Facile Synthesis of Fluorescent Hyper-Cross-Linked Î²-Cyclodextrin

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Facile Synthesis of Fluorescent Hyper-Crosslinked #Cyclodextrin-Carbon Quantum Dot Hybrid Nanosponges for Tumor Theranostic Application with Enhanced Antitumor Efficacy Mingliang Pei, Jui-Yu Pai, Pengcheng Du, and Peng Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00508 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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Molecular Pharmaceutics

Facile Synthesis of Fluorescent Hyper-Crosslinked β-Cyclodextrin-Carbon Quantum Dot Hybrid Nanosponges for Tumor Theranostic Application with Enhanced Antitumor Efficacy Mingliang Pei,a Jui-Yu Pai,b Pengcheng Du,a and Peng Liua,* a

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

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Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30043, Taiwan

ABSTRACT: Fluorescent hyper-crosslinked 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 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 feeding ratio of 1:5. The DOX@βCD-CQD theranostic nanomedicine, around 300 nm with DOX-loading capacity of 39.5% by loading Doxorubicin (DOX) via host-guest complexation, showed pH responsive controlled release, and released DOX in the simulated tumor microenvironment in a sustained release mode,

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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 enhanced antitumor efficacy than the free drug. Keywords: Tumor theranostics; hyper-crosslinked hybrid nanosponges; β-cyclodextrin; carbon quantum dots; pH-responsive controlled release; supramolecular complexation

INTRODUCTION Theranostic nanomedicines, in which diagnostic and therapeutic functions are combined in one dose, have attracted more and more interests for imaging-guided drug/gene delivery in cancer treatment recently.1 Carbon quantum dots (CQDs), a novel kind of fluorescent carbon nanomaterials possessing the unique advantages of high stability, remarkably biocompatibility, easy to 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 crosslinking agent18-20 or nanocomplex21 as nanomedicines with suitable size favoring the EPR effect.


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Cyclodextrins (CDs) are a series of natural and water-soluble 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 multi-hydroxyl groups, CD-based nanosponges have been designed for improving water-solubility of water insoluble molecules, protecting degradable substances and as creative carriers or supports in pharmaceutics, catalysts, environmental control,24 with various organic molecules such as epichlorohydrin,25 citric acid,26 tetrafluoroterephthalonitrile,27 4,4'-difluorodiphenylsulfone,28 ferrocene,29 glutathione-responsive crosslinker,30 etc. By now, the CD-based hyper-crosslinked hybrid nanosponge incorporating inorganic nanomaterials is undescribed. Recently, Ko et al designed graphene quantum dot (GQD)-based nanocarrier as a promising theranostics for breast cancer, by modifying the GQD-NH2 of 26 nm with β-CD and Herceptin.31 In the present work, the carboxyl-functionalized CQDs with high quantum yield and plentiful carboxyl groups on their surfaces are hypothesized as a multi-functional crosslinker for CDs, also endowing fluorescent function. Based on the hypothesis, fluorescent hyper-crosslinked βcyclodextrin-carbon quantum dot (β-CD-CQD) hybrid nanosponges were synthesized for tumor theranostic application by facile condensation polymerization of the carboxyl-functionalized carbon quantum dots (CQDs) and β-cyclodextrin as multi-functional monomers (Scheme 1), by integrating the fluorescent imaging performance of the CQDs and the drug delivery function of β-CD.


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Scheme 1. Preparation processes for CQDs, β-CD-CQD and DOX@β-CD-CQD hybrid nanosponges.

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 (β-CD, ≥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.


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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 ºC for 4 h. After cooling to room temperature, the CQDs were purified by dialysis (MW=1000 Da) in the dark for 3 days. Synthesis of β-CD-CQD hybrid nanosponges. The β-CD-CQD hybrid nanosponges were synthesized via a facile condensation polymerization of the carboxyl-functionalized CQDs and βCD, with different mass feeding ratios (CQDs:β-CD 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 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-CD-CQD) 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@βCD-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-CD-CQD 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), respectively. The dialysis tube was incubated into the corresponding buffer solution (110 mL) at 37 ºC with shaking (rpm of 120).


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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-CDCQD 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 DMEM containing 10% FBS and cultured in presence of the nanosponges with different concentrations at 37 ºC in 5% CO2 atmosphere. The culture medium was disposed and washed thrice 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 with DMSO (150 µL) to dissolve the crystal. The cell viability was measured with enzyme linked immunosorbent at 490 nm. The cellular uptake of the DOX@β-CD-CQD hybrid nanosponges into HepG2 cells were 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 nm (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 FLS920 fluorometer with an integrating sphere with excitation wavelength of 340 nm. 1

H NMR spectra were recorded on a JEOL Nuclear Magnetic Resonance (NMR) instrument at

400 MHz with dimethylsulfoxide-D6 (DMSO-D6) as solvent. The morphology and size of the CDs and β-CD-CQD hybrid nanosponges were analysed on a JEM-1200EX transmission electron microscopy (TEM) by depositing their aquesous dispersions


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on copper grid. The hydrodynamic diameter of the β-CD-CQD hybrid nanosponges was measured in aqueous dispersion with dynamic scattered light (DLS, BI-200SM) at room temperature.

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 carbon source in the presence of ammonium bicarbonate,32 with particle size < 2 nm (Fig. 1) and average hydrodynamic diameter (Dh) < 5 nm (Fig. 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 C=O bonds at 3404 and 1703 cm-1 in the FT-IR spectrum, respectively (Fig. S1).

Fig. 1. TEM images of the CQDs (left) and the β-CD-CQD hybrid nanosponges (right).


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The CQDs exhibited two notable peaks in UV-vis absorption originated from the carbogenic core and surface defects: the one at 235 nm of the π–π* transition of the C=C bond in the aromatic sp2 domains of CQDs, while the other transition centered at 335 nm of the n–π* transition of the C=O bond (Fig. 3),33 resulting in 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 (Fig. 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.


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Synthesis and characterization of β -CD-CQD hybrid nanosponges. Then the hypercrosslinked β-CD-CQD hybrid nanosponges were synthesized via facile condensation polymerization of the CQDs and β-CD as multi-functional monomers with different mass


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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 C=O stretching in ester group appeared in the FT-IR spectrum of the products (Fig. S1), indicating the successful esterification between the two multifunctional monomers. In the 1H NMR spectrum (Fig. S2), both 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 successfully 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:OH6: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 the 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 (Figs. 1 and 2). Such great swelling ratio should be resulted from the relatively lower crosslinking degree due to the big space hindrance between the CQDs. As for the others prepared with mass feeding ratios of 1:2 or 1:10 exhibited obvious aggregation with broader diameter range (Fig. S3). The unique particle size and distribution of the hyper-crosslinked β-CD-CQD hybrid nanosponges prepared at the mass feeding ratio of 1:5 indicated their potential application as 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 bright blue fluorescence (Fig. 3) and excellent emission performance (Fig. 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


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CQDs after crosslinking with β-CD.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 1H NMR spectrum of the DOX@β-CD-CQD (Fig. S2b). In the enlarged figure (the insert in Fig. S2b), the peaks of DOX at the chemical shifts around 8.00, 7.85, 7.59, and 5.27 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 (Fig. 2c), meaning a DOX content of 28.3%. The increased Dh also revealed the successful DOX-loading via the host-guest complexation. Furthermore, high drug encapsulation efficiency (DEE) of 79% was resulted, indicating a high formation constant in the supramolecular complexation. The in vitro controlled release behavior of the DOX@β-CD-CQD theranostic nanomedicine was investigated at pH 7.4 and 5.0, mimicking the normal physiological medium and the tumor intracellular micro-environment, respectively. At pH 7.4, the cumulative release increased quickly to about 13% within the first 12 h (Fig. 5a), due to the release of the DOX molecules loaded via the supramolecular complexation with the βCD units and/or the various non-covalent interaction (such as electrostatic interaction,


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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.43,44 120 (a)

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Fig. 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) and 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).

While at pH 5.0, DOX was released in a sustained mode with 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 basic media.45 The β-CD units in the β-CD-CQD hybrid nanosponges formed inclusion complexes with lipophilic DOX via non-covalent bonds, which are formed or broken during the guest–host complex formation, the complexes are in dynamic


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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 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. Interestingly, the release profile could be divided into three stages (Fig. 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 high release ratio was obtained than 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, favouring 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 (Fig. 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


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pH 7.4 and 5.0 respectively, indicating the nondiffusional release mechanism,50 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 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 (Fig. 5b). As increasing the dosage of the DOX@β-CD-CQD theranostic nanomedicine, 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 case, the cell viability (29%) was similar that with 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. The cellular uptake of the DOX@β-CD-CQD theranostic nanomedicine was investigated by fluorescence microscope. Obviously, the red fluorescence locations (DOX) were completely overlapped with the blue fluorescence locations (cell nuclei) in the merged image (Fig. 6), demonstrating the proposed DOX@β-CD-CQD theranostic


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nanomedicine could be internalized into HepG2 cells and the released DOX was accumulated into the cell nuclei.52

Fig. 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 flied and stained by the Hoechst, DOX fluorescence, and the merged image, respectively.

CONCLUSIONS In summary, the fluorescent hyper-crosslinked β-cyclodextrin-carbon quantum dot (β-CD-CQD) hybrid nanosponges, with 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 carboxyl-functionalized nitrogen-doped CQDs and β-CD as multifunctional monomers at 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


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supramolecular complexation, the final DOX@β-CD-CQD theranostic nanomedicine of about 300 nm was achieved with DOX content of 28.3%. The proposed theranostic nanomedicine showed pH responsive 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 enhanced antitumor efficacy than 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. Such features, easy synthesis, excellent biocompatibility, strong bright blue fluorescence emission, pH responsive sustained release, and enhanced anti-cancer efficacy, demonstrated a promising application of the proposed DOX@β-CD-CQD theranostic nanomedicine in future tumor treatment.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. FT-IR spectra of β-CD, CQDs, and β-CD-CQD hybrid nanosponges; 1H NMR 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; Fitted plots of the DOX release with Higuchi and Korsmeyer-Peppas models. AUTHOR INFORMATION Corresponding Author.


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* Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Program for New Century Excellent Talents in University of the Ministry of Education of China (Grant No. NCET-09-0441).

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Facile Synthesis of Fluorescent Hyper-Cross-Linked Î²-Cyclodextrin

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