Real-time self-tracking of an anticancer small molecule nanodrug based on colorful fluorescence variations

Abstract

A new self-tracking nanoscale drug delivery system has been developed to monitor drug delivery and release in tumor cells. The small molecule nanodrug was constructed via the conjugation and self-assembly of two widely used anticancer drugs, hydrophilic irinotecan (Ir) which displays blue fluorescence and hydrophobic doxorubicin (DOX) which displays red fluorescence, which produced colorful fluorescence variations during drug delivery and release in cells. Owing to the fluorescence resonance energy transfer (FRET), the Ir–DOX conjugate emitted strong red fluorescence when excited at a short wavelength. Benefiting from its amphiphilicity, the Ir–DOX conjugate self-assembled into micelles in aqueous medium and the fluorescence was quenched due to aggregation-caused quenching (ACQ). No obvious red or blue fluorescence was observed during a 12 h cell incubation with Ir–DOX, indicating that Ir–DOX entered cells in the form of micelles rather than free conjugate or free drugs. With increasing incubation time, the breaking of the Ir–DOX linkage resulted in the release of both free drugs, leading to the recovery of dual-color fluorescence. In vitro cytotoxicity studies showed that the Ir–DOX micelles could overcome the multidrug resistance (MDR) of tumor cells, resulting in a prominent growth inhibition against cancer cell proliferation. The Ir–DOX small molecule nanodrug provides a new design for real-time self-tracking of carrier-free and probe-free drug delivery systems in cancer treatment.

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Introduction

Cancer is the leading cause of death in many countries.¹ As an efficient method for cancer treatment, chemotherapy has been widely used in clinics.^2–4 However, direct administration of small molecular anticancer drugs suffers from several limitations, such as low bioavailability, nonspecific cytotoxicity, adverse side effects, rapid drug elimination, and severe multidrug resistance (MDR).⁵ With the emergence of nanotechnology, various nanoscale drug-carriers including liposomes, polymeric micelles, ceramic materials and metallic nanoparticles have been employed to solve these limitations.^6–11 The carrier-based delivery systems display better therapeutic efficacy and fewer side effects than free drugs. However, most of the drug-carriers have no therapeutic efficacy, which brings the problems of lower drug loading and unpredicted degradation and metabolism of the carriers in the body.^12,13 To avoid these annoying problems from drug-carriers, we have developed a carrier-free drug self-delivery system based on the self-assembly of an amphiphilic drug–drug conjugate (ADDC).^14–17 Direct conjugation of hydrophilic and hydrophobic anticancer drugs via a biodegradable bond or small link molecule results in the formation of pure drug micelles in aqueous solution, which makes the anticancer drugs deliver themselves into the sites of action without the help of any carriers. Once a drug–drug conjugate is biodegraded in the tissues or cells, free anticancer drugs are released to work on the diseased regions simultaneously, resulting in a high anticancer efficacy.

To further improve therapeutic effects, the monitoring and understanding of drug delivery and release behaviors of carrier-free small molecule nanodrugs is important. Among various monitoring techniques, fluorescence is one of the most powerful tools, and exhibits high sensitivity, multicolor labeling, great versatility, and a real-time monitoring ability.^18–21 Although fluorescent labeling can be readily achieved by grafting fluorescent probes onto conventional carrier-based drug delivery systems, it is not suitable for carrier-free small molecule nanodrugs. Apparently, the introduction of fluorescent probes onto a drug–drug conjugate will change the physical/chemical properties and self-assembly behavior of the small molecule nanodrugs. Therefore, real-time tracking of the delivery and release of small molecule nanodrugs becomes a challenge.

We note that some anticancer drugs show bright fluorescence by themselves, including the most common anticancer drugs, doxorubicin (DOX), camptothecin and their analogs,^22–27 providing a simple and feasible route for tracking drug delivery without the need of fluorescent labeling during tumor treatment. Especially, it can be imagined that if two fluorescent anticancer drugs are mixed or conjugated together, the colorful fluorescence variations can be designed and tracked during drug delivery and release. Following this idea, here we construct a new self-tracking amphiphilic drug–drug conjugate, which enables monitoring of the drug delivery and release in the tumor cells (Scheme 1). In details, the hydrophilic anticancer drug irinotecan (Ir) which displays blue fluorescence and hydrophobic anticancer drug DOX which displays red fluorescence are conjugated together via a simple carbamate bond, since the combined chemotherapy of Ir and DOX demonstrates a potential clinical benefit.^28,29 The excitation and emission wavelengths of Ir and DOX are at 370/450 nm and 480/590 nm, respectively, so there is a great overlap between the emission of Ir (donor) and the excitation of DOX (acceptor), leading to an efficient fluorescence resonance energy transfer (FRET). Thus, the Ir–DOX conjugate emits a strong red fluorescence when excited with a short wavelength in a good solvent (DMSO). Once the amphiphilic Ir–DOX conjugate self-assembles into micelles in aqueous medium, the fluorescence reduces significantly due to the serious π–π stacking of the rigid planar chromophores, i.e. aggregation-caused quenching (ACQ).^30–33 After cell internalization and intracellular responsiveness, both free Ir and DOX drugs are released from the Ir–DOX micelles, resulting in the appearance of strong blue Ir fluorescence and red DOX fluorescence. Benefitting from these fluorescence features, the intracellular drug delivery and release behaviors of the small molecule nanodrugs can be easily tracked by observing the fluorescence variations of the two anticancer drugs.

Scheme 1 Ir–DOX conjugate and the construction of the self-assembled micelles for self-tracking cancer therapy.

Experimental section

Materials

4-Nitrophenyl chloroformate (NPC, 98%) was purchased from Adams China Co., Ltd. Ir was obtained from Dalian Meilun Biology Technology Co., Ltd. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology Corporation. Pyridine (99.5%) was purchased from J&K Chemical Ltd. Triethylamine (TEA) was used as received without further purification. N,N-Dimethylformamide (DMF) and dichloromethane (CH₂Cl₂) were refluxed with calcium hydride and distilled before use.

Characterization

Nuclear magnetic resonance (NMR). ¹H and ¹³C NMR spectra were recorded using a Varian Mercury Plus 400 MHz spectrometer with dimethyl sulfoxide-d₆ (DMSO-d₆) as a solvent at 298 K. The chemical shifts were referenced to the residual peaks of the deuterated solvent: DMSO-d₆ (2.48 ppm).

Liquid chromatography-high resolution mass spectrometry (LC-HRMS). LC-HRMS was performed using a Waters ACQUITY UPLC system which consisted of a sample manager and a binary solvent delivery manager, coupled with a Waters Q-TOF Premier Mass Spectrometer (Waters Corporation, Milford, MA).

Fourier transform infrared (FTIR) spectroscopy. FTIR spectra were recorded using a Perkin Elmer Paragon 1000 spectrophotometer via a potassium bromide sample holder method.

Ultraviolet-visible (UV-Vis) absorption spectra. UV-Vis measurements were performed on a Thermo Evolution 300 UV-Vis spectrometer in the range of 200–700 nm.

Fluorescence spectra. The fluorescence measurements were carried out using a Perkin Elmer LS 50B fluorescence spectrometer.

Transmission electron microscopy (TEM). TEM tests were obtained using a JEOL JEM-100CX-II instrument at a voltage of 200 kV. Samples were prepared by drop-casting nano-aggregate solutions onto carbon-coated copper grids and then freeze-drying under vacuum before the measurements.

Dynamic light scattering (DLS). DLS measurements were performed using Malvern Zetasizer Nano ZS90 apparatus equipped with a 4.0 mW He–Ne laser operated at λ = 633 nm. All samples were tested at room temperature and a scattering angle of 90°.

Synthesis of Ir–DOX conjugate

The Ir–DOX conjugate was prepared by the conjugation of Ir and DOX via a carbamate linkage. The details of the synthesis and characterization are described in the ESI.†

Preparation of Ir–DOX micelles

Briefly, 4 mg of Ir–DOX was dissolved in 2 mL of DMSO and stirred uniformly at room temperature. Then, 3 mL of deionized water was added dropwise into the solution and stirred slightly for 2 h. After that, the solution was dialyzed in deionized water for 24 h (MWCO = 1000 g mol⁻¹) to remove the DMSO, during which time the water was renewed every 4 h. The appearance of turbidity in the aqueous solution indicated the formation of micelles.

Measurements of critical aggregation concentration (CAC)

The CAC values were calculated by measuring the fluorescence intensity of encapsulated Nile red at different Ir–DOX concentrations. A known amount of Nile red solution was added to a series of vials along with the Ir–DOX solution to give a final concentration of 6.0 × 10⁻⁷ M. The fluorescence intensities of all of the solutions were recorded using a Perkin Elmer LS 50B fluorescence spectrometer at 550 nm.

In vitro drug release study

A total of 2 mL of the Ir–DOX micelles was transferred into a dialysis bag (MWCO = 1000 g mol⁻¹). The solution was immersed in 50 mL of phosphate buffer (pH 7.4, pH 2.0) or acetate buffer (pH 5.0) solution and stirred slightly at 37 °C. At predetermined time intervals, 2 mL of the external buffer was withdrawn and replaced with an equal volume of fresh medium. The amount of released Ir and DOX was analyzed by UV measurement (Thermo Evolution 300 UV-Vis spectrometer, excitation at 370 nm/480 nm).

Cell culture

MCF-7/ADR cells (multidrug resistant human breast cancer cells) were cultured in DMEM with 10% FBS and antibiotics (50 units per mL of penicillin and 50 units per mL of streptomycin) at 37 °C in a humidified atmosphere containing 5% CO₂.

In vitro cytotoxicity assay

The anticancer activities of the Ir–DOX micelles against MCF-7/ADR cells were estimated using a MTT viability assay. In the MTT assay, the cells were seeded into 96-well plates with a density of 8 × 10³ cells per well in 200 μL of culture medium. The cell growth medium was removed after 24 h of incubation, and replaced with 200 μL of a medium containing serial dilutions of the Ir–DOX micelles, free Ir and DOX, or the Ir/DOX mixture. The cells were grown for another 72 h. Then, 20 μL of 5 mg mL⁻¹ MTT assay stock solution in PBS was added to each well. After incubating the cells for another 4 h, the medium containing the unreacted dye was removed carefully and 200 μL of DMSO was added to each well to dissolve the obtained blue formazan crystals. The absorbance was recorded using a BioTek Synergy H4 hybrid reader at a wavelength of 490 nm.

Cellular uptake of the Ir–DOX micelles

Flow cytometry was used to study the uptake of the Ir–DOX micelles into MCF-7/ADR cells. MCF-7/ADR cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well with 2 mL of DMEM and were allowed to attach at 37 °C for 24 h. Then, the diluted solution of the Ir–DOX micelles with the DMEM culture medium at a final concentration of 20 μM was added to different wells and the cells were incubated at 37 °C for 0.5, 1, 2, and 4 h. After that, the cell growth media were removed and the cells were rinsed with cold PBS three times and treated with trypsin to prepare for flow cytometry analysis. Data for 1.0 × 10⁴ gated events were recorded and analysis was performed using the measurements of a BD LSRFortessa flow cytometer.

Intracellular monitoring of drug release for the Ir–DOX micelles

MCF-7/ADR cells were seeded in 6-well plates at 5.0 × 10⁵ cells per well in 2 mL of complete DMEM and cultured for 24 h. The medium was carefully removed and this was followed by adding diluted Ir–DOX micelle solutions with DMEM at a final concentration of 20 μM. The cells were incubated at 37 °C at predetermined time intervals. After the incubation, the culture medium was removed and the cells were washed with cold PBS three times and fixed with 4% formaldehyde for 30 min at room temperature. Thereafter, the slides were rinsed with PBS three times. Finally, the slides were mounted and directly observed using a confocal laser scanning microscope (Nikon A1Si).

Results and discussion

The Ir–DOX conjugate was prepared in two steps, as described in Scheme 2. Firstly, Ir was reacted with NPC to give activated carbonate Ir–NPC. The chemical structure of Ir–NPC was confirmed using ¹H NMR and ¹³C NMR spectroscopy, as shown in Fig. S1.† For the ¹H NMR of Ir–NPC, the signal peak at 6.52 ppm attributed to the hydroxyl proton of free Ir disappears completely, and the peaks at 8.24 and 7.50 ppm are related to the Ar–H of NPC, confirming the successful linkage between Ir and NPC. The purity and molecular weight of Ir–NPC were further characterized using LC and HRMS techniques (Fig. S2†), which also confirm that Ir–NPC has been synthesized successfully.

Scheme 2 The synthetic route of the Ir–DOX conjugate.

Next, Ir–NPC was reacted with DOX·HCl to give Ir–DOX under alkaline conditions. The chemical structure of Ir–DOX was also confirmed using ¹H NMR and ¹³C NMR spectroscopy, as shown in Fig. 1. According to the ¹H NMR, the three proton signals at 0.86 (1), 1.26 (2) and 6.50 (3) ppm, attributed to methyl (CH₃CH₂–, lactonic ring, pyridone ring) and methylene (–CH₂–, pyridone ring) in Ir, shift to 0.76 (1′), 1.00 (2′) and 6.84 ppm (3′) in the ¹H NMR spectrum of the Ir–DOX conjugate. The proton signal shift of methyl from 3.96 ppm to 3.65 ppm for DOX also confirms the successful preparation of Ir–DOX. Comparing with the ¹³C NMR spectra of Ir and DOX in Fig. 1b, a new peak appears at 168.5 ppm corresponding to the –OCONH– group in the Ir–DOX conjugate. The purity and molecular weight of Ir–DOX were also characterized using LC and HRMS techniques (Fig. S3†). The LC profile gives the retention time of Ir–DOX as 4.06 min, indicating the high purity of Ir–DOX. The HRMS data show that the molecular weight of Ir–DOX (m/z, M + H⁺) is 1156.4500, which is consistent with the calculated value (m/z, M + H⁺, 1157.1915). The Ir–DOX conjugate was also characterized using FTIR spectroscopy and UV-Vis spectrophotometry (Fig. S4†). In the FTIR spectrum of Ir–DOX, the newly formed absorption band at 1723 cm⁻¹, which is ascribed to the vibration of the –OCONH– group, confirms the successful preparation of Ir–DOX. The structure of Ir–DOX is further confirmed by elemental analysis, and the details are given in the ESI.† From the UV-Vis spectra, the Ir–DOX conjugate exhibits two obvious absorption bands at around 364 nm and 482 nm. A 3 nm blue-shift of the Ir–DOX conjugate was observed compared to that of free Ir at 367 nm. A slight red-shift in the absorption of the Ir–DOX conjugate was also observed compared to that of free DOX at 480 nm.

Fig. 1 (a) ¹H NMR and (b) ¹³C NMR spectra of Ir, DOX, and the Ir–DOX conjugate in DMSO-d₆.

As shown in Scheme 2, the bipiperidine group of Ir–DOX is soluble in water, especially under mild acidic conditions; while the formation of a carbamate bond (OCONH) results in the loss of water solubility for DOX. Due to its amphiphilic nature, the Ir–DOX conjugate could self-assemble into micelles in aqueous solution. To confirm the formation of Ir–DOX micelles, the CAC value was measured using the fluorescence of encapsulated Nile red. Fig. S5† shows the relationship between the fluorescence intensity of the Nile red and the Ir–DOX concentration. At a low Ir–DOX concentration, the fluorescence intensity remains almost constant. However, when the Ir–DOX concentration reaches a certain value, the fluorescence intensity increases greatly, indicating that the hydrophobic Nile red molecules have transferred from the water environment to the hydrophobic micellar core; this proves that the Ir–DOX conjugate has formed micelles. According to the inflexion of the curve, the CAC value of the Ir–DOX conjugate is about 10 μg mL⁻¹.

Moreover, the size and morphology of the self-assembled Ir–DOX micelles were determined using DLS and TEM measurements. As shown in Fig. 2a, the DLS results reveal that the aqueous solution of Ir–DOX forms micelles with a unimodal size distribution. The average hydrodynamic diameter of these micelles is about 90.4 nm with a PDI of 0.36. Then, the TEM data were further taken to assess the size and morphology of the Ir–DOX micelles. According to the TEM photograph in Fig. 2b, the Ir–DOX conjugate forms approximately spherical micelles in aqueous solution with an average diameter of about 80 nm, which is close to that measured by DLS.

Fig. 2 (a) DLS results for the Ir–DOX micelles; (b) TEM photograph of the Ir–DOX micelles.

To assess the potential application of Ir–DOX in real-time monitoring of drug delivery and release, we examined the fluorescence behavior of Ir–DOX in DMSO and an aqueous medium. Firstly, fluorescence spectroscopy was employed to evaluate the fluorescence behavior of free Ir, DOX, an Ir/DOX mixture, and the Ir–DOX conjugate in DMSO. According to the fluorescence spectroscopy results shown in Fig. 3, free Ir shows an emission wavelength in the range of 400–500 nm with excitation at 370 nm, while free DOX shows a broad UV-Vis absorption peak in the range of 400–600 nm with the excitation wavelength at 480 nm. The UV-Vis absorption spectrum of DOX overlaps well with the fluorescence spectrum of Ir, which leads to an efficient FRET between Ir (donor) and DOX (acceptor). Thus, the Ir–DOX conjugate shows a fluorescence spectrum mainly in the range of 550–600 nm and the fluorescence intensity is raised greatly with excitation at 370 nm, similar to the 470 nm excitation of DOX. The different fluorescence behaviors of pure Ir, DOX, the Ir/DOX mixture and the Ir–DOX conjugate were visualized using a UV lamp with an excitation wavelength of 365 nm. As shown in Fig. 4, the Ir–DOX conjugate shows a different color compared to the mixture of Ir and DOX. In addition, the color of Ir–DOX and DOX is red, indicating that FRET occurs when Ir and DOX are conjugated together.

Fig. 3 Normalized excitation (black) and fluorescence emission (red) spectra of (a) Ir, excitation/emission = 370/450 nm and (b) DOX, excitation/emission = 470/590 nm. Fluorescence emission spectra of (c) the Ir/DOX mixture and (d) the Ir–DOX conjugate, excitation = 370 (black)/470 (red) nm.

Fig. 4 Photographs of (1) Ir, (2) DOX, (3) the Ir/DOX mixture and (4) the Ir–DOX conjugate under visible light (left) and under a UV lamp with laser excitation at 365 nm (right).

Using a solvent dependent aggregation method, we examined the dual-fluorescence quenching behavior of the Ir–DOX micelles. When the Ir–DOX conjugate was dissolved in DMSO, Ir–DOX exhibited intense fluorescence at 594 nm due to efficient FRET from Ir to DOX. However, with the addition of water, the fluorescence intensity decreased dramatically, as shown in Fig. 5a. When the water fraction (fw, water content in DMSO, vol%) reached 99.5%, the fluorescence intensity of Ir–DOX was only 1.8% of that in DMSO (Fig. 5b), confirming the occurrence of ACQ upon the formation of Ir–DOX micelles. Therefore, the emissive energy of Ir and DOX was greatly quenched by the effects of both ACQ and FRET, which led to the disappearance of the fluorescence of the two drugs. Moreover, the formation of FRET relies on there being a certain distance between the donor Ir and the acceptor DOX. Once the linkage in Ir–DOX was cleaved, the FRET was disrupted along with the fluorescence recovery of both Ir (blue fluorescence) and DOX (red fluorescence). These results demonstrate that Ir–DOX could be used for real-time monitoring of drug delivery and release in living tumor cells.

Fig. 5 Dual-fluorescence quenching behaviour of the Ir–DOX micelles. (a) Ir aggregation-caused quenching (ACQ) of the Ir–DOX micelle fluorescence in different fractions of water (fw) (fw means volume percentage of water in DMSO); (b) plot calculated from (a) showing the ACQ behaviour of the Ir–DOX micelles.

To verify the fluorescence recovery once Ir and DOX were released from the micelles, the in vitro release behavior of the Ir–DOX micelles was investigated. We incubated Ir–DOX micelles under simulated physiological conditions (PBS, pH = 7.4) and in an acidic environment (acetate buffer, pH = 5.0, pH = 2.0) at 37 °C. The release profiles of the free drugs from the micelles at different conditions are shown in Fig. 6a. The Ir–DOX micelles exhibit high stability in pH = 7.4 PBS solution, with about 30% drug release in 72 h; while in pH = 5.0, the release ratio increases to 45%. Further reducing the pH value to 2.0, the release ratio of free drugs increases to 70%. It can be inferred that the drug release becomes much faster in acidic conditions. These results show that the biodegradable bond of the carbamate linkage is pH-sensitive.

Fig. 6 In vitro drug release of the Ir–DOX micelles. (a) Cumulative release curves of the Ir–DOX micelles under different pH values (7.4, 5.0 and 2.0) at 37 °C and dual-fluorescence recovery of (b) Ir (excitation wavelength = 370 nm) and (c) DOX (excitation wavelength = 470 nm) in acidic conditions.

An in vitro assay was employed to further demonstrate the dual-fluorescence recovery of Ir and DOX in acidic conditions for 48 h. As shown in Fig. 6b and c, the fluorescence intensity of Ir and DOX recovers gradually with increasing time, which indicates that the ACQ phenomenon is disrupted as Ir and DOX are released from the Ir–DOX micelles.

Multidrug resistance (MDR) has been a growing concern for effective cancer chemotherapy, especially for small-molecule anticancer drugs. The main mechanism for MDR is drug efflux which is mediated by a membrane transport protein, P-glycoprotein (P-gp). P-glycoprotein can pump free small-molecule anticancer drugs out of tumor cells via an ATP-dependent route, leading to a reduction of drug accumulation in the tumor cells.^34,35 Fortunately, the micelles can bypass the P-gp efflux pump, accumulate themselves in cells, and deliver drugs into the cytoplasm efficiently.³⁶ Therefore, Ir–DOX drug micelles are expected to overcome the MDR of cancer cells. The cytotoxicity of free Ir, DOX, the Ir/DOX mixture and the Ir–DOX micelles was investigated using a MTT assay against MCF-7/ADR cells, as shown in Fig. 7a. Calculated by Graphpad Prism, the IC₅₀ values of free Ir, DOX and the Ir/DOX mixture are 27.3, 36.1 and 25.0 μM, respectively. However, the IC₅₀ of the Ir–DOX micelles in MCF-7/ADR cells is only 7.4 μM, which is far superior to that of MCF-7 (shown in Fig. S6 and Table S1†), demonstrating that the Ir–DOX micelles can overcome the MDR of cancer cells. These results demonstrate that the Ir–DOX micelles are promising for overcoming MDR and killing cancer cells efficiently.

Fig. 7 (a) Cell viability of MCF-7/ADR cells incubated with Ir, DOX, the Ir/DOX mixture and the Ir–DOX micelles after 72 h at various concentrations determined by the MTT assay. Values are presented as the average standard error (n = 3); (b) combination index (CI) of Ir and DOX combinations via different formulations.

To further investigate the synergy in the Ir/DOX mixture and in the Ir–DOX micelles, a combination index (CI) was carried out, based on the median-effect equation and derived from the mass-action law principle.^37,38 The values of the CI could provide qualitative information on the drug interaction nature, usually plotted against drug effect levels (IC_x values). Values of CI > 1 indicate antagonism, CI = 1 indicates an additive effect and CI < 1 indicates synergism. As shown in Fig. 7B, most of the CI values for the Ir–DOX micelles are under the line of CI = 1 after 72 h incubation of the drugs, while many data points of the Ir/DOX mixture are above or near the CI = 1 line. These results further show synergistic anticancer efficacy of the Ir–DOX micelles.

To confirm whether the Ir–DOX micelles could be effectively transported into tumor cells, a flow cytometry analysis was employed to evaluate the cellular uptake of the Ir–DOX micelles in MCF-7/ADR cells. DOX can produce red fluorescence by itself, thus the Ir–DOX itself could be used as a probe for the analysis of cell internalization. Fig. 8 shows that the relative geometrical mean fluorescence intensities of DOX gradually increase with incubation time, compared with the non-pretreated cells. These enhanced fluorescence signals demonstrate the effective internalization of the Ir–DOX micelles.

Fig. 8 Cellular uptake of the Ir–DOX micelles by MCF-7/ADR cells. (a) The relative geometrical mean fluorescence intensities of the Ir–DOX micelle pretreated cells. (b) Representative flow cytometry profiles of the control. (c) Representative flow cytometry profiles of MCF-7/ADR cells cultured with Ir–DOX micelles for 4 h.

Due to the special fluorescence feature, the Ir–DOX micelles should display the fluorescence of Ir and DOX after drug release. Hence, confocal laser scanning microscopy (CLSM) was conducted to investigate whether the fluorescence change in the Ir–DOX micelles could be correlated with the drug release and therapeutic effect. After treating MCF-7/ADR cells with different incubation times, the fluorescence change was observed using CLSM. As shown in Fig. 9, there is no obvious red or blue fluorescence before 12 h, confirming that Ir–DOX enters the cells in the form of micelles, but not free conjugate or free drugs. With the increase of incubation time, the blue fluorescence of Ir and red fluorescence of DOX are recovered, and the MCF-7/ADR cells exhibit distinct blue and red fluorescence after 48 h incubation. These results indicate that Ir and DOX have been released successfully in a time-dependent manner. Free Ir and DOX both perform specific activities in DNA, so we observe that the intensities of blue and red fluorescence in the nucleus increase gradually. However, after 72 h of incubation, the fluorescence turns dark, because many of the MCF-7/ADR cells are round-shaped and dead.

Fig. 9 Fluorescence images of MCF-7 cells treated with Ir–DOX micelles for 12 h, 24 h, 36 h, 48 h and 72 h. The scale bar represents 40 μm.

Conclusions

In summary, a new self-tracking amphiphilic drug–drug conjugate without the help of any carriers or fluorescent probes, was developed to monitor drug delivery and release in tumor cells. The Ir–DOX conjugate was constructed using hydrophilic Ir and hydrophobic DOX via a simple carbamate linkage and could self-assemble into micelles in aqueous solution. Owing to a special fluorescence feature from FRET and ACQ, the fluorescence of the Ir–DOX micelles was quenched. Once their linkage was broken in the tumor cells, the blue fluorescence of Ir and red fluorescence of DOX were recovered. Therefore, the double-color fluorescence change could be observed using CLSM. Moreover, the result of in vitro cytotoxicity assays showed that the Ir–DOX micelles could overcome the MDR of the tumor cells, which might result from their nanoscale characteristics. We anticipate that the Ir–DOX micelles can achieve self-tracking while treating cancer.

Acknowledgements

This work was financially supported by the National Basic Research Program of China (2015CB931801) and National Natural Science Foundation of China (51473093).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR and LC-HRMS spectra of Ir–NPC; FTIR and UV-Vis spectra for Ir, DOX, and Ir–DOX conjugate; the CAC value of Ir–DOX micelles. See DOI: 10.1039/c5ra24273h

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