579 3. J. Wang et al, (2012), J. Porphyrins Phthalocyanines 16, 802.

Contact Details : Dr. K. Premkumar, Mobile No : +91-8056589893, Email ID: prembdu@yahoo.com, kalpanahari.bio@gmail.com


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