A superior bright NIR luminescent nanoparticle preparation and indicating calcium signaling detection in cells and small animals

A superior bright NIR luminescent nanoparticle preparation and indicating calcium signaling... Background: Near-field fluorescence (NFF) effects were employed to develop a novel near-infrared (NIR) lumines- cent nanoparticle (LNP) with superior brightness. The LNP is used as imaging contrast agent for cellular and small animal imaging and furthermore suggested to use for detecting voltage-sensitive calcium in living cells and animals with high sensitivity. Results: NIR Indocyanine green (ICG) dye was conjugated with human serum albumin (HSA) followed by covalently binding to gold nanorod (AuNR). The AuNR displayed dual plasmons from transverse and longitudinal axis, and the longitudinal plasmon was localized at the NIR region which could efficiently couple with the excitation and emission of ICG dye leading to a largely enhanced NFF. The enhancement factor was measured to be about 16-fold using both ensemble and single nanoparticle spectral methods. As an imaging contrast agent, the ICG–HSA-Au complex (abbre- viate as ICG-Au) was conjugated on HeLa cells and fluorescence cell images were recorded on a time-resolved confo - cal microscope. The emission signals of ICG-Au complexes were distinctly resolved as the individual spots that were observed over the cellular backgrounds due to their strong brightness as well as shortened lifetime. The LNPs were also tested to have a low cytotoxicity. The ICG-Au complexes were injected below the skin surface of mouse showing emission spots 5-fold brighter than those from the same amount of free ICG–HSA conjugates. Conclusions: Based on the observations in this research, the excitation and emission of NIR ICG dyes were found to be able to sufficiently couple with the longitudinal plasmon of AuNRs leading to a largely enhanced NFF. Using the LNP with super-brightness as a contrast agent, the ICG-Au complex could be resolved from the background in the cell and small animal imaging. The novel NIR LNP has also a great potential for detection of voltage-gated calcium concentration in the cell and living animal with a high sensitivity. Keywords: Gold nanorod (AUNR), Indocyanine green (ICG), Dual-mode plasmons, Near-field fluorescence (NFF), Luminescent nanoparticle (LNP), Fluorescence imaging, Imaging contrast agent Background cells, voltage-gated calcium channels are coupled with Calcium is a well-known signaling ion in most eukary- the membrane depolarization due to the calcium influx, otes [1, 2]. A calcium concentration gradient across a which can significantly alter the cellular physiology [ 3, plasma membrane and intracellular organelle can fluxes 4]. Hence, it is of importance to understand the calcium dynamically via orchestrated channel openings, and fur- concentration gradient and fluctuation in the cells. This thermore generate tightly controlled spatial and tempo- study might also highlight the crucial role of calcium sin- ral patterns. In electrically excitable neurons and muscle gle at a cellular level as well as in living animals. Currently, the voltage-gated calcium channels in the cells, tissues and mediums are often measured by a fluo - *Correspondence: jianzhang@vigenebio.com rescence imaging [5, 6]. Typically, a fluorophore is used as Present Address: Vigene Biosciences Inc., 9430 Key W. Ave Suite 105, a calcium indicator to chelate with a calcium ion creating Rockville, MD 20850, USA Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 2 of 12 a fluorescence signal. With a change of concentration or range most of current NFF effects were tested using the 2+ environment of Ca ions in the cell, the fluorescence visible fluorophores, and only few using NIR fluoro - signal from the indicator is altered. This method can be phores [29–33]. Different from the spherical metal nano - also used for exploring the intracellular calcium concen- particles, the shaped metal nanoparticles, such as metal tration and gradient of calcium ion at the cellular level as nanoshells or nanorods, can display their surface plas- well as in the living animals [7, 8]. Actually, the monitor- mons at longer wavelength [34, 35]. For instance, the gold ing voltage-gated imaging calcium has become an impor- nanorods (AuNRs) can display their split dual plasmons tant topic in the calcium channel detection because the from the short (transverse) and long axis (longitudinal), calcium signals exert their highly specific functions in the respectively [36–38], and importantly, the longitudinal well-defined cells or/and small animals. plasmon can be tuned to the NIR region by adjusting the In the past decades, novel calcium indicators have been aspect ratio of AuNRs. Thus, the longitudinal plasmons synthesized as the organic compounds [9, 10]. Most of from the AuNRs are expected to be able to sufficiently these calcium indicators have their emission wavelengths couple with the excitation/emission of NIR fluorophores at visible region. It is known that the fluorescence signals leading to a strong NFF-induced fluorescence at the NIR at the visible region have severe interference from the region. Meanwhile, the NIR AuNRs remain have reason- strong backgrounds from cellular autofluorescence and able sizes. light scattering in the biological systems [11–17]. To sup- We are interested in developing novel NIR LNPs with press the interference, a near-infrared (NIR) fluorophore high brightness and furthermore using these LNPs as is suggested for using as imaging contrast agents [18–20]. imaging contrast agents, for determining the calcium Tissue and water have a window with a low background ions in the cells and living small animals. In this study, allowing a penetration of excitation light deeper into the NFF effect was employed to prepare the novel NIR the tissue and allow the detection of emissions from the LNPs. Indocyanine green (ICG) is a FDA-proved non- fluorophores with a better resolution with the cells and toxic NIR fluorophore for patient safety in ophthal - tissues. mology [39, 40], and also known as a voltage-sensitive However, as imaging contrast agents, the NIR fluo - fluorophore that can be used to determine the voltage- rophores have their two significant drawbacks: (1) low gated calcium channels by adding chelators on its chemi- absorption coefficients which may result in their low cal structure [41]. In this study, the ICG dye was bound brightness and (2) low photostability that results in their to AuNRs within a near-field distance to explore the NFF short bleaching time [21, 22]. To our knowledge, there at the NIR region. is still lack of an efficient contrast agent that allows the Briefly, the ICG dyes were first conjugated in human detection of calcium ions in the cells and small animals serum albumin (HSA) followed by covalently binding at the single molecule level. Hence, there is an essential the conjugates on the surfaces of AuNRs [42, 43]. Since need for a new approach that can greatly improve the flu - the HSA molecules have an average size of ca. 10  nm, orescence properties of NIR fluorophores particularly on the ICG dyes conjugated to the HSA molecules are dis- their brightness and photostability. tributed within a near-field distance from the surfaces of Near-field fluorescence (NFF) can improve the fluo - AuNRs. In addition, the excitation/emission of ICG dyes rescence properties of fluorophores [23]. Fundamentally, can sufficiently couple with the longitudinal plasmons of a metal nanoparticle can create a local electromagnetic AuNRs, and thus, a NFF from the bound ICG dyes on the field nearby as a light irradiation and the electromagnetic AuNRs was expected to occur. The ensemble and single field is confined into the metal plasmons [24–26]. When nanoparticle spectra were used to evaluate the change a fluorophore is localized within the near-field range of optical properties of ICG dyes prior to and after their from the metal nanoparticle surface, the excitation/emis- binding on the AuNRs. Using as a nanoparticle contrast sion of fluorophore can strongly couple with the light- agent, the ICG–HSA-Au (abbreviated as ICG-Au) com- induced plasmons on the metal nanoparticles [27, 28], plex was bound to HeLa cells and the fluorescence cell and the excitation or/and emission rates of fluorophore images were collected for evaluating the fluorescence can be significantly increased. As a result, the fluores - spectral properties at the single nanoparticle level. The cence properties of fluorophore can be greatly improved ICG-Au complex was also injected into the mouse for including (1) largely enhanced emission intensity and live animal fluorescence tomography. Comparing with quantum yield (2) extended photobleaching time and (3) the free ICG–HSA conjugates, the ICG-Au complex dis- reduced photoblinking of fluorophore [28]. played significantly improved properties for the live ani - Since the spherical metal nanoparticle with a reason- mal tomography uses [44–50]. able size displays its single-mode plasmons at the visible Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 3 of 12 Results ICG–HSA conjugates In this research, the NIR luminescent nanoparticle was developed using a strong NFF effect by binding the NIR ICG dyes on the surfaces of AuNRs within a near-field dis - tance. Thus, the ICG dyes were first conjugated with the HSA molecules to form the ICG–HSA conjugates, and the conjugates were then covalently bound onto the surfaces of AuNRs. In the experiments, the ICG and HSA were codissolved in an aqueous solution with a molar ratio of ICG/HSA = 4/1. After the reaction, the free ICG dyes were removed from solution by a dialysis against water. The fluorescence properties of ICG dyes before and after the conjugation were measured using ensemble spectroscopy. Upon excitation at 760 nm, the ICG–HSA conjugate was observed to exhibit an emission band cen- tered at 819, 7 nm shifting to shorter in comparison with the free ICG dyes in aqueous solution (Fig. 1a). The emis - sion band also became broader with the ICG conjugation, which may be due to the plasmons or the short emission wavelength of ICG. Covalently binding ICG–HSA conjugates on AuNRs The biological properties of nanoparticles, such as cell uptake and circulation time, are known to strongly rely on their surface properties [51, 52]. In this study, AuNRs were prepared with the protection of cetyltrimethylam- monium bromide (CTAB) monolayers on the surfaces. To improve their bioactivity, the CTAB-monolayers on Fig. 1 a Absorption spectra of AuNRs as CTAB-coated, PEG-coated, the AuNRs were replaced with the thiolate polyethylene and ICG–HSA conjugate-bound in a 10 mM PBS buffer solution. b glycol (PEG) monolayers via a surface substitution reac- Ensemble emission spectra from the ICG dyes as free, conjugates in tion on the nanoparticle. The free small molecules were HSA, and complexes with AuNRs in a 10 mM PBS buffer solution removed by a dialysis against water. Most CTAB mol- ecules on the AuNR surfaces were supposed to replace by the PEG molecules. The change of monolayers on the The ICG–HSA conjugates were covalently bound AuNR surfaces could be reflected by the solubility change on the AuNRs via the surface condensation of pri- of AuNRs in aqueous solution prior to and after the reac- mary amino moieties in the ICG–HSA conjugates tion. In addition, since these PEG molecules were bound with the carboxyl moieties on AuNRs in the presence on the AuNR surfaces via sulfur-metal bonds, much of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide stronger than the CTAB molecules via electrostatic inter- hydrochloride (EDC) as the condensation agent. The actions, the PEG-AuNRs should become more chemi- ICG–HSA conjugates were dissolved in excess amount cally stable in solution [51, 52]. in solution to avoid aggregation of nanoparticles To bind the ICG–HSA conjugates on the AuNRs, the through the crosslinking. The final AuNR product was PEG monolayers on the AuNRs were partially substi- recovered by a centrifugation and then purified by a tuted by thiolate carboxyl-ligand of N-(2-mercapto- dialysis against water. propinyl)glycine ligands to create the reactive sites on the AuNR surfaces via surface exchange reaction [53, 54]. Experimentally, the thiolate carboxyl-ligand was dis- Evaluation of ICG‑Au complex by microscope solved in solution with a molar ratio of carboxyl ligand/ and ensemble spectroscopy AuNR = 100/1. After the substitution reaction, the Tomography of AuNRs through the surface reactions unsubstituted ligands were removed by a dialysis against was evaluated using a transmission electron micro- water. scope (TEM). Representative images of AuNRs are Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 4 of 12 shown in Fig.  2a, b prior to and after the surface reac- time-resolved confocal microscope. Upon excitation with tions on the AuNRs. These AuNRs were observed to a 640  nm laser, both the emission intensities and life- have an average width of 10  nm and an average length times from the ICG-Au complexes (as shown in Fig.  3a) of 40  nm and the aspect ratio was calculated to be ca. were collected at single nanoparticle level [54]. As con- 4.0. There was no significantly change on the tomog - trol, the free ICG–HSA conjugates were also diluted in raphy with the three-step surface reactions on AuNRs, solution and cast on the coverslip. The emission signals reflecting that the surface reactions on the AuNRs only were collected with the same conditions on the confocal altered the monolayer composition on their surfaces microscope but with an excitation power of laser 10-fold but not on their metal cores. stronger. The collected emission spots from the free con - The plasmon absorption of metal nanoparticle is jugates were much dim as shown in Fig.  3b demonstrat- known to be sensitive to the composition of monolay- ing lower emission intensities of free conjugates. For each ers on the surface [30, 31]. In this study, the absorp- sample, at least 50 emission spots were collected, and the tion spectrum was used to measure the replacement of histogram of the intensities and lifetimes were obtained ligands on the AuNR surfaces. The AuNRs displayed a by fitting with a Gaussian distribution curve (Fig.  4a for dual plasmons from the short (transverse) and long axis the intensity and b for the lifetime), and the maximum (longitudinal) at 504 and 802 nm, respectively (Fig. 1a). values of curves were obtained to represent the emission Following the monolayer reactions on the surfaces of intensity and lifetime of sample, respectively. nanoparticles, the two plasmons bands were found to The near-field interaction of an excited fluorophore remain, but the maxima were slightly shifted to longer with a metal nanoparticle may increase the radiative rate at 511 and 807 nm (Fig. 1a), respectively. of fluorophore, and as a result, the lifetime of fluorophore Ensemble fluorescence spectra were also sensitive can be decreased [25]. Hence, the lifetime can be used as to the binding of ICG dyes on the metal nanoparticle an important parameter to evaluate the near-field inter - surfaces. It was shown that the emission band of ICG- action. Herein, the decays of excited ICG-Au complexes Au complexes was centered at 814  nm (Fig.  1b), 5  nm as well as ICG–HSA conjugates were recorded using the shifting to shorter in comparison with the free ICG– confocal microscope following by fitting with a Gauss - HSA conjugates. Fluorescence spectral shifts have been ian distribution curve (Fig.  4b). The maximum values of attractive to the wavelength dependent on the interac- lifetime of ICG dyes were obtained, showing a significant tion of metal nanoparticle and fluorophore [55– 57]. decrease of lifetime from 2.3  ns for the unbound ICG– HSA conjugates to 0.4 ns for the ICG-Au complexes. Evaluation of ICG‑Au complex by single nanoparticle spectroscopy In addition to the ensemble spectrum, NFF effect on Fluorescence cellular imaging the ICG-Au complexes could be evaluated using sin- To test the fluorescence properties of ICG-Au complexes gle nanoparticle spectral measurement. To prepare the for the cell imaging, the ICG-Au complexes were used as test samples, the ICG-Au complex was diluted to nM in an imaging contrast agent to conjugate with HeLa cells. aqueous solution and then cast a drop on a glass cover- Briefly, HeLa cells were cultured on coverslips followed slip followed by drying in air. With a low concentration by fixing using 4% paraformaldehyde. The cell-fixed in solution before drying, the ICG-Au complexes were coverslip were incubated with the ICG-Au complex for mostly existed as isolated particles on the coverslip. The 30  min and then completely washed with PBS buffer. single nanoparticle measurements were performed on a Fluorescence cell images were collected on the time- resolved confocal microscope in both the intensity and lifetime. A representative image was presented in Fig. 3c. It was shown that the ICG-Au complexes were presented as individual spots on the cells distinctly observable from the cellular backgrounds either due to their strong inten- sity and differentiated lifetime. As control, the ICG–HSA conjugates were also conju- gated with HeLa cells, and the cell images were recorded on the confocal microscopy with the same conditions (Fig.  3d). Comparing with the images of blank cells, the overall cell images became brighter, indicating that the ICG–HSA conjugates were indeed conjugated on the Fig. 2 TEM images of (a) CTAB-AuNRs and (b) ICG-Au complexes cells. But the emission signals from the single ICG–HSA Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 5 of 12 Fig. 3 Upper panels represent the emission imaging from (a) ICG-Au complexes and (b) ICG–HSA conjugates. Diagrams are 5 × 5 µm and resolutions are 100 × 100 pixels with an integration of 0.6 ms/pixel. Bottom panels represent fluorescence images from the cells conjugated with (c) ICG-Au complexes and (d) ICG–HSA conjugates. Diagrams are 50 × 50 µm and resolutions are 100 × 100 pixel with an integration of 0.6 ms/pixel. The samples were excited with a 640 nm laser. Note the different intensity scales. The images of a and c were collected with a laser power 10-fold less than the images of b and d conjugates could not be well resolved as individual spots nanoparticles (Fig.  5c) and as 13 in the presence of Au from the cellular backgrounds of cell images, which was nanoparticles (Fig.  5d), showing that the rates of viable due to their low brightness as well as a lifetime close to cells are 93.1 and 93.7%, respectively. The results in the the cellular background. presence of 0.3 and 3  nM as well as the control were listed in Fig. 6e reflecting that the presence of Au nano - Cytotoxicity measurements particles in the cell medium had only a slight influence Cytotoxicity of free conjugate and ICG-Au complex to the cells survival. It also demonstrates that the Au were tested on live HeLa cell using calcein AM assay. nanoparticles have very low cytotoxicity. The cell images at different time intervals were col - lected on the time-resolved confocal microscope as Fluorescence small animal imaging shown in Fig.  5. An area with a large number of cells Six 5–6  weeks nude mice were selected to test the opti- was selected for statistical analysis for the cell sur- cal properties of novel LNP by small animal fluores - vival. The live cells could be identified as stained with cence imaging. The mice were randomly divided into calcein AM (green cell viability stain) as shown in the two groups and each group had three mice. The mice image A when there was in the absence of nanoparticle were first euthanized under deep anesthesia following (294 cells) and in the image B when there was in the by injecting the ICG-Au complex below the skin sur- presence of 3 nM Au nanoparticles (207 cells) after the face of mouse [16, 17]. As control, the ICG–HSA con- treatment time of 24  h. The number of cell with high jugate solution containing the same amount of ICG dye autofluorescence was counted as 20 in the absence of was also injected in the same mouse at a different site. Fluorescence small animal imaging was performed on a Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 6 of 12 on the AuNRs [42, 43]. To achieve the ICG–HSA conju- gates with the maximal brightness, the molar ratio of ICG 0.30 ICG-HSA Conjugates over HSA in the conjugation was controlled to be 4/1 in the reaction. Too many ICG dyes on one HSA molecule ICG-Au Complexes 0.25 would result in self-quenching among the fluorophores and too few dyes in one HSA molecule would result in a 0.20 low brightness. 0.15 To improve the bioactivity of nanoparticles, the CTAB monolayers on the AuNRs were replaced by the 0.10 PEG monolayers via surface substitution reaction [51, 52]. Most of the CTAB molecules on the AuNRs were 0.05 believed to replace by the PEG molecules, and supported by a change on the solubility of AuNRs in aqueous solu- 0.00 tion prior to and after the replacement. Prior to the 0 100 200300 400 replacement, the AuNRs were found to have a very good b dispersion in water, whereas after the replacement, the Emission Intensity (counts) AuNRs were readily stuck on the wall of glass tube, which 0.25 ICG-Au Complexes was due to increased hydrophobicity of nanoparticle sur- ICG-HSA Conjugates faces by the PEG monolayers. In addition, with stronger 0.20 sulfur-metal covalent bonds of PEG with the AuNRs, the modified AuNRs were supposed to have an improved 0.15 chemical stability in solution [51, 52]. To covalently bind the ICG–HSA conjugates on the 0.10 AuNRs, the PEG monolayers on the AuNRs were par- tially substituted by N-(2-mercapto-propinyl)glycine to 0.05 create reactive sites on the nanoparticle surfaces. The ICG–HSA conjugates were then covalently bound on the AuNRs via a condensation reaction [53]. The binding of 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ICG–HSA conjugates on the AuNRs could be supported Lifetime (ns) by a change of absorption and fluorescence spectra prior Fig. 4 Histogram distributions of single sots of (a) emission to and after reactions as described early. The binding intensities and (b) lifetimes from the ICG–HSA conjugates as free and number of ICG–HSA on each AuNR could be measured bound on the AuNRs using a NaCN treatment method [53]. Typically, several drops of 0.1  N NaCN aqueous solution were added into 0.5  nM ICG-Au complex solution. It was observed that Xenogen IVIS-200 system and the representative images the plasmon color of solution disappeared progressively were shown in Fig. 6. An image from an untreated mouse with the time, showing that the metal nanoparticles were was also presented as control. The emission spots from dissolved by NaCN. As a result, the ICG–HSA conju- the injection sites by the ICG-Au complex and ICG–HSA gates were released from the nanoparticles as free into conjugate were observed to be significantly different: the the solution. The whole process could be monitored by spot by the ICG-Au complex was ca. 5-fold brighter than the ensemble fluorescence spectrum expressing a dra - the spot by the ICG–HSA conjugate. Since the two injec- matic decrease of emission intensity (Fig. 7) until satura- tion sites on the same mice were known to contain the tion. The ICG–HSA conjugates were released as free in same amounts of ICG dye, the difference on their bright - the solution completely lost the NFF effect leading to a ness should be due to their different emission intensities. dramatic decrease on the emission intensity [31]. Using In the other words, an enhanced fluorescence of NFF the saturated emission intensity, the concentration of from the ICG-Au complex results in increased brightness ICG–HSA conjugates in the solution was measured to of ICG dyes in the small animal imaging. −9 be 3 × 10   M. Since the amount of ICG–HSA was not significantly changed in solution prior to and after the Discussion NaCN treatment, according to a ratio of emission inten- In this study, a NIR LNP was designed and prepared on sity prior to the treatment over that after the treatment, the basis of NFF effect. The ICG dyes were conjugated the enhancement factor for the ICG dye on AuNR was with the HSA molecules followed by covalently binding calculated to be 16.3. Histogram Distribution (%) Histogram Distribution (%) Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 7 of 12 Fig. 5 The panel of cell images of live HeLa cells stained with calcein AM without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles. The images of calcein stained cells (a) and (b) were acquired after 24 h of nanoparticle treatments upon excitation with a 443 laser diode and at bandpass filter 514/30 nm. The images of c and d represent the autofluorescence of cells without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles after 24 h. The autofluorescence images of cells were collected upon an excitation at 640 nm and with a longpass filter of 655 nm. Cells with brighter autofluorescence in c and d are classified as dead. e represents rates of viable cells in the presence of 0.3 and 3 nM in the cell medium as well as in the absence of Au nanoparticle as the control at time interval = 0.5, 2, 12, 24 h To evaluate the NFF effect of NIR dyes on the AuNRs, microscope. The ICG-Au complexes were found to have 20 and 50  nm gold nanospheres were prepared follow- an intensity 10.5-fold higher than the free ICG–HSA con- ing by covalently binding with the ICG–HSA conju- jugates. Since the emission of ICG-Au complexes were gates via the same strategy. Using the NaCN treatment, collected upon excitation with a laser power of 10-fold the enhancement factor of ICG dyes on the 50  nm gold lower than those of ICG–HSA conjugates, the ICG-Au nanospheres was measured to be 2.3, much lower than complexes were calculated to be 105-fold brighter than that on the AuNRs, although a 50  nm gold nanosphere the ICG–HSA conjugates. Considering that one AuNR is almost 20-fold larger on the volume than a AuNR. The was averagely bound with 6 ICG–HSA conjugates, the ICG dyes on a 20  nm gold nanosphere, which has an enhancement factor per ICG molecule was estimated to approximately identical volume to a AuNR, resulted in an be 16.7, very close to the enhancement factor achieved insignificant NFF effect. The nanospheres did not display on the ensemble spectra. This enhancement factor is a plasmon band at the NIR region, and as a result, could also comparable with the value from some visible fluo - not sufficiently couple with the excitation and emission rophores on the metal nanospheres [41], representing a of ICG dyes. In contrast, the AuNRs displayed a NIR lon- sufficient NFF interaction of NIR fluorophores with the gitudinal plasmon band leading to their sufficiently cou - AuNRs. pling with the excitation and emission of ICG dyes, and Besides the emission intensity, the near-field effect of thus, resulted in a strong NFF effect. This result indicates a fluorophore with a metal nanoparticle may result in a that the longitudinal plasmon band from a shaped metal largely reduced lifetime [25]. In this study, the lifetimes nanoparticle is very important for its sufficient coupling were collected at single nanoparticle level on a confo- with a NIR fluorophore and brings up a strong NFF in the cal microscope, and the histogram of lifetimes was fitted NIR region. with a Gaussian distribution. A maximum was obtained The ICG-Au complexes were also evaluated at sin - at 0.4  ns (Fig.  4b), much shorter than the lifetime of gle nanoparticle level on a time-resolved confocal unbound ICG–HSA conjugates at 2.3  ns, supporting an Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 8 of 12 of novel NIR nanoparticle fluorophores from the cellular backgrounds on the time-resolved images [44, 45]. Using as an imaging contrast agent, the ICG-Au com- plexes were conjugated with HeLa cells for fluorescence cell imaging. Fluorescence cell images were recorded on the confocal microscope with both the intensity and life- time. It was shown that the emissions signals from the ICG-Au complexes were distinctly isolated as individual spots from the cellular backgrounds (Fig.  3c). The inten - sity ratio of signal/noise on the image was estimated to be approx. 63, much higher than the value from the most organic fluorophores or LNPs, which was due to high brightness of ICG-AuNRs [44, 45]. In addition, because of largely shortened lifetime, the emissions of ICG- AuNRs could be better resolved from the cellular back- grounds on the lifetime cell images. Fig. 6 In-vivo fluorescence tomography images of mice using the As control, the HeLa cells were also incubated with ICG-Au complexes as contrast agent injected below the skin of mice. the free ICG–HSA conjugates, and the cell images were The images were collected on a Xenogen IVIS-200 small animal recorded under the same conditions (Fig.  3d). It was tomography system with a bandpass filter from 665 to 695 nm for shown that the overall cell images became brighter than the background, a filter from 710 to 760 on the excitation side, and the images of blank cells without the treatment support- a filter from 810 to 875 nm on the emission side. A 750 nm laser was used as the excitation source. Total photon flux (photons/s) was ing that the ICG–HSA conjugates had been conjugated calculated and corrected for tissue depth by spectral imaging using with the cells. But the emissions from the ICG–HSA con- Living Image 3.0 software (Xenogen). The left image (a) was collected jugates could not be resolved as individuals from the cel- on a control mice and the right image (b) was collected by injection lular backgrounds, which was due to their low brightness with the ICG-Au complex, ICG–HSA conjugate, or a blank PBS buffer as well as close lifetime relative to the cellular autofluo - solution with the same volume on the same mice rescence at the backgrounds. In this study, the LNPs were not functionalized with the specific bioactive molecules. Thus the ICG-Au complexes were supposed to randomly distribute through the cells (Fig.  3c). On the other hand, because of bulky sizes of ICG-Au complexes and relatively short incubation time, these LNPs were observed to mostly attach on the cell surfaces, which could be the result of short incubation time with the cells. Our other experiments (not shown herein) also demonstrated that the metal nanoparticles of this size can penetrate through the cell membrane and enter the cells. We will use these LNPs as indicators to detect the calcium channels and concentration gradients by the fluorescence cell imaging. Once the nanoparti - cles are functionalized with the bioactive molecules and then enter the cells, they will become possible to label the target molecules with a higher efficiency because of the Fig. 7 Emission spectral change of ICG-Au complex in 10 mM PBS presence of multiple functional groups on their surfaces. buffer solution before and after a NaCN treatment Cytotoxicity of ICG-Au complexes was tested on live HeLa cell using a calcein AM assay showing that the luminescent nanoparticles have only slight or even insig- efficient near-field coupling of ICG molecules with the nificant cytotoxicity to the live HeLa cell. It is known that AuNRs. It was interesting to notice that the lifetime of the cytotoxicity of metal nanoparticles strongly relies on ICG-Au complex was beyond the range of autofluores - the coating layers on the metal cores. For a relatively low cence (2–5  ns) in the lifetime fluorescence cell images, cytotoxicity of metal nanoparticles in this study, it can which would beneficiate to isolate the emission signals be described by two possible factors. First, polyethylene glycol layers were coated on the Au cores via covalent Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 9 of 12 bonds. These covalent bonds are much stronger than the insight the target calcium ions and their activities with a statistic interactions that the nanoparticles are generally better resolution and a larger depth of tissue layer. bound by leading to the current ICG-Au complexes are Because of instrumentation limitations, the small ani- more chemically stable in the cell medium or animal bod- mal image could not be recorded in time-resolved model ies. Second, the ICG dyes have low toxicity. Hence, the on the current imaging system. But we expect that with ICG-Au complexes can affect insignificantly or slightly largely enhanced fluorescence and unique lifetime, the the cells viability as observed in this study. ICG-Au complex can provide us an opportunity to To test the imaging function, the ICG-Au complex observe the target molecules and their activities with a was injected to the skin surface of mice for the fluores - better resolution and a larger depth of tissue layer in the cence small animal imaging [16, 17]. It was shown that time-resolved small animal imaging. the emission spot from the injection site by the ICG- Au complex was ca. 5-fold brighter than the site by the Conclusions ICG–HSA conjugate. Since the two injection sites con- Due to a longitudinal plasmon at the NIR region, AuNRs tained the same amounts of ICG dye, the difference of were demonstrated to sufficiently couple with the exci - the brightness of two spots over the mice image should tation/emission of NIR fluorophores leading to largely be due to the different brightness between the ICG-Au enhanced NFF effect. NFF could be well evaluated with complex and free ICG–HSA conjugate. both ensemble and single nanoparticle spectroscopy. However, it was noticed that 5-fold increased fluores - Considering each AuNR was averagely bound by six cence intensity of LNP over the free ICG–HSA conjugate ICG–HSA conjugates, a single ICG-Au complex was on the mouse was smaller than the enhancement factor over 100-fold brighter than a single ICG–HSA conjugate. of 16-fold for the ICG dyes on the AuNRs. This value was Strong near-field interactions could also result in short - also much less than the difference of brightness for the ened lifetime which is distinguished from the lifetime LNP over the free ICG–HSA conjugates in the fluores - range of cellular autofluorescence in the fluorescence cell cence cellular imaging. It was probably due to a much and small animal images. Because of its unique lifetime, stronger interference of autofluorescence background in the ICG-Au complex can provide us an opportunity to the small animal imaging. observe the target molecules and their activities with a We are interested in developing novel NIR LNP and better resolution and a larger depth of tissue layer in the using it for determining the target molecules in both time-resolved small animal imaging. The novel NIR nan - the cell and small animal. The immunohistochemistry of oparticle fluorophores will be used as calcium indicators ICG-Au complex in the organs of mouse were not per- to efficiently determine voltage-sensitive fluorescence formed in this study, and thus, the information on the calcium signal in-vivo at single cellular level and in living toxicity of ICG-Au complex to the small animals is not small animals. available in this paper. But it is also noticed that the mice kept good health after 1 week of ICG-Au complex injec- Methods tion, indicating that the ICG-Au complexes have a rela- All chemical reagents and spectroscopic grade solvents tively low toxicity to these mouse [58, 59]. More research were used as received from Fisher or Sigma/Aldrich. Car- on this aspect will be conducted in our laboratory. diogreen (indocyanine green, ICG) and human serum In this study, a superior bright NIR LNP was developed albumin (HSA) were available from Sigma/Aldrich. The for determining the cell membrane specific targets in the gold nanorods (AuNRs) and gold nanospheres were cells and small animals. We are interested in the voltage- purchased from Sigma/Aldrich. RC dialysis membrane gated calcium channels in the cell, tissue, and medium (MWCO 4000) was obtained from Spectrum Labora- −1 as well as in the small animals. The ICG-Au complex tories, Inc. Nanopure water (> 18.0  MΩ  cm ) purified will be used as the fluorescence indicator to explore the on Millipore Milli-Q gradient system was used in all change of calcium ion in the cells and furthermore the experiments. intracellular calcium concentrations or gradients at the cellular level as well as in the small animals. It is impor- Preparing ICG–HSA conjugates and binding conjugates tant to use this LNP for single molecule detection at the on gold nanorods cell level. But due to the strong backgrounds, the emis- Indocyanine green (ICG) was first conjugated in human sion signals of single nanoparticles become very difficult serum albumin (HSA). The ICG and HSA were codis - to resolve on the small animal imaging. However, with solved in 10  mM phosphate buffered saline (PBS buffer) largely enhanced fluorescence and shortened lifetime, the solution at pH = 7.4. The molar ratio of ICG over HSA ICG-Au complexes can offer us a greater opportunity to was 4/1 in solution. The solution was stirred at room Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 10 of 12 temperature for 24  h. Free ICG dyes in solution were mirror, focused onto a 75-µm pinhole for a spatial filter - removed by dialysis against 10 mM PBS buffer. ing, and recorded on a single photon avalanche diode The ICG–HSA conjugates were covalently bound (SPAD) (SPCM-AQR-14, Perkin-Elmer Inc.). A longpass on the gold nanorods (AuNRs). Three-step chemi - filter over 750 nm was used to eliminate the residual exci - cal reaction on the AuNR surface was employed. First, tation signals. The data were collected with a TimeHarp the CTAB monolayers on the AuNRs were replaced by 200 board and stored in time-tagged time-resolved mode hexa(ethyleneglycol)mono-11-(acetylthio)undecyl ether, (TTTR). −11 a polyethylene glycol (PEG) ligand. 5 × 10   M com- mercially available AuNRs were dispersed in an aque- Conjugation of ICG‑Au complexes with cells and their −5 ous solution containing 1 × 10   M hexa(ethyleneglycol) cytotoxicity mono-11-(acetylthio)undecyl ether. The solution was ICG-Au complexes were conjugated on HeLa cells for continuously stirred for 12  h, and the AuNRs were fluorescence cell imaging. The HeLa cells were dispersed recovered by centrifugation. Second, the PEG mon- in Dulbecco’s modified Eagle’s medium (DMEM), sup - olayers on the AuNRs were partially substituted with plemented with 10% fetal bovine serum (FBS), and sub- N-(2-mercapto-propinyl)glycine via surface substitution sequently grown on a 6-well glass coverslip incubated at −11 reaction.  5 × 10 M PEG-AuNRs were dispersed in 37 °C/5% CO /95% humidity for 48 h. The cells were then −9 an aqueous solution containing 5 × 10   M N-(2-mer- fixed with 4% paraformaldehyde in 10 mM PBS buffer at capto-propinyl)glycine. The solution was continuously pH 7.4 for 30  min at 4  °C. The fixed cells were washed stirred for 24  h. The AuNRs were recovered by configu - twice with 10 mM PBS buffer followed by incubating with ration. Finally, the ICG–HSA conjugates were covalently 0.5  nM ICG-Au in 10  mM PBS buffer for 30  min. The bound on the AuNRs via N-hydroxysuccinimide (NHS) samples were rinsed with 10  mM PBS-Mg buffer, dried −11 condensation reaction.  5 × 10 M PEG-AuNRs were in air, and stored at 4 °C. The imaging of LNP conjugated- dispersed in 10  mM PBS buffer solution at pH 8.2 con - cell samples were performed on the time-resolved confo- −9 taining 5 × 10 M ICG–HSA conjugates. Subsequently, cal microscope. −6 −6 1 × 10 M  N-hydroxy-succinimide (NHS) and 1 × 10 Cytotoxicity was tested on live HeLa cell using calcein M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide AM assay. Briefly, the HeLa cells were grown in a 6-well hydrochloride (EDC) were added in solution. The solu - glass coverslips for 48  h as described. The cells were tion was stirred for 12  h. The final AuNR product was washed twice with 10 mM PBS buffer followed by adding recovered by configuration and dispersed in 10 mM PBS 1  μM Calcein AM solution. 0.3 and 3  nM Au nanopar- buffer at pH 7.4. ticle solution was added and the cells were continuously cultured in the incubator. The images of live HeLa cells Nanoparticle characterization stained with calcein AM were acquired on the confocal Transmission electron microscopy (TEM) images were microscope at different time intervals with a bandpass taken with a side-entry Philips electron microscope at filter of 514/30 nm using a 443 nm laser diode as excita - 120  keV. The AuNRs were diluted to nanomolar con - tion source. The images of dead cells were identified by centrations followed by casting onto the copper grids their stronger autofluorescence on other channel with a (200  mesh) with standard carbon-coated Formvar films longpass filter of 655/20  nm using a 640  nm laser diode (200–300 Å). The samples were dried in air for the TEM as excitation source. The cell images were counted at sin - measurements. The distributions of nanoparticle sizes gle cell level and analyzed for the cell viability. were analyzed with Scion Image Beta Release 2. Absorption spectra were recorded on a Hewlett Pack- Small animal tomography measurements ard 8453 spectrophotometer. Ensemble fluorescence The ICG-Au complexes were tested as imaging contrast spectra were recorded on a Cary Eclipse Fluorescence agents for fluorescence small animal imaging. Typically, Spectrophotometer. 5–6 weeks nude mice were first euthanized under deep Fluorescence imaging measurements were conducted anesthesia. Removing the hair on the belly, the mice was on a time-resolved scanning confocal microscope (Micro- injected by 0.1  mL of 10  mM PBS buffer solution con - Time 200, PicoQuant) which consists of an inverted taining 0.5 nM ICG-Au complexes below the surface of confocal microscope coupled to a high-sensitivity detec- the mice skin. Subsequently, the same volumes of ICG- tion setup. A single mode pulsed laser diode (470  nm, HSA conjugate (concentration = 3  nM) in 10  mM PBS 100  ps, 40  MHz) was used as the excitation source. An buffer solution and blank in 10 mM PBS buffer solution oil immersion objective (Olympus, 100×, 1.3 NA) was were also respectively injected at different sites of same used to focus the laser beam on the sample and to collect mice. Fluorescence small animal imaging were per- the emission. 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A superior bright NIR luminescent nanoparticle preparation and indicating calcium signaling detection in cells and small animals

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Life Sciences; Cell Biology; Microbiology; Stem Cells; Neurobiology; Proteomics
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Abstract

Background: Near-field fluorescence (NFF) effects were employed to develop a novel near-infrared (NIR) lumines- cent nanoparticle (LNP) with superior brightness. The LNP is used as imaging contrast agent for cellular and small animal imaging and furthermore suggested to use for detecting voltage-sensitive calcium in living cells and animals with high sensitivity. Results: NIR Indocyanine green (ICG) dye was conjugated with human serum albumin (HSA) followed by covalently binding to gold nanorod (AuNR). The AuNR displayed dual plasmons from transverse and longitudinal axis, and the longitudinal plasmon was localized at the NIR region which could efficiently couple with the excitation and emission of ICG dye leading to a largely enhanced NFF. The enhancement factor was measured to be about 16-fold using both ensemble and single nanoparticle spectral methods. As an imaging contrast agent, the ICG–HSA-Au complex (abbre- viate as ICG-Au) was conjugated on HeLa cells and fluorescence cell images were recorded on a time-resolved confo - cal microscope. The emission signals of ICG-Au complexes were distinctly resolved as the individual spots that were observed over the cellular backgrounds due to their strong brightness as well as shortened lifetime. The LNPs were also tested to have a low cytotoxicity. The ICG-Au complexes were injected below the skin surface of mouse showing emission spots 5-fold brighter than those from the same amount of free ICG–HSA conjugates. Conclusions: Based on the observations in this research, the excitation and emission of NIR ICG dyes were found to be able to sufficiently couple with the longitudinal plasmon of AuNRs leading to a largely enhanced NFF. Using the LNP with super-brightness as a contrast agent, the ICG-Au complex could be resolved from the background in the cell and small animal imaging. The novel NIR LNP has also a great potential for detection of voltage-gated calcium concentration in the cell and living animal with a high sensitivity. Keywords: Gold nanorod (AUNR), Indocyanine green (ICG), Dual-mode plasmons, Near-field fluorescence (NFF), Luminescent nanoparticle (LNP), Fluorescence imaging, Imaging contrast agent Background cells, voltage-gated calcium channels are coupled with Calcium is a well-known signaling ion in most eukary- the membrane depolarization due to the calcium influx, otes [1, 2]. A calcium concentration gradient across a which can significantly alter the cellular physiology [ 3, plasma membrane and intracellular organelle can fluxes 4]. Hence, it is of importance to understand the calcium dynamically via orchestrated channel openings, and fur- concentration gradient and fluctuation in the cells. This thermore generate tightly controlled spatial and tempo- study might also highlight the crucial role of calcium sin- ral patterns. In electrically excitable neurons and muscle gle at a cellular level as well as in living animals. Currently, the voltage-gated calcium channels in the cells, tissues and mediums are often measured by a fluo - *Correspondence: jianzhang@vigenebio.com rescence imaging [5, 6]. Typically, a fluorophore is used as Present Address: Vigene Biosciences Inc., 9430 Key W. Ave Suite 105, a calcium indicator to chelate with a calcium ion creating Rockville, MD 20850, USA Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 2 of 12 a fluorescence signal. With a change of concentration or range most of current NFF effects were tested using the 2+ environment of Ca ions in the cell, the fluorescence visible fluorophores, and only few using NIR fluoro - signal from the indicator is altered. This method can be phores [29–33]. Different from the spherical metal nano - also used for exploring the intracellular calcium concen- particles, the shaped metal nanoparticles, such as metal tration and gradient of calcium ion at the cellular level as nanoshells or nanorods, can display their surface plas- well as in the living animals [7, 8]. Actually, the monitor- mons at longer wavelength [34, 35]. For instance, the gold ing voltage-gated imaging calcium has become an impor- nanorods (AuNRs) can display their split dual plasmons tant topic in the calcium channel detection because the from the short (transverse) and long axis (longitudinal), calcium signals exert their highly specific functions in the respectively [36–38], and importantly, the longitudinal well-defined cells or/and small animals. plasmon can be tuned to the NIR region by adjusting the In the past decades, novel calcium indicators have been aspect ratio of AuNRs. Thus, the longitudinal plasmons synthesized as the organic compounds [9, 10]. Most of from the AuNRs are expected to be able to sufficiently these calcium indicators have their emission wavelengths couple with the excitation/emission of NIR fluorophores at visible region. It is known that the fluorescence signals leading to a strong NFF-induced fluorescence at the NIR at the visible region have severe interference from the region. Meanwhile, the NIR AuNRs remain have reason- strong backgrounds from cellular autofluorescence and able sizes. light scattering in the biological systems [11–17]. To sup- We are interested in developing novel NIR LNPs with press the interference, a near-infrared (NIR) fluorophore high brightness and furthermore using these LNPs as is suggested for using as imaging contrast agents [18–20]. imaging contrast agents, for determining the calcium Tissue and water have a window with a low background ions in the cells and living small animals. In this study, allowing a penetration of excitation light deeper into the NFF effect was employed to prepare the novel NIR the tissue and allow the detection of emissions from the LNPs. Indocyanine green (ICG) is a FDA-proved non- fluorophores with a better resolution with the cells and toxic NIR fluorophore for patient safety in ophthal - tissues. mology [39, 40], and also known as a voltage-sensitive However, as imaging contrast agents, the NIR fluo - fluorophore that can be used to determine the voltage- rophores have their two significant drawbacks: (1) low gated calcium channels by adding chelators on its chemi- absorption coefficients which may result in their low cal structure [41]. In this study, the ICG dye was bound brightness and (2) low photostability that results in their to AuNRs within a near-field distance to explore the NFF short bleaching time [21, 22]. To our knowledge, there at the NIR region. is still lack of an efficient contrast agent that allows the Briefly, the ICG dyes were first conjugated in human detection of calcium ions in the cells and small animals serum albumin (HSA) followed by covalently binding at the single molecule level. Hence, there is an essential the conjugates on the surfaces of AuNRs [42, 43]. Since need for a new approach that can greatly improve the flu - the HSA molecules have an average size of ca. 10  nm, orescence properties of NIR fluorophores particularly on the ICG dyes conjugated to the HSA molecules are dis- their brightness and photostability. tributed within a near-field distance from the surfaces of Near-field fluorescence (NFF) can improve the fluo - AuNRs. In addition, the excitation/emission of ICG dyes rescence properties of fluorophores [23]. Fundamentally, can sufficiently couple with the longitudinal plasmons of a metal nanoparticle can create a local electromagnetic AuNRs, and thus, a NFF from the bound ICG dyes on the field nearby as a light irradiation and the electromagnetic AuNRs was expected to occur. The ensemble and single field is confined into the metal plasmons [24–26]. When nanoparticle spectra were used to evaluate the change a fluorophore is localized within the near-field range of optical properties of ICG dyes prior to and after their from the metal nanoparticle surface, the excitation/emis- binding on the AuNRs. Using as a nanoparticle contrast sion of fluorophore can strongly couple with the light- agent, the ICG–HSA-Au (abbreviated as ICG-Au) com- induced plasmons on the metal nanoparticles [27, 28], plex was bound to HeLa cells and the fluorescence cell and the excitation or/and emission rates of fluorophore images were collected for evaluating the fluorescence can be significantly increased. As a result, the fluores - spectral properties at the single nanoparticle level. The cence properties of fluorophore can be greatly improved ICG-Au complex was also injected into the mouse for including (1) largely enhanced emission intensity and live animal fluorescence tomography. Comparing with quantum yield (2) extended photobleaching time and (3) the free ICG–HSA conjugates, the ICG-Au complex dis- reduced photoblinking of fluorophore [28]. played significantly improved properties for the live ani - Since the spherical metal nanoparticle with a reason- mal tomography uses [44–50]. able size displays its single-mode plasmons at the visible Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 3 of 12 Results ICG–HSA conjugates In this research, the NIR luminescent nanoparticle was developed using a strong NFF effect by binding the NIR ICG dyes on the surfaces of AuNRs within a near-field dis - tance. Thus, the ICG dyes were first conjugated with the HSA molecules to form the ICG–HSA conjugates, and the conjugates were then covalently bound onto the surfaces of AuNRs. In the experiments, the ICG and HSA were codissolved in an aqueous solution with a molar ratio of ICG/HSA = 4/1. After the reaction, the free ICG dyes were removed from solution by a dialysis against water. The fluorescence properties of ICG dyes before and after the conjugation were measured using ensemble spectroscopy. Upon excitation at 760 nm, the ICG–HSA conjugate was observed to exhibit an emission band cen- tered at 819, 7 nm shifting to shorter in comparison with the free ICG dyes in aqueous solution (Fig. 1a). The emis - sion band also became broader with the ICG conjugation, which may be due to the plasmons or the short emission wavelength of ICG. Covalently binding ICG–HSA conjugates on AuNRs The biological properties of nanoparticles, such as cell uptake and circulation time, are known to strongly rely on their surface properties [51, 52]. In this study, AuNRs were prepared with the protection of cetyltrimethylam- monium bromide (CTAB) monolayers on the surfaces. To improve their bioactivity, the CTAB-monolayers on Fig. 1 a Absorption spectra of AuNRs as CTAB-coated, PEG-coated, the AuNRs were replaced with the thiolate polyethylene and ICG–HSA conjugate-bound in a 10 mM PBS buffer solution. b glycol (PEG) monolayers via a surface substitution reac- Ensemble emission spectra from the ICG dyes as free, conjugates in tion on the nanoparticle. The free small molecules were HSA, and complexes with AuNRs in a 10 mM PBS buffer solution removed by a dialysis against water. Most CTAB mol- ecules on the AuNR surfaces were supposed to replace by the PEG molecules. The change of monolayers on the The ICG–HSA conjugates were covalently bound AuNR surfaces could be reflected by the solubility change on the AuNRs via the surface condensation of pri- of AuNRs in aqueous solution prior to and after the reac- mary amino moieties in the ICG–HSA conjugates tion. In addition, since these PEG molecules were bound with the carboxyl moieties on AuNRs in the presence on the AuNR surfaces via sulfur-metal bonds, much of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide stronger than the CTAB molecules via electrostatic inter- hydrochloride (EDC) as the condensation agent. The actions, the PEG-AuNRs should become more chemi- ICG–HSA conjugates were dissolved in excess amount cally stable in solution [51, 52]. in solution to avoid aggregation of nanoparticles To bind the ICG–HSA conjugates on the AuNRs, the through the crosslinking. The final AuNR product was PEG monolayers on the AuNRs were partially substi- recovered by a centrifugation and then purified by a tuted by thiolate carboxyl-ligand of N-(2-mercapto- dialysis against water. propinyl)glycine ligands to create the reactive sites on the AuNR surfaces via surface exchange reaction [53, 54]. Experimentally, the thiolate carboxyl-ligand was dis- Evaluation of ICG‑Au complex by microscope solved in solution with a molar ratio of carboxyl ligand/ and ensemble spectroscopy AuNR = 100/1. After the substitution reaction, the Tomography of AuNRs through the surface reactions unsubstituted ligands were removed by a dialysis against was evaluated using a transmission electron micro- water. scope (TEM). Representative images of AuNRs are Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 4 of 12 shown in Fig.  2a, b prior to and after the surface reac- time-resolved confocal microscope. Upon excitation with tions on the AuNRs. These AuNRs were observed to a 640  nm laser, both the emission intensities and life- have an average width of 10  nm and an average length times from the ICG-Au complexes (as shown in Fig.  3a) of 40  nm and the aspect ratio was calculated to be ca. were collected at single nanoparticle level [54]. As con- 4.0. There was no significantly change on the tomog - trol, the free ICG–HSA conjugates were also diluted in raphy with the three-step surface reactions on AuNRs, solution and cast on the coverslip. The emission signals reflecting that the surface reactions on the AuNRs only were collected with the same conditions on the confocal altered the monolayer composition on their surfaces microscope but with an excitation power of laser 10-fold but not on their metal cores. stronger. The collected emission spots from the free con - The plasmon absorption of metal nanoparticle is jugates were much dim as shown in Fig.  3b demonstrat- known to be sensitive to the composition of monolay- ing lower emission intensities of free conjugates. For each ers on the surface [30, 31]. In this study, the absorp- sample, at least 50 emission spots were collected, and the tion spectrum was used to measure the replacement of histogram of the intensities and lifetimes were obtained ligands on the AuNR surfaces. The AuNRs displayed a by fitting with a Gaussian distribution curve (Fig.  4a for dual plasmons from the short (transverse) and long axis the intensity and b for the lifetime), and the maximum (longitudinal) at 504 and 802 nm, respectively (Fig. 1a). values of curves were obtained to represent the emission Following the monolayer reactions on the surfaces of intensity and lifetime of sample, respectively. nanoparticles, the two plasmons bands were found to The near-field interaction of an excited fluorophore remain, but the maxima were slightly shifted to longer with a metal nanoparticle may increase the radiative rate at 511 and 807 nm (Fig. 1a), respectively. of fluorophore, and as a result, the lifetime of fluorophore Ensemble fluorescence spectra were also sensitive can be decreased [25]. Hence, the lifetime can be used as to the binding of ICG dyes on the metal nanoparticle an important parameter to evaluate the near-field inter - surfaces. It was shown that the emission band of ICG- action. Herein, the decays of excited ICG-Au complexes Au complexes was centered at 814  nm (Fig.  1b), 5  nm as well as ICG–HSA conjugates were recorded using the shifting to shorter in comparison with the free ICG– confocal microscope following by fitting with a Gauss - HSA conjugates. Fluorescence spectral shifts have been ian distribution curve (Fig.  4b). The maximum values of attractive to the wavelength dependent on the interac- lifetime of ICG dyes were obtained, showing a significant tion of metal nanoparticle and fluorophore [55– 57]. decrease of lifetime from 2.3  ns for the unbound ICG– HSA conjugates to 0.4 ns for the ICG-Au complexes. Evaluation of ICG‑Au complex by single nanoparticle spectroscopy In addition to the ensemble spectrum, NFF effect on Fluorescence cellular imaging the ICG-Au complexes could be evaluated using sin- To test the fluorescence properties of ICG-Au complexes gle nanoparticle spectral measurement. To prepare the for the cell imaging, the ICG-Au complexes were used as test samples, the ICG-Au complex was diluted to nM in an imaging contrast agent to conjugate with HeLa cells. aqueous solution and then cast a drop on a glass cover- Briefly, HeLa cells were cultured on coverslips followed slip followed by drying in air. With a low concentration by fixing using 4% paraformaldehyde. The cell-fixed in solution before drying, the ICG-Au complexes were coverslip were incubated with the ICG-Au complex for mostly existed as isolated particles on the coverslip. The 30  min and then completely washed with PBS buffer. single nanoparticle measurements were performed on a Fluorescence cell images were collected on the time- resolved confocal microscope in both the intensity and lifetime. A representative image was presented in Fig. 3c. It was shown that the ICG-Au complexes were presented as individual spots on the cells distinctly observable from the cellular backgrounds either due to their strong inten- sity and differentiated lifetime. As control, the ICG–HSA conjugates were also conju- gated with HeLa cells, and the cell images were recorded on the confocal microscopy with the same conditions (Fig.  3d). Comparing with the images of blank cells, the overall cell images became brighter, indicating that the ICG–HSA conjugates were indeed conjugated on the Fig. 2 TEM images of (a) CTAB-AuNRs and (b) ICG-Au complexes cells. But the emission signals from the single ICG–HSA Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 5 of 12 Fig. 3 Upper panels represent the emission imaging from (a) ICG-Au complexes and (b) ICG–HSA conjugates. Diagrams are 5 × 5 µm and resolutions are 100 × 100 pixels with an integration of 0.6 ms/pixel. Bottom panels represent fluorescence images from the cells conjugated with (c) ICG-Au complexes and (d) ICG–HSA conjugates. Diagrams are 50 × 50 µm and resolutions are 100 × 100 pixel with an integration of 0.6 ms/pixel. The samples were excited with a 640 nm laser. Note the different intensity scales. The images of a and c were collected with a laser power 10-fold less than the images of b and d conjugates could not be well resolved as individual spots nanoparticles (Fig.  5c) and as 13 in the presence of Au from the cellular backgrounds of cell images, which was nanoparticles (Fig.  5d), showing that the rates of viable due to their low brightness as well as a lifetime close to cells are 93.1 and 93.7%, respectively. The results in the the cellular background. presence of 0.3 and 3  nM as well as the control were listed in Fig. 6e reflecting that the presence of Au nano - Cytotoxicity measurements particles in the cell medium had only a slight influence Cytotoxicity of free conjugate and ICG-Au complex to the cells survival. It also demonstrates that the Au were tested on live HeLa cell using calcein AM assay. nanoparticles have very low cytotoxicity. The cell images at different time intervals were col - lected on the time-resolved confocal microscope as Fluorescence small animal imaging shown in Fig.  5. An area with a large number of cells Six 5–6  weeks nude mice were selected to test the opti- was selected for statistical analysis for the cell sur- cal properties of novel LNP by small animal fluores - vival. The live cells could be identified as stained with cence imaging. The mice were randomly divided into calcein AM (green cell viability stain) as shown in the two groups and each group had three mice. The mice image A when there was in the absence of nanoparticle were first euthanized under deep anesthesia following (294 cells) and in the image B when there was in the by injecting the ICG-Au complex below the skin sur- presence of 3 nM Au nanoparticles (207 cells) after the face of mouse [16, 17]. As control, the ICG–HSA con- treatment time of 24  h. The number of cell with high jugate solution containing the same amount of ICG dye autofluorescence was counted as 20 in the absence of was also injected in the same mouse at a different site. Fluorescence small animal imaging was performed on a Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 6 of 12 on the AuNRs [42, 43]. To achieve the ICG–HSA conju- gates with the maximal brightness, the molar ratio of ICG 0.30 ICG-HSA Conjugates over HSA in the conjugation was controlled to be 4/1 in the reaction. Too many ICG dyes on one HSA molecule ICG-Au Complexes 0.25 would result in self-quenching among the fluorophores and too few dyes in one HSA molecule would result in a 0.20 low brightness. 0.15 To improve the bioactivity of nanoparticles, the CTAB monolayers on the AuNRs were replaced by the 0.10 PEG monolayers via surface substitution reaction [51, 52]. Most of the CTAB molecules on the AuNRs were 0.05 believed to replace by the PEG molecules, and supported by a change on the solubility of AuNRs in aqueous solu- 0.00 tion prior to and after the replacement. Prior to the 0 100 200300 400 replacement, the AuNRs were found to have a very good b dispersion in water, whereas after the replacement, the Emission Intensity (counts) AuNRs were readily stuck on the wall of glass tube, which 0.25 ICG-Au Complexes was due to increased hydrophobicity of nanoparticle sur- ICG-HSA Conjugates faces by the PEG monolayers. In addition, with stronger 0.20 sulfur-metal covalent bonds of PEG with the AuNRs, the modified AuNRs were supposed to have an improved 0.15 chemical stability in solution [51, 52]. To covalently bind the ICG–HSA conjugates on the 0.10 AuNRs, the PEG monolayers on the AuNRs were par- tially substituted by N-(2-mercapto-propinyl)glycine to 0.05 create reactive sites on the nanoparticle surfaces. The ICG–HSA conjugates were then covalently bound on the AuNRs via a condensation reaction [53]. The binding of 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ICG–HSA conjugates on the AuNRs could be supported Lifetime (ns) by a change of absorption and fluorescence spectra prior Fig. 4 Histogram distributions of single sots of (a) emission to and after reactions as described early. The binding intensities and (b) lifetimes from the ICG–HSA conjugates as free and number of ICG–HSA on each AuNR could be measured bound on the AuNRs using a NaCN treatment method [53]. Typically, several drops of 0.1  N NaCN aqueous solution were added into 0.5  nM ICG-Au complex solution. It was observed that Xenogen IVIS-200 system and the representative images the plasmon color of solution disappeared progressively were shown in Fig. 6. An image from an untreated mouse with the time, showing that the metal nanoparticles were was also presented as control. The emission spots from dissolved by NaCN. As a result, the ICG–HSA conju- the injection sites by the ICG-Au complex and ICG–HSA gates were released from the nanoparticles as free into conjugate were observed to be significantly different: the the solution. The whole process could be monitored by spot by the ICG-Au complex was ca. 5-fold brighter than the ensemble fluorescence spectrum expressing a dra - the spot by the ICG–HSA conjugate. Since the two injec- matic decrease of emission intensity (Fig. 7) until satura- tion sites on the same mice were known to contain the tion. The ICG–HSA conjugates were released as free in same amounts of ICG dye, the difference on their bright - the solution completely lost the NFF effect leading to a ness should be due to their different emission intensities. dramatic decrease on the emission intensity [31]. Using In the other words, an enhanced fluorescence of NFF the saturated emission intensity, the concentration of from the ICG-Au complex results in increased brightness ICG–HSA conjugates in the solution was measured to of ICG dyes in the small animal imaging. −9 be 3 × 10   M. Since the amount of ICG–HSA was not significantly changed in solution prior to and after the Discussion NaCN treatment, according to a ratio of emission inten- In this study, a NIR LNP was designed and prepared on sity prior to the treatment over that after the treatment, the basis of NFF effect. The ICG dyes were conjugated the enhancement factor for the ICG dye on AuNR was with the HSA molecules followed by covalently binding calculated to be 16.3. Histogram Distribution (%) Histogram Distribution (%) Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 7 of 12 Fig. 5 The panel of cell images of live HeLa cells stained with calcein AM without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles. The images of calcein stained cells (a) and (b) were acquired after 24 h of nanoparticle treatments upon excitation with a 443 laser diode and at bandpass filter 514/30 nm. The images of c and d represent the autofluorescence of cells without (a) the Au nanoparticle and (b) in the presence of 3 nM of Au nanoparticles after 24 h. The autofluorescence images of cells were collected upon an excitation at 640 nm and with a longpass filter of 655 nm. Cells with brighter autofluorescence in c and d are classified as dead. e represents rates of viable cells in the presence of 0.3 and 3 nM in the cell medium as well as in the absence of Au nanoparticle as the control at time interval = 0.5, 2, 12, 24 h To evaluate the NFF effect of NIR dyes on the AuNRs, microscope. The ICG-Au complexes were found to have 20 and 50  nm gold nanospheres were prepared follow- an intensity 10.5-fold higher than the free ICG–HSA con- ing by covalently binding with the ICG–HSA conju- jugates. Since the emission of ICG-Au complexes were gates via the same strategy. Using the NaCN treatment, collected upon excitation with a laser power of 10-fold the enhancement factor of ICG dyes on the 50  nm gold lower than those of ICG–HSA conjugates, the ICG-Au nanospheres was measured to be 2.3, much lower than complexes were calculated to be 105-fold brighter than that on the AuNRs, although a 50  nm gold nanosphere the ICG–HSA conjugates. Considering that one AuNR is almost 20-fold larger on the volume than a AuNR. The was averagely bound with 6 ICG–HSA conjugates, the ICG dyes on a 20  nm gold nanosphere, which has an enhancement factor per ICG molecule was estimated to approximately identical volume to a AuNR, resulted in an be 16.7, very close to the enhancement factor achieved insignificant NFF effect. The nanospheres did not display on the ensemble spectra. This enhancement factor is a plasmon band at the NIR region, and as a result, could also comparable with the value from some visible fluo - not sufficiently couple with the excitation and emission rophores on the metal nanospheres [41], representing a of ICG dyes. In contrast, the AuNRs displayed a NIR lon- sufficient NFF interaction of NIR fluorophores with the gitudinal plasmon band leading to their sufficiently cou - AuNRs. pling with the excitation and emission of ICG dyes, and Besides the emission intensity, the near-field effect of thus, resulted in a strong NFF effect. This result indicates a fluorophore with a metal nanoparticle may result in a that the longitudinal plasmon band from a shaped metal largely reduced lifetime [25]. In this study, the lifetimes nanoparticle is very important for its sufficient coupling were collected at single nanoparticle level on a confo- with a NIR fluorophore and brings up a strong NFF in the cal microscope, and the histogram of lifetimes was fitted NIR region. with a Gaussian distribution. A maximum was obtained The ICG-Au complexes were also evaluated at sin - at 0.4  ns (Fig.  4b), much shorter than the lifetime of gle nanoparticle level on a time-resolved confocal unbound ICG–HSA conjugates at 2.3  ns, supporting an Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 8 of 12 of novel NIR nanoparticle fluorophores from the cellular backgrounds on the time-resolved images [44, 45]. Using as an imaging contrast agent, the ICG-Au com- plexes were conjugated with HeLa cells for fluorescence cell imaging. Fluorescence cell images were recorded on the confocal microscope with both the intensity and life- time. It was shown that the emissions signals from the ICG-Au complexes were distinctly isolated as individual spots from the cellular backgrounds (Fig.  3c). The inten - sity ratio of signal/noise on the image was estimated to be approx. 63, much higher than the value from the most organic fluorophores or LNPs, which was due to high brightness of ICG-AuNRs [44, 45]. In addition, because of largely shortened lifetime, the emissions of ICG- AuNRs could be better resolved from the cellular back- grounds on the lifetime cell images. Fig. 6 In-vivo fluorescence tomography images of mice using the As control, the HeLa cells were also incubated with ICG-Au complexes as contrast agent injected below the skin of mice. the free ICG–HSA conjugates, and the cell images were The images were collected on a Xenogen IVIS-200 small animal recorded under the same conditions (Fig.  3d). It was tomography system with a bandpass filter from 665 to 695 nm for shown that the overall cell images became brighter than the background, a filter from 710 to 760 on the excitation side, and the images of blank cells without the treatment support- a filter from 810 to 875 nm on the emission side. A 750 nm laser was used as the excitation source. Total photon flux (photons/s) was ing that the ICG–HSA conjugates had been conjugated calculated and corrected for tissue depth by spectral imaging using with the cells. But the emissions from the ICG–HSA con- Living Image 3.0 software (Xenogen). The left image (a) was collected jugates could not be resolved as individuals from the cel- on a control mice and the right image (b) was collected by injection lular backgrounds, which was due to their low brightness with the ICG-Au complex, ICG–HSA conjugate, or a blank PBS buffer as well as close lifetime relative to the cellular autofluo - solution with the same volume on the same mice rescence at the backgrounds. In this study, the LNPs were not functionalized with the specific bioactive molecules. Thus the ICG-Au complexes were supposed to randomly distribute through the cells (Fig.  3c). On the other hand, because of bulky sizes of ICG-Au complexes and relatively short incubation time, these LNPs were observed to mostly attach on the cell surfaces, which could be the result of short incubation time with the cells. Our other experiments (not shown herein) also demonstrated that the metal nanoparticles of this size can penetrate through the cell membrane and enter the cells. We will use these LNPs as indicators to detect the calcium channels and concentration gradients by the fluorescence cell imaging. Once the nanoparti - cles are functionalized with the bioactive molecules and then enter the cells, they will become possible to label the target molecules with a higher efficiency because of the Fig. 7 Emission spectral change of ICG-Au complex in 10 mM PBS presence of multiple functional groups on their surfaces. buffer solution before and after a NaCN treatment Cytotoxicity of ICG-Au complexes was tested on live HeLa cell using a calcein AM assay showing that the luminescent nanoparticles have only slight or even insig- efficient near-field coupling of ICG molecules with the nificant cytotoxicity to the live HeLa cell. It is known that AuNRs. It was interesting to notice that the lifetime of the cytotoxicity of metal nanoparticles strongly relies on ICG-Au complex was beyond the range of autofluores - the coating layers on the metal cores. For a relatively low cence (2–5  ns) in the lifetime fluorescence cell images, cytotoxicity of metal nanoparticles in this study, it can which would beneficiate to isolate the emission signals be described by two possible factors. First, polyethylene glycol layers were coated on the Au cores via covalent Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 9 of 12 bonds. These covalent bonds are much stronger than the insight the target calcium ions and their activities with a statistic interactions that the nanoparticles are generally better resolution and a larger depth of tissue layer. bound by leading to the current ICG-Au complexes are Because of instrumentation limitations, the small ani- more chemically stable in the cell medium or animal bod- mal image could not be recorded in time-resolved model ies. Second, the ICG dyes have low toxicity. Hence, the on the current imaging system. But we expect that with ICG-Au complexes can affect insignificantly or slightly largely enhanced fluorescence and unique lifetime, the the cells viability as observed in this study. ICG-Au complex can provide us an opportunity to To test the imaging function, the ICG-Au complex observe the target molecules and their activities with a was injected to the skin surface of mice for the fluores - better resolution and a larger depth of tissue layer in the cence small animal imaging [16, 17]. It was shown that time-resolved small animal imaging. the emission spot from the injection site by the ICG- Au complex was ca. 5-fold brighter than the site by the Conclusions ICG–HSA conjugate. Since the two injection sites con- Due to a longitudinal plasmon at the NIR region, AuNRs tained the same amounts of ICG dye, the difference of were demonstrated to sufficiently couple with the exci - the brightness of two spots over the mice image should tation/emission of NIR fluorophores leading to largely be due to the different brightness between the ICG-Au enhanced NFF effect. NFF could be well evaluated with complex and free ICG–HSA conjugate. both ensemble and single nanoparticle spectroscopy. However, it was noticed that 5-fold increased fluores - Considering each AuNR was averagely bound by six cence intensity of LNP over the free ICG–HSA conjugate ICG–HSA conjugates, a single ICG-Au complex was on the mouse was smaller than the enhancement factor over 100-fold brighter than a single ICG–HSA conjugate. of 16-fold for the ICG dyes on the AuNRs. This value was Strong near-field interactions could also result in short - also much less than the difference of brightness for the ened lifetime which is distinguished from the lifetime LNP over the free ICG–HSA conjugates in the fluores - range of cellular autofluorescence in the fluorescence cell cence cellular imaging. It was probably due to a much and small animal images. Because of its unique lifetime, stronger interference of autofluorescence background in the ICG-Au complex can provide us an opportunity to the small animal imaging. observe the target molecules and their activities with a We are interested in developing novel NIR LNP and better resolution and a larger depth of tissue layer in the using it for determining the target molecules in both time-resolved small animal imaging. The novel NIR nan - the cell and small animal. The immunohistochemistry of oparticle fluorophores will be used as calcium indicators ICG-Au complex in the organs of mouse were not per- to efficiently determine voltage-sensitive fluorescence formed in this study, and thus, the information on the calcium signal in-vivo at single cellular level and in living toxicity of ICG-Au complex to the small animals is not small animals. available in this paper. But it is also noticed that the mice kept good health after 1 week of ICG-Au complex injec- Methods tion, indicating that the ICG-Au complexes have a rela- All chemical reagents and spectroscopic grade solvents tively low toxicity to these mouse [58, 59]. More research were used as received from Fisher or Sigma/Aldrich. Car- on this aspect will be conducted in our laboratory. diogreen (indocyanine green, ICG) and human serum In this study, a superior bright NIR LNP was developed albumin (HSA) were available from Sigma/Aldrich. The for determining the cell membrane specific targets in the gold nanorods (AuNRs) and gold nanospheres were cells and small animals. We are interested in the voltage- purchased from Sigma/Aldrich. RC dialysis membrane gated calcium channels in the cell, tissue, and medium (MWCO 4000) was obtained from Spectrum Labora- −1 as well as in the small animals. The ICG-Au complex tories, Inc. Nanopure water (> 18.0  MΩ  cm ) purified will be used as the fluorescence indicator to explore the on Millipore Milli-Q gradient system was used in all change of calcium ion in the cells and furthermore the experiments. intracellular calcium concentrations or gradients at the cellular level as well as in the small animals. It is impor- Preparing ICG–HSA conjugates and binding conjugates tant to use this LNP for single molecule detection at the on gold nanorods cell level. But due to the strong backgrounds, the emis- Indocyanine green (ICG) was first conjugated in human sion signals of single nanoparticles become very difficult serum albumin (HSA). The ICG and HSA were codis - to resolve on the small animal imaging. However, with solved in 10  mM phosphate buffered saline (PBS buffer) largely enhanced fluorescence and shortened lifetime, the solution at pH = 7.4. The molar ratio of ICG over HSA ICG-Au complexes can offer us a greater opportunity to was 4/1 in solution. The solution was stirred at room Zhang and Lakowicz Cell Biosci (2018) 8:37 Page 10 of 12 temperature for 24  h. Free ICG dyes in solution were mirror, focused onto a 75-µm pinhole for a spatial filter - removed by dialysis against 10 mM PBS buffer. ing, and recorded on a single photon avalanche diode The ICG–HSA conjugates were covalently bound (SPAD) (SPCM-AQR-14, Perkin-Elmer Inc.). A longpass on the gold nanorods (AuNRs). Three-step chemi - filter over 750 nm was used to eliminate the residual exci - cal reaction on the AuNR surface was employed. First, tation signals. The data were collected with a TimeHarp the CTAB monolayers on the AuNRs were replaced by 200 board and stored in time-tagged time-resolved mode hexa(ethyleneglycol)mono-11-(acetylthio)undecyl ether, (TTTR). −11 a polyethylene glycol (PEG) ligand. 5 × 10   M com- mercially available AuNRs were dispersed in an aque- Conjugation of ICG‑Au complexes with cells and their −5 ous solution containing 1 × 10   M hexa(ethyleneglycol) cytotoxicity mono-11-(acetylthio)undecyl ether. The solution was ICG-Au complexes were conjugated on HeLa cells for continuously stirred for 12  h, and the AuNRs were fluorescence cell imaging. The HeLa cells were dispersed recovered by centrifugation. Second, the PEG mon- in Dulbecco’s modified Eagle’s medium (DMEM), sup - olayers on the AuNRs were partially substituted with plemented with 10% fetal bovine serum (FBS), and sub- N-(2-mercapto-propinyl)glycine via surface substitution sequently grown on a 6-well glass coverslip incubated at −11 reaction.  5 × 10 M PEG-AuNRs were dispersed in 37 °C/5% CO /95% humidity for 48 h. The cells were then −9 an aqueous solution containing 5 × 10   M N-(2-mer- fixed with 4% paraformaldehyde in 10 mM PBS buffer at capto-propinyl)glycine. The solution was continuously pH 7.4 for 30  min at 4  °C. The fixed cells were washed stirred for 24  h. The AuNRs were recovered by configu - twice with 10 mM PBS buffer followed by incubating with ration. Finally, the ICG–HSA conjugates were covalently 0.5  nM ICG-Au in 10  mM PBS buffer for 30  min. The bound on the AuNRs via N-hydroxysuccinimide (NHS) samples were rinsed with 10  mM PBS-Mg buffer, dried −11 condensation reaction.  5 × 10 M PEG-AuNRs were in air, and stored at 4 °C. The imaging of LNP conjugated- dispersed in 10  mM PBS buffer solution at pH 8.2 con - cell samples were performed on the time-resolved confo- −9 taining 5 × 10 M ICG–HSA conjugates. Subsequently, cal microscope. −6 −6 1 × 10 M  N-hydroxy-succinimide (NHS) and 1 × 10 Cytotoxicity was tested on live HeLa cell using calcein M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide AM assay. Briefly, the HeLa cells were grown in a 6-well hydrochloride (EDC) were added in solution. The solu - glass coverslips for 48  h as described. The cells were tion was stirred for 12  h. The final AuNR product was washed twice with 10 mM PBS buffer followed by adding recovered by configuration and dispersed in 10 mM PBS 1  μM Calcein AM solution. 0.3 and 3  nM Au nanopar- buffer at pH 7.4. ticle solution was added and the cells were continuously cultured in the incubator. The images of live HeLa cells Nanoparticle characterization stained with calcein AM were acquired on the confocal Transmission electron microscopy (TEM) images were microscope at different time intervals with a bandpass taken with a side-entry Philips electron microscope at filter of 514/30 nm using a 443 nm laser diode as excita - 120  keV. The AuNRs were diluted to nanomolar con - tion source. The images of dead cells were identified by centrations followed by casting onto the copper grids their stronger autofluorescence on other channel with a (200  mesh) with standard carbon-coated Formvar films longpass filter of 655/20  nm using a 640  nm laser diode (200–300 Å). The samples were dried in air for the TEM as excitation source. The cell images were counted at sin - measurements. The distributions of nanoparticle sizes gle cell level and analyzed for the cell viability. were analyzed with Scion Image Beta Release 2. Absorption spectra were recorded on a Hewlett Pack- Small animal tomography measurements ard 8453 spectrophotometer. Ensemble fluorescence The ICG-Au complexes were tested as imaging contrast spectra were recorded on a Cary Eclipse Fluorescence agents for fluorescence small animal imaging. Typically, Spectrophotometer. 5–6 weeks nude mice were first euthanized under deep Fluorescence imaging measurements were conducted anesthesia. Removing the hair on the belly, the mice was on a time-resolved scanning confocal microscope (Micro- injected by 0.1  mL of 10  mM PBS buffer solution con - Time 200, PicoQuant) which consists of an inverted taining 0.5 nM ICG-Au complexes below the surface of confocal microscope coupled to a high-sensitivity detec- the mice skin. Subsequently, the same volumes of ICG- tion setup. A single mode pulsed laser diode (470  nm, HSA conjugate (concentration = 3  nM) in 10  mM PBS 100  ps, 40  MHz) was used as the excitation source. An buffer solution and blank in 10 mM PBS buffer solution oil immersion objective (Olympus, 100×, 1.3 NA) was were also respectively injected at different sites of same used to focus the laser beam on the sample and to collect mice. Fluorescence small animal imaging were per- the emission. 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Cell & BioscienceSpringer Journals

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