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Design and Investigation of Core/Shell GQDs/hMSN Nanoparticles as an Enhanced Drug Delivery Platform in Triple-Negative Breast Cancer Dongzhi Yang, Xinyue Yao, Jingjing Dong, Na Wang, Yan Du, Shian Sun, Liping Gao, Yuanyuan Zhong, Chuntong Qian, and Hao Hong Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00399 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Bioconjugate Chemistry

Design and Investigation of Core/Shell GQDs/hMSN Nanoparticles as an Enhanced Drug Delivery Platform in Triple-Negative Breast Cancer Dongzhi Yang1,2∗, Xinyue Yao2, Jingjing Dong2, Na Wang2, Yan Du2, Shian Sun4, Liping Gao5, Yuanyuan Zhong2, Chuntong Qian2, Hao Hong3∗ 1

Department of Pharmaceutical Analysis, Xuzhou Medical University, Xuzhou, Jiangsu 221004,

China 2

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University,

Xuzhou, Jiangsu 221004, China 3

Department of Radiology, University of Michigan, Ann Arbor, Michigan 48109-2200, United

States 4

Xuzhou Air Force College, Xuzhou, Jiangsu 221000, China

5

Xuzhou Cancer Hospital, Xuzhou, Jiangsu 221000, China

* (D.Y.) Tel: 86-516-63262138; E-mail: [email protected] (H.H.) Tel: 1-734-615-4634; E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Due to the excellent photoluminescent properties and singlet oxygen (1O2) generating efficiency, graphene quantum dots (GQDs) with maximal emission in near-infrared region (NIR) exhibited great potentials in cancer imaging and therapy. However, GQDs can be cleared quickly via the renal system in vivo because of their ultra-small size, which leads to the compromised cancer cell killing efficacy. Here, we report a hybrid nanoplatform, where GQDs were incorporated into the cavity of hollow mesoporous silica nanoparticles (hMSN) to form GQDs@hMSN-PEG nanoparticles (NPs). Optical characterization indicated that GQDs@hMSN-PEG NPs still maintained good absorption and emission properties from GQDs, and the composite NPs still possessed similar 1O2 generating efficiency. GQDs@hMSN-PEG NPs exhibited good biocompatibility in vitro and in vivo. High cargo-loading

efficiency

were

achieved

for

doxorubicin

(DOX),

and

the

formed

GQDs@hMSN(DOX)-PEG NPs showed the feasibility of tumor-oriented drug delivery. The extended retention time in tumor and good drug loading efficacy confirmed that GQDs@hMSN-PEG could serve as one promising candidate for combinational cancer treatment where photodynamic therapy and chemotherapy modules can be integrated into one system.

INTRODUCTION At present, the drug resistance caused by chemotherapy1, as well as adverse effects resulting from chemotherapeutic agents (e.g. liver and kidney damage, hair loss, nausea, cardiac toxicity etc.)2, promotes to develop more optimized anticancer strategies for cancer therapy. Over the past decade, nanostructure-based cancer therapeutics have been extensively explored and some nanomaterials have been confirmed to possess intrinsic anticancer properties. For example, Au nanostructures and fullerene-based nanomaterials were among the most investigated materials with those properties. Various types of Au nanostructures with tunable size and shapes exhibit strong light absorption and rapid heat conversion, which makes Au nanostructures attractive photothermal agents used in cancer ablation

3-7

. Fullerene-based nanomaterials such as Gd@C82(OH)22 were also described to have the

antioxidant properties and serve as the effective reactive oxygen species scavenger8. Fullerenol also has strong interactions with nitric oxide (NO) 9 synthesized by the nitric oxide synthases (NOS), which makes fullerene-based nanomaterial useful for cancer therapy. For those materials, however, ACS Paragon Plus Environment

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metal contamination is one major safety concern since metal ions (such as silver or copper) are always used during the synthesis procedures to control their morphology or structures10,11. Thus, it is very important to develop some functional materials without metal-mediated toxicity. Among all the available choices, carbon-based graphene quantum dots (GQDs) have been used in various biological applications due to their excellent water solubility and good luminescent/excitation properties12,13. It was reported that GQDs passivated with polyethylene glycol (PEG) derivatives could generate 1O2 upon irradiation with blue light, ready for therapeutic purposes14. By optimizing the synthetic route via a hydrothermal method with polythiophene derivatives as the carbon source, water-dispersible GQDs with a very high quantum yield (~1.3, approximately twice as high as that of most the state-of-the-art photodynamic therapy agents) can be synthesized15,16. In these two studies, researchers have confirmed that GQDs can produce single oxygen via the energy transfer from the GQDs to oxygen, instead of the common electron transfer. Those attractive properties suggest that the optimized GQDs could be used for synergistic imaging and therapeutic applications. Two major factors should be considered before a given nanomaterial can be used for imaging or therapeutic purposes: in vivo retention time and biocompatibility. Nanoparticles (NPs) like GQDs with ultra-small (less than 10 nm) size are usually cleared through the renal system promptly, resulting in minimal interactions with the diseased area. As critical as maintaining the original properties of GQDs, increasing the accumulation of GQDs in diseased regions is also indispensable before they can be used as biomedical agents. In previous research reports, GQDs were not good drug carriers due to their limited drug loading capacity or unoptimized drug release profile. Furthermore, how to effectively integrate both photodynamic therapy and chemotherapy modules into one system while having longer the retention time in vivo remains a challenge. NPs smaller than 200 nm can preferably enter the tumor region via a passive diffusion mechanism due to the existence of large gaps between the endothelial cells of the cancer tissue (100-600 nm), a phenomenon called enhanced permeation and retention (EPR) effect17,18. Thus, loading GQDs into biocompatible materials to adjust their circulation time maybe a feasible approach. Silica is classified as “generally recognized as safe” (GRAS) by the FDA and used frequently in cosmetics and as a food-additive. Owing to the high drug-loading capacity, biocompatibilities and ease of surface functionalization, one of the most popular silica materials series, mesoporous silica nanoparticles (MSN) have been continuously explored as drug delivery vectors19,20. Different materials can be loaded inside or on the ACS Paragon Plus Environment


surface of MSN for cancer imaging or therapy, where nanocomposites usually possess the properties from both MSN and NPs21-23. Hollow mesoporous silica nanoparticles (hMSN) with a large cavity inside MSN has gained increasing interests due to ultrahigh cargo holding capacity24-26. The porous and hollow structure makes hMSNs an ideal supporting system for GQDs, to not only adjust their retention time in vivo but also provide extra space for accommodating therapeutic agents. Here, we developed a graphene quantum dots / hollow mesoporous silica nanoparticle (GQDs@hMSN-PEG NPs) by loading GQDs into the cavity of hMSN. The 1O2 generation capacity of GQDs did not change significantly after being loaded into the cavity of hMSN. The GQDs@hMSN-PEG NPs still exhibit a broad absorption and strong emission spectra with the peak at 570-620 nm. After loading DOX into the nanoplatform, almost 2-fold higher tumor uptake (at 2 h post-injection [p.i.]) was observed in the GQDs@hMSN(DOX)-PEG NPs group when compared with that from the GQDs@hMSN-PEG NPs group, which indicated the potent cargo and material accumulation in tumor. With the growing development of carbon materials in biological applications, this study may pave the way for future cancer theranostics using GQDs derivatives. RESULTS AND DISCUSSION Materials synthesis and characterization. The GQDs prepared can be easily dissolved in an aqueous solution. Different synthetic methods were attempted for getting the GQDs@hMSN NPs. In method 1, the GQDs and hMSN were prepared separately before mixing these two solutions together. After being stirred for 24 h at room temperature (RT), the solution was rotary evaporated and freeze dried to get the NPs powder. Before studying the characteristics, the NPs were dissolved in phosphate-buffered solution (PBS) solution. In method 2, the GQDs and dSiO2 were separately synthesized. Tetraethyl orthosilicate (TEOS) was added into the mixture solution of GQDs and dSiO2 to form the original shell of hMSN. In method 3, the fresh GQDs was used as the seed to synthesize the dSiO2 NPs to form the GQDs@dSiO2 NPs. After being coated with another SiO2 layer, the complex GQDs@dSiO2/SiO2 NPs were etched in alkaline environment, where GQDs still keep the original characteristics, while dSiO2 was removed. The particle size distribution of the nanoparticles in each synthetic step was provided by dynamic light scattering (DLS) shown in Fig. S1. As shown in Fig. 1a, transmission electron microscope (TEM) images of NPs synthesized by the method 1 indicated that GQDs were not able to enter the cavity of hMSN through the relatively long surface channels with small pore size of hMSN being another limiting factor. By adopting ACS Paragon Plus Environment

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method 2, the mixture of GQDs and dSiO2 were used as the seed to form dSiO2 NPs. Fig. 1b showed that only very few GQDs was present in the cavity of hMSN because mesoporous SiO2 layer was formed more easily on dSiO2 instead of GQDs during the synthetic procedure of GQDs@dSiO2/SiO2 NPs. In method 3, GQDs were firstly prepared and was used as the seed to produce dSiO2. Fig. 1(c and e) indicated that GQDs@hMSN NPs were successfully formed, where GQDs were in the cavity of hMSN, and it maintained the porous and hollow structure of hMSN. The etching time is also very critical for the synthesis of hMSN. As shown in Fig. 1(d and f), the inner dSiO2 was not removed completely when the etching time was shorter than 40 min, while the outer layer structure would lose the uniformity and structural integrity when the etching time was longer than 50 min. So, in this work, the etching time was chosen to be in the range of 40-50 min. Owing to the good stability of GQDs in different solutions, the morphological characteristics of GQDs did not change in the etching and washing procedures. By measuring the fluorescence intensity of excessive GQDs, the loading efficiency of 0.26 mg GQDs/mg hMSN was calculated.

Fig. 1 The TEM images of GQDs@hMSN NPs prepared by different methods NPs prepared by different methods: a. Method 1; b. Method 2; c. Method 3; In method 3, GQDs@dSiO2/SiO2 NPs were etched for different time: d. 30 min; e. 45 min; f. 60 min.



Fig. 2 is the structure characteristics of the prepared GQDs@hMSN-PEG NPs. From the atomic force microscope (AFM) and TEM images, the height of GQDs was lower than 6.2 nm and the size was about 4.5 nm. From the TEM images, the size of GQDs@hMSN-PEG NPs is about 130 nm. The size obtained from DLS curve was bigger than that from TEM images, which resulted from the hydration of NPs in aqueous solution. After modification of 3-Aminopropyl trimethoxysilane (APS) hydrolysate, the amine group concentration determined by ninhydrin coloration method was about 200 nmol/mL. The successful surface engineering was validated in ζ-potential measurements, shown in Table S1. Compared with as-synthesized GQDs@hMSN, significant change of surface charge was observed after SCM-PEG5k-Mal coating (ζ-potential: from 11.86 ± 0.3 mV to -2.20 ± 0.02 mV). TGA curve was shown in Fig. 2g, where the GQDs@hMSN-PEG exhibited a slow decomposition upon heating, reaching a plateau at 300-500℃ with a 25% weight loss. Thus, the relative amount of PEG grafted onto GQDs@hMSN was calculated from TGA results, to be ∼0.25 mg of SCM-PEG5K-Mal loaded per mg of GQDs@hMSN.

Fig. 2 The structural and morphological characteristics of the prepared GQDs@hMSN-PEG NPs a. TEM image of GQDs; b. TEM image of hMSN; c. TEM image of GQDs@hMSN-PEG NPs; d. The single and magnification of GQDs@hMSN-PEG NPs. e. AFM image of GQDs; f. DLS of GQDs@hMSN, GQDs@hMSN-NH2 and GQDs@hMSN-PEG NPs; g. TGA curve of GQDs@hMSN-PEG NPs. The optical characteristics of GQDs and GQDs@hMSN nanoconjugates were measured by


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ultraviolet-visible (UV-Vis) and fluorescence (FL) spectroscopy. Fig. 3a showed the UV-Vis absorption spectra of the GQDs and GQDs@hMSN conjugates, illustrating that both GQDs and GQDs@hMSN conjugates exhibited broad absorption at the range of 200-500 nm. Characteristic absorption peak of GQDs is about 450 nm, while it can undergo blue shift to about 425 nm for GQDs@hMSN and its derivatives. It is consistent with that of PL spectra in Fig. 3c, where the emission wavelength of GQDs@hMSN NPs also exhibited blue shift from its corresponding GQDs, resulting from the quantum confinement effect after coating with hMSN. When the maximum emission wavelength was fixed, the same maximum excitation wavelength for GQDs and GQDs@hMSN derivations could be observed in the excitation spectra in Fig. 3b, indicating that the emission characteristics were primarily attributed to GQDs. During the transformation from polythiophene (PT2) to GQDs, the heating time is the vital factor for the emission performance of GQDs. By changing the heating time, GQDs and GQDs@hMSN-PEG with different emission wavelength can be obtained. As shown in Fig. 3d and Fig. 3e, the GQDs emitted fluorescence from 610 nm to 690 nm (between the red and near infrared [NIR] region) with a full width at half-maximum (FWHM) of ~ 70 nm. Compared with GQDs, the fluorescence spectra of GQDs@hMSN-PEG exhibited a ~40 nm blue shift with an emission wavelength of 570-620 nm. By using rhodamine 6G as the reference, the fluorescence yields (QYs) was up to 34.2% and 18.3% for GQDs and GQDs@hMSN-PEG, respectively, calculated from the slopes of the absorbance at 350 nm and the area of the emission spectra excited at 350 nm at different concentrations.



Fig. 3 The optical properties of GQDs and GQDs@hMSN conjugates a. UV-Vis absorbance spectra of GQDs and GQDs@hMSN conjugates; b. Excitation spectra of GQDs and GQDs@hMSN conjugates; c. Emission spectra of GQDs and GQDs@hMSN conjugates; d. Fluorescence spectra of GQDs; e. Fluorescence spectra of GQDs@hMSN-PEG. Stability evaluation and singlet oxygen leasing. The changes in the fluorescent intensity of GQDs and GQDs/hMSN-PEG NPs in an aqueous solution (PBS) and serum solution (10% fetal bovine serum [FBS], 90% PBS) over time were measured to investigate the stability of GQDs/hMSN-PEG NPs. The GQDs@hMSN-PEG could be well dispersed in different media/buffers (e.g. water, PBS, Roswell Park Memorial Institute [RPMI] 1640 medium and FBS) without observable aggregation for up to 2 weeks. Based on DLS measurements, no significant morphological alteration of GQDs@hMSN-PEG was identified post FBS incubation for 2 weeks. After being incubated in PBS for 2 weeks, the materials are centrifuged and the fluorescence spectra of supernatant and precipitate were both obtained. As shown in Fig. S2a, the steady fluorescence properties including the emission wavelength and fluorescence intensity confirmed that GQDs@hMSN-PEG were useful for in vivo applications. The hydrodynamic diameter of GQDs@hMSN-PEG was found to be 180 ± 10 nm (Fig. S2b). Previous report suggested that GQDs could produce single oxygen (1O2) under laser irradiation16. Here, the fluorescence properties under light irradiation and its correlation with oxygen


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level were studied. Results shown in Fig. 4a and Fig. 4b exhibited that the fluorescence intensity decreased slightly after increasing irradiation time and the fluorescence still maintained at 92% of original level after irradiation for 30 min. The fluorescence properties of GQDs were also affected by the oxygen concentration in the solution. Results indicated that the fluorescence intensity decreased with continuous oxygen filling over the time, which could reach 75% of original intensity. The decrease of fluorescence intensity was also consistent with that reported in literature16, which confirmed that the energy transfer (ET) from the GQDs to oxygen should be responsible for the excitation

of

ground-state

oxygen.

A

chemical

trapping

method

with

disodium

9,10-anthracendipropionic acid (Na2-ADPA) as the trapping agent and Rose Bengal (RB) as the standard photosensitizer (1O2 quantum yield ΦRB=0.75 in water) was used to assess the ability of GQDs and GQDs@hMSN-PEG to generate 1O2. Furthermore, the 1O2 generation ability of prepared GQDs were also compared with that of the GQDs synthesized in traditional method (GQDs’) with blue fluorescence properties. As illustrated in Fig. 4c, the absorbance of Na2-ADPA decreased in the presence of GQDs29, GQDs@hMSN-PEG and RB with prolonged irradiation time, while it still keeps stable for the GQDs’. Furthermore, the degradation of GQDs and GQDs@hMSN-PEG is larger than that from the RB, which indicated that the 1O2 quantum yields for GQDs and GQDs@hMSN-PEG are higher than that of RB. Compared with RB, the 1O2 quantum yields were 1.2 and 0.9 for GQDs and GQDs@hMSN-PEG, respectively. The 1O2 generation producing capacity in different media were evaluated. Results shown in Fig. S3 indicated that PBS, FBS and cells culture media had no effect on the characteristics of GQDs.

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a. Fluorescence spectra of GQDs with increasing light irradiation time; b. Fluorescence intensity variation with increasing light irradiation time; c. The 1O2 generation ability of GQDs, GQDs@hMSN-PEG, RB and GQDs’ with different oxygen level (indicated by oxygen filling time). DOX loading and release. DOX was loaded into GQDs@hMSN-PEG NPs to serve as a model anticancer drug to test the drug loading capacity. Our results indicate that GQDs@hMSN-PEG possess a very high DOX loading capacity. Based on the absorbance measurement at 488 nm for DOX, the amount of DOX loaded into GQDs@hMSN-PEG was calculated to be 0.780 mg DOX/mg GQDs@hMSN-PEG. The DOX release profile from GQDs@hMSN(DOX)-PEG was tested under simulated physiological condition at the pH of 5.0, 6.5 and 7.4 at 37℃. As shown in Fig. S4, at pH 7.4, 15.8% of DOX (0.12 mg DOX/mg GQDs@hMSN-PEG) could be released after 4 days, which suggested that loaded DOX within GQDs@hMSN-PEG was relatively stable under this condition. In contrast, when media pH was decreased to 5.0 (mimics endocytic compartments where the pH ranges from 4.5 to 6.5), the amount of released DOX was evaluated to approximately 57.6% (0.45 mg DOX/mg GQDs@hMSN-PEG) after 4 days. The results confirmed that the DOX can be loaded into GQDs@hMSN-PEG with high efficiency and the released behavior was pH-dependent. Safety/toxicity evaluation. For cytotoxicity assessment of the GQDs@hMSN-PEG samples in vitro, cells viability is examined to evaluate the effect of GQDs. The impact of GQDs@hMSN-NH2 and GQDs@hMSN-PEG on cells growth was tested in 4T1 cells (triple-negative breast tumor cells) and L929 cells (fibroblasts as the normal control). Fig. 5a indicated that over 86% of L929 and 4T1 cells remain alive after 24 h of incubation with GQDs. Similar cell activity profiles were found for those treated with GQDs@hMSN-NH2 and GQDs@hMSN-PEG (Fig 5b and 5c). For example, a cell viability of 84% was shown under the treatment of GQDs@hMSN-NH2 (150 µg/mL). These results revealed that GQDs, GQDs@hMSN-NH2 and GQDs@hMSN-PEG had good biocompatibility and imposed very low toxicity to L929 and 4T1 cells. At the same time, the effect of GQDs@hMSN(DOX)-PEG on the cells viability were also determined. Compared with the free DOX, the GQDs@hMSN(DOX)-PEG exhibited comparable cell toxic effect (in Fig. 5d), which proved the effective DOX release from the nanoplatform in tumor microenvironment. A relative long-term toxicity assay (72 h) of 2 µg/mL GQDs@hMSN(DOX)-PEG on 4T1 and L929 cells was evaluated. As shown in Fig. S5, the cell viability decreased over incubation time, and distinct cell viability difference between L929 and 4T1 cells could be witnessed after 60 h treatment of ACS Paragon Plus Environment

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GQDs@hMSN(DOX)-PEG, which may be due to the decreased efflux of DOX from 4T1 cells.

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Fig. 5 In vitro cytotoxicity of GQDs and GQDs@hMSN conjugates a. In vitro cytotoxicity of GQDs; b. In vitro cytotoxicity of GQDs@hMSN-NH2 NPs; c. In vitro cytotoxicity of GQDs@hMSN-PEG NPs; d. In vitro cytotoxicity of free DOX and released DOX from GQDs@hMSN(DOX)-PEG NPs A standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide (MTT) assay was performed to quantitatively evaluate the PDT efficiency and cytotoxicity of the GQDs@hMSN-PEG. 4T1 cells were irradiated for a constant concentration of 3 µg/mL GQDs@hMSN-PEG in PBS with during time from 1 min to 6 min, as illustrated in Fig. S6, respectively. A cell viability of 78% was observed with the irradiation time of 1 min; this value decreased with increasing irradiation time, decreasing to 26% for the time of 6 min. However, GQDs@hMSN-PEG has little effect on the survival of 4T1 cells in the dark even for the irradiation time of 6 min, indicating the low cytotoxicity and good biocompatibility of GQD@hMSN-PEG. A high dose of 50 mg/kg GQDs@hMSN-PEG solution was intravenously (i.v.) injected into



healthy Balb/c mice, and these animals were monitored for up to 30 days to assess the in vivo toxicity from GQDs@hMSN-PEG. As shown in Fig. S7, no noticeable signs of toxicity or side effects were found based on the body weight measurement compared to the control group, and in each case the mice survived. The main organs (heart, liver, spleen, lung, kidney, intestine, and stomach) of mice were collected post 1, 7 and 30 days of injection with the nanomaterials. Histological assessment was carried out on hematoxylin and eosin (H&E) stained organ slides, from which no observable changes were found indicating chronic (30 days p.i.) toxicity caused by GQDs@hMSN-PEG (Fig. 6b). To further study the potential toxic effect of GQDs@hMSN-PEG on the treated mice, mini chemistry panel test was also carried out to determine the general health status of GQDs@hMSN-PEG injected mice (Fig. 6a). Inspiringly, all measured biochemical parameters such as glutamic pyruvic transaminase (GPT), alkaline phosphatase (AKP), albumin (ALB), creatinine (Cr), urea nitrogen (BUN) fell within the normal ranges on day 1, day 7, and day 30 postinjection of GQDs@hMSN-PEG, which indicated that the primary function of liver and kidney were not impaired post GQDs@hMSN-PEG treatment. The oxidative stress index variation was also evaluated post GQD@hMSN-PEG treatment in vivo, which including total protein (TP), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and catalase (CAT). As shown in Fig. S8 and S9, the values of ALT, AST, BUN, and CREA fluctuate a little compared to the control groups but they remained within the normal ranges. The results of blood biochemistry, hematology analysis and oxidative stress index detection suggest no obvious toxicity of GQDs@hMSN-PEG.


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Fig. 6 In vivo toxicity of GQDs@hMSN-PEG a. Biochemical parameters on day 1, day 7, and day 30 postinjection of GQDs@hMSN-PEG. Healthy female Balb/c mice with the intravenous injection of GQD@hMSN-PEG were sacrificed on 1, 7, and 30 days p.i. for blood collection. Untreated healthy mice were used as the control; b. H&E stained major organ slides collected from healthy Balb/c mice and GQDs@hMSN-PEG injected mice on day 30 post injection (n = 5). Scale bar: 100 µm. Organ distribution profile in vivo. To investigate the distribution of GQDs@hMSN(DOX)-PEG in different tissues/organs, we demonstrated the feasibility of drug delivery and imaging in vivo using DOX loaded GQDs@hMSN-PEG, denoted as GQDs@hMSN(DOX)-PEG. 4T1 tumor-bearing mice were injected with free DOX and GQDs@hMSN(DOX)-PEG (5.0 mg GQDs@hMSN-PEG/kg, 0.4 mg DOX/kg for both groups). The mice were then sacrificed at 2 h p.i., and the major organs were collected and imaged in the NightOWL II system (ex/em=485/630 for the mouse imaging and ex/em= 485/570 nm for the organs imaging) to detect the tissue presence of GQDs@hMSN-PEG and DOX. From fluorescence imaging observation (Fig. 7), significant fluorescence was found in the



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tumor with GQDs@hMSN-PEG, while no signal was found for free DOX. For the organs imaging, higher

fluorescence

(about

2-fold

of

that

of

free

DOX

group)

were

shown

in

GQDs@hMSN(DOX)-PEG group than that of free DOX group, which demonstrated the feasibility of enhanced tumor target drug delivery in vivo using GQDs@hMSN-PEG.

Fig. 7 In vivo accumulation of free DOX, GQDs@hMSN-PEG and GQDs@hMSN(DOX)-PEG T. 4T1 tumor nodules; L1. liver; L2. lung; K. kidney; H. heart; S. spleen; M. muscle; B. bone; I. intestine CONCLUSIONS In conclusion, core/shell GQDs@hMSN-PEG NPs were designed and synthesized for enhanced drug delivery and combinational tumor treatment. The uniform shape and size, excellent biocompatibility and stability at physiological condition indicate that GQDs@hMSN-PEG NPs are the promising and applicable drug delivery vehicle. Relatively high amount of DOX (0.78 mg DOX/mg GQDs@hMSN-PEG) can be loaded onto GQDs@hMSN-PEG conjugates, and a pH- responsively DOX release behavior was detected. Large dose of GQDs@hMSN-PEG did not impose significant in vivo toxicity. Although it only relied on passive targeting of tumor, the accumulation of GQDs@hMSN and enhanced DOX delivery to 4T1 tumors in vivo were demonstrated in tumor-bearing mice. Further optimization of the tumor uptake was under way by combination with a


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tumor-targeting agent, and the potential of these nano-conjugates for combinational drug/GQD/ photodynamic therapy will be investigated in the future. EXPERIMENTAL SECTION Chemicals and reagents. 4-bromobenzyl bromide, thiophene-3-boronic acid, Pd(PPh3)4 and N, N-dimethyldodecylamine were purchased from Alfa Aesar (China). SCM-PEG5k-Mal were purchased from YareBio (Shanghai, China), APS and hexadecyl trimethyl ammonium chloride (CTAC) were acquired from J&K Scientific (Beijing, China). DOX, TEOS, triethylamine (TEA), phenol, potassium cyanide and ninhydrin were all purchased from Aladdin (Shanghai, China). Test kits for GPT, AKP, ALB, Cr, BUN, TP, SOD, GSH-PX and CAT were all acquired from Jiancheng Bioengineering, Inc. (Nanjing, China). RPMI 1640 cell culture medium, FBS and trypsin were purchased from Vicmed (Xuzhou, China). All the other reagents were acquired from Sinopharm Chemical reagent (Beijing, China). All reagents were directly used without further purifications following manufacturer's instructions. All buffers were prepared from Millipore-grade water. L929 cells were got from Pharmacy College of Shenyang Pharmaceutical University. 4T1 cells were purchased from SIBS (Shanghai, China). 6-Week-old Balb/c mice were purchased from SLAC Laboratory (Shanghai, China). Synthesis of GQDs@hMSN-PEG nanoparticles. As shown in Scheme 1, the GQDs were synthesized in accordance with previously reported method27,16 with minor modifications. In brief, 4-bromobenzyl bromide and N, N-dimethyldodecylamine with the molar ratio of 1:1.3 were reacted in a mixed solvent (CH2Cl2/CH3OH=3/2) under nitrogen protection for 12 h by nucleophilic addition to form compound 1. Then, thiophene-3-boronic acid was catalyzed by Pd(PPh3)4 by a Suzuki reaction to get compound 2. Subsequently, compound 2 was catalyzed by FeCl3 in a hydrous CHCl3 under nitrogen to form PT2 by an oxidative polymerization. The GQDs were then prepared by hydrothermal treatment of PT2. In a typical synthesis process, 30 mg of PT2 was dispersed in 40 ml of NaOH solution (0.5 mM). The mixture was treated ultrasonically for 30 min and then transferred into an autoclave for heating at 160 ℃ for a period of 24 h. After cooling to room temperature, the resulting GQDs were collected by filtering using 0.22 µm membranes (to remove large aggregates), and then dialyzed against distilled water several times to remove the residual NaOH. The GQDs were dispersed in water for further characterization and use. The prepared GQDs was firstly dispersed in water and the outer layer hMSN is grown following ACS Paragon Plus Environment


previously established method28. 35.7 mL of absolute ethanol was mixed with 5 mL water and 0.8 mL of ammonia and stirred at room temperature, followed by addition of TEOS (1 mL). The mixture was allowed to react at room temperature for 1 h and subsequently washed with water and ethanol and suspended in 20 mL water to form the dense silica nanoparticles (dSiO2) coated GQDs (donated by GQDs@dSiO2) complex. In the following step, GQDs&dSiO2 complex was coated with a layer of mesoporous silica and etched by mild base to form GQDs@hMSN. CTAC (2 g) and TEA (20 mg) were dissolved in 20 mL of deionized water and added to 10 mL of GQDs@dSiO2 water solution. The mixture was stirred at room temperature for 1.5 h, followed by the addition of TEOS (0.15 mL). The mixture was stirred for 1 h at 80 °C. The last step involved etching of inner dSiO2 from GQDs@dSiO2/SiO2 NPs to obtain GQDs@hMSN. 636 mg of Na2CO3 was added to the reaction mixture, which was stirred continuously at 50 °C for 30 min. Finally, CTAC was removed by NaCl methanol (1%) extraction. After functionalization with the hydrolysate of APS, the surface of GQDs@hMSN NPs were enriched with amine groups. To decrease the absorption by mononuclear phagocyte system (MPS) in vivo, PEG was coupled on the surface via amide bond to form GQDs@hMSN-PEG NPs. The obtained GQDs@hMSN-NH2 was subsequently reacted with SCM-PEG5k-Mal at pH 8.5 at a molar ratio of 1:25 for 2 h. After purifying by centrifugation filtration using 50 kDa cut off Amicon filters, the resulting reaction product GQDs@hMSN-PEG NPs were obtained.

Scheme 1. Schematic synthesis route of GQDs@hMSN NPs Characteristics of GQDs@hMSN nanoparticles. The morphology of GQDs, hMSN and GQDs@hMSN nanoparticles were evaluated by G2T12 transmission electron microscope (FEI,


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USA). In addition, their hydrodynamic size and size distribution, as well as ζ-potentials were determined by DLS (380 ZLS, Nicomp, USA) at the concentration of 0.05 mg/mL (based on GQDs@hMSN). Thermogravimetric analysis was carried out in a TGA 8000 thermogravimetric analyzer (Perkin-Elmer) with a scan rate of 5 ℃/min. UV-Vis and FL spectra were recorded using Hitachi U-3010 and F-4600 spectrophotometers, respectively. Drug loading/releasing measurement. DOX was used as a model drug to test the drug loading capacity of GQDs@hMSN-PEG NPs. Briefly, DOX was mixed with GQDs@hMSN-PEG at pH 8.0 overnight. Unbound excess DOX was removed by centrifuging with repeated rinsing of PBS. The resulting GQDs@hMSN(DOX) was resuspended in PBS and stored at 4℃. Unbounded DOX and drug release were quantified by UV-Vis spectrometry with DOX at known concentration as the reference. Drug loading capacity was calculated from the ratio of DOX amount loaded into GQDs@hMSN-PEG to the weight of GQDs@hMSN-PEG. Drug release was evaluated at 37℃ in an acetate buffer (pH 5.0) and phosphate buffer (pH 6.5 and 7.4). GQDs@hMSN(DOX)-PEG dispersed in the buffer was placed in a dialysis bag with a molecular weight cut-off of 3 kDa. The dialysis bag was immersed in release medium and kept in a shaker (150 rpm) under room temperature. Samples were periodically removed and the same volume of fresh medium was added. The amount of released DOX was analyzed with a spectrometer at 488 nm. The experiments were performed in triplicate for each time point. Cell line and animal model. Cells culture: 4T1 and L929 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Regular cell culturing condition was applied (37℃ with 5% CO2). Cytotoxicity: Cells were seeded in a 96-well plate for 24 h before GQDs@hMSN-PEG treatment. GQDs@hMSN-PEG with known concentrations (via serial dilution) were added into cells. After 24 h incubation, the relative viabilities of cell samples were determined by a cell titer 96 kit following vendor’s protocols. The percentages of viable cells relative to the untreated control were plotted against GQDs@hMSN-PEG concentrations. Animal model: Female Balb/c mice (6 weeks, 18-22 g) were operated under protocols approved by the Animal Ethics Committee of Xuzhou Medical University. Tumors were established by subcutaneous injection of 1×106 of 4T1 cells suspended in 50 µL of PBS into the Balb/c mice. The tumor sizes were monitored every other day and the mice were subjected to imaging studies when the tumor diameter reached 5-8 mm. ACS Paragon Plus Environment


Blood analysis and histology examinations: Twenty healthy female Balb/c mice were injected with a dose of 20 mg/kg GQDs@hMSN-PEG and sacrificed at various time points after injection (1, 7, and 30 days, five mice per time point). Untreated healthy Balb/c mice were used as the control (5 mice per group). An approximately 0.8 mL portion of blood from each mouse was collected for the blood chemistry test and complete blood panel analysis before the mouse was euthanatized.

In vivo drug delivery evaluation. For in vivo drug delivery study, GQDs@hMSN-PEG and GQDs@hMSN(DOX)-PEG (5 mg/kg GQDs@hMSN-PEG) were injected intravenously into 4T1 tumor-bearing mice. The mice were sacrificed at 2 h p.i., with tumor and other major organs taken for ex vivo fluorescence imaging in the Berthold LB983 NightOWL II system (ex/em=485/630 nm for GQDs@hMSN, and 485/570 nm for DOX). Histology. To confirm the in vivo biocompatibility of GQDs@hMSN conjugates, the mice were sacrificed after each treatment for the hematoxylin and eosin (H&E) analysis. Major organs from those mice were harvested, fixed in 4% neutral buffered formalin, transferred routinely into paraffin, sectioned into 8 µm thick slices, stained with H&E and examined by an IX73 digital microscope (Olympus, Japan) with a magnitude of 200×. Examined tissues include lung, liver, kidneys, spleen, heart and intestine. AUTHOR INFORMATION Corresponding authors E-mail: [email protected] (Yang D.Z.) E-mail: [email protected] (Hong H.) ORCID information Dongzhi Yang: 0000-0001-6796-1613 Hao Hong: 0000-0002-9730-9367 Conflict of interest The authors declare no competing financial interest. All authors have no potential conflict of interest. ACKNOWLEDGMENTS This work is supported by Natural Science Foundation of Jiangsu Province, China (BK20161173) and the Program for Distinguished Talents of Six Domains in Jiangsu Province, China (2014-SWYY-007)， Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX17-1709). ACS Paragon Plus Environment

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed material characterization, single oxygen producing capacity, drug release analysis, phototoxicity, and in vivo biological assays.

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hMSN Nanoparticles as

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