Fluorescence Resonance Energy Transfer Based Highly Efficient

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FRET Based Highly Efficient Theranostic Nanoplatform for Two-Photon Bio-Imaging and Two-Photon Excited Photodynamic Therapy of MDRB Aruna Vangara, Avijit Pramanik, Ye Gao, Kaelin Gates, Salma Begum, and Paresh Chandra Ray ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00071 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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FRET Based Highly Efficient Theranostic Nanoplatform for Two-Photon BioImaging and Two-Photon Excited Photodynamic Therapy of MDRB Aruna Vangara, Avijit Pramanik, Ye Gao, Kaelin Gates, Salma Begum and Paresh Chandra Ray* Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS, USA; Email:[email protected]; Fax: +16019793674 ABSTRACT: Near-infrared (NIR) light between 700-2500 nm, which are in the range of the 1st, 2nd and 3rd biological windows have the capability to penetrate biological tissues and blood, which provides a huge advantages of higher penetration depth. However, due to the lack of available biocompatible single photon probes in NIR window, there is an urgent need for new theranostic material which could be used for two-photon bioimaging as well as for two-photon photodynamic therapy (PDT) in biological window. Driven by the need, the current manuscript reports gold nanoclusters (GNCs) attached graphene quantum dot (GQD) based two-photon excited theranostic nanoplatform with high two-photon absorption, very strong two-photon luminescence, as well as twophoton stability in NIR region. Experimental result shows strong two-photon luminescence and two-photon- induced PDT, which is based on fluorescence resonance energy transfer FRET mechanism, where graphene quantum dots with very high two-photon absorption act as two-photon donors and gold nanoclusters act as acceptors. Reported data indicate that 1O2 generation efficiency enhances tremendously due to the FRET process, which increases the two-photon excited PDT efficiency for multiple drug resistance bacteria (MDRB). Reported data indicate that the nanoplatform has the capability for bright two-photon bioimaging and two-photon photodynamic therapy for MRSA and carbapenem-resistant (CRE) Escherichia coli. Reported nanoplatform is a promising candidate to serve as a contrast agent for multiphoton imaging as well as for two-photon excited PDT agent to eliminate multidrug-resistant strains. KEYWORDS: gold nanoclusters attached graphene quantum dot based nanoprobes, two photon induced FRET, multiple drug resistance MRSA and Escherichia coli, two-photon luminescence image, two-photon photodynamic therapy for MDRB. rial for imaging as well as for image guided therapy2129 .

1. INTRODUCTION In the last two decades, multiphoton laser-scanning microscopy has opened up dramatic new capabilities for breakthrough discovery in living organisms 1-5. Since the presence of NIR window light between 7002500 nm is essential for biomedical imaging to minimize absorption by biological tissues and blood and reducing scattering, as well as auto fluorescence, there is a huge demand for the development of biocompatible probe which can be used in ‘biologically transparent NIR window’ in clinical diagnostics and interventions1-11. Recently, there is a huge effort to develop NIR probe which can be used as a theranostics agent with capability for simultaneous diagnostic via fluorescence imaging and therapeutic via converting absorbed photon energy into heat or for generation of reactive oxygen species (ROS)12-20 However, due to the lack of single photon NIR probes which are capable of bioconjugation to targeting ligands and can produce ROS for therapy, clinical research has prevented the use of NIR light based mate-

Two-photon excited theranostic material with the capability for two-photon imaging as well as for image guided two-photon excited photodynamic therapy is one of the most promising applications in clinical research11-29. The efficiency of two-photon excited theranostic material is highly dependent on the 2PA cross-section (σ2)9-29. However, due to rapid photobleaching and low two-photon absorption crosssection (50 Goeppert-Mayer (GM)) 9-29, prevent the use of FDA approved organic dyes such as Photofrin, ICG or methylene blue as a two-photon excited theranostic material in clinics 12-29. To overcome the above problem, in this manuscript we have reported gold nanoclusters (GNCs) attached graphene quantum dot (GQD) based two-photon excited theranostic nanoplatform with high two-photon absorption, very strong two-photon luminescence, as well as twophoton excited ROS generation capability in NIR region, as shown in Scheme 1.

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Scheme 1: Scheme shows FRET based two-photon excited theranostic nanoplatform using gold nanoclusters attached graphene quantum dot for two-photon imaging and two-photon induced PDT killing of MDRB. In last decade, we and other groups have shown that one can engineer the bandgap into graphene quantum dots (GQDs) and they are excellent candidates for opto-electronic applications due to quantum confinement and edge effect9,11,12,14,30-38. Due to good solubility in water and buffer, excellent photostability and biocompatibility, GQDs are demonstrated to be excellent bioimaging agents30-38. We and other groups have reported very strong two-photon absorption from graphene quantum dots with absorption cross section of ~105 GM, where −50 4 9,11,23,26,28 (1 GM = 10 cm s/photon) . On the other hand, ultra-small gold nanoclusters exhibit a range of unique quantum confinement and photophysical properties, as we and others reported before29-32. Several groups have demonstrated that photophysical proerties of gold nanoclusters are very different from that of their largersized nanoparticle counterparts or that of the bulk parent material 29-32,36,38-39. Due to small size, near-IR emission, high photostability and biocompatibility, gold nanoclusters have stimulated a lot of interest for biological applications.32,36,,38-39. Several groups have reported that the two-photon absorption cross section from gold cluster with absorption cross section is~103 GM29-32,. Recent reports indicate that gold nanoclusters have long lived triplet excited states, which help to generate ROS29-32,36,. To improve ROS formation and increase two-photon excited PDT efficiency, we have designed gold nanoclusters attached graphene quantum dot based two-photon excited theranostic nanoplatform as shown in Scheme 1A. In our design, graphene quantum dots with very high two-photon absorption act as two-photon donors and gold nanoclusters act as acceptors.

Scheme 2: A) Scheme shows the synthetic pathway we have used to develop acid chloride functionalized grapheme quantum dots (GQDs.) B) Scheme shows the synthetic pathway we have used to develop hexamethylenediamine functionalized grapheme quantum dots from acid chloride functionalized graphene quantum dots. C) Scheme shows the synthetic pathway we have used to develop dihydro α-Lipoic acid conjugated gold nanoclusters (GNCs). D) Scheme shows the synthetic pathway we have used to develop GNCs conjugated GQDs.

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Due to the fluorescence resonance energy transfer (FRET) process, the two-photon luminescence intensity from gold cluster improved significantly, which allows better bright two-photon imaging. It also enhances the ROS formation. As a result, we have observed much higher two-photon excited 1O2 formation than when we use gold nanocluster or GQD alone. According to the World Health Organization (WHO), multiple drug resistance bacteria (MDRB) could be responsible for 10 million deaths per year due to the lack of availability of new antibiotics 40. Methicillinresistant Staphylococcus aureus (MRSA) and Enterobacteriaceae including carbapenem-resistant (CRE) Escherichia coli are the priority 1 critical class bacterial pathogens for our society41-50. As a result, developing alternative approach for killing these MDRB without antibiotics is very urgent for our society41-50. In this current manuscript, we have demonstrated that two-photon excited theranostic platform designed by us can be used for targeted two-photon imaging and two-photon excited PDT killing of 100% of MDRB. Reported data demonstrate that 100% of Escherichia coli and MRSA can be killed using our two-photon excited theranostic platform at NIR light excitation.

Figure 1A shows the TEM image of freshly prepared dihydro α-Lipoic acid stabilized GNCs, which indicates that Lipoic acid attached GNCs are about 4±2 nm in size. We have also performed dynamic light scattering (DLS) which indicates that the average size of dihydro α-Lipoic acid stabilized GNC is about 5±2 nm. Inserted HRTEM image, as reported in Figure 1A shows the crystallinity of dihydro α-Lipoic acid stabilized GNCs, which is due to the face-centered cubic (fcc) of Au. XRD spectra, as reported in Figure 1G, shows index of diffractions from the (111), (200), (220) facets of fcc Au. Figure 1I shows the red emitted fluorescence from dihydro α-Lipoic acid stabilized GNCs in the presence of UV light. Figure 2A shows the emission spectra from dihydro α-Lipoic acid stabilized GNCs at 380 nm excitation, which shows that the λmax for emission is around 680 nm. Using quinine sulfate as a standard, we have determined the photoluminescence quantum yield for dihydro α-Lipoic acid stabilized GNCs. Experimental details have been reported before36. The photoluminescence quantum yield for dihydro α-Lipoic acid stabilized GNCs was determined to be 10.2%. 2.1.2. Synthesis and Characterization of hexamethylenediamine functionalized grapheme quantum dots (GQDs)

2. RRSULTS & DISCUSSIONS 2.1. Development of GNC conjugated GQDs based theranostic platform 2.1.1. Synthesis and Characterization of α-Lipoic acid stabilized gold nanoclusters (GNC) For the development of the GNC conjugated GQDs based thranostic platform, at first we have synthesized α-Lipoic acid stabilized gold nanoclusters using our and other reported methods 32.36. As shown in Scheme 2C, we have synthesized gold nanoclusters (GNCs) using α-Lipoic acid, HAuCl4, 3H20 and NaBH4. Synthetic details have been discussed in experimental section, which is reported in supporting information. At the end, synthesized GNCs were purified using the centriplus centrifugal filter device (regenerated cellulose 3,000 MWCO) at 7500 rpm for 45 mins. After that, the purified particles were characterized by Xray powder diffraction (XRD), high-resolution tunneling electron microscopy (HR-TEM), energydispersive X-ray (EDX) spectroscopy, Time-resolved fluorescence spectroscopy (TRFS) and dynamic light scattering (DLS).

In the next step, we have synthesized hexamethylenediamine functionalized graphene quantum dots (GQDs) as shown in Scheme 2A. For this purpose, at first, we have synthesized graphene oxide (GO) from natural graphite powder using the modified Hummers method, as we and others have reported before 91.11.12.14,34-35 . Experimental details have been reported in supporting information. Next, we have performed hydrothermal reaction for producing graphene quantum dots from GO. Experimental details have been reported in the supporting information. For this purpose, 0.15g of synthesized graphene oxide (GO) was dispersed in 30 ml anhydrous DMF and was ultrasonicated for an hour. This mixture was transferred into a stainless steel autoclave with a teflon liner and heated at 200oC for 6 hours. After that, the product GQD was dialyzed by snake skin dialysis tubing (MWCO=3500D) against water at room temperature. The pure graphene quantum dots (GQDs) were obtained successfully by column chromatography on a silica gel using THF/water (1:2 v/v) as an eluent.



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of C and O. Inserted HRTEM image shows the crystal lattice fringe for hexamethylenediamine functionalized graphene quantum dots (GQDs). D) TEM image shows the morphology of hexamethylenediamine functionalized GQDs attached αlipoic acid stabilized GNCs, which indicates that GNC conjugated GQD sizes are 10±3 nm. Inserted EDX data clearly show the presence of Au, C and O. Inserted HRTEM image shows the crystal lattice fringe for hexamethylenediamine functionalized GQD attached α-lipoic acid stabilized GNCs. E1-3) DLS data show the histogram of the size distribution of the freshly prepared nanodots, E1) αlipoic acid stabilized GNCs, E2) hexamethylenediamine functionalized GQDs and E3) hexamethylenediamine functionalized GQDs attached αlipoic acid stabilized GNCs, which indicates that the size distribution is around 10 ± 4 nm. F) FTIR spectra from hexamethylenediamine functionalized GQD attached α-lipoic acid stabilized GNC shows characteristic amide –O-H, -S-H, amide-I, amide-II , C=O and –C-S peaks. G) XRD spectra from hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNCs shows diffractions peaks due to the (111), (200), (220), facets of Au and a broad diffraction peak due to the graphite (002) planes. H) Raman spectrum from hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNC shows the presence of D and G bands due to the GQD. Higher D bands indicate the high defect due to the presence of GNCs. I) Fluorescence in the presence and absence of UV light for hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs and hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNCs.

Figure 1 A) TEM image shows the morphology of dihydro α-lipoic acid stabilized gold nanoclusters (GNCs), which indicate GNCs sizes are 4±2 nm. Inserted EDX data clearly show the presence of Au. Inserted HRTEM image shows the crystal lattice fringe for dihydro α-Lipoic acid stabilized gold nanoclusters. B) Absorption spectra for freshly prepared α-Lipoic acid stabilized GNCs. C) TEM image shows the morphology of hexamethylenediamine functionalized grapheme quantum dots (GQDs), which indicate GQDs sizes are 2±1 nm. Inserted EDX data clearly show the presence

The solid GQDs were collected after evaporation using a rotary evaporator. As shown in Scheme 2B, after that we have synthesized hexamethylenediamine functionalized GQDs from graphene quantum dots (GQDs). Experimental details have been reported in supporting information. For this purpose, 20 mg of purified GQD was dissolved in 5 ml of dry THF in 50 ml round bottom flask. Then 5 ml oxalyl chloride was added to it and refluxed at 80 oC for 24 hours. After completion of the reaction, the obtained acid chloride derivative of GQDs were centrifuged using the centriplus centrifugal filter device (regenerated cellulose 10,000 MWCO) at 1000 rpm for 20 minutes to remove the excess reactants. Next, 25 mg acid chloride derivative of GQDs were dispersed in 10 ml dry THF



and 500 µl of hexamethylene diamine was added and the reaction mixture was refluxed at 80oC for 48 hours. The final product was obtained through centrifugation followed by washing with dry THF for several times to remove excess of un-reacted reagents. Then it was stored at 4 oC for future use. Figure 1C shows the TEM image of our hexamethylenediamine functionalized GQDs which indicates that the average size is about 2±1 nm. We have also performed DLS measurement which indicates that the average size of our hexamethylenediamine functionalized GQDs is about 2±1 nm. Inserted HRTEM image, as reported in Figure 1C shows the crystallinity of hexamethylenediamine functionalized GQDs, with lattice spacing around 0.31 nm. Lattice spacing for hexamethylenediamine functionalized GQDs was found to be very close to the 0.32 nm interlayer spacing of bulk graphite. XRD spectra, as reported in Figure 1G, shows broad diffraction peak which can be due to the graphite (002) planes. Figure 1I shows the bluish-green emitted fluorescence from hexamethylenediamine functionalized GQDs in the presence of UV light. Figure 2A shows the emission spectra from hexamethylenediamine functionalized GQDs at 380 nm excitation, which shows that the λmax for emission is around 480 nm. Using quinine sulfate as a standard, we have determined the photoluminescence quantum yield for hexamethylenediamine functionalized GQDs. The photoluminescence quantum yield for hexamethylenediamine functionalized GQDs was determined to be 21.3%. 2.1.3. Synthesis and Characterization of hexamethylenediamine functionalized grapheme quantum dots (GQDs) attached GNCs At the end, we have synthesized hexamethylenediamine functionalized GQD attached GNCs, as reported in Scheme 2D. For this purpose, 5 ml of water dispersed GNC was mixed with 5 ml of hexamethylenediamine functionalized GQDs in 50 ml beaker at room temperature. Then 400 µl of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC, 1mg/ml) and 100 µl Nhydroxysulfosuccinimide (NHS, 1mg/ml) were added to the reaction mixture with constant stirring for 24 hours. Then the product was dialyzed by pur-A-Lyzer mega 1000 dialysis kit (MWCO = 1000D) for 4 hours to remove the excess reactants and were stored at 4oC. The pure GNCs conjugated hexamethylenediamine functionalized GQD was characterized by X-ray powder diffraction (XRD), high-resolution tunneling

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electron microscopy (HR-TEM), energy-dispersive Xray (EDX) spectroscopy, Time-resolved fluorescence spectroscopy (TRFS), Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy and dynamic light scattering (DLS). Figure 1D shows the TEM image of our hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNCs which indicates that the average size is about 10±4 nm. We have also performed DLS measurement, as reported in Figure 1E, which indicates that the average size for our hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNCs is about 10±4 nm. Inserted HRTEM image, as reported in Figure 1D shows the crystallinity of hexamethylenediamine functionalized GQDs attached α-lipoic acid stabilized GNCs. As reported in Figure 1G, XRD spectra from GNCs conjugated hexamethylenediamine functionalized GQD show diffractions peaks due to the (111), (200), (220), facets of Au and a broad diffraction peak due to the graphite (002) planes. As shown in Figure 1F, FTIR spectra from hexamethylenediamine functionalized GQDs attached GNCs shows characteristic amide –O-H, -S-H, amide-I, amide-II, C=O and –C-S peaks. Similarly, as reported in Figure 1H, the Raman spectrum from hexamethylenediamine functionalized GQDs attached GNCs shows the presence of D and G bands due to GQD. Higher D bands indicate high defect due to the presence of GNCs. Figure 1I shows the strong red emitted fluorescence from hexamethylenediamine functionalized GQDs attached GNCs in the presence of UV light. Figure 2A shows the emission spectrum from hexamethylenediamine functionalized GQDs attached GNCs at 380 nm excitation, which shows two completely separated emission peaks. The first peak is around 470 nm which is due to the hexamethylenediamine functionalized GQDs and the second peak is around 680 nm, which is due to the α-lipoic acid stabilized GNCs. It is very interesting to note from Figure 2A that due to the higher quantum yield, the photoluminescence intensity is much higher for hexamethylenediamine functionalized GQDs than that of for α-lipoic acid stabilized GNCs. On the other hand, in case of hexamethylenediamine functionalized GQDs attached GNCs, the photoluminescence peak for GQDs quenched very highly, where as photoluminescence peak for GNCs enhanced abruptly, which is due to the fluorescence resonance energy transfer (FRET). In this FRET platform, GCD has been used as the energy acceptor because it possesses good absorbance in the emission wavelength of the donor (GQD). Also due to


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the high quantum yield as well as strong photoluminescence from GQD, GQD has been used as a donor. The FRET between GQD donors and GCD acceptors results the quenching of the GQD fluorescence in the presence of GCD. Also FRET process will decrease the GQD donor fluorescence lifetime. In our study, the FRET process was confirmed through measuring the GQD donor fluorescence intensity in the presence of different concentration of the acceptor GCD. As shown in Figure 2F, our experimental data clearly shows that GQD donor fluorescence intensity decreased abruptly, as we have increased the concentration of GCD acceptor. On the other hand, as reported in Figure 2G, GCD acceptor fluorescence intensity increases abruptly, as we have increased the concentration of GQD donor. To find the efficiency of FRET process, we have used time-resolved fluorescence measurements, as shown in Figure 2E. To measure the fluorescent lifetime for GQD and GQD-GCD, we have used Horiba Jobin Yvon fluorolog instrument attached with Fluorohub single photon counting. From the theoretical fitting data, have determined the average life time for GQD donor is 2.8 ns and the average life time for GQD-GCD complex is 1.2 ns. The FRET efficiency (E) was calculated using equation 1 as described below, E= (τD - τD-A)/τD

(1)

where τD and τD-A are the lifetimes of the donor and donor-acceptor complex respectively. From the experimental lifetime values, FRET efficiency between GQD and GCD was determined to be 57 %. 2.2. Determining the two-photon absorption properties and two-photon excitation FRET for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform Since near-infrared (NIR) light between 700-2500 nm in the range of the first, second and third biological windows have the capability to provide enormous advantages of deeper penetration depth 1-10, we have explored the two-photon absorption capability for our hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs and for hexamethylenediamine functionalized GQDs attached GNCs separately. Experimental details for two-photon absorption experiment have been reported before6.7,9,11. In brief, for the measurement of two-photon emission from hexamethylenediamine functionalized GQDs , α-lipoic acid stabilized GNCs and for hexamethylenediamine

functionalized GQDs attached GNCs, we have used an 80 MHz Ti-sapphire laser as an excitation source with 100 fs pulse width and 80 MHz repetition rate6.7,9,11. We have used optical parametric amplifier to generate 860 nm excitation wavelength, as we have described before6.7,9,11. Figures 2B shows the two photon photoluminescence spectra from hexamethylenediamine functionalized GQDs and hexamethylenediamine functionalized GQDs attached GNCs separately, when we have used 860 nm excitation wavelength for two-photon experiment. Similarly, Figures 2C shows the two-photon photo-luminescence spectra from αlipoic acid stabilized GNCs and hexamethylenediamine functionalized GQDs attached GNCs separately, where we have used 860 nm excitation wavelength for the two-photon experiment. Figure 2D shows the excitation wavelength power dependent plot for hexamethylenediamine functionalized GQD attached GNCs at 720 nm emission. Experimental data, as reported in Figure 2D, clearly show that the photoluminescence intensity from hexamethylenediamine functionalized GQDs attached GNCs at 720 nm is proportional to the square of the 860 nm excitation light intensity. Observed linear plots clearly confirm that the observed photoluminescence at 720 nm is indeed a two-photon process. For the measurement of two-photon luminescence quantum yield, we have used fluorescein as the reference, whose quantum yield is known to be as 0.9. Using fluorescein as the reference, the two-photon luminescence quantum yield for our hexamethylenediamine functionalized GQDs was determined to be 0.38. Similarly, using fluorescein as the reference, the two-photon luminescence quantum yield for our α-lipoic acid stabilized GNC was determined to be 0.08. Also, using fluorescein as the reference, the two-photon luminescence quantum yield for our hexamethylenediamine functionalized GQDs attached GNCs was determined to be 0.52. As we know, two-photon luminescence intensity from hexamethylenediamine functionalized GQDs or αlipoic acid stabilized GNCs is highly dependent on the two-photon absorption cross-section (σ2p) and two photon luminescence quantum yield of hexamethylenediamine functionalized GQDs or α-Lipoic acid stabilized GNCs respectively.



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Figure 2: A) Single-photon photo-luminescence spectra from hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs and for hexamethylenediamine functionalized GQDs attached GNCs separately. We have used 380 nm light for the single photon experiment. B) Twophoton photo-luminescence spectra from hexamethylenediamine functionalized GQDs and hexamethylenediamine functionalized GQDs attached GNCs separately. We have used 860 nm light. C) Two-photon photo-luminescence spectra from αlipoic acid stabilized GNCs and hexamethylenediamine functionalized GQDs attached GNCs separately. We have used 860 nm light. D) Plot shows how the two-photon luminescence intensity from


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hexamethylenediamine functionalized GQDs attached GNCs at 720 nm varies with the square of intensity of 860 nm incident light. The observed linear plot for hexamethylenediamine functionalized GQDs attached GNCs indicates that photoluminescence is indeed a two-photon process. E) Life-time decay of photoluminescence from GQDs and GQD-GCDs. The average photoluminescence lifetime decreases from ∼2.8 ns for GQD to ∼1.2 ns for GQD-GCD. F) Plot shows how the luminescence intensity from hexamethylenediamine functionalized GQDs varies in the presence of different concentration of α-lipoic acid stabilized GCDs. G) Plot shows how the luminescence intensity from α-lipoic acid stabilized GCDs varies in the presence of different concentration of hexamethylenediamine functionalized GQDs. As a result, we have used experimental two-photon luminescence spectra from hexamethylenediamine functionalized GQDs or α-lipoic acid stabilized GNCs to determine the two-photon absorption crosssection for each separately. Details of the calculation have been reported before by our group and other groups6.9,11. Using fluorescein as a reference, we have determined the two-photon absorption cross-sections for hexamethylenediamine functionalized GQDs to be 5.6 x 104 Goeppert-Mayer (GM) units, which is only 11 GM for fluorescein at 860 nm excitation. Similarly, using fluorescein as a reference, we have determined the two-photon absorption cross-sections for αlipoic acid stabilized GNCs to be 1.6 x103 GM at 860 nm excitation. It is very interesting to note from Figures 2B and 2C that in case of hexamethylenediamine functionalized GQDs attached GNCs, the two-photon photoluminescence peak for GQDs quenched very highly, whereas two-photon photoluminescence peak for GNCs enhanced abruptly, which is due to twophoton excited fluorescence resonance energy transfer (FRET). In our two-photon excited FRET platform, graphene quantum dots with very high two-photon absorption, as well as two photon quantum yield, GQD acts as two-photon donors. On the other hand, gold nanoclusters act as acceptors. Due to the fluorescence resonance energy transfer (FRET) process, the two-photon luminescence intensity from gold cluster improved significantly, which will allow better bright two-photon imaging.

ylenediamine functionalized GQDs attached GNCs based nanoplatform As we have discussed before, two-photon NIR light theranostic material is the next generation targeted bio-imaging and therapy material. We have explored whether hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform can be used for two-photon NIR light excited imaging as well as therapy probe. Since methicillin-resistant Staphylococcus aureus (MRSA) and Enterobacteriaceae, including carbapenem-resistant (CRE) Escherichia coli are the priority 1 critical class bacterial pathogens for our society, we have explored the possible use of antiMRSA antibodies or anti-E.coli antibodies attached hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform for live drug resistance bacteria imaging as well as for PDT killing. For this purpose, at first hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform were modified with acid-terminated polyethylene glycol (PEG) for high stability in physiological solutions using our reported method. After that, anti-MRSA antibodies or anti-E. coli antibodies were attached with nanoplatform via PEG for targeted imaging. Anti-MRSA antibodies or anti-E. coli antibodies were attached to the PEG coated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform via EDC/NHS chemistry, as we and others have reported before6.9,11,21,32. As it is well documented that photostability and biocompatibilities are the most important parameter for two-n excited theranostic material, in the next step we have found out about photostability and biocompatibilities for antibody conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. For this purpose, we have performed continuous femtosecond laser illumination experiment on antibodyconjugated nanoplatform using 860 nm light for 90 minutes. As reported in Figure 3A, the two-photon luminescence intensity from antibody-conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform at 530 and 720 nm remained unchanged till 90 minutes of illuminations. Our experimental data show very good photostability for antibody-conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform as a two-photon excited theranostic material.

2.3. Finding two-photon excited singlet oxygen generation capability, photo-stability and biocompatibility for antibody-conjugated for hexameth-



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Figure 3: A) Plot shows how the two-photon luminescence intensity for hexamethylenediamine functionalized GQDs attached GNCs varies with time. We have measured two-photon luminescence intensity at 530 and 720 nm separately. Reported data at both wavelengths indicate very good photo-stability for anti-MRSA antibodies connected hexamethylenediamine functionalized GQDs attached GNCs. B) Plot shows the biocompatibility for hexamethylenediamine functionalized GQDs attached GNCs platform against MRSA drug resistance bacteria and human skin HaCaT keratinocytes cells. Reported data indicate that hexamethylenediamine functionalized GQDs attached GNCs platform are not toxic even after 24 hours incubation. C) Two-photon excited 1O2 emissions at ~1270 nm induced by the hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs, anti-MRSA antibodies connected hexamethylenediamine functionalized GQDs attached GNCs platform and MDRB conjugated nanoplatform under excitation with a 860nm laser. D) Two-photon excited singlet oxygen generation capability by the hexamethylenediamine functionalized GQDs, α-Lipoic acid stabilized GNCs, anti-MRSA antibodies connected hexamethylenediamine functionalized GQDs attached GNCs platform, MDRB conjugated nanoplatform and rose Bengal (RB), measured using Na2-ADPA as the 1O2-trapping agent. E) Plot shows how twophoton excited singlet oxygen generation capability for hexamethylenediamine functionalized GQDs vary with the addition of different concentration ofα α-Lipoic acid stabilized GNCs. Next, we have performed biocompatibility experiments for antibody-conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. For the above experiment, 6.2 × 105


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cells/mL HaCaT normal cells and 6.2 × 105 cells/mL MRSA were incubated with antibody-conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform for different time intervals separately. After 24 hours of incubation, the cell viability for HaCaT normal cells was measured using MTT colorimetric test. Experimental details haves been reported in the supporting information. Similarly, we have used colony counter to find the amount of live MRSA bacteria. Experimental details haves been reported in the supporting information44,47-50. Figure 3B shows the experimentally observed colony counting for MRSA and MTT test for HaCaT normal cells, which clearly indicates that even after 24 h of incubation more than 98% cell viability was observed. The above reported data show very good biocompatibility for antibody-conjugated for hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform developed by us. Next to understand the two-photon excited PDT capability of antibody-conjugated hexamethylenediamine functionalized GQDs attached GNC based nanoplatform, we have measured the 1O2 emission signals from nanoplatform at 860 nm excitation using a NIR detector. Figure 3C shows the comparative 1 O2 emission signals from hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs, hexamethylenediamine functionalized GQD attached GNC based nanoplatform and MDRB attached nanoplatform. Reported data in Figure 3C clearly shows that 1O2 formation capability for hexamethylenediamine functionalized GQD attached GNC based nanoplatform is much higher than that of hexamethylenediamine functionalized GQDs or α-lipoic acid stabilized GNCs. Experimental reported data also indicate that 1O2 formation capability for MDRB attached nanoplatform is slightly lower than only nanoplatform, but it is much higher than that of hexamethylenediamine functionalized GQDs or α-lipoic acid stabilized GNCs. To understand better, we have also performed the 1O2 quantum yield measurement using a chemical trapping method via disodium 9,10anthracendipropionic acid. In this case, we have used Na2-ADPA as the 1O2-trapping agent. We have used the absorption of Na2-ADPA to find the 1O2 formation at various irradiation times. Figure 3D clearly shows that 1O2 formation capability for hexamethylenediamine functionalized GQD attached GNC based nanoplatform is much higher than that of hexamethylenediamine functionalized GQDs or α-lipoic acid stabilized GNCs. Figure 3D also indicates that

1

O2 formation capability for MDRB attached nanoplatform is slightly lower than only nanoplatform, but it is much higher than that of hexamethylenediamine functionalized GQDs or α-lipoic acid stabilized GNCs. Next to determine the two-photon excited singlet oxygen generation efficiency, we have used Rose Bengal (RB) as the standard photosensitizer , whose 1 O2 quantum yield is known to be ΦRB=0.75 in water 17-24. For this purpose, we have used Na2-ADPA as the 1O2-trapping agent. As shown in Figure 3D, 1 O2 formation capability for hexamethylenediamine functionalized GQD attached GNC based nanoplatform is much higher than that of RB. Using RB as standard, we have determined 1O2 quantum yield for α-lipoic acid stabilized GNCs as .15 and .44 for hexamethylenediamine functionalized GQDs. Similarly, 1 O2 quantum yield is determined to be .97 for hexamethylenediamine functionalized GQD attached GNC based nanoplatform and .88 for MDRB conjugated nanoplatform. To confirm that the enhancement of 1O2 generation efficiency was resulted from the FRET process, we have measured the 1O2 formation capability for hexamethylenediamine functionalized GQD donor in the presence of different concentration of α-lipoic acid stabilized GNC acceptor, as reported in Figure 3E. Reported experimental data clearly shows that 1 O2 formation capability enhances abruptly, as we have increased the concentration of GNC acceptor. As shown in Figure 2F, our experimental data clearly shows that FRET process enhances abruptly, as we have increased the concentration of GCD acceptor. Those two reported data clearly shows that the enhancement of 1O2 generation efficiency is mainly due to the FRET process. As we have discussed before, due to the two-photon excited FRET process between GQD and GNC, ROS formation has been enhanced significantly. As a result, we have observed much higher two-photon excited 1O2 formation than when we used gold nanocluster or GQD alone. To determine whether antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform could be used for imaging and PDT killing of different MDRBs, 103 CFU/mL of each of carbapenem-resistant (CRE) Escherichia coli and MRSA were mixed with antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform separately.



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Figure 4: A-B) Two-photon luminescence image of MRSA attached nanoplatform at different depths. To mimic the 3D biological environment, for this experiment we have embedded MRSA in a collagen matrix. A) MRSA were observed from 75 µm depth and B) MRSA were observed from 50 µm depth. C) TEM image of image of antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound methicillin-resistant Staphylococcus aureus (MRSA). D) TEM image of image of antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound carbapenemresistant (CRE) Escherichia coli before PDT E) TEM image of antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound carbapenem-resistant (CRE) Escherichia coli after PDT. It shows damage on the bacterial surface morphology and surrounding biological substrates via PDT. 2.4. Use of antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform for two-photon excited MDRB imaging and PDT killing After that the mixture was gently shaken for over 40 minutes. In the next step, antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound MDRBs were separated from unbound antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform, by centrifugation. Finally, we have used TEM and two-photon luminescence mapping analysis for the characterization of antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound MDRBs as shown in Fig-


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ure 4. Since two-photon excitation has the advantage of deeper penetration of biological tissue, we have performed two-photon excitation images of MRSA at different depth as shown in Figures 4A and 4B. To mimic the 3D biological environment, we have embedded MRSA in a collagen matrix. Figure 4A shows the TPL image where MRSA were observed from 75 µm depth. Similarly Figure 4B shows the TPL image where MRSA were observed from 50 µm depth. Reported results in Figure 4A and 4B indicate that antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform can be used TPL image and clear localization of MRSA. TEM images, as reported in Figures 4C and 4D, indicate that that antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform can bind with different MDRBs via antigen-antibody interaction. As a result, we are able to use bright two-photon imaging of MDRB using antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. Next, to find out whether our antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform can be used for two-photon excited PDT killing of MDRB, we performed NIR irradiation experiments using 860 nm excitation light. For this purpose, antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound methicillinresistant Staphylococcus aureus (MRSA) and antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform bound carbapenem-resistant (CRE) Escherichia coli were exposed to two-photon excited 860 nm NIR light at different time intervals. After that, we have used colony counting technique to find the bacteria viability as reported in Figure 5A. We have also used molecular Probes’ LIVE/DEAD® BacLightTM Bacterial Viability Kits (from Fisher Scientific) to determine the amount of MDRBs dead during two-photon excited PDT as reported in Figures 5C-5E. This kit is well known to provide a two-color fluorescence assay, which utilizes mixtures of our SYTO® 9 greenfluorescent nucleic acid stain which can bind with the live bacteria and the red-fluorescent nucleic acid stain, propidium iodide, which is known to bind with dead bacteria. Since when red and green combine, the result is yellow, we have also observed yellow fluorescence due to the overlap of fluorescence from live and dead bacteria, as reported in Figure 5D.



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ence of 860 nm NIR light without antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform. D) 3 minutes of exposure to 860 nm NIR light with antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform. E) 10 minutes of exposure to 60 nm NIR light with antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. F) Comparison of the carbapenem-resistant (CRE) Escherichia coli killing efficiency using 860 nm NIR light without nanoplatform, NIR light with only GNCs, NIR light with only GQDs & NIR light with hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. All the reported data is for 10 minutes of exposure to 860 nm NIR light. G) Comparison of the MRSA killing efficiency using 860 nm NIR light without nanoplatform; NIR light with only GNCs; NIR light with only GQDs & NIR light with hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. All the reported data is for 10 minutes of exposure to 860 nm NIR light. H) Viability of carbapenem-resistant (CRE) Escherichia coli bacteria during two-photon excited PDT, at different time interval.

Figure 5: A, B) MRSA killing efficiency measured using colony counting technique; A) In the presence of 860 nm NIR light without antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform; B) using 860 nm NIR light with antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. C, D & E) Viability of carbapenem-resistant (CRE) Escherichia coli bacteria before and after two-photon excited PDT. Live/Dead kit was used to stain bacteria. Green fluorescence indicates live-bacteria and red fluorescence indicates dead bacteria. C) In the pres-

So in the microscopy image, as reported in Figures 5C-5E, green fluorescence indicates live-bacteria and red fluorescence indicates dead bacteria. Figure 5H shows the viability of carbapenem-resistant Escherichia coli bacteria during two-photon excited PDT, at different time interval, which clearly indicate that 100% of carbapenem-resistant (CRE) Escherichia coli can be killed by just using 10 minutes of 860 nm NIR light exposure. The observed 100% killing efficiency for different types of MDRB is mainly due to two-photon excited PDT killing mechanism in the presence of NIR light. To understand better, we have also performed experiments to find the MDRB killing efficiency using antibody conjugated hexamethylenediamine functionalized GQD and antibody conjugated dihydro α-lipoic acid stabilized GNC. After that, we have compared the result with antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform. As shown in Figures 5F and 5G, around 15% of MRSA and carbapenemresistant (CRE) Escherichia coli MDRBs were killed in the presence of 860 nm NIR light after 10 minutes of exposure and when antibody conjugated dihydro αlipoic acid stabilized GNCs are present. On the other hand, around 50% of MRSA and carbapenem-


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resistant (CRE) Escherichia coli MDRBs were killed in the presence of 860 nm NIR light after 10 minutes of exposure and when antibody conjugated hexamethylenediamine functionalized GQDs are present. Whereas, around 100% of MRSA and carbapenemresistant (CRE) Escherichia coli MDRBs were killed in the presence of 860 nm NIR light after 10 minutes exposure and when antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatforms are present. Experimentally observed trend of two-photon excited MDRB killing matches very well with the experimental observation of two-photon excited 1O2 emissions at ~1270 nm induced by the hexamethylenediamine functionalized GQDs, α-lipoic acid stabilized GNCs and anti-MRSA antibodies connected hexamethylenediamine functionalized GQD attached GNC platform under excitation with a 860-nm laser as reported in Figure 3C. We have also performed high-resolution TEM experiment for MDRB after 10 minutes of exposure with 860 nm NIR light in the presence of antibody conjugated hexamethylenediamine functionalized GQDs attached GNCs based nanoplatform. As shown in Figure 4E, HRTEM data clearly show that antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform has the capability to damage the bacterial surface morphology and surrounding biological substrates via PDT.

luminescence intensity from gold cluster improved significantly, which allows better bright two-photon imaging. Reported experimental data indicate that 1O2 generation efficiency enhances tremendously due to the FRET process, which increases the two-photon excited PDT efficiency for antibody conjugated theranostic nanoplatforms. Reported results indicate that around 15% MDRBs were killed in the presence of 860 nm NIR light after 10 minutes of exposure and when antibody conjugated dihydro α-lipoic acid stabilized GNCs are present. On the other hand, around 50% MDRBs were killed in the same condition when antibody conjugated hexamethylenediamine functionalized GQDs are present. Whereas, 100% of MDRBs were killed in same condition when antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatforms are present. After proper engineering, the reported two-photon excited theranostic nanoplatform has a good potential for benefitting the multiphoton diagnosis and two-photon excited PDT killing of MDRB in the clinical setting. 4. SUPPORTING INFORMATION AVAILABLE Detailed synthesis and characterization of theranostic material and other experiments are available as a supporting information. This information is available free of charge via the internet at http://pubs.acs.org/.

5. ACKNOWLEDGEMENT

3. CONCLUSIONS In conclusion, in this article, we have reported a new class of two-photon excited theranostic material using antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform, for multiphoton bioimaging and two-photon excited PDT killing of 100% MDRB, using 860 nm NIR light. We have demonstrated that our two-photon excited theranostic nanoplatform has the capability for bright two-photon bioimaging and two-photon photodynamic therapy for drug resistance bacteria like MRSA and carbapenem-resistant (CRE) Escherichia coli. Rported experimental data show strong two-photon luminescence and two-photon induced PDT from antibody conjugated hexamethylenediamine functionalized GQD attached GNC based nanoplatform is based on fluorescence resonance energy transfer (FRET) mechanism. In the reported design, graphene quantum dots with very high two-photon absorption (5.6 x 104 Goeppert-Mayer (GM) unit), as well as two -photon quantum yield (0.38), act as two-photon donors and gold nanoclusters act as acceptors. Experimental data show that due to the FRET process, the two-photon

Dr. Ray thanks NSF-PREM grant # DMR-1205194 for material development and NIH RCMI grant (#G12RR013459-13) for bio-imaging funding.

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7. Graphical Abstract


Fluorescence Resonance Energy Transfer Based Highly Efficient

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