Multifunctional near-infrared fluorescent nanoclusters for simultaneous targeted cancer imaging and photodynamic therapy

Accepted Manuscript Title: Multifunctional Near-Infrared Fluorescent Nanoclusters for Simultaneous Targeted Cancer Imaging and Photodynamic Therapy Author: Jun Ai Jing Li Lu Ga Guohong Yun Li Xu Erkang Wang PII: DOI: Reference:

S0925-4005(15)30330-0 http://dx.doi.org/doi:10.1016/j.snb.2015.09.026 SNB 19011

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

4-6-2015 1-9-2015 6-9-2015

Please cite this article as: J. Ai, J. Li, L. Ga, G. Yun, L. Xu, E. Wang, Multifunctional Near-Infrared Fluorescent Nanoclusters for Simultaneous Targeted Cancer Imaging and Photodynamic Therapy, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.09.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Manuscript Click here to view linked References

Multifunctional Near-Infrared Fluorescent Nanoclusters

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Photodynamic Therapy

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for Simultaneous Targeted Cancer Imaging and

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Jun Ai1a,c, Jing Li1c, Lu Gab, Guohong Yuna, Li Xu c, Erkang Wangc*

College of Chemistry and Environmental Science, Inner Mongolia Normal University, 81 zhaowudalu,

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,

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College of Pharmacy, Inner Mongolia Medical University, Jinchuankaifaqu, Hohhot 010110, China.

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Hohhot 010022, China.

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Chinese Academy of Sciences, Changchun 130022, Jilin, China. 1 Jun Ai and Jing Li contributed equally to this work. *Corresponding authors: Erkang Wang E-mail: [email protected]

Tel: +86-431-85262003; Fax: +86-431-85689711

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ABSTRACT

In this paper, the water-soluble DNA encapsulated silver nanoclusters with near-infrared (NIR) fluorescence were prepared, which can be used for the simultaneous targeted cancer imaging and

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enhanced photothermal therapy (PTT) or photodynamic therapy (PDT) due to the synergistic effects between the photodynamic agent and NIR silver nanoclusters. AS1411 was rationally connected with

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DNA scaffold of the silver nanoclusters and retained its secondary structure to load photosensitizer

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efficiently. The binding affinity of AS1411 to the nucleolin with high fluorescence of silver nanoclusters provided opportunities for their application in the intracellular imaging and nuclear staining. Meanwhile,

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the PPIX (Protoporphyrin IX) can also be captured by AS1411 to yield the multifunctional nanoconjugates (MF-NACs), which can be used for the cancer therapy under irradiation of light. More

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interesting, enhanced photodynamic efficiency towards the colon cancer cells was achieved based on the synergetic effect of PDT of PPIX and photothermal therapy of NIR fluorescent probe.

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Keywords: silver nanoclusters, NIR, DNA aptamer, cancer imaging, PDT

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1. INTRODUCTION Cancer is still one of the most causes of mortality in the world to date although rapid advances have been made in the diagnostic and therapeutic methods [1]. Exploration of novel approaches with accurate tumor detection and targeted therapy is extremely urgent. In recent years, silver nanoclusters have been

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considered as a desirable choice for targeted and non-invasive strategies for early diagnosis [2-4], owing to the ultrasmall size, outstanding photostability and good biocompatibility. Especially, using DNA

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encapsulated silver nanoclusters as cancer targeting probe provided a simple and efficient strategy since

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they can be easily grafted with specific recognized elements. As for the therapeutic methods, photodynamic therapy (PDT) as a useful tool offered a light-activated, noninvasive, and economical

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modality for treatment of cancers [5]. Protoporphyrin IX (PPIX) was widely used as the photodynamic agent in cancer therapy. However, the poor solubility, limited tumor or cancer cell localization of PPIX

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and the quantum yields of reactive oxygen species (ROS) [6] inspire people to explore more efficient carrier of PPIX for the targeted PDT.

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AS1411, a 26-base guanine-rich oligonucleotide aptamer, is the first nucleic acid aptamer applied for

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the treatment of tumor in humans [7]. It can be used as carrier to load the PPIX with high efficiency. On

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the other hand, a favorable feature of AS1411 is that it can bind to nucleolin (a protein over-expressed in multiple cancer cells) with high affinity and specificity [8] for the targeted PDT. While for the targeted cancer imaging, AS1411 can be easily grafted on the template of the silver nanoclusters. Based on the above consideration, a multifunctional near-infrared (NIR) fluorescent nanoplatform was designed for the simultaneous targeted cancer imaging and PDT was fabricated. As shown in Scheme 1, the DNA strand including AS1411 sequence was designed as the template to synthesize NIR-AgNCs-AS1411 for bioimaging[9], followed by conjugating the NIR-AgNCs-AS1411 with photosensitizer of PDT- PPIX to obtain the multifunctional nanoconjugates (MF-NACs). PPIX is efficiently captured onto NIR-AgNCsAS1411 via interaction between PPIX and AS1411 G-quadruplex in the presence of KCl and can be used for PDT. Meanwhile, the loaded AS1411 ligands can achieve the precise location for targeted cancer therapy via the nucleolin-mediated endocytosis. More interesting, MF-NACs exhibited the Page 3 of 17

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photothermal therapy (PTT) with enhanced phototoxicity towards cancer cell combined with the PDT efficiency of PPIX. This PPIX-mediated NIR PDT is ideal for high tissue penetration and provides a

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promising tool for cancer treatment.

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Scheme 1 Principle demonstration of MF-NACs for bioimaging and PDT in HeLa cells. AS1411 was

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linked with oligonucleotide templated AgNCs.

2. MATERIALS AND METHODS 2.1 Material:

Silver nitrate (Sigma-Aldrich, 99.9999%) and sodium borohydride (Aldrich, 98%) were used without further purification. The sequence of oligodeoxynucleotide used in the present study is 5’GGTGGTGGTGGTTGTGGTGGTGGTGGTTTCCCTAACTCCCC-3’.

The

oligonucleotide

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purified by HAP and synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). All solutions were prepared with water purified by a Milli-Q system (Millipore, Bedford, MA, USA) and stored at 4 °C.

2.2 Synthesis and Characterization MF-NACs

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The DNA were allowed to dissolve in a 20 mM potassium phosphate (PBS) buffer (pH 7.4) containing 100 mM K+. The DNA solution were then heated at 90°C for 10 min and cooled gradually to room temperature. The concentrations of oligonucleotides were quantified by using UV/Vis/near IR spectrophotometer and determined using the 260 nm UV absorbance and the corresponding extinction coefficient: A=15400, C=7400, G=11500, T=8700. NIR-AgNCs was synthesized via direct reduction of AgNO3 using NaBH4[1]. Briefly, The DNA and AgNO3 solutions with a molar ratio 1:6 were mixed and

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cooled to 0°C with ice. After 15 minutes, the solutions were reduced with NaBH4 (molar ratio of AgNO3: NaBH4=1:1) and shaken intensively to form silver nanoclusters (termed as NIR-AgNCs). It should be

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noted that NaBH4 was dissolved in deionized water and added within 30 seconds. The MF-NACs was

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obtained by mixing NIR-AgNCs with PPIX.

Transmission electron microscope (TEM) was performed with a TECNAI G2 high-resolution TEM (Philips Electronic Instruments Co., The Netherlands) operating at 200 kV. Circular dichroism spectra

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were recorded with a 1 mm optical path length-quartz cell using a JASCO J-820 spectropolarimeter (Tokyo, Japan) to characterize the G-rich quadruplexes structure change by taking the average of three

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times scans made from 210 to 340 nm. Fluorescence measurements were recorded at room temperature using a Perkin-Elmer LS 55 luminescence spectrometer. UV/Vis absorption spectra were carried out by a CARY 500 UV/Vis/near-IR spectrophotometer (Varian). The sample for cell imaging was obtained by

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incubating the cells with NIR-AgNCs on a 35 mm tissue culture dish and the fluorescence images were

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acquired using LEICA TCS SP2 laser scanning confocal microscope with a 40ｘ immersion objective. Briefly, Stock solutions of MF-NACs dissolved in PBS buffer were prepared with the concentrations of

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10 μM. Hela cells and Romas cells were employed onto 35 mm glass chamber slides. Diluted solutions in complete growth medium were then freshly prepared and placed over the cells for 2 h. All cells were washed with PBS buffer (3×) at room temperature. After that, cells imaging was scanned by LEICA TCS SP2 laser scanning confocal microscope. 2.3 MTT assay

In order to evaluate the MF-NACs dose on cellular toxicity, the complex treated cells were illuminated using serials of concentration. After treatment, cells were incubated in fresh medium for 24 h. The culture medium was replaced by 100μL fresh medium containing 0.5 mg/mL 3-[4,5dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide assays reagent, and then incubated at 37 °C and 5% CO2 for 4 h. Then, the MTT containing medium was added with 100 μL of acid/isopropanol solution, in which the concentration of HCl was 0.04 M to dissolve the MTT product, formazan. Viability of non- MF-NACs-treated control cells was arbitrarily defined as 100%. Finally, the absorption

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at 490 nm of each well was measured by EL808ultramicroplate reader. The relative cell viability was recorded. 3. RESULTS AND DISCUSSION

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3.1 Characterization of DNA Capped Silver Nanoclusters

Fig.1. Fluorescence spectra of NIR-AgNCs with the excitation at 705 nm (A) and 585 nm (B); C. TEM of NIR-AgNCs (Inset: HRTEM data); D. CD spectra of MF-NACs (red) and NIR-AgNCs (black).

Fig.1 shows the fluorescence emission spectra of the prepared NIR-AgNCs with the excitation at 585 nm and 705 nm, respectively. NIR emission at 762 nm and visible red emission at 653 nm were observed using NIR-AgNCs, close to the previous report [9], indicating the formation of fluorescent nanoclusters. The size and shape of the NIR-AgNCs were further characterized using TEM and Dynamic light scattering particle size distribution analyzer. The TEM image (Fig.1C) reveals that the Page 6 of 17

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spherical nanoparticles can be clearly distinguished due to the high electron density of the molecule, and the size of NIR-AgNCs is smaller than 2 nm (Fig.1C and Fig.S2), which is consistent with fluorescent silver nanoclusters. CD experiments were performed to confirm the secondary structure of AS1411 grafted on NIR-AgNCs. Fig.1D shows the CD spectra of MF-NACs and NIR-AgNCs in the presence of

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0.1 M K+. The positive peak at 264 nm and the negative peak at 240 nm demonstrated the formation of the parallel G-quadruplex structures. It should be noted that the incorporation of the PPIX could not

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induce the structure change (black line), which provides a basis for targeting over-expressed nucleolin

3.2 DNA Capped Silver Nanoclusters for bioimaging

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on the cancer cells using the AS1411 as recognized elements.

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Owing to its favorable spectroscopic properties, DNA capped silver nanoclusters were used for fluorescence imaging in living tumor cells. HeLa cells were stained with a 10 μM solution of NIR-

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AgNCs-AS1411 and MF-NACs in PBS buffer for 30 min at 37 ◦C and 5% CO2, respectively. Cells incubated with 10 μM solution of acridine orange (AO) was used as control. Then, laser scanning

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confocal microscope (LSCM) was used for bioimaging. Cellular internalization of NIR-AgNCs-AS1411

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was evident from fluorescence imaging with an obvious fluorescence in the intracellular area (Fig.2).

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Having confirmed the cellular uptake the HeLa cells (Fig.2.A), Romas cells (Fig.2.B) were also employed for cell imaging using NIR-AgNCs-AS1411, known for its efficiency in culture dish staining. The images were also acquired via confocal microscopy and shown in Fig. 2B. Similar behavior was obtained using these two kinds of cells. It is indicated that AS1411 was efficiently captured by the nucleolin on the cell membrane and it can be acted as a predecessor of tetrahydrofolate in the nucleus [10-12]. The Fluorescence images of Hek293 cells incubated with MF-NACs were given in the Fig S3. It can be clearly seen that the normal cell cannot be stained by the MF-NACs. These phenomena further demonstrated that the NIR-AgNCs-AS1411 could bind with nucleolin of HeLa cells, which holds great potential for diagnosis of cancer cells.

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Fig. 2 Fluorescence images of live HeLa cells (A) and Romas cells (B) incubated with NIR-AgNCs、 AO and PPIX for 30 min at 37 °C ([NIR-AgNCs-AS1411] = 10 μM、[AO] = 10 μM and [PPIX] = 10

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μM) fluorescence image of Hela cell incubated with NIR-AgNCs-AS1411、AO and PPIX on glass chamber slides. Confocal spectroscopy scanning image was obtained by collecting fluorescence of NIR-

at 37℃ and 5℅ CO2.

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AgNCs enfold by AS1411-nucleolin with excitation at 488 nm (AO) and 543 nm (NIR-AgNCs-AS1411)

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In addition, we also investigated the bioimaging performance of MF-NACs. Fig. 3 records the fluorescence images of live HeLa cells using MF-NACs with excitation at 405 nm and 543 nm in cell system. As displayed in Fig. 3, after capturing PPIX, fluorescence images with two different emissions were observed, one was from the silver nanoclusters and the other was from the loaded PPIX. These results indicated that the secondary structure of AS1411 grafted on NIR-AgNCs-AS1411 was not changed after incorporation of PPIX, which can also be employed as targeted recognized elements for bioimaging and early diagnose.

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Fig. 3 Fluorescence images of HeLa cells incubated with nanoclusters (without PPIX) (A), MF-NACs

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(B) and AO for 30 min at 37 °C ([MF-NACs] = 10 μM and [AO] = 10 μM) fluorescence image of

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HeLa cell incubated with MF-NACs and AO on glass chamber slides. Confocal spectroscopy scanning

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image was obtained by collecting fluorescence of MF-NACs enfold by AS1411-nucleolin with excitation at 488 nm , 543 nm and 405 nm at 37℃ and 5℅ CO2.

3.4 DNA Capped Silver Nanoclusters for PDT PPIX is a well-known photodynamic agent in cancer, therefore, we further studied the application in the PDT system using PPIX conjugated NIR-AgNCs-AS1411 (MF-NACs). For this purpose, HeLa cell was first incubated with MF-NACs for 30 min and then treated under irradiation with excitation source. Cell viability was assessed 24 h after PDT treatment. The cell viability of non-transfected cells was set as 100%. The PDT is expressed as a difference of the percentage cell viability between the dark toxicity and photodynamic toxicity after light irradiation. As shown in Fig. 4, the results reveal that the NIRAgNCs-AS1411 exhibited minimal anti-proliferative effects without light irradiation at the maximum Page 9 of 17

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concentration of 2 µM. Under light illumination, equivalent of the NIR-AgNCs-AS1411 without the conjugation of PPIX exhibited significant phototoxicity to cancer cell. It may be attributed to the PTT induced from the NIR-AgNCs-AS1411. However, when PPIX was captured with NIR-AgNCs-AS1411 to form MF-NACs for PDT, an enhanced photodynamic efficacy towards the colon cancer cells was

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achieved with approximately 80% mortality of cell. The enhanced PDT efficiency may be attributed to the synergetic effect of PDT of PPIX and PTT of NIR fluorescent probe. These combined results

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confirm the effectiveness of MF-NACs for simultaneous targeting cancerous cells and efficient cancer

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therapy.

Fig. 4 Cytotoxicity of the MF-NACs on HeLa cells was measured by MTT assays (n= 8, mean ± S.D.).After being treated with various concentrations of MF-NACs (0 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1μM and 2 μM, respectively) and additionally being exposed to radiation for 30 min (green and blue). Untreated cells without the light illumination were severed as the control, in which the viability was set as 100%. Page 10 of 17

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4. CONCLUSIONS In conclusion, NIR silver nanoclusters as probe for targeting cancerous cells and efficient cancer therapy were prepared through a one-step process. AS1411 was rationally connected with DNA scaffold of the silver nanoclusters and retained its secondary structure to load photosensitizer efficiently. The

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binding affinity of AS1411 to the nucleolin combined with the high fluorescence of silver nanoclusters provided opportunities for their application in the intracellular imaging and nuclear staining. Meanwhile,

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the PPIX can captured by AS1411 to yield the multifunctional nanoconjugates (MF-NACs) can be used

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for the cancer therapy under irradiation of light. More interesting, the enhanced photodynamic efficiency towards the colon cancer cells was achieved based on the synergetic effect of PDT of PPIX and PTT of

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NIR fluorescent probe. These favorable characteristics together with the facile synthesis and assembly procedure of the MF-NACs will potentially broaden the applications of silver nanoclusters in the

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biological area.

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ACKNOWLEDGEMENTS

This work was supported by the National Natural Science Foundation of China (Grant No. 21190040,

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21427811 and 11072104), the Natural Science Foundation of Inner Mongolia (Grant No. 2013MS0217), and the Program of Higher-level talents of Inner Mongolia University (Grant No. 135118). This research work was supported by the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201503).

Supporting Information Available Supplementary data associated with this article is available via the Internet at doi:

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Supplementary Material Click here to download Supplementary Material: sp.doc

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*Author Biographies

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Jun Ai

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Jun Ai obtained his B.S. and M.S. degree of Chemistry in 2000 and 2004 from Inner Mongolia Normal University and Inner Mongolia University. Then he worked in Inner Mongolia Normal University and at present he is associateprofessor. In 2009, he joined Prof. ErkangWang’s group, and received his Ph.D. degree ofanalytical chemistry in January, 2013 from Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. His researches are centered on biosensor construction and fabrication of functional nano-biomaterials.

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Prof. Erkang Wang

Erkang Wang is a professor of chemistry at Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. He is Academicians of the Chinese Academy of Sciences and the Third World Academy of Sciences. He obtained his B.S. degree from University of Shanghai in 1952 and his Ph.D. degree from Czechoslovak Academy of Sciences in 1959 under the direction of Professor J. Heyrovsky (Nobel Laureate). He published over 800 papers in international SCI journals cited over 22, 000 times by others with h-index of 76. His research interests lie in the fields of nanomaterials/nanotechnology, biosensors, electrochemistry and electrochemiluminescence.

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