Cancer theranostics, which combine bioimaging diagnostics and cancer therapy, have the potential to help millions of people in typically fatal situations [1
]. Over the last decade, this field has witnessed the rapid development of nanotheranostic devices. These devices are constructed using both organic and inorganic nanomaterials to integrate both therapeutics and bioimaging agents into one entity, simultaneously realizing their functionalities [2
In theranostic systems, a pivotal concern is the choice of imaging techniques to accurately disclose the location of tumors for a specific diagnosis. Several methods, such as computed tomography (CT), fluorescence (FL) imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), photoacoustic (PA) imaging and upconversion luminescence (UCL) imaging have been applied [4
]. However, each technique has its own inherent limitations. To address their shortcomings, tremendous efforts have been made in the development of multimodal imaging, which can take advantage of different techniques to properly meet clinic requirements [7
For instance, despite the excellent sensitivity of FL imaging, its limited tissue penetration depth [10
] and the finite information derived from fluorescence intensity micrographs of cells have compromised its diagnostic abilities. Meanwhile, fluorescence lifetime imaging microscopy (FLIM) has been proven as a highly advanced spectroscopic method for biological and biomedical applications [12
]. The excellent performance of FLIM can contribute to high contrast images that are independent of excitation intensity and fluorophore concentration [13
]. Moreover, it provides both temporal and spatial information of intracellular structures labeled fluorescently by detecting changes in the fluorescence lifetime (FLT) [14
]. Thus, the integration of FLIM with FL imaging could be more favorable for accurate cancer diagnosis. Therefore, there is a need to find more appropriate fluorescent probes for FLIM and FL imaging.
Fluorescent probes, such as organic fluorescent dyes and quantum dots (QDs), have been widely explored in biomedical fields for imaging [15
]. However, the biocompatibility, photo-bleaching and photo scintillation of some probes hinder these applications [19
]. In contrast, carbon dots (CDs) are ideal candidates for biological applications due to their biocompatibility, chemical inertness, as well as strong fluorescence performance, photochemical stability, and easy functionalization [21
]. In the current era, a range of research aims to investigate the potential of CDs as biocompatible nanoprobes for targeting cancer cells in vitro. For example, CDs doped with heteroatoms (such as N, P, and S) were widely studied for fluorescence imaging in cells [27
]. However, CDs are rarely regarded as a therapeutic agent [30
]. Moreover, a variety of photothermal agents (e.g., layered double hydroxides, gold nanorods, chlorin e6) were combined with CDs and the prepared hybrid system was successfully used as a theranostic agent [31
]. Therefore, photothermal agents can be integrated with CDs to achieve multifunctional cancer theranostics.
In photothermal therapy (PTT), near-infrared (NIR) light is applied for the generation of localized heat energy from specifically-designed nanomaterials, which can cause hyperthermia and hence the apoptosis or necrosis of cancer cells [30
]. The unique surface plasmon resonance (SPR) of noble metal nanoparticles, especially gold nanoparticles, promote their ability to quickly and effectively convert absorbed photon energy into heat in the picosecond time domain [37
]. In this catalog, gold nanorods (GNRs) have been extensively studied due to their facile synthesis and surface modification, biocompatibility, superior tunable optical properties and photostability, good cellular affinity and long blood circulation [38
]. One unique advantage of GNRs is their longitudinal SPR peak can be adjusted to the NIR region by modulating the aspect ratio (length/width). NIR is known to have optimal light penetration in biological tissues due to its minimal absorption by chromophores and water [39
]. Furthermore, the high scattering cross sections of GNRs render them good contrast agents for dark field microscopy imaging.
Early diagnosis and definitive therapy can be integrated into an unprecedented nanoplatform to break the limitations of individual functionality. To date, GNRs were integrated with fluorescent dyes [14
] or quantum dots (QDs) [40
], either by electrostatic interaction [32
] or covalent linkages [41
], for their utility as an imaging contrast agent. However, there is still a need for new agents with a stable structure, excellent biocompatibility, and high therapeutic efficiency to meet the demands for clinical applications. Polyethylene glycol (PEG) is a biocompatible polymer that has been used for an extremely wide range of products, ranging from skin care products to tablet formulations, laxatives, and food additives [42
]. Thus, PEG is the most suitable material for latent clinical applications. In addition, these biocompatible PEG chains can be easily functionalized as a covalent linker, which can improve the chemical stability of the hybrids in physiological environments, as well as prevent absolute quenching of CD fluorescence when bound to GNRs.
In this paper, we report the construction of a covalently-linked nanohybrid as a novel nanoplatform for dual-modal imaging and phototherapy made up of GNRs and CDs bridged with PEG (GNR–PEG–CDs), and these luminescent nanohybrid materials express excellent fluorescence features for FLIM and confocal FL imaging, leading to good spatial resolutions and a strong response to PTT. In addition, they have a low toxicity and good biocompatibility. This study exhibits a new multifunctional nanoplatform, i.e., GNR–PEG–CDs, for cancer diagnostics and therapeutics.
2. Materials and Methods
HAuCl4·3H2O and sodium borohydride (NaBH4), were purchased from J&K Chemical. Co. (Beijing, China). Cetyltrimethyl Ammonium Bromide (CTAB), L-ascorbic acid, HCl, silver nitrate (AgNO3) and p-phenylenediamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Thiolated and methoxyl terminated polyethylene glycol (mPEG-SH, molecular weight 1000 Da), Thiolated and carboxyl terminated polyethylene glycol (SH-PEG-COOH, molecular weight 1000 Da), were purchased from Peng Shuo Biological Technology Co., Ltd. (Shanghai, China). 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), were purchased from Aladdin (Shanghai, China). All reagents were of analytical grade and used without further purification. High glucose Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8) was obtained by Dojindo China Co., Ltd. (Shanghai, China). HeLa cells were obtained from the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences (Beijing, China).
2.2. Preparation of Gold Nanorods
The seed-mediated growth method was utilized to prepare GNRs according to Nikoobakht’s method, with some modifications [43
]. In this seed solution, ice-cold aqueous NaBH4
solution (0.01 M, 0.6 mL) was added to an aqueous mixture solution, which was composed of HAuCl4
(0.01 M, 0.25 mL) and CTAB (0.1 M, 9.75 mL), followed by rapid mixing for 2 min. Then, the seed solution was kept at 27 °C for 2 h for use in growth solution. The growth solution was composed of CTAB (0.1 M, 200 mL), HAuCl4
(0.01 M, 10 mL), AgNO3
(0.01 M, 2.6 mL), HCl (1 M, 5 mL) and L-ascorbic acid (0.1 M, 2.4 mL). Next, 240 µL seed solution was injected into the growth solution, and was allowed to incubate at 30 °C for a period of 24 h. The resultant GNRs solution was then centrifuged and redispersed in water several times to remove the unbound excess surfactant. Finally, the GNRs were obtained and resuspended in water for further use.
2.3. Preparation of GNR–PEG
The protocol was adapted and modified from previous reports [14
]. In short, 90% thiolated mPEG (mPEG–SH) and 10% thiolated PEG–COOH (SH–PEG–COOH) were added to a solution of GNR and allowed to incubate for 2 h at room temperature before removing excess thiolated PEG by centrifugation to afford GNR-PEG. Three different molecular weights of PEG (400, 600, 1000) were implemented in this process.
2.4. Preparation of GNR–PEG–CDs Nanohybrids
The CDs were prepared using a modified protocol by Jiang et al. [44
] in which 0.5 g p
-phenylenediamine was dissolved in 20 mL of water at 180 °C for 12 h in a polytetrafluoroethylene-lined stainless autoclave and then purified using silica gel column chromatography after cooling to room temperature. The resultant CDs were dispersed in water and stored at 4 °C for the further use. Subsequently, 100 μL of 10 mM EDC and NHS were added to 10 mL of the GNR–PEG solution and mixed for 15 mins; then, 1 mL of CD solution was added. The solution was left to stir for 12 h at room temperature. In addition, the aqueous solution was dialyzed against 2000 mL of water for 48 h with 6 changes.
2.5. Measurement of Photothermal Performance
A MDL-III-808 nm model laser was used in the photothermal experiment, in which the light source was a NIR laser of 808 nm. The specific experimental steps and parameters were as follows: 1.5 mL of each of phosphate-buffered saline (PBS), an aqueous solution of CDs, GNR–PEG and GNR–PEG–CDs were introduced in a quartz cuvette and this was followed by irradiation treatments with the 808 nm laser, in sequence. The laser power output at the light source was 1.5 W cm−2. The temperature of the liquid in the quartz cuvette was recorded with a digital thermometer equipped with a thermocouple probe under irradiation. In addition, to prove the thermal and mechanical stability of the hybrid, we provide the electron microscopic images of the hybrid after irradiation and ultrasound for 10 min. An ultrasonic machine (KQ5200DE) (Kunshan Ultrasonic Instruments Co., Ltd., Kunshan, China) was used to perform the ultrasound.
HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells (1 × 104 cells per well) were seeded into a 96-well plate for 24 h at a humidified atmosphere maintaining 5% CO2 at 37 °C. Then, various concentrations of sample the solutions were added to replace the culture media and subsequently incubated at 37 °C. After further incubation for 24 h, the medium was replaced with 10% CCK-8 reagent solution and incubated for 2 h at 37 °C. The cell viability was calculated as the ratio of the absorbance of the wells. The absorbance at 450 nm was measured by a microplate reader (Multiskan FC, Thermo Scientific, Waltham, MA, USA).
2.7. Confocal Fluorescence Imaging Measurements
Confocal fluorescence imaging measurements were performed on HeLa cells with Hoechst staining. Hoechst is a nuclear dye of which optimal excitation wavelength is 350 nm, and the emitted wavelength is 460 nm. Briefly, HeLa cells (1 × 105 cells per well) were seeded onto a plate for 24 h at 37 °C, followed by incubation with pristine CDs and GNR–PEG–CDs for another 24 h. Subsequently, the cells were washed with PBS three times and stained with Hoechst. Subsequently, CDs and GNR–PEG–CDs were cultured with HeLa cells to perform confocal imaging measurements. A dual-channel imaging mode was used in this test. Channel I used a 405-nm wavelength laser as the excitation light to excite Hoechst, and the location information of the nuclei stained by Hoechst was collected. Channel II was stimulated by a laser of 485 nm wavelength to get information of CDs and GNR–PEG–CDs.
2.8. FLIM Measurements
The GNR–PEG–CDs sample was measured in terms of HeLa cells to observe the changes in fluorescence lifetime and imaging effect. In this test, the dual-channel imaging mode was used, that is, Channel I used the 405-nm wavelength laser as the excitation light, and an emission light of 482 nm (±35 nm) was collected; Channel II was stimulated by a laser with a 485-nm wavelength and an emission light of 550 nm (±49 nm) was collected.
2.9. Sample Characterization
Transmission electron microscopy (TEM) images and high-resolution (HR) TEM were obtained using a HT7700 biological transmission electron microscope (Hitachi, Tokyo, Japan) with an accelerating voltage of 100 kV, and a JEM-2100F high-resolution transmission electron microscope (JEOL, Tokyo, Japan) with an accelerating voltage of 200 kV. Fourier transform infrared (FT-IR) spectra were obtained by an Excalibur HE 3100 (Varian, Palo Alto, CA, USA) in the range from 4000 to 400 cm−1
. The ultraviolet (UV–vis) absorption spectra were obtained from a U-3000 (Hitachi, Tokyo, Japan). A Cary Eclipse spectrophotometer (Varian, Palo Alto, CA, USA) was used to record the fluorescence spectra and relative quantum yield (QY) values. Rhodamine B (RhB) was used as reference to measure the QY values of CDs and the hybrids. More details about the method can be found in Reference [44
] and the Supplementary Materials (SM)
. Confocal fluorescence imaging and fluorescence lifetime imaging were performed using confocal laser scanning microscopy (CLSM, Nikon, Tokyo, Japan) and a Nikon two-photon fluorescence lifetime imaging microscope (ARsiMP-LSM-Kit-Legend Elite-USX, Picoquant, Berlin, Germany).