Non-invasive imaging of breast cancer using RGDyK functionalized fluorescent carbonaceous nanospheres

Abstract

Fluorescent carbonaceous dots (CDs) have attracted much attention due to their unique properties. However, their application in non-invasive imaging of diseased tissues was restricted by the short excitation/emission wavelength and the poor targeting efficiency of CDs. In this study, CDs were prepared from sucrose and glutamic acid with a particle size of 57.5 nm. An obvious emission could be observed at 600 nm to 700 nm when excited at around 500 nm. This property enabled CDs with a capacity for deep tissue imaging with low background adsorption. RGD, a ligand which could target most tumor and neovasculature cells, was anchored onto CDs after PEGylation. The product, RGD–PEG–CDs could accumulate in MCF-7/ADR xenografts at high intensity, which was 1.65-fold higher than that of PEG–CDs. Furthermore, RGD–PEG–CDs showed good colocalization with neovasculature. Thus, RGD–PEG–CDs could be used for non-invasive MCF-7/ADR tumor imaging. CDs functionalized with other ligands may also be used as a non-invasive probe for many kinds of tumor imaging.

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Introduction

Fluorescent probes have been widely used for particle tracking and labelling, virus labelling, and small compound tracking.^1,2 Among the probes, fluorescent carbonaceous dots (CDs) have gained extensive attention due to not only their unique characteristics, such as green synthetic route, optical stability, good compatibility and low toxicity, but also their great potential for application in detection and catalysis.^3–6 To obtain CDs, many methods were developed, including thermal/hydrothermal oxidation, electrochemical synthesis and microwave/ultrasonic synthesis.⁴ Among which, one-step hydrothermal/thermal treatment methods have been widely used in producing CDs from various biomaterials, such as silk, juice, glucose, or other synthetic polymers.^7–9 However, Most of these produced CDs showed short excitation/emission wavelength (lower than 500 nm for excitation wavelength and 600 nm for emission wavelength), which restricted the application in bio-imaging of diseases such as tumor, especially in the non-invasive whole body imaging, owing to the high background adsorption.^10,11 Thus preparing CDs with near-infrared (NIR) fluorescence spectrum is critical for expanding the application of CDs to non-invasive whole-body imaging. Unfortunately, few CDs displayed strong emission intensity when changed the excitation wavelength from blue or green to red or NIR region and no studies have reported non-invasive whole body imaging of diseased tissue using these CDs.^3,12–14

Besides the property of CDs, the physical barrier of the diseased tissue is another obstacle that restricted the application in non-invasive imaging, and rear studies have used CDs in biological imaging.^15,16 Tumor is one of the biggest threats for human being. Multidrug resistance further restricted distribution of drugs or probes in the tumor cells, resulting poor tumor treatment outcome and low tumor/normal tissue ratio.^17,18 Nanoparticulated systems could passive target to these tumors utilizing enhanced permeability and retention (EPR) effect.^19–23 Surface modification of these systems with tumor homing ligands could further improve the accumulation in tumor site and the internalization by tumor/tumor associated cells, leading to better drug delivery efficiency and higher imaging ability.^24–27 Integrin, such as α_vβ₃, is overexpressed on many kinds of tumor cells and tumor neovascular cells.^28,29 The corresponding ligand, RGD, was widely used as a specific ligand to improve the tumor and neovasculature targeting efficiency of nanoparticulated systems.^30–32 Thus, RGD was selected to be anchored CDs for non-invasive cancer imaging.

In this study, a new method was developed to produce CDs with relatively long excitation/emission wavelength. The characteristics of the CDs were evaluated by several experiments. To evaluate the targeting efficiency of RGD modified CDs (RGD–PEG–CDs), a kind of multidrug resistance breast cancer cell line, MCF-7/ADR, was used in this study.

Materials and methods

Materials

RGDyK–NH₂ was synthesized by GL Biochem Co.,Ltd. (Shanghai, China). PEG–NH₂ (Mw = 5000) and HOOC–PEG–NH₂ (Mw = 5000) were purchased from Seebio (Shanghai, China). Glutamic acid and sucrose were purchased from Sinopharm Chemical Reagent (Shanghai, China). Dulbecco's Modified Eagle Medium (high glucose) cell culture medium (DMEM) and FBS were purchased from Life Technologies (NY, USA). Cell culturing dishes and plates were purchased from Cyagen Biosciences Inc. (Guangzhou, China). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) were purchased from Sigma (MO, USA). DAPI was purchased from Beyotime (Haimen, China). Rabbit anti-CD31 antibody was purchased from Abcam (Hong Kong, China). Alexafluor 594-conjugated donkey anti rabbit antibody was purchased from Jackson Immunoresearch Laboratories, Inc. (PA, USA). LysoTracker red was purchased from Life Technologies (NY, USA). HUVEC was purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). MCF-7/ADR cells were kindly donated from school of Pharmacy, Peking University (China).

BALB/c nude mice (male, 4–5 weeks, 18–22 g) were obtained from the Animal Research Center of Sichuan University (Chengdu, China) and maintained under standard housing conditions. All animal experiments were carried out in accordance with protocols evaluated and approved by the ethics committee of Sichuan University.

Preparation and characterization of RGD–PEG–CDs

As shown in Fig. 1, a dry flask (100 mL) was heated to 250 °C, and then 1 g of glucose was added. One minute later, 0.1 g of glutamic acid was added and the heater was removed. When the temperature of the flask decreased to approximately 50 °C, 5 mL of deionized water was added to suspend the produced CDs.

Fig. 1 (A) Elucidation of the preparation procedure of RGD–PEG–CDs. (B) Elucidation of transportation and uptake procedure of RGD–PEG–CDs.

To prepare PEGylated CDs (PEG–CDs), CDs were activated by EDC/NHS for 0.5 h, followed with the adding of PEG–NH₂. After 6 h incubation, the PEG–CDs could be obtained after dialysis (MW = 10000D). To prepare RGD–PEG–CDs, CDs was firstly reacted with HOOC–PEG–NH₂ then the production was further activated by EDC/NHS and RGDyK–NH₂ was added into the solution. Another 6 h later, the RGD–PEG–CDs could be obtained after dialysis (MW = 10000D).

The particle size and morphology were observed through transmission electron microscope (TEM) (H-600, Hitachi, Japan). The hydrated particle size and zeta potential were determined by a zeta/particle sizer (Malvern, NanoZS, Germany). To determine the surface density of PEG, the unconjugated PEG was removed by dialysis and recovered by freeze-drying. Then the unconjugated PEG was dissolved and determined by barium chloride method. Briefly, 0.6 mL of PEG contained solution was added with 1.2 mL of 5% barium chloride. After reaction, 60 μL of 0.025 mol L⁻¹ I₂ was added and absorbance at 385 nm was determined. The surface density of RGD was further determined, the unconjugated RGD was removed by dialysis and recovered by freeze-drying. Then the unconjugated RGD was dissolved and determined by BCA method. Briefly, 0.1 mL of RGD contained solution was added with 2 mL of BCA working regent. The solution was incubated at 60 °C for 30 min and determined at 562 nm. Fluorescence spectrum was evaluated using a RF-5301 PC spectrofluorophotometer (Shimadzu, Japan). X-ray photoelectron spectroscopy (XPS) experiments were performed on an AXIS Ultra DLD (Kratos UK) with Mg Ka radiation (hν = 1486.6 eV), with a chamber pressure of 2.2 × 10⁻⁹ Torr. The source power and high voltage was set at 150 W and 15 kV, and pass energies of 40 eV for survey scans was used. The analysis spot size was 300 × 700 μm. The data was analyzed by PHI-MATLAB software with C1s = 284.6 eV as a benchmark for the binding energy correction. Fluorescence quantum yields were evaluated using a RF-5301 PC spectrofluorophotometer (Shimadzu, Japan).

Serum stability and safety

The stability of CDs was evaluated in PBS with different concentrations of FBS. CDs, PEG–CDs and RGD–PEG–CDs were suspended in 0%, 10% or 50% FBS and incubated in a 37 °C incubator. The absorption at 560 nm was determined by a microplate reader (Multiskan MK3, Thermo, USA) at 0, 1, 2, 3, 4, 6, 8, 12 and 24 h.

To evaluate the hemocompatibility, whole blood was collected from mice using heparin as anticoagulant. After centrifugation at 1500 rpm for 5 min, the red blood cells were resuspended in PBS at the final density of 2%. Different concentrations of CDs, PEG–CDs and RGD–PEG–CDs were added into cell suspension and incubated at 37 °C for different periods of time.³³ After incubation, cell suspension was centrifuged at 1500 rpm for 5 min, and the adsorption of supernatant at 560 nm was determined by a microplate reader (Thermo Scientific Varioskan Flash, USA). 1% of Triton X-100 was used as positive control and PBS was used as negative control.

The cytotoxicity of different formulations was evaluated by MTT assay. HUVEC cells and MCF-7/ADR cells (2 × 10⁴ cells per mL) were seeded in 96-well plates. Twenty-four hours later, CDs, PEG–CDs and RGD–PEG–CDs were added into wells with a serial concentrations ranging from 5 mg mL⁻¹ to 1 μg mL⁻¹. Twenty-four hours later, 100 μL of MTT solution was added into each well and incubated for 4 h. After replacing the medium with DMSO, the 490 nm absorption was observed by a microplate reader (Thermo Scientific Varioskan Flash, USA).

In vitro cellular uptake and subcellular localization

MCF-7/ADR cells were seeded in 6 cm glass-bottom dishes at a density of 1 × 10⁴ cells per mL and incubated at 37 °C for 24 h. After 5 min incubation in PBS, the cells were treated with different concentrations of PEG–CDs and RGD–PEG–CDs for different times. After incubation, the cells were washed, fixed and stained with 0.5 μg mL⁻¹ of DAPI. Images were captured with a confocal microscope (LSM710, Carl Zeiss, Germany).

To determine the subcellular colocalization with endosomes, MCF-7/ADR cells were seeded into dishes as described above and incubated with 125 μg mL⁻¹ of PEG–CDs or RGD–PEG–CDs for 0.25 h or 4 h. Thirty minutes before the incubation ended, Lysotracker Red (100 nmol L⁻¹) was added into the wells. After incubation, the cells were washed, fixed and stained with 0.5 μg mL⁻¹ of DAPI. Images were captured with a confocal microscope (LSM710, Carl Zeiss, Germany).

Uptake mechanism

MCF-7/ADR cells were seeded in 12-well plates at a density of 5 × 10⁴ cells per mL and incubated for 24 h. After a 20 min pre-incubation in DMEM, the cells were treated with 125 μg mL⁻¹ of PEG–CDs or RGD–PEG–CDs at the presence of various inhibitors for 1 h at 37 °C, respectively: PBS (control), 20 μg mL⁻¹ chlorpromazine, 10 μg mL⁻¹ filipin, 0.1% w/v sodium azide, 20 μg mL⁻¹ nocodazole, 25 μg mL⁻¹ colchicine, 400 μg mL⁻¹ poly-L-lysine and 200 μg mL⁻¹ RGDyK. Cells incubated with PEG–CDs or RGD–PEG–CDs at 4 °C were also used to determine the uptake mechanism. After washing with ice-cold PBS 3 times, the cells were digested and the mean fluorescence intensity was observed by flow cytometry (FACS Aria Cell Sorter, BD, USA).

In vivo and ex vivo imaging and slice distribution

The MCF-7/ADR xenografts bearing mice were established through injecting of 1 × 10⁷ MCF-7/ADR cells into the right flank of the mice as described previously.³⁴ The length and width of the tumor were determined every two days using a caliper, and the volume was calculated using following equation: tumor volume = (length × width²)/2. After the volume of the tumor was approximately 100 mm³, mice were i.v. administered 50 mg kg⁻¹ of PEG–CDs or RGD–PEG–CDs through tail vein. Then the whole body fluorescent distribution was observed through an in vivo imaging system (IVIS Spectrum, Caliper, USA) 5, 15, 30, 60, 120, 180 and 240 min after injection. Then, the mice were sacrificed, and their tissues were subjected to ex vivo fluorescence imaging. After fixed with 4% paraformaldehyde, organs were further dehydrated by 15% sucrose followed with 30% sucrose. Consecutive frozen sections of 10 μm thicknesses were prepared and then stained by 0.5 μg mL⁻¹ DAPI for 5 min. For tumor slices, microvessel was stained with rabbit anti-CD31 antibody (1 : 200) followed with Alexafluor 594 conjugated donkey anti rabbit antibody according to previous established procedure.³⁵ The distribution of fluorescence was observed by a confocal microscope (LSM710, Carl Zeiss, Germany).

Statistical analysis

Data were presented as mean ± SD. Statistical differences in the experiments were determined by the Student t test.

Results and discussion

Characterization of RGD–PEG–CDs

CDs were prepared from sucrose and glutamic acid through thermal treatment. The hydrated particle size of CDs was 57.5 nm with a polydispersity index of 0.147 (Fig. 2A), while the zeta potential was −23.5 mV. According to TEM (Fig. 2D), the particles were well dispersed with spherical shape. There were plenty of carboxyl group on surface of CDs,¹⁶ which could be used for PEGylation and other modification. After modification with PEG and RGD, the density of PEG and RGD was 4.04 ± 0.02 μg mg⁻¹ CDs and 1.91 ± 0.02 μg mg⁻¹ CDs respectively. The hydrated particle sizes of PEG–CDs and RGD–PEG–CDs were changed to 63.2 nm and 67.4 nm (Fig. 2B and C. Table 1). The elevation in particle sizes was contributed by the surface modification. However, the zeta potential was not considerably changed.

Fig. 2 DLS data of CDs (A), PEG–CDs (B) and RGD–PEG–CDs (C). (D) TEM image of CDs, bar represents 50 nm, inner picture was semi-quantitative data of TEM image. (E) Fluorescent spectrum of CDs at excitation wavelength from 340 nm to 560 nm.

Table 1 Particle size and zeta potential of PEG–CDs and An-PEG–CDs (n = 3)

Formulations	Particle size (nm)	PDI	Zeta potential (mV)
CDs	57.5 ± 1.4	0.147 ± 0.078	−23.5 ± 2.7
PEG–CDs	63.2 ± 3.1	0.181 ± 0.105	−20.3 ± 1.1
RGD–PEG–CDs	67.4 ± 4.6	0.197 ± 0.028	−22.8 ± 1.5

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The highest absorbance of CDs was 340 nm according to UV-vis spectrum (ESI Fig. S1†). Interestingly, when we increased the excitation wavelength of the CDs from 340 nm to 440 nm, both the emission wavelengths and fluorescent intensity increased (Fig. 2E), which was significantly different from previous reported CDs.^9,36 The highest emission wavelength was 552 nm while the excitation wavelength was 440 nm, which was longer than recently reported CDs possessing highest excitation/emission wavelength of 420 nm and 520 nm.¹² Additionally, further increasing the excitation wavelength could also expand the emission wavelength, displaying a broad excitation and emission range. Although the intensity peak of emission decreased with increasing of excitation wavelengths from 440 nm to 560 nm, there were still apparently emission peaks. The fluorescent property of the CDs was useful for in vivo imaging because longer excitation/emission wavelength could endow the CDs with the ability to penetrate deeper tissues. The quantum yields of CDs and RGD–PEG–CDs were 37.5% and 21.7% respectively, which were higher than many other reports.^15,37

Surface state of CDs was further characterized by XPS. The XPS overall spectrum showed three main peaks at 284.6, 399.7, 531.7 eV (ESI Fig. S2A†), contributed to the C1s, N1s, O1s respectively. The C1s spectrum (ESI Fig. S2B†) displayed four peaks at 284.60, 285.11, 286.06, 288.31 eV, which were attributed to C–C, C–N, C–O and C=N/C=O groups, respectively.^16,38 The N1s spectrum (ESI Fig. S2C†) displayed three peaks at 399.05, 399.81 and 400.85 eV, which were attributed to C–N–C, C–N and N–H groups, respectively.³⁹ The O1s spectrum (ESI Fig. S2D†) exhibited two peaks at 531.56 and 532.80 eV, which were owing to C=O and C–OH/C–O–C groups, respectively.¹⁶ There results demonstrated that the surface of CDs possessed plentiful oxygen and nitrogen functional groups, which was consistent with previous studies and useful for further modification. Similar peaks were observed in the spectrum of PEG–CDs (ESI Fig. S2E–H†). The concentration of C in the surface of PEG–CDs was higher than that of CDs, contributing to the higher C concentration in PEG, suggesting the PEG was successfully anchored onto CDs. Furthermore, the spectrum of RGD–PEG–CDs also showed the similar peaks (ESI Fig. S2I–L†). The concentration of C and O in the surface RGD–PEG–CDs was close to that of PEG–CDs, while the concentration of N was relatively higher than that of PEG–CDs, indicating that the RGD was successfully conjugated onto PEG–CDs. Until recently, the actual photoluminescence mechanism of CDs remained unclear. It supposed that the surface-defect based mechanism might contribute to the luminescence of CDs.⁷ In this study, amine groups were doped in the surface of CDs, which may also contribute to the luminescence of CDs.³⁶

Evaluation of CD serum stability, hemocompatibility and cytotoxicity

In order to be used in bio-systems, the particles should be stable in the blood circulation. Thus serum stability was evaluated by incubation with different concentrations of serum for different periods of time. The adsorption of protein onto particles could increase the turbidity leading to the elevating of absorption at 560 nm (Fig. 3). The absorption of CDs at 560 nm significantly increased during the increase of incubation time, suggesting the CDs could considerably adsorb serum protein at a time-dependent manner. Comparatively, after PEGylation, the absorption of both PEG–CDs and RGD–CDs were significantly lower than that of CDs, demonstrating the PEGylation could inhibit the protein adsorption and increase the serum stability.⁴⁰ Incubation of CDs, PEG–CDs and RGD–PEG–CDs with different concentrations of serum also demonstrated CDs could adsorb significantly more proteins than the amount adsorbed by PEG–CDs and RGD–PEG–CDs (Fig. 3B).

Fig. 3 Serum stability of CDs, PEG–CDs and RGD–PEG–CDs in serum. (A) Time related absorption of 100 μg mL⁻¹ of CDs, PEG–CDs and RGD–PEG–CDs after incubation with 50% FBS. (B) Absorption of 100 μg mL⁻¹ of CDs, PEG–CDs and RGD–PEG–CDs after 24 h incubation with different concentrations of FBS. *p < 0.05.

Hemocompatibility was carried out using fresh 2% blood red cells. Although the hemolysis rate increased over time, the rates of PEG–CDs and RGD–PEG–CDs were significantly lower than that of CDs (Fig. 4A). For example, at the concentration of 5 mg mL⁻¹, the hemolysis rate of CDs was 21.1% after 12 h incubation (Fig. 4B). However, the rates of PEG–CDs and RGD–PEG–CDs were only 13.6% and 14.6% respectively, which were similar to that of PBS. The images of blood cells after 8 h incubation also demonstrated the lower hemolysis rate of PEG–CDs and RGD–PEG–CDs compared to CDs (Fig. 4C). These results indicated the PEGylation could improve the compatibility, which was consistent with previous results.¹⁶

Fig. 4 Hemocompatibility of CDs, PEG–CDs and RGD–PEG–CDs. (A) Time-related hemolysis rates of 5 mg mL⁻¹ of CDs, PEG–CDs and RGD–PEG–CDs. *p < 0.05 vs. PEG–CDs and RGD–PEG–CDs. (B) Concentration related hemolysis rates of different concentrations of CDs, PEG–CDs and RGD–PEG–CDs incubated with red blood cells for 8 h. (C) Image of red blood cells incubated with 5 mg mL⁻¹ of CDs, PEG–CDs and RGD–PEG–CDs for 8 h.

MTT assay was performed on both HUVEC and MCF-7/ADR cells. The cell viabilities of both MCF-7/ADR cells and HUVEC did not significantly decrease after 24 h incubation with CDs, PEG–CDs or An-PEG–CDs at concentrations lower than 1 mg mL⁻¹ (Fig. 5). Thus it could be concluded that CDs displayed low toxicity, which was consistent with previous reports.¹¹

Fig. 5 MTT assay of different concentrations of CDs, PEG–CDs and RGD–PEG–CDs on MCF-7/ADR cells (A) and HUVEC cells (B).

Cellular uptake

To determine their potential application in tumor imaging, in vitro cellular uptake was carried out. The fluorescent intensity of cells increased when elevating the concentration of PEG–CDs or expanding the incubation time, suggesting the uptake of PEG–CDs by MCF-7/ADR cells was concentration- and time-dependent (Fig. 6A and B), which was consistent with other particles.^11,41 RGD modification could apparently increase the cellular uptake compared to PEG–CDs, owing to the highly expressed α_vβ₃ on tumor cells such as MCF-7/ADR cells,²⁹ which was the specific receptor of RGD.⁴² These results demonstrated RGD–PEG–CDs could target to MCF-7/ADR cells, thus they might be used for breast tumor imaging. Additionally, the expression of P-gp on MCF-7/ADR cells lead to the cells hard to be killed by chemotherapeutics.⁴³ In this study, it was showed MCF-7/ADR cells could obviously uptake of RGD–PEG–CDs with high intensity, suggesting the RGD–PEG–CDs could be served as carriers to deliver chemotherapeutics into MCF-7/ADR cells to conquer the drug resistance.

Fig. 6 Cellular uptake of PEG–CDs and RGD–PEG–CDs by MCF-7/ADR cells. (A) Concentration related uptake by MCF-7/ADR cells after 1 h incubation, bar represents 10 μm. (B) Time related uptake by MCF-7/ADR cells after incubation with 125 μg mL⁻¹ of PEG–CDs and RGD–PEG–CDs, bar represents 20 μm.

Uptake mechanism

To determine the uptake pathways that involved in the uptake procedure of PEG–CDs and RGD–PEG–CDs, various inhibitors were used. Obviously, the uptake of both PEG–CDs and RGD–PEG–CDs by MCF-7/ADR cells was energy-dependent, because the uptake was significantly reduced to 39.3% and 46.1% respectively after energy depletion by sodium azide and to 41.3% and 31.1% respectively after incubation at 4 °C (Fig. 7). Colchicine and nocodazole, the inhibitors of macropinocytosis, significantly decreased the cellular uptake of PEG–CDs to 31.2% and 40.6% respectively and decreased the cellular uptake of RGD–PEG–CDs to 33.1% and 38.9% respectively, suggesting that macropinocytosis was involved in the uptake of both PEG–CDs and RGD–PEG–CDs. Filipin, a special inhibitor of caveolae-mediated endocytosis,⁴⁴ significantly decreased the uptake of PEG–CDs and RGD–PEG–CDs to 59.5% and 60.7% respectively, suggesting caveolae-mediated endocytosis was also considerably involved in the uptake procedure. The positive charge inhibitor PLL,⁴⁵ could only inhibited the uptake of PEG–CDs and RGD–CDs to approximately 70.3% and 67.0% respectively, indicating the internalization of these two particles were not mediated by charge interaction. Additionally, free RGD significantly inhibited the uptake of RGD–PEG–CDs to 41.8%, while RGD could not considerably inhibit the uptake of PEG–CDs, suggesting the internalization of RGD–PEG–CDs was mediated by specific receptor of RGD, which was consistent with previous studies.⁴⁶

Fig. 7 MCF-7/ADR uptake of PEG–CDs and RGD–CDs at the presence of various inhibitors.

In vivo and ex vivo imaging

In vivo imaging is one of the most common and powerful non-invasive diagnosis methods. After intravenous injection of PEG–CDs and RGD–PEG–CDs, obvious fluorescent distribution could be observed in the whole body especially in the liver (Fig. 8A), because the liver is the main organ to eliminate foreign materials from the blood circulation. However, the distribution in MCF-7/ADR xenografts was different between PEG–CDs and RGD–PEG–CDs. The highest distribution of PEG–CDs in tumor was observed at 30 min after intravenous injection, and then the fluorescent intensity gradually decreased during the time expanding, which was contributed to EPR effect.¹⁹ Comparatively, the distribution of RGD–PEG–CDs in tumor was much higher than that of PEG–CDs at almost all time points, suggesting functionalization with RGD could improve the tumor targeting efficiency of PEG–CDs, contributing to the targeting effect of RGD that mediated by highly expressed α_vβ₃ on tumor,⁴⁷ which was consistent with in vitro cellular uptake results. Ex vivo imaging of various tissues further demonstrated the distribution of RGD–PEG–CDs was much higher than that of PEG–CDs (Fig. 8B), and the former was approximately 1.7-fold higher than the latter (Fig. 8C). However, the distribution of RGD–PEG–CDs in lung and kidney was also higher than that of PEG–CDs (Fig. 8B and C). These results demonstrated that nanoscale CDs with PEG modification possess photolumiscence property, which was due to the quantum confinement effect.⁴⁸ After modification with PEG, it could reduce the adsorption by plasma proteins and significantly improve the cycle time. However, it is insufficient to deliver the CDs to tumor site after conjugation with PEG. To further improve the delivery and target efficiency, it is necessary to conjugate PEG–CDs with specific ligand. Thus, RGD, a specific ligand targeted to integrin receptor was conjugated to the PEG–CDs, which could significantly deliver the CDs to tumor site and improve bio-imaging efficiency.²⁵ In addition, the relative long excitation and emission wavelength was also important for the application of CDs on bio-imaging.²

Fig. 8 In vivo and ex vivo imaging of PEG–CDs and RGD–PEG–CDs treated MCF-7/ADR xenografts bearing mice. (A) in vivo imaging of MCF-7/ADR xenografts bearing mice at different times after injected with PEG–CDs and RGD–PEG–CDs. (B) Ex vivo imaging of tissues at 4 h after injected with PEG–CDs and RGD–PEG–CDs. (C) Semi-quantitative fluorescent intensity of different kinds of organs. *p < 0.05.

Tissue distribution

Microvessels were stained using anti-CD31 antibody to elucidate the targeting ability of RGD–PEG–CDs. The distribution of PEG–CDs in tumor was relative low and showed poor colocalization with microvessel (Fig. 9). In contrast, the RGD–PEG–CDs showed well colocalization with microvessel, suggesting RGD–PEG–CDs could target the microvessel, which overexpressed α_vβ₃. Thus RGD–PEG–CDs could be used for delivering anti-angiogenesis drugs to the endothelial cells of microvessel, resulting in disrupting of blood supply to tumor.

Fig. 9 Distribution of PEG–CDs and RGD–PEG–CDs in tumor slices. neovessels were stained by anti-CD31 antibody. Blue represents nuclei which stained by DAPI and bar represents 100 μm.

The distribution in slices of normal tissues were similar to the results of ex vivo imaging (Fig. 10). In most tissues, there was no obvious difference between PEG–CDs and RGD–PEG–CDs. But in kidney, the distribution of RGD–PEG–CDs was higher than that of PEG–CDs, which was consistent with previous studies that showed higher accumulation in kidney of RGD modified probes.⁴⁹ Additionally, the modification of RGD also could elevate the distribution in brain, which was due to the expression of α_vβ₃ on brain endothelial cells.⁴⁶

Fig. 10 Distribution of PEG–CDs and An-PEG–CDs in normal tissue slices. Bar represents 100 μm.

Conclusion

In this study, a kind of CDs was prepared with long excitation/emission wavelength. After surface modification, RGD–PEG–CDs possessed well plasma stability, well hemocompatibility and low cytotoxicity. In vitro, RGD–PEG–CDs could be taken up by MCF-7/ADR cells at a density higher than PEG–CDs through a time-, concentration- and energy-dependent manner. The uptake of RGD–PEG–CDs was involved in endosomes, mainly contributing to macropinocytosis and caveolae mediated pathway. In vivo, RGD–PEG–CDs could target to MCF-7/ADR xenografts, which was significantly better than PEG–CDs. Fluorescent distribution in tumor slices further demonstrated the RGD–PEG–CDs could target to microvessel, owing to the highly expressed α_vβ₃ on microvessel. These results demonstrated RGD–PEG–CDs were a promising probe for MCF-7/ADR tumor imaging.

Acknowledgements

The work was granted by National Natural Science Foundation of China (81402866, 81301974) and the Sichuan University Starting Foundation for Young Teachers (2014SCU11044).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra00099h

‡ Contributed equally to this work.

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