Non-polymeric pH-sensitive carbon dots for treatment of tumor

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Non-polymeric pH-sensitive carbon dots for treatment of tumor Jeongdeok Seo, Jonghwan Lee, Chaebin Lee, Soo Kyung Bae, and Kun Na Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00813 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 12, 2019

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Bioconjugate Chemistry

Non-polymeric pH-sensitive carbon dots for treatment of tumor Jeongdeok Seo†, Jonghwan Lee†, Chae Bin Lee‡, Soo Kyung Bae‡ and Kun Na*,† †Department

of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Bucheon-si, Gyeonggi-do 14662, Republic of Korea

‡College

of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The

Catholic University of Korea, 43 Jibong-ro, Bucheon-si Gyeonggi-do 14662, Republic of Korea *Correspondence

should be addressed to K. N. (email: [email protected])

ACS Paragon Plus Environment

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Non-polymer, pH-sensitive carbon dots (pSCDs) was developed to overcome the disadvantage of pH-sensitive polymers such as inevitable synthesis, wide distribution of molecular weight, uncontrolled loading and release rate of drug, and toxicity by biodegradation. The pSCDs were synthesized by one spot synthesis for 3 min using citric acid (CA) and 1-(3-aminopropyl) imidazole (API). Imidazole groups were present on pSCD surfaces and facilitated DOX loading via hydrophobic interactions (loading efficiency: 78.55%). The DOX-loaded pSCDs collapsed at tumoral pH (pH ~6.5) due to protonation of the imidazole groups, and DOX was released about 7 times higher than the control group. The therapeutic effect was confirmed in vitro using HCT-116 (human colon cancer), PANC-1 (human pancreatic cancer) and SKBR-3 (human breast cancer) cells. Additionally, the DOX-loaded pSCDs successfully inhibited tumor growth in an HCT-116bearing mouse model and did not show toxicity. These results indicate that non-polymeric, pSCDs platform has the potential to be used as a cancer targeting therapeutic materials.

KEYWORDS: pH-sensitive materials, carbon dot, non-polymeric, drug delivery, cancer therapy


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Introduction pH-sensitive polymers are stimuli-responsive materials that can respond to specific pH through changes in structure and properties such as surface activity, chain form, solubility and composition(1). pH-sensitive polymers generally very useful in a variety of applications such as drug delivery(2), gene delivery(3), sensor(4) and membranes(5). Especially, pH-sensitive polymers widely be used in the treatment of cancer due to tumor microenvironments (pH 6.8)(6) and endosomes (pH 5.5)(7) that are more acidic than normal cells (extracellular pH 7.4 and intracellular pH 7.2)(6, 8-11). As the pH-sensitive polymers, chitosan(12), piperidine(13), imidazole(14), and amino acid-containing polymers (histidine(15), lysine(16, 17), glutamate(18)) are mainly used. In addition, pH-sensitive polymer that act by breaking bonds in the acidic environment of cancer have been studied by utilizing hydrazine(19), oxime (20), and acetal(21) bonds, which are sensitive to acid. However, synthesis is inevitable in order to possess the property of loading and delivery to the target region of the drug using pH-sensitive polymers. These should maintain the stability of the pH-sensitive region of materials and be solubilized during the synthesis process. This causes a complicated synthesis processes and, thereby, time and cost problems(22-24). Furthermore, polymers have a wide distribution of molecular weight, so even if a pH-sensitive polymer is synthesized, the molecular weight is not uniform (25). As a result, it is not possible to obtain a change characteristic at a desired pH and there is a problem that the efficiency of drug encapsulation varies. In addition, drug delivery system (DDS) using a pH-sensitive polymer, there is a problem that the pH-sensitive region is inside in the carrier and may not response at a desired pH (26), and the degradation of the pH-sensitive polymer produced the toxically residue(27, 28). Therefore, pH-sensitive polymer utility enhances therapeutic and diagnostic advantages over


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conventional medicines, but still necessary to improve and verify the functionality, stability, and toxicity. Carbon dots (CDs) have a prominent characteristic of fluorescence, and much research has been conducted over the past decade due to their easy synthesis, low cost and high biocompatibility(2932). CDs with quasi-spherical morphology based on carbon and sp2 and sp3 chemical bonds and can be manufactured through a simple process(33, 34). CDs are produced through organic materials containing carbon, and among the production methods are hydrothermal (35, 36), microwave (37, 38), pyrolysis (39), electrochemical methods (40), and acid vapor cutting (41). These methods are capable of easily mass-producing(42) the nanoparticles in a short period of time. In particular, in the microwave-assisted synthesis process, microwave irradiation results in the materials align to dipoles in the external field. At the same time, the electric field causes strong agitation by changing the direction of molecules, which causes intense internal heating. Consequently, microwave-assisted pyrolysis can substantially reduce manufacturing energy and time cost by heating the substance almost instantly in a homogeneous and optional way. Additionally, the microwave-assisted synthesis process has the feature that various functional groups can be imparted to the dot surface, depending on the materials used. Here, we developed non-polymeric, pH-sensitive DDSs to treatment of colon cancer using CDs through a simple manufacturing process. Cancer growing in the wall of the colon may be curable with surgery, but widely spread cancers are usually not curable. Although, DDSs has been applied for the treatment of colon cancer, this is the first research to delivery non-polymeric, doxorubicin loaded pH sensitive carbon dot (DOX-pSCDs) for the treatment of colon cancers. The nonpolymeric pSCDs were manufacturing via microwave using citric acid (CA) and 1-(3-aminopropyl) imidazole (API) (Scheme 1A). DOX can be encapsulated by imidazole group present in the CDs,


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and can be released safely and effectively in the cancer by the tumor environment pH. After in vitro experiments with DOX-pSCDs, we evaluated and discussed delivery and therapeutic effect for the treatment of cancer in mice.

Results and discussion Preparation and Characterization of pSCDs. Before the pSCDs were prepared, the time required for the microwave-assisted synthesis was determined using CA and API synthesized in a 1:4 molar ratio via microwave for 1 min, 3 min and 5 min. As shown in Figure S1A in Supporting Information (SI), the CDs synthesized for 3 min had a high fluorescence property, but the CDs synthesized for 1 min and 5 min showed almost no fluorescence properties. In addition, the surface charge of CDs synthesized for 1 min and 5 min was not changed to a positive charge at the pH of the tumoral environment (~ pH 6.5), but the surface charge of CDs synthesized for 3 min was changed to a positive charge at tumoral pH (Figure S1B in SI). As a result, when the microwaveassisted synthesis is applied for 5 min, the structure of the imidazole group in API collapsed, and the pH-sensitive property disappeared. Since the C-H bond energy of the imidazole group in API (119 kcal/mol)(43) is higher than the C-H bond energy of CA (89 kcal/mol)(44), CA becomes the material of the carbon dot, and imidazole group is present on the surface of CDs. Therefore, pSCDs were synthesized for 3 min in the microwave by different ratios of API (Table S1 in SI). The three types of pSCDs were prepared at different concentrations of API (1, 2, and 4 M) and designated as pSCD1, pSCD2, and pSCD4, respectively. When the reaction mixture was heated in the microwave, the solution turned into a sticky solid, and the color turned to brown-yellow. The reaction flask was naturally cooled to room temperature and then placed in a dialysis bag (MWCO


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500) and dialyzed against deionized water for 1 day (exchanged twice) to remove small molecules. Finally, the dialyzed solution was freeze-dried, and we obtained 0.92 g of brown-yellow pSCDs. The UV-vis absorption property of pSCD1, pSCD2 (Figure S2A in SI) and pSCD3 (Figure 1A) showed a 340 nm absorption band that the n-π* transition, and pSCD2 and pSCD3 had similar absorption intensities. From the result of the n-π* transition, pSCDs emitted bright blue light at 365 nm (Figure S2B in SI). The fluorescence intensity of pSCD3 (Figure 1B) indicated that the emission wavelength of pSCD3 showed a maximum emission wavelength at an excitation wavelength of 380 nm, and the fluorescence intensities of pSCD2 and pSCD3 was higher than that of pSCD1 (Figure S2C and D in SI). Free API did not show the fluorescence (Figure S2E in SI), unlike pSCDs. In the literature, carbon dots prepared with N-containing carbon materials facilitate high fluorescence(45-48). N-containing carbon materials have been used for intrinsically doping N atoms into the carbon core to change the fluorescence intensity properties of the resultant carbon dots (49). The fluorescence intensity of pSCD3 at pH 5 to 10 (Figure S3A in SI) was constant and unchanged. The fluorescence properties, according to the concentration of pSCD3 in aqueous solution, confirmed that the fluorescence pSCD3 was quenched at 2.5 mg/ml or more (Figure S3B in SI). The surface charge of pSCD3 was 0.9 mV and -9.2 mV at pH 6.5 and 7.4, respectively (Figure 1C). On the other hand, the charge of pSCD1 and pSCD2 (Figure S4A in SI) did not change to a positive charge at pH 6.5, making these ill-suited as pH-sensitive DDSs for cancer therapy. The structure and morphology of pSCD3 were analyzed by transmission electron microscopy (TEM). The TEM images (Figure 1D) showed that the size of pSCD3 was distributed in the range from 3 to 6 nm. Likewise, the DLS analysis of pSCD3 showed an average size of 5.01 nm, and pSCD1 and pSCD2 had an average size of 4.80 and 4.91 nm, respectively (Figure S4B and C in SI). The X-ray photoelectron spectroscopy (XPS) spectra of the pSCDs (Figure 1E, Figure S5A,


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B and C in SI) exhibit three peaks at 284.1, 398.6, 531.8 eV, which are originated to C 1s, N 1s, and O 1s, respectively. The XPS spectrum of C 1s (Figure S5D, E and F in SI) could be deconvoluted into three surface components and exhibited three peaks at C-C, with binding energies of 284.4 eV, C-O/C=N at 285.8 eV and C=O at 287.7 eV. The high-resolution XPS spectra of N 1s (Figure 1F and Figure S6A and B in SI) exhibited three peaks at C=N-C at a binding energy of 398.1 eV, N-H at 399.8 eV, and N-H (pyrrole) at 401.2 eV. On the other hand, the XPS spectra CDs not containing N exhibited two peaks at 284.1 and 531.8 eV (Figure S6C in SI), which are originated to C 1s and O 1s, respectively. The XPS spectrum of C 1s (Figure S6D in SI) showed three peaks at C-C at binding energies of 284.4 eV, C-O at 285.7 eV, and C=O at 287.7 eV. In addition, as shown in Figure S6E in SI, the content of N was 3.2% in pSCD1, 6.9% in pSCD2 and 12.1% in pSCD3. On the other hand, the content of O was 16.6% in pSCD1, 19.9% in pSCD2 and 9.9% in pSCD3, but the content of C did not change significantly. This is indicative of the API doped onto the surface of pSCDs, and the doped imidazole of API facilitated increasing fluorescence and pH sensitivity. The XRD patterns of pSCD3 (Figure S7A in SI) displayed one peak at 3.3 Å, which was originated to dis-ordered C element and the graphite lattice spacing. FTIR spectra (Figure S7B in SI) were used to identify the surface functional groups present on pSCD3. The two absorption bands at 2900 and 3800 cm-1 were assigned to stretching vibrations of N-H and O-H, and the bands at 1640 cm-1 were attributed to the vibrational absorption band of C=O. The bands at 1570 and 1387 cm-1 were from the banding vibrations of NH and C-NH, respectively. This indicated that there are lots of N containing groups on the surface of pSCD3. The peaks at 1050 and 1105 cm-1 are related to the C-OH stretching vibrations and suggest the presence of many hydroxyl groups. These functional groups improve the hydrophilicity and stability of pSCD3 in an aqueous system.


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Preparation and Characterization of DOX-loaded pSCDs The DOX-loaded pSCDs were prepared through self-assembly of DOX and pSCD. Unreacted free DOX was removed using the dialysis method (MWCO 3000 Da). DOX was incorporated into the complex core through hydrophobic interactions with imidazole. To determine optimal pSCD needed to prepare DOX-loaded pSCDs, three types of pSCDS were compared with different DOX feeding ratio; the CD ratio was fixed to 1.0:13.0. At each ratio, DOX loading contents and loading efficiency varied by approximately 5-13% and 4-78%, respectively (Table S2 in SI). As the N element ratio increases (Figure S6E in SI), the loading efficiency and loading contents of DOX increase, which is due to the increase of the imidazole groups. The hydrodynamic size and morphology of the DOX-loaded pSCDs was confirmed by DLS, and DOX-loaded pSCD3 were confirmed via transmission electron microscopy (TEM). As described in Figure S8A, B and C in SI, the average size of DOX-loaded pSCD3 was 71.36 nm, and those of DOX-loaded pSCD1 and DOX-loaded pSCD2 were 88.6 and 85.3 nm, respectively (Table S2 in SI). According to DOX loading efficiency and loading contents results, we decided to use DOX-loaded pSCD3 for the subsequent experiments. The TEM image of DOX-loaded pSCD3 showed that the size was approximately 50 nm at pH 7.4 (Figure 2A). At pH 6.5, we observed that the DOX-loaded pSCD3 was collapsed (Figure 2B), which indicates that the deprotonated imidazole was protonated at pH 6.5, and the hydrophobic property changed to a hydrophilic core DOX-loaded pSCD3. In addition, the DOX-loaded pSCD3 surface presented imidazole group protonation or deprotonation, so DOX-loaded pSCD3 size was reversible (Figure 2C), and below pH 6.5, DOX was released and precipitated (Figure S9 and Figure S10 is SI). The fluorescence properties were compared between free DOX and DOX-loaded pSCD3 in water and DMSO to confirm that the DOX was loaded successfully. Based on the results


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(Figure S11 in SI), the DOX fluorescence intensity of free DOX and DOX-loaded pSCD3 was not displayed in water, but the DOX fluorescence intensity was same in the DMSO. This means that the hydrophobic DOX was well-loaded in the DOX-loaded pSCD3 core. The zeta potential of DOX-loaded pSCD3 was -27.5 mV at pH 7.4 and converted to approximately 0 mV at pH 6.4 (Figure 2D). The protonation of the imidazole group below pH 6.5 induced an increase in the zeta value. To confirm the critical micelle concentration (CMC) of pSCD3, CMC was investigated using Nile red. Nile red is a fluorescent dye that is strongly dependent on the polarity of its environment (50, 51). When the amount of pSCD3 was increased at pH 7.4, the Nile red fluorescence intensity was increased (Figure S12A in SI) due to increased non-polarity. On the other hands, the fluorescence intensity of pSCD3 (x-axis) at pH 6.5 was decreased (Figure S12B in SI) due to the increase polarity of Nile red. This is related to insolubility, and fluorescence is redshifted but strongly quenched(51). As a result, the CMC value of pSCD3 at pH 7.4 and 6.5 was 0.958 mg/ml and 4.090 mg/ml, respectively (Figure 2E and F). This demonstrates that pSCD3 can be used for hydrophobic drug loading and release at a specific pH via protonation or deprotonation of imidazole. In vitro drug release of DOX-loaded pSCD3 pH-sensitive, controlled release of DOX-loaded pSCD3 was investigated. Only 10% of DOX from DOX-loaded pSCD3 was released after 3 days of incubation in pH 7.4 buffer (Figure 3), because hydrophobic interactions between DOX and imidazole of pSCD3 prohibits the efficient release of DOX. On the other hand, DOX was released up to nearly 80% in pH 6.5 buffer after 3 days (Figure 3A). Furthermore, the DOX-loaded pSCD3 showed the fastest release after exchange of buffer from pH 7.4 to 6.5, leading to rapid collapse (Figure 3B) through protonation of imidazole in pSCD3, which enables DOX release.


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In vitro cellular uptake and cytotoxicity of DOX-loaded pSCD3 The cytotoxicity of pSCD3 was confirmed before the cell uptake and cytotoxicity of DOX-loaded pSCD3 was confirmed in HCT-116 (Figure S13A in SI). After treatment with pSCD3, no cytotoxicity was observed when treated with pH 7.4 media. On the other hand, treatment with pSCD3 at pH 6.5 showed slight cytotoxicity at the concentration of 1 mg/ml. Likewise, pSCD3 showed a slight cytotoxicity at pH 6.5 in PANC-1 and SKBR-3 cells (Figure S13B and C in SI). The cytotoxicity is due to the protonation of imidazole present in the pSCDs at pH 6.5. The protonated imidazole has the cationic charge of pSCDs, which causes cell membrane disruption and, thereby, cytotoxicity(52, 53). The cellular internalization behavior of DOX-loaded pSCD3 was investigated by confocal microscopy with human colon cancer (HCT-116), human pancreatic cancer (PANC-1), and human breast cancer (SKBR-3) cells. When HCT-116 cells were treated with DOX-loaded pSCD3, DOX fluorescence intensity showed a different tendency depending on the pH. As shown in Figure 4A, the fluorescence intensity of pSCD3 and DOX in the treated DOXloaded pSCD3 in pH 6.5 media was higher than DOX-loaded pSCD3 in pH 7.4 media. Similarly, the Z-stack image (Figure 4B and C) showed that treatment with DOX-loaded pSCD3 in pH 6.5 media led to more penetration and fluorescence of DOX and pSCD3 than DOX-loaded pSCD3 in pH 7.4 media. In the cellular internalization of DOX-loaded pSCD3, the PANC-1 (Figure S15 in SI) and SKBR-3 (Figure S16 in SI) cells showed the same result as HCT-116. The difference in cellular internalization by pH is indicative of the pH sensitivity of pSCDs. When DOX-loaded pSCD3 was exposed to the cancer pH environment (~6.5), DOX was burst released momentarily due to the protonation of the imidazole group in pSCDs, and the released DOX precipitated via charge interactions with pSCDs (Figure S9 in SI). As a result, precipitated DOX and pSCD3 were internalized to the cultured cell in plate. When the DOX-loaded pSCD3 arrived at the tumor region,


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DOX was burst released to the tumor. Then, released DOX, precipitated DOX and pSCD3 showed an effect of anti-cancer effect via internalization to the cell membrane. To evaluate the in vitro toxicity, the HCT-116, PANC-1 and SKBR-3 cells were incubated with free DOX and DOXloaded pSCD3 in pH 7.4 and 6.5 media. The in vitro cytotoxicity was confirmed by methyl thiaolyl tetrazolium (MTT) assay. As shown Figure 5A, there was no difference in the cytotoxicity of DOX in the HCT-116 cells treated with free DOX at pH 7.4 and 6.5. However, when the DOX-loaded pSCD3 was treated at pH 6.5, the cytotoxicity was higher, with a drug concentration at least two times lower than pH 7.4 in HCT-116 cells (Figure 5B). Likewise, the cytotoxicity of DOX in the PANC-1 (Figure S14A in SI) and SKBR-3 (Figure S14B in SI) cells showed no difference in pH 6.5 and pH 7.4 compared with free DOX. On the other hand, the DOX-loaded pSCD3 showed higher cytotoxicity at pH 6.5 than pH 7.4 (Figure S14C and D in SI). Based on the cellular internalization experiments, the cytotoxicity of the drug is higher at a pH than 7.4 because the cell internalization on the drug is high at pH 6.5. In vivo chemotherapeutic effect of DOX-loaded pSCD3 in HCT-116 tumor-bearing mice To evaluate the feasibility of DOX-loaded pSCD3 for tumor growth inhibition, we evaluated the tumor therapeutic effect on HCT-116 tumor bearing mice. Generally, the colon cancers are difficult to treat only by chemotherapy and there are some limitations related to dose-limiting toxicities (DLTs) (54). The PBS, pSCD3, free DOX, and DOX-loaded pSCD3 were injected intravenously into mice and tumor volume change was confirmed over time (Figure 6A and B). The PBS-treated mouse tumors, which served as a control group, showed 2.7-fold more growth than the sample injected mice. Meanwhile, the pSCD3-treated mice showed lower cancer growth than the PBS group, indicating that injected pSCD3 affects the cancer cell membrane due to cationic charge by protonation of imidazole in the tumor region. Mice treated with free DOX


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initially did not show tumor growth, but confirmed the tumors grew 4 days later. Likewise, the body weight of the mice was reduced in the initial body weight, but the body weight recovered after 4 days (Figure 6C). This suggests that the anti-tumor effect of free DOX is slight but has toxicity. However, the tumors of treated with DOX-loaded pSCD3 showed dramatic tumor suppression efficacy than other groups, especially about 7 times smaller than the PBS group. No significant body weight decrease was detected throughout the treatments in the PBS, pSCD3 and DOX-loaded pSCD3 groups, although there was a slight decrease in the free DOX group (Figure 6C). These results indicate that the DOX-loaded pSCD3 is safe for use in tumor treatment and it is applicable to pH-sensitive DDSs. After treatment, tumors and organs were isolated from the mice and hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were conducted.As shown in Figure 7, free DOX and DOXloaded pSCD3 groups demonstrated the destruction of tumor cells. However, the DOX-loaded pSCD3 group produced more necrotic cells and showed better therapeutic effect than the free DOX group. The treatment of DOX-loaded pSCD3 significantly reduced the number of cancer cells and increased the number of TUNEL-positive tumor cells, indicating that the DOX-loaded pSCD3 effectively inhibited cell proliferation and increased anti-tumor efficiency leading to apoptosis. H&E analyses of major organ results are presented in Figure S17 in SI. Treatments with pSCD3, free DOX and DOX-loaded pSCD3 did not cause significant morphological changes in liver, heart, lung, spleen, and kidney compared to the PBS group. Thus, these results indicate that DOX-loaded pSCD3 leads to significant tumor growth inhibition that is superior to free DOX due to pH sensitivity. Tissue distribution of DOX and DOX-loaded pSCD3


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To investigate the distribution of DOX, the distribution of DOX and doxorubicinol in major organ along with different tissues were determined after intravenous injection of free-DOX and DOXloaded pSCD3 (10 mg/kg DOX equivalent, each) in male rats. Figure 8A and B show various tissue concentrations of DOX and doxorubicinol at 48 h post intravenous dosing. Concerning the tissue distribution, at 48 h post dosing, DOX-loaded pSCD3 led to 11.4- and 1.96-fold increases of the concentration of DOX in liver and spleen, respectively, compared to the free-DOX (Figure 8A), whereas doxorubicinol was not detectable in both tissues (Figure 8B). In contrast, DOX and doxorubicinol concentrations in heart, colon, and kidney in DOX-loaded pSCD3 treated rats were significantly lower than with free-DOX (Figure 8A and B). Notably, we also observed much lower conversion ratios of doxorubicin to doxorubicinol in all tested five tissues, liver, spleen, heart, colon, and kidney, when DOX-loaded pSCD3 is administered in comparison to free-DOX (Figure 8A and B). Doxorubicinol is a highly cytotoxic metabolite and might be responsible for adverse effects, especially on the cardiotoxicity (55) . These results suggest that the pSCD3 enabled lower exposures of doxorubicinol and lower conversions of doxorubicin to doxorubicinol in tissues, thereby supporting the reduced tissue toxicity.

CONCLUSION In summary, we demonstrated a non-polymeric, pH-sensitive DDSs for the controlled release of anticancer drugs and treatment of cancer by taking advantage of the acidic tumor environment. In the designed DOX-loaded pSCDs, imidazole was present on the carbon dot surface, and DOX was encapsulated via hydrophobic interactions. In the tumor microenvironment (~ pH 6.5), protonation of imidazole in pSCDs was induced, thereby triggering the release of DOX. In vitro and in vivo


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experiments demonstrated that treatment with DOX-loaded pSCD3 resulted in higher anticancer efficacy and safety compared to free DOX. Importantly, this study can be used for pH-sensitive DDSs for cancer therapy as a new platform for facile synthesis of non-polymeric, pH-sensitive materials.


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EXPERIMENTAL SECTION Materials: Doxorubicin hydrochloride was purchased from MedKoo Biosciences (Morrisville, NC, USA). Citric acid (CA) and 1-(3-aminopropyl) imidazole (API) were purchased from Sigma Aldrich (St. Louis, Mo, USA). The 0.5 kDa dialysis membrane was obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA), and the 3.5 kDa membrane was purchased form Membrane Filtration Products, Inc. (Sequin, TX, USA). Phosphate-buffered saline, 1× (PBS), was purchased from Welgene (Daegu, Korea). RPMI 1640, DMEM medium, antibiotics (penicillin/streptomycin), fetal bovine serum (FBS), and DPBS were purchased from Gibco BRL (Invitrogen Corp., CA, USA). All of the other chemicals and solvents were analytical grade. Preparation of non-polymeric pSCDs First, 1 g of CA was dissolved with 5 ml distilled water in an Erlenmeyer flask (200 mL) and then added with different amounts of API (1 M – 4 M) under vigorous stirring. Then, the clear solution was put into a domestic microwave oven (1100 W) and heated for 3 min. After cooling to room temperature, the obtained red-brown, sticky liquid was dissolved in distilled water. Then, it was dialyzed against distilled water through a dialysis membrane (MWCO of 500) for 2 days (exchanged twice) to eliminate free CA and API, then freeze-dried for use in further experiments. As a comparative group, CDs not containing of N were prepared by the same method using citric acid (1 g) and glycerol (10 ml). Characterization of pSCDs UV-Vis absorption was measured on a UV-Visible spectrophotometer (UV-2350, Shimadzu, Japan).

Photoluminescence

(PL)

property

measurements

were

performed

using

a

Spectrofluorophotometer (RF-5301, Shimadzu, Japan). The XPS spectra of the pSCDs were


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measured on an electron spectroscope (SIGMA PROBE, Thermo Fisher Scientific, UK). The structure and morphology of the pSCDs were confirmed by transmission electron microscopy (TEM), (JEM-2100, JEOL Ltd, Japan) with an accelerating voltage of 200 kV. The samples for TEM were prepared by dropping an aqueous solution onto a copper grid-coated carbon film. The zeta potential and size distribution of pSCDs at various pH conditions were analyzed using DLS (Zetasizer Nano ZS, Malvern Instruments Ltd, UK) at 25 ℃. X-ray diffraction (XRD) profiles of the pSCDs were analyzed on an X-ray diffractometer (MiniFlex600, Rigaku, Japan) equipped with CuKα radiation at a scanning speed of 2°/min in the range from 10° to 80°. The FT-IR spectra of the pSCDs were measured on a FT-IR spectroscope (IRAffinity-1, Shimadzu, Japan). Preparation and Characterization of DOX-loaded pSCDs The DOX-loaded pSCDs were prepared by a dialysis method. In brief, purified pSCDs were dissolved in DMSO (13 mg/ml) and added with desalted DOX (1 mg/ml in DMSO), then dialyzed against distilled water (pH 7.5) through a dialysis membrane (MWCO of 3500) for 1 day. Then, the solution was purified with a syringe filter (0.45 μm, Millipore) to eliminate the precipitated materials. Additionally, the solution was purified using Amicon® Ultra Filter (MWCO of 10000, 3500 rpm for 25 min) to eliminate of free pSCD and DOX. To confirm the DOX loading efficiency (LE) and loading contents (LC) in DOX-loaded pSCDs, the contents of DOX was quantified via a standard curve. The samples dissolved in DMSO and water (8:2 ratio). The excitation and emission wavelengths of DOX were set to 490 and 590 nm, respectively. The LE and LC of DOX in DOX-loaded pSCDs were calculated as the following equations: LE (%) =

[

𝐷𝑂𝑋𝑖𝑛

] × 100

(1)

𝐷𝑂𝑋𝑡𝑜𝑡𝑎𝑙


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LC (%) =

[

𝐷𝑂𝑋𝑖𝑛 𝑇𝑀

] × 100

(2)

DOXin is the contents of DOX in DOX-loaded pSCDs, DOXtotal is the feeding contents of DOX for the preparation of DOX-loaded pSCDs, and TM is the total mass of DOX-loaded pSCDs containing all carbon dots and drugs used for micelle preparation. The size distribution and zeta potential of DOX-loaded pSCDs at varying pH were analyzed using a DLS. The morphological measurements were confirmed by TEM at pH 7.4 and 6.5. The sample for TEM was prepared by dropping an aqueous solution onto a copper grid-coated carbon film. The critical micelle concentration (CMC) of DOX-loaded pSCDs at different molar ratios in water was measured at room temperature using a Spectrofluorophotometer (RF-5301, Shimadzu, Japan), with Nile red as a fluorescent probe. The final concentration of Nile red was 5 X 10-5 M in acetone, and the solution was divided into 20 ml vials. The acetone was evaporated to make a thin film of Nile red. Then, a solution was added in which various concentration (10 – 0.039063 mg/ml) of pSCDs were dissolved at pH 7.4 and 6.5. The excitation and emission fluorescence intensity of nile red was measured at 520 and 618 nm, respectively. And, the slit widths were measured at ex = 5 nm and em = 5 nm. In vitro drug release test The drug release test of DOX-loaded pSCDs were evaluated using 0.01 M PBST (0.1% Tween 80, pH 7.4 and 6.5) using a drug release test kit (MWCO = 3.5 kDa). DOX-loaded pSCDs were transferred to kit tube and then parallel shaken in a water bath (50 rpm, 37 ℃). To evaluate drug release behavior based on pH change, pH 7.4 PBST was replaced with pH 6.5 PBST after 12 hours for evaluation of drug release behavior. At a predetermined sampling time, all PBST was removed and replaced with fresh PBST. The concentration of DOX in the buffer was determined using a


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solution (DMSO/DI water; 8:2 ratio). The DOX was quantified by fluorescence at 490 and 590 nm of excitation and emission wavelength, respectively. Cell culture and Incubation conditions. HCT-116 (human colon cancer), PANC-1 (human pancreatic cancer), SKBR-3 (human breast cancer) and L929 (mouse fibroblast) cells were obtained from the Korean Cell Lind Bank. Cells were cultured in RPMI-1640 or DMEM supplemented with 1% antibiotics solution and 10% heat inactivated FBS at 37 ℃ (100% humidity and 5% CO2). Cells were sub-cultured every 2-3 days. The pSCDs were suspended in serum-free (SF) medium. The free DOX was dissolved in DMSO and then mixed with SF medium (the DMSO concentration 397.10 for doxorubicin, 546.15 > 399.15 for doxorubicinol, and 528.00 > 321.10 for the internal standard. The calibration curves were linear (r ≥ 0.994) over the concentration range of 10–5000 ng/g for doxorubicin and 1– 500 ng/g for doxorubicinol. The within- and between-batch precisions and the accuracy were within the acceptable limits of ±15%. Statistical Analysis Data are expressed as the mean ± standard deviation. Student’s t test was used for statistical analysis.

ASSOCIATED CONTENT Supporting Information Available: The prepared ratio of pSCD synthesis and DOX-loaded pSCDs; DOX loading efficiency and loading contents of DOX-loaded pSCDs; additional characterization of pSCDs with fluorescence property, size, zeta potential, XRD, FTIR and XPS analysis; the size distribution of DOX-loaded pSCDs; physicochemical properties of DOX-loaded pSCD3; the cytotoxicity of pSCD3 in HCT-116 cells and DOX-loaded pSCD3 in SKBR-3 and


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PANC-1 cells; confocal images of DOX-loaded pSCD3 in SKBR-3 and PANC-1 cells; H&E staining of major organs. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (K. Na)

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2015R1A4A1042350), the Strategic Research through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. NRF-2017R1A2B3010038)


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Scheme 1. Schematic synthesis of (A) the pSCDs and preparation of (B) DOX-loaded pSCDs. (C) Schematic illustration for the drug delivery process of DOX-loaded pSCDs: (1) DOX-loaded pSCDs were delivered via the enhanced permeability and retention (EPR) effect and accumulated in the tumor region, (2) protonation of imidazole on the surface of pSCDs, (3) DOX released from DOX-loaded pSCDs.


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Figure 1. Characterization of pSCDs. (A) UV spectrum of API, CA and pSCD3. (B) the fluorescence intensity (C) and zeta potential of pSCD3 with various wavelength (pH adjusted via 1 N HCl or NaOH). (D) the TEM image and size distribution of pSCD3. (E) the full scan XPS spectrum of and (F) N1s spectrum of pSCD3.


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Figure 2. Characterization of DOX-loaded pSCD. The TEM image of DOX-loaded pSCD3 in (A) pH 7.4 and (B) pH 6.5. (C) the reversible size of DOX-loaded pSCD dispersed in pH 7.4 and pH 6.5 (interval time of pH change; 10 min, n=3). (D) the zeta potential of DOX-loaded pSCD at various pH. The critical micelle concentration (CMC) determination of pSCD3. (E) Maximum fluorescence intensity of Nile red in pH 7.4 pSCD3 solution and (F) pH 6.5 pSCD3 solution. All pH adjusted via 1N HCl and NaOH.


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Figure 3. (A)The DOX release behavior of DOX-loaded pSCD3 at pH 7.4 and 6.5 PBST and (B) change with pH 6.5 PBST after 12 hours of drug release in pH 7.4 PBST. (PBST, 0.01 M, 0.1% Tween 80, n=3).


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Figure 4. (A) The confocal image of HCT-116 cells with DOX-loaded pSCD3 in pH 7.4 and 6.5. (B) the Z-stack image of DOX-loaded pSCD3 treated HCT-116 in pH 7.4 and (c) pH 6.5. Scale bar: 100 μm.


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Figure 5. In vitro cytotoxicity of HCT-116 cells. MTT assay of (A) free DOX and (B) DOXloaded pSCD3 at pH 7.4 and pH 6.5 media (n=3, **P < 0.01).


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Figure 6. In vivo tumor therapeutic efficacy of HCT-116 subcutaneous mice model by intravenous injection with various samples. (A) The photograph of tumor growth inhibition test after tail vein injection of PBS, pSCD3, free DOX and DOX-loaded pSCD3. (B) the graph of tumor growth inhibition test and (C) body weight measurement of HCT-116 tumor bearing mice (n = 6, ***P < 0.001)


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Figure 7.

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Histological observation of the tumor tissues stained with H&E after 14 days of

intravenous injection of PBS, pSCD3, Free DOX and DOX-loaded pSCD3. Nuclei were stained blue, and the extracellular matrix and cytoplasm were stained red in H&E staining. Brown color indicates apoptotic cells after a TUNEL assay. Scale bars are 100 μm.


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Figure 8. Tissue concentrations of doxorubicin (A) and doxorubicinol (B) in rats treated with freeDOX and DOX-loaded pSCD at 48 h after intravenous injection. *P < 0.05; **P < 0.01; ***P < 0.001, as compared with free-DOX. Each point and bar represent the mean ± SD (n = 4).


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Table of Contents


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Non-polymeric pH-sensitive carbon dots for treatment of tumor

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