Article Cite This: Mol. Pharmaceutics 2018, 15, 4084−4091
pubs.acs.org/molecularpharmaceutics
Facile Synthesis of Fluorescent Hyper-Cross-Linked β‑CyclodextrinCarbon Quantum Dot Hybrid Nanosponges for Tumor Theranostic Application with Enhanced Antitumor Efficacy Mingliang Pei,† Jui-Yu Pai,‡ Pengcheng Du,† and Peng Liu*,† †
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State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ‡ Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30043, Taiwan S Supporting Information *
ABSTRACT: Fluorescent hyper-cross-linked β-cyclodextrin-carbon quantum dot (β-CD-CQD) hybrid nanosponges of about 200 nm with excellent biocompatibility and strong bright blue fluorescence excited at 365 nm with a high photoluminescence quantum yield (PLQY) of 38.0% were synthesized for tumor theranostic application by facile condensation polymerization of carbon quantum dots (CQDs) with β-cyclodextrin (β-CD) at a feeding ratio of 1:5. The DOX@β-CD-CQD theranostic nanomedicine, around 300 nm with DOX-loading capacity of 39.5% by loading doxorubicin (DOX) via host−guest complexation, showed a pH responsive controlled release and released DOX in the simulated tumor microenvironment in a sustained release mode, owing to the formation constant in the supramolecular complexation of DOX with the β-CD units in the β-CD-CQD nanosponges. The proposed DOX@β-CD-CQD theranostic nanomedicine could be internalized into HepG2 cells, and the released DOX was accumulated into the cell nuclei, demonstrating an antitumor efficacy more enhanced than that of the free drug. KEYWORDS: tumor theranostics, hyper-cross-linked hybrid nanosponges, β-cyclodextrin, carbon quantum dots, pH-responsive controlled release, supramolecular complexation
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nanocomplex21 as nanomedicines with a suitable size favoring the EPR effect. Cyclodextrins (CDs) are a series of natural and watersoluble cyclic oligosaccharides with a hydrophilic exterior surface and a hydrophobic interior cavity, as a truncated cone containing six, seven, or eight D(+)-glucose units via α-1,4 linkages for α-CD, β-CD, or γ-CD, respectively. The primary and secondary hydroxyl groups in their outer surface endow easy functionalization, while the lipophilic central cavities could be used for the inclusion complexation with lipophilic molecules. Therefore, CDs have been widely used for the environmental and pharmaceutical applications.22,23 Owing to their surface multihydroxyl groups, CD-based nanosponges have been designed for improving the water solubility of water insoluble molecules; protecting degradable substances; and as creative carriers or supports in pharmaceutics, catalysts, and environmental control,24 with various organic molecules, such as epichlorohydrin,25 citric acid,26 tetrafluoroterephthaloni-
INTRODUCTION
Theranostic nanomedicines, in which diagnostic and therapeutic functions are combined in one dose, have attracted more and more interest for imaging-guided drug/gene delivery in cancer treatment recently.1 Carbon quantum dots (CQDs), a novel kind of fluorescent carbon nanomaterial possessing the unique advantages of high stability, remarkable biocompatibility, easy synthesis and surface functionalization, and comparable optical characteristics, have been extensively studied,2,3 especially for bioimaging application owing to their tunable strong fluorescence emission property.4 Although various synthetic approaches have been established for the production of GQDs via top-down or bottom-up strategies, their size and surface functionalization have been recognized as the key factors affecting their biological applications.5 Due to their small particle size, which can be eliminated through the kidneys, 6 the clinical application of the theranostics based on single CQD is restricted, despite the intense investigation recently.7−13 Nonetheless, the CQDs could be utilized for the imaging-guided delivery drugs or genes in tumor treatment, after being been modified14−17 or introduced into nanohydrogels as a cross-linking agent18−20 or © 2018 American Chemical Society
Received: Revised: Accepted: Published: 4084
May 15, 2018 June 30, 2018 July 24, 2018 July 24, 2018 DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
Article
Molecular Pharmaceutics Scheme 1. Preparation Processes for CQDs, β-CD-CQD, and DOX@β-CD-CQD Hybrid Nanosponges
Synthesis of β-CD-CQD Hybrid Nanosponges. The βCD-CQD hybrid nanosponges were synthesized via a facile condensation polymerization of the carboxyl-functionalized CQDs and β-CD, with different mass feeding ratios (CQDs:βCD of 1:2, 1:5, or 1:10). In short, 10 mL of CQDs solution (containing of 18.5 mg CQDs), EDC·HCI (0.109 g), and DMAP (0.070 g) were added into 30 mL of DMF to the active carboxyl group of the CQDs. After 30 min, β-CD (92.5 mg) was added, and the mixture was stirred at room temperature in the dark for 24 h. The β-CD-CQD hybrid nanosponges were collected by dialyzing (WMCO = 3500 Da) against water and freeze-drying. Preparation of DOX@β-CD-CQD Theranostic Nanomedicine. The DOX-loaded β-CD-CQD (DOX@β-CDCQD) hybrid nanosponges were prepared at room temperature by the following procedure: β-CD-CQD hybrid nanosponges (10.0 mg) and DOX (5.0 mg) were added into 10 mL of water. After ultrasonication for 4 h, the mixture was stirred in the dark for 12 h. The DOX@β-CD-CQD hybrid nanosponges were collected through dialyzing (WMCO = 3500 Da) against water for 3 days and freeze-drying. The DOX-loading capacity was calculated by measuring the DOX concentration in the dialysate on a Lambda 35 UV−vis spectrometer at 480 nm. In Vitro Controlled Release Profiles. The DOX@β-CDCQD hybrid nanosponges (10 mg) were dispersed into phosphate buffer solution (PBS, pH 7.4, 10 mL) or acetic acid buffer solution (ABS, pH 5.0, 10 mL) in a dialysis tube (MWCO = 14000 Da). The dialysis tube was incubated into the corresponding buffer solution (110 mL) at 37 °C with shaking (rpm of 120). After a certain interval, the dialysate (5 mL) was taken out to measure the DOX concentration on a Lambda 35 UV−vis spectrometer, and the same volume of fresh buffer was added. Cytotoxicity and Cellular Uptake. The cellular toxicity of the blank and DOX-loaded β-CD-CQD hybrid nanosponges were evaluated against HepG2 cells by the MTT assay. The HepG2 cells were seeded into the 96-well plate (1 × 105 cells per well) in 100 μL of DMEM containing 10% FBS and cultured in the presence of the nanosponges with different concentrations at 37 °C in a 5% CO2 atmosphere. The culture
trile,27 4,4′-difluorodiphenylsulfone,28 ferrocene,29 glutathioneresponsive cross-linker,30 etc. By now, the CD-based hypercross-linked hybrid nanosponge incorporating inorganic nanomaterials is not described. Recently, Ko et al. designed a graphene quantum dot (GQD)-based nanocarrier as a promising theranostics for breast cancer, by modifying the GQD-NH2 of 26 nm with βCD and herceptin.31 In the present work, the carboxylfunctionalized CQDs with a high quantum yield and plentiful carboxyl groups on their surfaces are hypothesized as a multifunctional cross-linker for CDs, also endowing fluorescent function. On the basis of the hypothesis, fluorescent hypercross-linked β-cyclodextrin-carbon quantum dot (β-CD-CQD) hybrid nanosponges were synthesized for tumor theranostic application by facile condensation polymerization of the carboxyl-functionalized CQDs and β-cyclodextrin as the multifunctional monomers (Scheme 1) by integrating the fluorescent imaging performance of the CQDs and the drug delivery function of β-CD.
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EXPERIMENTAL SECTION Materials and Reagents. Analytical graded sodium citrate and NH4HCO3 were obtained from Tianjin Chemical Reagent Co. 1-(3-Dimethylaminoproyl)-3-ethylcarbodiimide hydrochloride (EDC·HCI, 98%) and 4-dimthylaminopyridine (DMAP) were purchased from Aladdin Chemistry Co., Ltd. and J&K Chemical Co., Ltd., respectively. β-Cyclodextrin (βCD, ≥ 99%) was obtained from Tianjin Guangfu Fine Chemical Research Institute. Doxorubicin hydrochloride (DOX, 99.4%) was provided from Beijing Huafang United Technology Co., Ltd. All other reagents and solvents were of analytical grade and used without further purification. Double distilled water was used throughout. Synthesis of Carboxyl-Functionalized CQDs. The carboxyl-functionalized CQDs were prepared as reported previously.32 Briefly, the mixture of sodium citrate (1.40 g), NH4HCO3 (10.5 g), and 70 mL of water was sealed in an autoclave and then heated at 180 °C for 4 h. After being cooled to room temperature, the CQDs were purified by dialysis (MW = 1000 Da) in the dark for 3 days. 4085
DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
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Molecular Pharmaceutics
bond in the aromatic sp2 domains of CQDs, while the other transition was centered at 335 nm of the n−π* transition of the CO bond (Figure 3),33 resulting in a strong bright blue fluorescence excited at 365 nm. The PL emission intensity at 440 nm increased in the excitation range of 310−350 nm and then decreased gradually, with a Stokes shift of 90 nm (Figure 4). The absolute photoluminescence quantum yield (PLQY) was calculated as 49.0% in solution, owing to the surface passivation and N-doping,34,35 from the PL emission measurement. The absolute QY value was higher than those reported previously.36−38 The excellent PL property demonstrated their promising application in fluorescent imaging-guided therapy. Synthesis and Characterization of β-CD-CQD Hybrid Nanosponges. Then the hyper-cross-linked β-CD-CQD hybrid nanosponges were synthesized via facile condensation polymerization of the CQDs and β-CD as multifunctional monomers with different mass feeding ratios, via the esterification between the surface carboxyl groups of the CQDs and the hydroxyl groups in β-CD molecules. The characteristic absorbance at 1735 cm−1 of CO stretching in the ester group appeared in the FT-IR spectrum of the products (Figure S1), indicating the successful esterification between the two multifunctional monomers. In the 1H NMR spectrum (Figure S2), both of the chemical shifts of β-CD and the protons connected with the unsaturated carbons in the CQDs (δ = 8.10, 7.96, and 6.63 ppm) could be seen obviously,39 also demonstrating the successful condensation polymerization between the CQDs and β-CD. Furthermore, the integration ratio of the protons H-1 to OH-6 and OH-2 and OH-3 (H-1:OH-6:OH-2,3) changed from 1:1:2 in β-CD to 1:0.85:1.77 in the β-CD-CQD hybrid nanosponges, indicating that all three kinds of hydroxyl groups in β-CD had partially participated in the condensation polymerization. The product prepared at the mass feeding ratio of 1:5 showed a particle size of 20−40 nm and Dh of 200 nm with narrow distribution from the TEM and DLS analysis (Figures 1 and 2). Such a great swelling ratio could have resulted from the relatively lower cross-linking degree due to the big space hindrance between the CQDs. The others prepared with mass feeding ratios of 1:2 or 1:10 exhibited obvious aggregation with a broader diameter range (Figure S3). The unique particle size and distribution of the hyper-cross-linked β-CD-CQD hybrid nanosponges prepared at the mass feeding ratio of 1:5 indicated their potential application as a drug delivery system (DDS) for antitumor drugs favoring the EPR effect. Moreover, the one prepared at the mass feeding ratio of 1:5 also maintained the optical property of the CQDs, with a bright blue fluorescence (Figure 3) and excellent emission performance (Figure 4). The β-CD-CQD hybrid nanosponges remained high quantum yield with an absolute PLQY of 38.0%, although it was lower than that of the CQDs, maybe due to the fluorescence quenching by π−π interactions because of the close contacts of the CQDs after cross-linking with βCD.40 It demonstrated that the esterification of the surface carboxyl groups in the CQDs did not affect their PL emission property. So they were selected for the further investigation for tumor theranostic application. DOX-Loading and in Vitro Controlled Release. After DOX-loading, no obvious change was found for DOX in chemical shifts of 0−5 ppm but in the range of 5−8 ppm in the 1 H NMR spectrum of the DOX@β-CD-CQD (Figure S2b). In the enlarged figure (the inset in Figure S2b), the peaks of DOX at the chemical shifts around 8.00, 7.85, 7.59, and 5.27
medium was disposed and washed three times with PBS (0.01 mol/L, pH 7.4), after 48 h of incubation. Then MTT liquor (20 μL, 5 mg/mL in PBS) was added to each well, followed by DMSO (150 μL) to dissolve the crystal. The cell viability was measured with an enzyme-linked immunosorbent at 490 nm. The cellular uptake of the DOX@β-CD-CQD hybrid nanosponges into HepG2 cells was exhibited with a DM 4000B fluorescence microscope, after 24 h of incubation. After the cell nuclei were stained with the Hoechst 33258, the location of cellular fluorescence was validated with excitation wavelength of 480 (DOX) and 405 nm (Hoechst 33258), respectively. Analysis and Characterization. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet 360 FT-IR spectrometer with the KBr pellet technique. The steady-state fluorescence emission spectra of the CQDs and β-CD-CQD hybrid nanosponges in aqueous dispersion were detected on a Hitachi F-7500 fluorescence spectrometer. The absolute photoluminescence quantum yield (PLQY) was measured on an FLS920 fluorometer with an integrating sphere with an excitation wavelength of 340 nm. 1 H NMR spectra were recorded on a JEOL nuclear magnetic resonance (NMR) instrument at 400 MHz with dimethyl sulfoxide-D6 (DMSO-D6) as the solvent. The morphology and size of the CDs and β-CD-CQD hybrid nanosponges were analyzed on a JEM-1200EX transmission electron microscopy (TEM) by depositing their aqueous dispersions on the copper grid. The hydrodynamic diameter of the β-CD-CQD hybrid nanosponges was measured in an aqueous dispersion with dynamic scattered light (DLS, BI-200SM) at room temperature.
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RESULTS AND DISCUSSION Synthesis and Characterization of Luminescent Carboxyl-Functionalized Nitrogen-Doped CQDs. The fluorescent carboxyl-functionalized nitrogen-doped CQDs were obtained via a hydrothermal route with sodium citrate as the carbon source in the presence of ammonium bicarbonate,32 with a particle size < 2 nm (Figure 1) and an
Figure 1. TEM images of the CQDs (left) and the β-CD-CQD hybrid nanosponges (right).
average hydrodynamic diameter (Dh) < 5 nm (Figure 2), due to swelling in water, indicating their surface hydrophilic functional groups (−OH and −COOH). It was also revealed by the characteristic absorbance of −OH and CO bonds at 3404 and 1703 cm−1 in the FT-IR spectrum, respectively (Figure S1). The CQDs exhibited two notable peaks in the UV−vis absorption originated from the carbogenic core and surface defects: the one at 235 nm of the π−π* transition of the CC 4086
DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
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Figure 2. Typical hydrodynamic diameter distributions of CQDs (a), β-CD-CQD (b), and DOX@β-CD-CQD (c) nanosponges.
tumor intracellular microenvironment, respectively. At pH 7.4, the cumulative release increased quickly to about 13% within the first 12 h (Figure 5a), due to the release of the DOX molecules loaded via the supramolecular complexation with the β-CD units and/or the various noncovalent interaction (such as electrostatic interaction, hydrogen bond, and π−π stacking) with the CQDs units in the surface layer of the DOX@β-CD-CQD nanosponges.42 Then, the releasing rate slowed down within the following 90 h, with a final cumulative release of 19%. Owing to the supramolecular complexation in the theranostic nanomedicine, the premature drug leakage in the normal physiological medium was much lower than the reported works, in which DOX was loaded on the surface of CQDs via the weak electrostatic interaction.43,44 While at pH 5.0, DOX was released in a sustained mode with a cumulative release of 61% within 100 h, mainly due to the lower inclusion constant in the supramolecular complexation in the acidic media than that in the basic media.45 The βCD units in the β-CD-CQD hybrid nanosponges formed inclusion complexes with lipophilic DOX via noncovalent bonds, which are formed or broken during the guest−host complex formation, the complexes are in dynamic equilibrium with free drug and β-CD units,46 showing an affinity-driven regulated release behavior.47 It is to say that there should be a balance between the free released DOX and the encapsulated DOX molecules in the β-CD units via supramolecular complexation,41 which actually controlled the DOX release although DOX could be protonated in the acidic media. Such an accelerated DOX release in the simulated tumor microenvironment but only a minimal release in the normal physiological condition is favorable to achieve high antitumor efficacy and minimize cytotoxicity in healthy cells.
Figure 3. UV−vis spectra of DOX, CQDs, β-CD-CQD, and DOX@ β-CD-CQD in aqueous dispersion. Inset: (a) CQDs and (b) β-CDCQD excited by daylight and at 365 nm UV.
downshifted to 7.89, 7.63, 7.33, and 5.24, respectively, although the former two overlapped with the protons linked with the unsaturated carbons in the CQDs. The phenomena revealed the inclusion position between β-CD and the anthracene ring of DOX, indicating the DOX-loading via host−guest inclusion.41 A DOX-loading capacity of 39.5% was obtained for the DOX@β-CD-CQD theranostic nanomedicine with Dh of 309 nm (Figure 2c), meaning a DOX content of 28.3%. The increased Dh also revealed the successful DOXloading via the host−guest complexation. Furthermore, a high drug encapsulation efficiency (DEE) of 79% resulted, indicating a high formation constant in the supramolecular complexation. The in vitro controlled release behavior of the DOX@β-CDCQD theranostic nanomedicine was investigated at pH 7.4 and 5.0, mimicking the normal physiological medium and the
Figure 4. PL emission spectra of CQDs (left) and β-CD-CQD (right) in an aqueous dispersion under different excitation wavelengths; Inset: the normalized PL intensity. 4087
DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
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Figure 5. In vitro DOX release profiles for DOX@β-CD-CQD at pH 5.0 and pH 7.4. Data present mean ± SD (n = 3) (a). In vitro cell viability of HepG2 cells incubated with different concentrations of free DOX, β-CD-CQD, and DOX@β-CD-CQD nanosponges for 24 h. Data present mean ± SD (n = 5) (b).
Figure 6. Fluorescent images of HepG2 cells stained by Hoechst, incubated without (a) or with DOX@β-CD-CQD theranostic nanomedicine (b) for 24 h. For each panel, images from left to right show the cell nuclei in bright field and stained by the Hoechst, DOX fluorescence, and the merged image, respectively.
indicating the nondiffusional release mechanism50 due to the formation constant. Cellular Uptake and Cytotoxicity. The in vitro cellular toxicity of the β-CD-CQD and DOX@β-CD-CQD nanosponges to HepG2 cells were evaluated by the MTT assay. The blank β-CD-CQD hybrid nanosponges exhibited no obvious toxicity (cell viability >84%) at 0−20 μg/mL after 24 h of incubation (Figure 5b). As the dosage of the DOX@β-CDCQD theranostic nanomedicine was increased, the cell viability decreased distinctly, although the data were higher than those with the free DOX in the whole dosage range. Considering the DOX content of 0.283 g DOX/g nanomedicine and the sustained release behavior (accumulative release less than 30% within 24 h), the DOX concentration gradient increased to 1.7 μg/mL with the DOX@β-CD-CQD theranostic nanomedicine concentration of 20 μg/mL after 24 h of incubation. Under such a case, the cell viability (29%) was similar to that with the free DOX of 10 μg/mL. The results demonstrated that the DOX@β-CD-CQD theranostic nanomedicine exhibited an enhanced anticancer efficacy rather than the free DOX.51 A half maximal inhibitory concentration (IC50) of about 5.00 μg/ mL was found for the proposed DOX@β-CD-CQD theranostic nanomedicine, in comparison to the free DOX of 2.26 μg/mL.
Interestingly, the release profile could be divided into three stages (Figure 5a): within the first 30 h, the loaded DOX molecules in the surface layer of the DOX@β-CD-CQD theranostic nanomedicine were released, similar as that in the pH 7.4 media. A release ratio was obtained that was higher than that in the pH 7.4 media, owing to the high DOX solubility in acidic media due to protonation, which would decrease the inclusion constant in the supramolecular complexation of DOX with the β-CD units. For the loaded DOX inner layer, they were released slowly, because of the high formation constant.48,49 Furthermore, the hydrophobic nature of the DOX-complexed β-CD went against the diffusion of the protonated DOX. After 12 h of releasing with accumulative release near 50%, the hydrophilic outer shells were formed due to the DOX release, favoring the diffusion of the protonated DOX out of the theranostic nanomedicine. Thus, a faster sustained DOX release was observed. The Higuchi and Korsmeyer−Peppas equations were used to investigate the release mechanism. The correlation coefficients (R2) in the Higuchi theory were much higher than that from the Korsmeyer−Peppas one (Figure S4), indicating the release mechanism could be well explained with the Higuchi model. The values of the release rate constant (k) were fitted as 0.2486 and 0.6298 at pH 7.4 and 5.0 respectively, 4088
DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of Gansu Province (Grant No. 18JR3RA271) and the Program for New Century Excellent Talents in University of the Ministry of Education of China (Grant NCET-090441).
The cellular uptake of the DOX@β-CD-CQD theranostic nanomedicine was investigated by a fluorescence microscope. Obviously, the red fluorescence locations (DOX) were completely overlapped with the blue fluorescence locations (cell nuclei) in the merged image (Figure 6), demonstrating the proposed DOX@β-CD-CQD theranostic nanomedicine could be internalized into HepG2 cells, and the released DOX was accumulated into the cell nuclei.52
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00508. FT-IR spectra of β-CD, CQDs, and β-CD-CQD hybrid nanosponges; 1NMR spectra of β-CD, β-CD-CQD nanosponges, and DOX@β-CD-CQD nanosponges; DLS analysis and TEM images of the β-CD-CQD hybrid nanosponges prepared with other mass feeding ratios; and fitted plots of the DOX release with Higuchi and Korsmeyer−Peppas models (PDF)
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REFERENCES
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CONCLUSIONS In summary, the fluorescent hyper-cross-linked β-cyclodextrincarbon quantum dot (β-CD-CQD) hybrid nanosponges, with a high photoluminescence quantum yield (PLQY) of 38.0% and average hydrodynamic diameter of about 200 nm, were synthesized for the first time for tumor theranostic application, by facile condensation polymerization with the carboxylfunctionalized nitrogen-doped CQDs and β-CD as the multifunctional monomers at a mass feeding ratio of 1:5. They exhibited excellent biocompatibility and strong bright blue fluorescence excited at 365 nm, demonstrating their potential application in tumor imaging. After loading DOX via supramolecular complexation, the final DOX@β-CD-CQD theranostic nanomedicine of about 300 nm was achieved with a DOX content of 28.3%. The proposed theranostic nanomedicine showed a pH response-controlled release and could release DOX in the simulated tumor microenvironment in a sustained release mode, owing to the formation constant in the supramolecular complexation of DOX with the β-CD units in the β-CD-CQD hybrid nanosponges. They possessed an IC50 value of 5.00 μg/mL to HepG2 cells, demonstrating an antitumor efficacy more enhanced than that of the free drug. The fluorescence microscope analysis indicated that the proposed theranostic nanomedicine could be effectively internalized into HepG2 cells, and the released DOX was accumulated into the cell nuclei. Features, such as easy synthesis, excellent biocompatibility, strong bright blue fluorescence emission, pH responsive sustained release, and enhanced anticancer efficacy, demonstrated a promising application of the proposed DOX@β-CD-CQD theranostic nanomedicine in future tumor treatment.
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AUTHOR INFORMATION
Corresponding Author
*Tel./Fax: 86 0931 8912582; Email:
[email protected]. ORCID
Peng Liu: 0000-0003-1198-4925 Notes
The authors declare no competing financial interest. 4089
DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
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DOI: 10.1021/acs.molpharmaceut.8b00508 Mol. Pharmaceutics 2018, 15, 4084−4091
