Bei Liua,
Lirong Suna,
Xijian Lua,
Yuping Yanga,
Hongshang Penga,
Zhaogang Sun*b,
Juan Xu*c and
Hongqian Chu*b
aCollege of Science, Minzu University of China, Beijing, 100081, China
bTranslational Medicine Center, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, China. E-mail: sunzhaogang@bjxkyy.cn; chuhongqian@bjxkyy.cn
cNational Research Institute for Family Planning, Beijing, 100005, China. E-mail: xujuan@nrifp.org.cn
First published on 8th April 2022
Real-time monitoring of drug release behaviors over extended periods of time is critical in understanding the dynamics of drug progression for personalized chemotherapeutic treatment. In this work, we report a metal–organic framework (MOF)-based nanotheranostic system encapsulated with photothermal agents (CuS) and therapeutic drug (DOX) to achieve the capabilities of real-time drug release monitoring and combined chemo-photothermal therapy. Meanwhile, folic acid-conjugated polyethylene glycol (FA-PEG) antennas were connected to the MOF through coordination interactions, endowing the MOF with an enhanced active targeting effect toward cancer cells. It is anticipated that such a theranostic agent, simultaneously possessing tumor-targeting, real-time drug monitoring and effective treatment, will potentially enhance the performance in cancer therapy.
So far, several DDSs with drug release monitoring have been developed.10–19 These DDSs can provide opportunities to evaluate the therapeutic effectiveness, and export useful information for dose adjustment and prognosis for personalized medicine. For example, Zheng and co-workers18 used carbon nanodots as both fluorophore and drug nanocarrier for real-time monitoring of drug release on the basis of the Förster resonance energy transfer (FRET) signal. Xing et al.19 conjugated photoactivatable platinum(IV) prodrugs and caspase imaging peptides on the silica-coated upconversion-luminescent nanoparticles, achieving the real-time and in situ reporting of drug activation. Though effective, these approaches are often limited by the expensive, time-consuming and ineffective post-modification or conjugation steps. Design of a simple but effective strategy for synthesizing smart DDS with real-time monitoring of drug release is highly desirable.
Metal–organic frameworks (MOFs), constructed by self-assembly of organic linkers and metal or metal-oxo nodes, have showed a great promise in the field of gas storage/separations, catalysis and nanomedicine.20–28 Especially, the tumor microenvironment (TME)-responsive MOFs, including hypoxia-sensitive Cu-MOF20 and acidic pH-degradable zeolitic imidazolate framework-8 (ZIF-8),21,22 have been attracting emerging research efforts as ideal drug carriers. These MOFs possess the capability of efficient drug encapsulation and excellent controllable release property under the given stimulation, thus leading to a remarkably enhanced drug-delivery efficiency and minimal cytotoxicity upon degradation. However, construction of the TME-responsive MOFs that can simultaneously achieve real-time monitoring of drug release remains a great challenge. Moreover, the low specific tumor recognition together with the complexed preparation protocol also limited the future medical usage and clinical translation of these TME-responsive MOFs.
Herein, a new kind of “Smart” DDS with real-time drug release monitoring was developed based on the acidic pH-degradable MOFs. These nanotheranostics, referred to as CuS/D@Z-FA, were fabricated by encapsulating functional guests of photothermal agents (CuS) and therapeutic drug (DOX) within a well-defined ZIF-8 host, followed by the surface modification of folic acid-conjugated polyethylene glycol (FA-PEG) antennas (Scheme 1). Upon the active accumulation of CuS/D@Z-FA in tumor cells through FA-receptor-mediated endocytosis, the coordination between zinc and imidazolate ions of ZIF-8 dissociated, resulting in the on-demand release of CuS and DOX for combined photothermal therapy (PTT) and chemotherapy (CT). Additionally, the FRET-quenched fluorescence of DOX in the nanoparticles can be gradually recovered upon the dissociation of ZIF-8, enabling the real-time drug release monitoring. These results highlight that this MOF-based nanoplatform is promising for high-performance cancer therapy.
Notably, the synthesis approach allows to encapsulate CuS and DOX in an ultra-efficient and precisely controllable manner (Fig. 1d–f). CuS/D@Z NP in Fig. 1e were chosen for the following experiments, which can not only retain the hexagon shape and size uniformity of ZIF-8, but also possess reasonable amounts of CuS (59.375%, w/w) and DOX (2.083%, w/w) for further combined CT-PTT therapy. UV-vis-NIR absorption measurement (Fig. 1g, and S2†) confirms the successful encapsulation of CuS and DOX in CuS/D@Z owning to the existence of characteristic peaks of DOX centered at ∼560 nm and a broad absorbance by CuS NPs (700–1100 nm). Note that the absorption spectrum of CuS/D@Z showed a remarkable broadening and redshift of DOX or CuS characteristic absorbance, mainly owing to the self-aggregate of DOX or CuS during the nanoparticle formation.
In order to prolong the blood circulation time and enhance the active tumor-targeting effect of nanoparticles, FA-PEG antennas were further functionalized on the surface of CuS/D@Z through coordination interaction of its carboxy groups with coordinatively unsaturated metal sites of Zn2+ on the surface of ZIF-8.30,31 As shown in Fig. S3,† the resulting CuS/D@Z-FA NPs remained monodisperse in size without obvious shape change and aggregation. Zeta potential analysis (Fig. 1h) showed that CuS/D@Z were positively charged (+24.5 mV), and became negatively charged (−21.1 mV) after the attachment of FA-PEG. Additionally, the absorption peak at ∼300 nm was the characteristic peak of FA, further confirming the successful functionalization of FA on the surface of CuS/D@Z (Fig. 1i).
The UV-vis-NIR absorption spectra of CuS/D@Z-FA showed a high absorbance in the NIR range (800–1100 nm), and the absorption intensity increased steadily with the increasing concentrations of CuS/D@Z-FA (Fig. S4†). The significant absorption of CuS/D@Z-FA in the NIR window motivated us to investigate their photothermal properties. As shown in Fig. 2a, the temperature of CuS/D@Z-FA solution (40 μg mL−1) increased rapidly with increased exposure time to the 808 nm NIR laser (1.0 W cm−2), indicating that the NIR light harvesting by the CuS/D@Z-FA enabled effective heat generation. As a control, DI water or DOX@ZIF-8-FA (labeled as D@Z-FA) presented no significant increase of temperature under the same irradiation conditions. Moreover, CuS, CuS/D@Z and CuS/D@Z-FA NPs presented similar temperature profiles (Fig. 2a), suggesting the negligible impact of the ZIF-8 coating, DOX loading or FA-PEG modification on the photothermal effect of CuS.
The temperature variation curves of different concentrations of CuS/D@Z-FA were also conducted under the same 808 nm irradiation (1.0 W cm−2). As expected, the temperature increase of CuS/D@Z-FA presented a concentration-dependent photothermal behavior (Fig. 2b). The photothermal conversion efficiency (η) of CuS/D@Z-FA was calculated as 38.11% according to the as-obtained data (Fig. 2b and c), which is comparable to that in previous reports.32 Then the photothermal stability of CuS/D@Z-FA NPs was investigated by exerting four cycles of 808 nm NIR laser irradiation on CuS/D@Z-FA aqueous solution. As shown in Fig. 2d, CuS/D@Z-FA NPs remained a robust photothermal property. Therefore, the as-synthesized CuS/D@Z-FA NPs hold a great potential as promising candidates for PTT.
Since the coordination interaction between Zn2+ and 2-methylimidazolate ligand dissociates at pH 5.0–6.0 owing to the protonation effect, ZIF-8 NPs can degrade in acidic lysosomes of tumor cells (pH ∼ 5.5, Fig. 3a), while stay stable under normal physiological conditions (pH ∼ 7.4). Based on this line, pH-responsive release of DOX from CuS/D@Z-FA NPs were explored by immersing CuS/D@Z-FA NPs in PBS solutions with different pH values (pH 7.4 and 5.5). As illustrated in Fig. 3b, the DOX release profiles of CuS/D@Z-FA were obviously pH-dependent: the cumulative release amount of DOX could reach 60.1% at pH 5.5, which is much higher than that at pH 7.4 (17.3%). Considering that the tumor microenvironment of many solid tumors is mildly acidic, such pH-sensitive drug release property of CuS/D@Z-FA does great benefit for tumor-specific therapy. To be specific, the accumulated release ratio at pH 5.5 condition increased from 60.1 to 64.0% with the 808 nm laser irradiation, and a similar variation tendency was found at pH 7.4. Such enhancement of drug release mainly attributed to the increasing temperature generated by the CuS, which can accelerate the dissociation of CuS/D@Z-FA to release DOX. These results suggested that the as-synthesized CuS/D@Z-FA NPs had a pH-responsive and NIR-induced drug release behavior, which do a great benefit to enhance the cytotoxicity toward tumor cells selectively.
Interestingly, when CuS and DOX simultaneously encapsulated in ZIF-8 host, FRET occurred between DOX and CuS due to the close proximity of the two, leading to a low fluorescence signal background. To prove it, the fluorescence signal of DOX in both D@Z-FA and CuS/D@Z-FA were tested. Fig. S5† showed a strong depression of DOX fluoresce after co-encapsulation of CuS and DOX in ZIF-8 host, suggesting an efficient energy transfer process has occurred from the DOX to the CuS. Upon accumulation of CuS/D@Z-FA in the acidic environment (e.g., pH = 5.5), the coordination between zinc and imidazolate ions of ZIF-8 dissociated, leading to the on-demand release of CuS and DOX to eliminate LRET. As a result, the fluorescence signal of DOX enhanced simultaneously (Fig. 3c and d), demonstrating the great potential of this nanoconstruct to monitor the drug release in real-time.
Then we verified the real-time monitoring capability of CuS/D@Z-FA via live-cell confocal laser scanning microscopy (CLSM). MCF-7 cells were incubated with CuS/D@Z-FA for 0.5 h, washed with PBS, and further cultured at 37 °C under 5% CO2 for 1 h and 3 h. As presented in Fig. 4a and b, a negligible DOX fluorescence can be detected after 0.5 h incubation, indicating a limited DOX was released from CuS/D@Z-FA after 0.5 h incubation. With the extension of the culture time, the fluorescence intensity of DOX increased gradually due to the decreased FRET effect from DOX to CuS in living MCF-7 cells. Such time-dependent increasement of DOX fluorescence help to track the release behavior of drug, which plays an important role in avoiding insufficient or excess drug dosing for cancer therapy.
The specific cancer cell recognition and cellular uptake of NPs is an important but formidable challenge for drug delivery systems. Herein, we introduced PEG to enhance the biostability of NPs,21 and decorated FA at the end of PEG chain as a targeted recognition for cancer cells. To prove it, the intracellular uptake efficiency of DOX@Z and DOX@Z-FA to MCF-7 cells was demonstrated by CLSM. Note that DOX@Z and DOX@Z-FA were chosen for the experiments in order to exclude the FRET effect from DOX to CuS. As shown in Fig. 5a, for DOX@Z-FA treated group, the cells exhibited more red-fluorescence signal of DOX than those incubated with DOX@Z. Additionally, flow cytometry analysis (Fig. 5b and c) showed that the fluorescence intensity of cells treated with DOX@Z-FA was 3.1-fold higher than those treated with DOX@Z, indicating the enhanced cellular uptake of DOX@Z-FA NPs via FA-receptor-mediated endocytosis.
The in vitro cytotoxicity of the nanosystem against MCF-7 cells was then evaluated by CCK-8 assay. As presented in Fig. 6a, treatment with the only NIR light irradiation, CuS@ZIF or CuS@ZIF-FA did not decrease the cell viability significantly, demonstrating the negligible toxicity of the light irradiation or these NPs to MCF-7 cells. The cells treated with CuS@ZIF + NIR or CuS/D@Z showed a significant cytotoxicity compared with the control groups, confirming their PTT or CT effect as anti-tumor agents. Notably, photo-irradiated CuS@Z-FA showed higher cytotoxicity (59.0%) compared to that of the cells treated with CuS@Z + NIR (39.2%), indicating an enhanced potency could be achieved through the folic acid targeting effect. As expected, incubation of the cells with CuS/D@Z-FA followed by light irradiation led to a highest cell cytotoxicity (82.5%) due to the combinational effects of FA receptor-mediated targeting, PTT and CT. Similar results can also be observed by the calcein AM/propidium iodide (PI) assay. As shown in Fig. 6b, the calcein AM/PI staining results demonstrated that the MCF-7 cells treated with CuS/D@Z-FA show apparent fluorescence change from green to red color when compared with the control groups, implying the efficient killing effect of CuS/D@Z-FA on MCF-7 cells.
Fig. 6 (a) Viability of MCF-7 cells after different treatments. (b) Confocal fluorescence images of cell apoptosis by staining with Annexin V-FITC and PI after different treatments. Scale bar: 50 μm. |
Inspired by the good performance of the nanoconstruct in vitro, we further explored the in vivo activity of CuS/D@Z-FA. Firstly, the metabolic kinetics of NPs were investigated. Cy5 labeled CuS/D@Z and CuS/D@Z-FA were administrated to MCF-7 tumor-bearing mice through the tail vein. Ex vivo fluorescence imagings of various harvested organs and tumors upon necropsy at different time points after injection were measured using in vivo imaging system (IVIS). As shown in Fig. S6,† the fluorescent intensity increased gradually and reached highest at 6 h postinjection, which may attribute to the degradation of ZIF-8 and the sustained release of Cy5 over time. Then the fluorescent intensity decreased, indicating the gradually clearance of NPs out of the body. Remarkably, CuS/D@Z-FA treated mice showed a much higher fluorescence intensity than CuS/D@Z-treated mice, demonstrating the satisfactory targeting ability of folic acid.
Then in vivo therapeutic efficacy of CuS/D@Z-FA NPs was evaluated. MCF-7 tumor-bearing mice were randomly divided into 5 groups (5 mice per group), treated with PBS, CuS@Z-FA, CuS@Z-FA + NIR, CuS/D@Z-FA, CuS/D@Z-FA + NIR, respectively. As presented in Fig. 7a, fast tumor growth was observed in the PBS or CuS@Z-FA groups. Treatment with CuS@Z-FA + NIR or CuS/D@Z-FA had moderate antitumor capability because of the PTT or CT. Notably, a strongest anti-tumor effect was achieved in the CuS/D@Z-FA + NIR group, implying that the combination of PTT and CT was more effective than either modality alone. Moreover, the final tumors of each group were dissected, and the weights and size of excised tumors were presented in Fig. 7b. As expected, mice treated with CuS/D@Z-FA + NIR showed a smaller tumor size compared with the other groups. The hematoxylin and eosin (H&E) stained sections of tumors were also shown in Fig. 7c. The results demonstrated the PTT/CT combined therapy can lead to a higher level of nucleus dissociation and necrosis than other groups.
The toxicity of CuS/D@Z-FA was further evaluated in vivo. As presented in Fig. S7,† the body weights of mice in all groups are increased steadily with time, implying little adverse side effect of NPs. Then the morphology of sectioned organs from mice after different treatments was studied. Fig. S8† showed that the main organs (kidney, heart, lung, liver and spleen) experienced no obvious changes in cellular integrity and tissue morphology, indicating the negligible systematic toxicity of CuS/D@Z-FA NPs. The results of serum biochemistry (Fig. S9†) also show that no significant changes of serum parameters occurred between the control group and the treatment groups, including the liver function markers of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the kidney function indicators of blood urea nitrogen (BUN) and creatinine (CRE).
2-Methyl imidazole methanol solution (6 mL, 8 mg mL−1), CuS solution (3 mL) and DOX (2 mL, 1 mg mL−1) were mixed with at room temperature, followed by the addition of 14 mL of Zn (NO3)2·6H2O methanol solution. After a quick vortex, the well-mixed solution was allowed to stand at room temperature for 24 hours. The as-obtained CuS/D@Z NPs were collected by centrifugation, washed with DMSO for three times and re-dispersed in DMSO for future use. CuS@Z NPs were synthesized under the same reaction conditions but without adding DOX solution.
The CuS/D@Z-FA NPs were prepared by the functionalization with PEG-FA on the surface of CuS/D@Z through the formation of coordination bonds with Zn2+. Briefly, CuS/D@Z NPs were dispersed in PEG-FA solution and then stirred for 48 h under room temperature. Then the final CuS/D@Z-FA NPs were obtained by centrifugation, washed three times with DMSO to completely wash off the free PEG-FA.
Footnote |
† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d1ra09320g |
This journal is © The Royal Society of Chemistry 2022 |