Hydrophobicity-Adaptive Nanogels for Programmed Anticancer Drug

Nov 19, 2018 - ... and 1H nuclear magnetic resonance (NMR) spectroscopy (Figure S2), ..... to permeate the cell membrane and accumulate in the spheroi...
0 downloads 0 Views 1MB Size
Download PDF
Subscriber access provided by Kaohsiung Medical University

Communication

Hydrophobicity-Adaptive Nanogels for Programmed Anticancer Drug Delivery Hao Yang, Qin Wang, Zifu Li, Fuying Li, Di Wu, Man Fan, Anbi Zheng, Bo Huang, Lu Gan, Yuliang Zhao, and Xiangliang Yang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03828 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27 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

Nano Letters

Hydrophobicity-Adaptive Nanogels for Programmed Anticancer Drug Delivery Hao Yang,‡,# Qin Wang,†,# Zifu Li,‡ Fuying Li,‡ Di Wu,† Man Fan,† Anbi Zheng,† Bo Huang,⊥,∇ Lu Gan,*,‡ Yuliang Zhao,*,§ and Xiangliang Yang*,‡ ‡National

Engineering Research Center for Nanomedicine, College of Life Science and

Technology, Huazhong University of Science and Technology, Wuhan 430074, China †Hubei

Key Laboratory of Bioinorganic Chemistry and Materia Medica, School of

Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China §CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience and Technology of China, Beijing 100190, China ⊥

National Key Laboratory of Medical Molecular Biology, Department of Immunology,

Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China ∇Department

of Biochemistry & Molecular Biology, Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430074, China

*E-mail:

[email protected]

(L.

Gan);

[email protected]

[email protected] (Y. Zhao) Tel: +86-27-87792147 Fax: +86-27-87792234

1 ACS Paragon Plus Environment

(X.

Yang);

Nano Letters 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

Page 2 of 27

ABSTRACT: Reconciling the conflicting needs for prolonged circulation time, enhanced cellular uptake by bulk tumor cells and cancer stem cells (CSCs), and extensive tumor tissue penetration remains a major challenge for current nano drug delivery systems. Here we describe

smart

poly(N-isopropylacrylamide)-based

nanogels

with

fast

adaptive

hydrophobicity to solve these contradictory requirements for enhanced cancer chemotherapy. The nanogels are hydrophilic in the blood to prolong their circulation time. Once they accumulate at tumor sites, they rapidly become hydrophobic in response to tumor extracellular acidity. The adaptive hydrophobicicty of the nanogels facilitates tumor accumulation, deep tumor penetration and efficient uptake by bulk tumor cells and CSCs, resulting in greater in vivo enrichment in tumor cells and side population cells. Together with lysosomal pH-regulated charge reversal and redox-responsive intracellular drug release, the nanogels escape from lysosomes and release their cargo doxorubicin. Thus, the nanogels significantly improve the in vivo anticancer efficacy and decrease side effects of doxorubicin. Strikingly, the ratio of CSCs is greatly decreased after treatment with the nanogels loaded with doxorubicin. Our current study provides new insights into designing effective anticancer drug delivery systems.

KEYWORDS: Hydrophobicity-adaptive nanogels, pH-responsiveness, tumor penetration, cancer chemotherapy

2 ACS Paragon Plus Environment

Page 3 of 27 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

Nano Letters

Insufficient in vivo drug enrichment in tumor cells, especially cancer stem cells (CSCs) that are responsible for drug resistance, recurrence and metastasis,1,2 is one of the major obstacles facing cancer chemotherapy.3 Although nanotechnology-based drug delivery systems (NDDSs) exhibit promising therapeutic efficacy and reduced side effects due to the enhanced permeability and retention (EPR) effect,4-7 they still encounter a series of physiological barriers in their transport from the intravenous injection site to the tumor, limiting their enrichment in tumor cells and CSCs.8-11 The ideal NDDS would possess the following general features including prolonged circulation time in blood, enhanced tumor accumulation and penetration, efficient internalization by bulk tumor cells and CSCs, effective lysosomal escape and intracellular drug release.12-16 Owing to the aberrant vascular structure, high interstitial fluid pressure (IFP) and dense extracellular matrix in tumor tissues, nanoparticles have been found to be strongly restricted to the vicinity of tumor vessels.17-19 The limited penetration of nanoparticles into the tumor parenchyma lowers the possibility of delivering anticancer drugs to cancer cells, especially CSCs located within the hypoxic core of tumor tissues distal from tumor vessels,20-22 ultimately resulting in failure to eradicate CSCs and tumor relapse. Therefore, enhancing the tumor penetration capacity of nanoparticles remains a crucial goal in improving cancer treatment efficiency. It is widely known that the physiochemical properties of nanoparticles, such as size,23-25 surface charge,26,27 shape,28,29 and hydrophobicity,30-32 have profound impacts on their transport processes in the body affecting the antitumor activity. However, conflicting features of nanoparticles need to be balanced to overcome various physiological barriers. Recently, several studies have explored the effects of size and surface charge of nanoparticles on their tumor penetration capacity and therefore developed stimuli-responsive size- or surface charge-switchable nanoparticles to improve anticancer efficacy.25,26,33 In addition, stimuliresponsive hydrophilic/hydrophobic reversible nanoparticles, such as the nanoparticles 3 ACS Paragon Plus Environment

Nano Letters 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

shedding a surface hydrophilic layer (e.g., polyethylene glycol) in response to unique properties of the tumor microenvironment (e.g., enzymes and tumor pH),34-37 have been exploited to solve the need for both prolonged circulation time in blood and efficient cellular uptake by cancer cells. These optimized nanoparticles were hydrophilic for prolonged blood circulation, but switched to a hydrophobic nature for efficient cellular internalization at tumor tissues. Unfortunately, the chemical reaction removing the hydrophilic layer takes hours,38 diminishing the treatment efficacy. Additionally, how the hydrophobic characteristics impact the behavior of nanoparticles in tumor stroma, and whether the hydrophilic/hydrophobic reversible nanoparticles in response to the tumor microenvironment can enable deep tumor penetration into the parenchymal tissues and subsequent internalization into CSCs still remain to be elucidated. Here, we report poly(N-isopropylacrylamide) (PNIPAM)-based nanogels with rapid adaptive hydrophobicity to efficiently harmonize the contradictory needs for prolonged circulation and efficient internalization into bulk tumor cells and CSCs, and more importantly, to enhance tumor penetration. Moreover, the nanogels are further endowed with pH-regulated charge reversal and GSH-responsive drug release properties to release an anticancer drug intracellularly. The nanogels are prepared via a simple precipitation polymerization method by using NIPAM, pH-responsive N-methylallylamine (MAA) and the betaine-based zwitterionic monomer sulfobetaine methacrylate (SBMA) as comonomers, and the disulfide bond-containing N, N'-bis(acryloyl) cystamine (BAC) as a crosslinker (Figure 1A). Poly(NIPAM)-based nanogels show typical thermosensitive properties and their volume phase transition temperature (VPTT) can be finely tuned by adding comonomers.31,32 Poly(SBMA), similar to polyethylene glycol, resists protein adsorption and is relatively stable in the circulation in vivo.39 MAA, with a pKb of 3.89, is adopted to adjust the surface charge and VPTT of the nanogels.40 Thus, nanogels with tailored VPTT and surface charge can be synthesized by controlling the molar ratio of these comonomers. The constructed nanogels 4 ACS Paragon Plus Environment

Page 4 of 27

Page 5 of 27 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

Nano Letters

with adaptive hydrophobicity are hydrophilic in blood (pH 7.4, 37 °C) for prolonged circulation, but switch to a hydrophobic character with nearly unchanged size and surface charge in the acidic tumor microenvironment (around pH 6.5,41 37 °C) within minutes. This rapid switching characteristic of the nanogels facilitates tumor accumulation, deep tumor penetration and efficient cellular uptake by bulk tumor cells and CSCs, resulting in enhanced in vivo enrichment in bulk tumor cells and side population cells with properties of CSCs.42 Furthermore, the nanogels switch to being positively charged under lysosomal conditions (around pH 4.541), which enables effective lysosomal escape, leading to enhanced release of doxorubicin (DOX) triggered by the high GSH level in the cytosol (around 10 mM41) to induce cytotoxicity against bulk tumor cells and CSCs (Figure 1B). After optimization of the formula design, nanogels with the tailed hydrophobicity and surface charge at 37 °C at different pH values were synthesized with optimal molar ratios of comonomers. Particularly, the nanogels with feeding molar ratio of NIPAM to SBMA to MAA of 85.7 : 4.8 : 9.5 were capable of undergoing hydrophilic/hydrophobic reversal at pH 6.5 and charge reversal at pH 4.5 (designated as SNG). As a control, nanogels with the molar ratio of NIPAM to SBMA to MAA of 81.8 : 4.6 : 13.6 exhibited only a charge switch at pH 4.5, yet no change in hydrophobicity at pH 6.5 (designated as ING). The chemical structures of the nanogels were confirmed through analyses by Fourier transform infrared (FTIR) spectroscopy (Figure S1) and 1H nuclear magnetic resonance (NMR) spectroscopy (Figure S2), indicating that comonomers of NIPAM, SBMA, MAA and the crosslinker BAC were successfully incorporated into the nanogels. ING and SNG were nearly monodispersed and had similar sizes, as seen by dynamic light scattering (DLS, Figures 2A and S3A) and transmission electron microscopy (TEM) analyses (Figures 2B and S3B) at pH 7.4. The anticancer drug DOX was loaded into ING and SNG by the solvent evaporation method (designated as DOX@ING and DOX@SNG, respectively) and the diameters of the nanogels were not significantly impacted after the drug loading process (Figures 2A, 2B, S3A and 5 ACS Paragon Plus Environment

Nano Letters 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

S3B). The drug loading capacities of SNG and ING were determined to be 4.0% and 3.6%, respectively. To assess the pH-triggered adaptive hydrophobicity of SNG, we determined the VPTT of SNG at pH 7.4, 6.5 or 4.5 (mimicking the pH values in blood and normal tissues, the tumor extracellular microenvironment and lysosomes, respectively). The VPTT of SNG at pH 7.4, 6.5 and 4.5, based on the optical transmittance change, was 37.5, 36.1 and 34.3 °C, respectively (Figure 2C). Meanwhile, the insets in Figure 2C showed that SNG dispersions in PBS containing 10% FBS at 37 °C were transparent at pH 7.4, yet became turbid at pH 6.5 and 4.5. These results suggest that SNG is hydrophilic in blood and normal tissues owing to its VPTT above physiological temperature (37 °C) at pH 7.4, whereas it switches to a hydrophobic nature in tumor tissues and lysosomes on account of its VPTT below the physiological temperature at pH values corresponding to those compartments. The pHtriggered adaptive hydrophobicity of SNG was further confirmed by determining its VPTT through the change in the diameters of the nanogels using DLS (Figure 2D). Considering the acidic property of the tumor extracellular microenvironment, we also confirmed that SNG turned to hydrophobic at pH 6.7 (Figure S4A). In particular, the transmittance of SNG remained nearly unchanged at physiological pH 7.4 and 37 °C, while its transmittance at pH 6.7, 6.5 and 4.5 at 37 °C dropped sharply with 11, 11 and 3 min incubation, respectively (Figures 2E and S4B), which confirms there is a pH-triggered instantaneous switch from hydrophilic to hydrophobic. In contrast, the VPTT of ING at pH 7.4, 6.5 and 4.5 was all above 37 °C (Figure S3C and S3D) and all ING dispersions in PBS containing 10% FBS at pH 7.4, 6.5 and 4.5 were transparent at 37 °C (Figure S3C), indicating that ING does not possess the pH-triggered adaptive hydrophobicity. Consistently, the transmittance of ING incubated at pH 7.4, 6.5 and 4.5 at 37 °C remained almost the same (Figure S3E). The zeta potentials of ING and SNG at pH 7.4 and 6.5 were negative, but shifted to positive at pH 4.5 (Figure S5A and S3F), suggesting a surface charge reversal under lysosomal pH. 6 ACS Paragon Plus Environment

Page 6 of 27

Page 7 of 27 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

Nano Letters

The zeta potential values of SNG at pH 7.4, 6.5 and 4.5 were -14, -11 and +10 mV, respectively. Moreover, acid-base titration results showed that the pKa of SNG (equal to the pH at the dissociation degree α of 0.5) was approximately 6.0 and the percentage of protonated amino groups at pH 4.5 was 96.9% (Figure S5B and S5C). These results indicate that SNG is protonated in lysosomes and escaped from the lysosomes to the cytoplasm through proton sponge effect, where hydrophobic SNG quickly reversed to the hydrophilic form (Figure S6). One possible mechanism accounting for the change of hydrophobicity and zeta potential of SNG at different pH values is the following: SNG is negatively charged at pH 7.4 (Figure S5A), which may be due to the existence of sulfate groups on the surface of nanogels.43-46 These sulfate groups could come from the radical initiator potassium persulfate (KPS) that decomposed during the polymerization process, or from the residual surfactant sodium dodecyl sulfate (SDS).43,44 Due to the existence of these sulfate groups and MAA comonomers in the nanogels, the pKa of SNG nanogels was approximately 6 (Figure S5C). As such, when additional acid was added to adjust the pH value of the nanogels, the secondary amine groups of MAA gradually became protonated and interacted with some sulfate groups through electrostatic interactions, leading to a decrease in the absolute value of the negative charge (such as at pH 6.5, Figure S5A) and even reversal to a lower positive zeta potential (such as at pH 4.5, Figure S5A). As a result, the VPTT of SNG decreases from 40.0 °C at pH 7.4 to 36.8 °C at pH 6.5 (Figure 2D). Thus, SNG is hydrophilic at pH 7.4 and switches to hydrophobic at pH 6.5 at physiological temperature (37 °C). Next, we determined the release profiles of DOX from DOX@SNG at different redox and pH conditions at 37 °C (Figure S7). SNG exhibited increased DOX release at 10 mM GSH (comparable to the GSH concentration in tumor cells) and pH 4.5, revealing that an intracellular reducing environment and lysosomal pH promoted drug release. However, DOX release at both pH 4.5 and 10 mM GSH did not exhibit synergistic effects compared to that at pH 7.4 and 10 mM GSH, which may be due to the strong hydrophobicity of SNG at 4.5 7 ACS Paragon Plus Environment

Nano Letters 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

hindering the interaction between GSH and the disulfide bond inside SNG. These results reveal that DOX may be partially released from DOX@SNG in lysosomes after internalization and then further released in response to high cytoplasmic GSH (pH 7.4) after escape from lysosomes. It is known that hydrophilic nanoparticles exhibit decreased protein adsorption and phagocytosis by the reticuloendothelial system (RES), potentially lending the nanoparticles prolonged circulation time.30 To determine whether the pH-triggered adaptive hydrophobicity of the nanogels could affect their blood circulation, we first evaluated the interaction between the nanogels and fetal bovine serum (FBS) at pH 6.5 or 7.4 (Figure S8A). At physiological pH 7.4, both SNG and ING exhibited little protein adsorption. SNG showed markedly enhanced protein adsorption at pH 6.5 compared with that at pH 7.4. In contrast, no significant difference in protein adsorption by ING was found between pH 7.4 and 6.5. Consistently more SNG, but not ING, was phagocytosed by RAW264.7 macrophages at pH 6.5 than at pH 7.4 (Figure S8B). These results are likely to be a result of the pH-triggered hydrophilic-to-hydrophobic switch of SNG at pH 6.5, implying that the hydrophilicity of these nanogels at pH 7.4 endows them with prolonged circulation time. We next evaluated the in vivo pharmacokinetics of the nanogels (Figure 3A and Table S1). The Cmax of DOX@SNG and DOX@ING in the plasma was 3.96- and 3.30-fold higher than that of free DOX, respectively. Meanwhile, the areas under curve (AUCs) of DOX@SNG and DOX@ING were 3.7- and 4.0-fold greater than that of free DOX, respectively. In addition, SNG and ING extended the terminal elimination half-life (t1/2β) of DOX up to 3.7- and 5.1fold, respectively. These data confirm that these nanogels are capable of reducing their nonspecific interaction with blood proteins and phagocytosis by RES owing to their hydrophilicity at pH 7.4, resulting in a prolonged circulation in blood. Meanwhile, DOXloaded nanogels were stable under physiological conditions, as evidenced by low DOX release and unchanged diameter in PBS medium containing 10% FBS at pH 7.4 and 37 °C 8 ACS Paragon Plus Environment

Page 8 of 27

Page 9 of 27 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

Nano Letters

(Figure S9). Although the hydrophilicity of the nanoparticles can contribute to prolonged blood circulation, it preventes the nanoparticles from interacting with tumor cells and inhibited cellular uptake.30,31 Therefore, changing the nanoparticles’ hydrophilic surface to a hydrophobic one is essential to affect tumor inhibition. To verify whether the nanogels with adaptive hydrophobicity can be efficiently internalized by cancer cells under the acidic tumor microenvironment, the cellular uptake of DOX@SNG was evaluated at different pH values (Figures 3B and S10). HepG2 and H22 cells were incubated with DOX@SNG or DOX@ING at either pH 7.4 or 6.5 for 2 h, and the intracellular DOX fluorescence intensity was determined by flow cytometry. As expected, more DOX@SNG was internalized into HepG2 and H22 cells at pH 6.5 than that at pH 7.4. However, no significant difference in the cellular uptake of DOX@ING was observed between pH 7.4 and 6.5. Consequently, DOX@SNG, but not DOX@ING, induced more cytotoxicity in HepG2 cells at pH 6.5 than that at pH 7.4 (Figure S11). Furthermore, we determined the cellular uptake of DOX@SNG by CSCs at different pH values. For these experiments, we used a mechanics-based method to select CSCs by culturing single cancer cells in fibrin matrices of about 100 Pa in stiffness.47,48 Subcutaneous injection of 100 selected cells in syngeneic mice induced the formation of solid tumors much more efficiently than control cancer cells selected using conventional surface marker methods.47,48 Consistently, much stronger intracellular fluorescence was detected when H22 CSCs were incubated with DOX@SNG for 6 h at pH 6.5 than that at pH 7.4 (Figure 3C). Meanwhile, treatment with DOX@SNG at pH 6.5 for both 24 and 48 h significantly decreased the colony number (Figure S12) of living tumor spheroids compared with that at pH 7.4. In addition, the shedding of cell fragments from the surface of tumor spheroids of CSCs was observed upon treatment with DOX@SNG at pH 6.5 for 48 h (Figure S13). The shedding was likely caused by DOX@SNG-induced apoptosis. In contrast, no distinctive change was observed in the colony number or morphology of tumor spheroids 9 ACS Paragon Plus Environment

Nano Letters 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

treated with DOX@ING at pH 7.4 and 6.5 (Figure S12 and S13). These results indicate that the pH-triggered adaptive hydrophobicity of SNG can enhance cellular uptake by bulk tumor cells and CSCs, thus overcoming the contradiction of the need for a long circulation time with efficient cancer cell uptake. Achieving long circulation of nanoparticles may contribute to their accumulation in tumor tissues by the EPR effect.4-7 Furthermore, it has been reported that nanoparticles that can be efficiently internalized by cancer cells are not readily removed from tumor tissues.27 With the confirmed prolonged blood circulation and enhanced cellular uptake of SNG under an acidic environment, we then assessed the accumulation and retention of DOX@SNG in tumor tissues. The DOX content of tumor tissues was evaluated at different intervals after intravenous injection of free DOX, DOX@ING or DOX@SNG into H22 hepatoma-bearing mice (Figure 3D). DOX in the tumor tissue of the mice administrated with DOX@SNG was 1.6- and 6.4-fold at 24 h, and 3.9- and 5.4-fold at 72 h higher than those administrated with DOX@ING and free DOX, respectively, demonstrating that SNG accumulates and retains more DOX in tumor tissues than ING. Furthermore, we determined the in vivo enrichment in total tumor cells at 24 h after H22-bearing mice were intravenously injected with free DOX, DOX@ING or DOX@SNG (Figure 3E). The tumor tissues were digested into single cells and the intracellular DOX fluorescence in the tumor cells was measured. As expected, DOX@SNG exhibited 1.7- and 4.0-fold higher DOX fluorescence in total tumor cells than DOX@ING and free DOX, respectively. Moreover, we isolated side population cells that possess some properties of CSCs,42 in H22 tumor tissues by flow cytometry and then quantified intracellular DOX fluorescence (Figure 3F). Consistently, more DOX was observed in side population cells when the mice were administrated with DOX@SNG compared to those treated with DOX@ING or free DOX. These results reveal that more DOX@SNG is enriched in not only total tumor cells but also side population cells in vivo. The superior intratumoral penetration of nanoparticles may provide a greater opportunity to 10 ACS Paragon Plus Environment

Page 10 of 27

Page 11 of 27 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

Nano Letters

approach tumor cells, especially CSCs located in the hypoxic center of tumors, and promote cellular uptake in vivo.20 To determine the effects of pH-triggered adaptive hydrophobicity of DOX@SNG on tumor penetration capacity, tumor spheroids formed in 3D fibrin gels, akin to in vivo tumors, were first used to investigate the tumor penetration of DOX@SNG at different pH values (Figure 4A). After tumor spheroids were incubated with free DOX, DOX@ING or DOX@SNG for 24 h, greater amount of DOX@SNG was found to penetrate into the 3D tumor spheroids at pH 6.5 than that at pH 7.4. Moreover, bright red DOX fluorescence was scattered in most regions of DOX@SNG-treated tumor spheroids at pH 6.5 even at a 50 µm depth from the spheroid surface, while only weak DOX fluorescence was detected at the edge of DOX@SNG-treated tumor spheroids at pH 7.4. In contrast, no remarkable difference in the penetration of DOX@ING into 3D tumor spheroids was observed between pH 7.4 and 6.5. Our finding that a higher amount of free DOX penetrated into 3D tumor spheroids at pH 7.4 than at pH 6.5 may indicate that the protonation of the weak base of DOX at pH 6.5 was not sufficient to allow the drug to permeate the cell membrane and accumulate in the spheroids.49 DOX fluorescence analysis of the interior area of the spheroids at a 50 µm depth confirmed that the fluorescence of DOX@SNG, but not DOX@ING or free DOX, at pH 6.5 was significantly higher than at pH 7.4. The tumor penetration capability was further investigated by incubating ex vivo tumor tissues in cell culture media containing free DOX, DOX@SNG or DOX@ING at different pH values (Figure 4B). Consistent with the results using 3D tumor spheroids, DOX@SNG was mostly distributed around the tumor periphery at pH 7.4, while more DOX@SNG was spread inside the tumor tissue at pH 6.5. In contrast, DOX@ING was mostly found to be scattered around the edge of the tumor tissues at both pH 7.4 and 6.5. These results demonstrate that the pH-triggered adaptive hydrophobicity of the nanogels promotes tumor penetration. The enhanced tumor penetration of DOX@SNG was further verified by fluorescent microscopic images of H22 tumor sections following intravenous injection of free DOX, DOX@ING or DOX@SNG into mice bearing the tumors (Figure 4C). 11 ACS Paragon Plus Environment

Nano Letters 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

At 24 h post-injection, DOX fluorescence was scattered throughout the entire tumor section in mice administrated with DOX@SNG, while free DOX and DOX@ING were mostly retained in the blood vessels based on their strong colocalization with CD31-labeled endothelial cells. The distance-dependent DOX fluorescence intensity also confirmed the superior tumor penetration of DOX@SNG. The enhanced tumor penetration of pH-triggered hydrophobicityadaptive nanogels may be because the hydrophobicity increases the internalization of the nanogels by cells, which facilitates transcellular penetration of the nanogels through transcytosis and thus favor deeper tumor penetration.12,50 The detailed mechanism needs to be further clarified in the future research. Although our data indicate that DOX@SNG highly accumulates in tumor tissues, deeply penetrates into tumor tissues and efficiently enters into bulk tumor cells and CSCs due to the pH-triggered adaptive hydrophobicity, the nanogels still need to escape from lysosomes and then release DOX to exert cytotoxicity. Therefore, we evaluated the lysosomal escape capacity of the nanogels after DOX@SNG was internalized into tumor cells by caveolin- and clathrin-dependent endocytosis (Figure S14). HepG2 cells were exposed to a 2 h pulse of Alexa Fluor® 405-labeled SNG and then chased with nanoparticle-free fresh media for different time intervals. The internalized SNG was found to be sequestered in lysosomes after a 2 h chase, while increasing amounts of SNG appeared outside lysosomes over time (Figure S15), suggesting that SNG can indeed escape from lysosomes after internalization. Furthermore, a gradual reduction in the colocalization of DOX with lysosomes was detected as HepG2 cells and H22 CSCs were treated with DOX@SNG for longer intervals, while elevating levels of DOX were found in the nucleus with increasing time (Figure S16). These results imply that the high positive charge of SNG at lysosomal pH allows it to translocate from lysosomes to the cytosol, where SNG can disintegrate in response to the high cytoplasmic GSH concentration and then release DOX, entering the nucleus to exert cytotoxicity in both bulk tumor cells and CSCs. We found that little DOX was translocated 12 ACS Paragon Plus Environment

Page 12 of 27

Page 13 of 27 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

Nano Letters

into the nucleus in HepG2 cells incubated with DOX@ING (Figure S17), possibly because that the positive surface charge of ING at lysosomal pH is lower than that of SNG (Figure S3F), resulting in an insufficient lysosomal escape of ING and subsequent DOX release. Since SNG can efficiently accumulate in and penetrate tumor tissues, be internalized into bulk tumor cells and CSCs, escape from lysosomes and intracellularly release the encapsulated drug, we evaluated the in vivo anticancer activity of DOX@SNG. DOX@SNG consistently elicited a more significant inhibition of tumor growth than DOX@ING or free DOX (Figure 5A). The tumor growth rate in DOX@SNG-treated group was decreased by about 70% compared to 50% in DOX@ING-treated group and 56% in the mice treated with free DOX. The average weight of the tumor tissues excised at the end of treatment also exhibited the same trend (Figure 5B). Furthermore, treatment with DOX@SNG markedly inhibited the percentage of Ki67-positive proliferating tumor cells and augmented the number of TUNEL-positive apoptotic tumor cells (Figures 5C, 5D and S18). Moreover, we evaluated the in vivo inhibitory effects of DOX@SNG on CSCs considering that DOX@SNG was highly enriched in side population cells (Figure 3F). After administration with free DOX, DOX@ING or DOX@SNG once every two days for 5 times, the tumor tissues were digested into single cells and the proportion of side population cells was measured by flow cytometry (Figures 5E and S19). Fewer side population cells were detected in the DOX@SNG-treated group, about 14.2% and 28.2% of those in the free DOX- and DOX@ING-treated groups, respectively. We simultaneously seeded 1000 single tumor cells isolated from the tumor tissues in soft 3D fibrin gels and the samples were incubated for 5 days. Significantly fewer colony numbers (Figure 5F) were formed in the DOX@SNG-treated group compared with the free DOX- and DOX@ING-treated groups, further confirming that DOX@SNG can effectively kill CSCs. No significant difference in body weights was found in mice administrated with DOX@SNG, DOX@ING or free DOX (Figure S20). The histologic analysis by hematoxylin-eosin (H&E) staining indicated that DOX@SNG did not apparently 13 ACS Paragon Plus Environment

Nano Letters 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

induce toxicity in any major organs, including the heart, liver, spleen, lung and kidney, although free DOX induced cardiotoxicity (Figure S21). Together, our results reveal that DOX@SNG exhibits enhanced anticancer activity with few side effects. In summary, we have successfully developed smart nanogels with rapid pH-triggered adaptive hydrophobicity for programmed anticancer drug delivery. These nanogels are designed to be hydrophilic in the blood to prolong their circulation time on account of the decreased protein opsonization and phagocytosis by macrophages. The nanogels automatically become hydrophobic in response to tumor acidity within minutes at tumor sites, leading to enhanced tumor accumulation, deep tumor penetration and efficient internalization into bulk tumor cells and CSCs. Inside the tumor cells, the nanogels become positively charged in response to the lysosomal pH and escape from the lysosomes to enter the cytosol, where they further release DOX in response to high intracellular GSH. Therefore, superior in vivo tumor inhibition and CSCs killing efficiency of DOX@SNG are achieved with few side effects. The pH-triggered adaptive hydrophobic strategy not only successfully harmonizes the contradictory needs of nanoparticles to possess both prolonged blood circulation and efficient cellular uptake by bulk tumor cells and CSCs, but also enables deep tumor penetration. In combination with the pH-regulated charge reversal and GSH-responsive intracellular drug release, this delivery system surmounts key physiological barriers after systemic administration in a harmonized manner, providing an efficient platform to achieve better therapeutic effects in cancer chemotherapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 14 ACS Paragon Plus Environment

Page 14 of 27

Page 15 of 27 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

Nano Letters

Materials and methods and supplementary Figures S1-21 (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; Phone: +86-27-87792147; Fax: +86-27-87792234 *E-mail: [email protected] *E-mail: [email protected] Author Contributions #H.

Yang and Q. Wang contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (2018YFA0208903 and 2015CB931802) and National Natural Science Foundation of China (81672937, 81473171, 81372400, 81773653 and 81627901). We also thank the Analytical and Testing Center of Huazhong University of Science and Technology for related analysis.

REFERENCES (1) Visvader, J. E.; Lindeman, G. J. Nat. Rev. Cancer 2008, 8, 755−768. (2) Beck, B.; Blanpain, C. Nat. Rev. Cancer 2013, 13, 727−738. (3) Guillemard, V.; Saragovi, H. U. Curr. Cancer Drug Targets 2004, 4, 313−326. (4) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat. Nanotech. 2007, 2, 751–760. (5) Maeda, H.; Nakamura, H.; Fang, J. Adv. Drug Deliv. Rev. 2013, 65, 71–79. 15 ACS Paragon Plus Environment

Nano Letters 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

(6) Ye, S.; Rao, J.; Qiu, S.; Zhao, J.; He, H.; Yan, Z.; Yang, T.; Deng, Y.; Ke, H.; Yang, H.; Zhao, Y.; Guo, Z.; Chen, H. Adv. Mater. 2018, 30, 1801216. (7) Tang, Y.; Yang, T.; Wang, Q.; Lv, X.; Song, X.; Ke, H.; Guo, Z.; Huang, X.; Hu, J.; Li, Z.; Yang, P.; Yang, X.; Chen, H. Biomaterials 2018, 154, 248−260. (8) Chauhan, V. P.; Jain, R. K. Nat. Mater. 2013, 12, 958−962. (9) An, X.; Zhu, A.; Luo, H.; Ke, H.; Chen, H.; Zhao, Y. ACS Nano 2016, 10, 5947−5958. (10) Chen, H.; Xiao, L.; Anraku, Y.; Mi, P.; Liu, X.; Cabral, H.; Inoue, A.; Nomoto, T.; Kishimura, A.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2014, 136, 157−163. (11) Guo, Z.; Zou, Y.; He, H.; Rao, J.; Ji, S.; Cui, X.; Ke, H.; Deng, Y.; Yang, H.; Chen, C.; Zhao, Y.; Chen, H. Adv. Mater. 2016, 28,10155–10164. (12) Ju, C.; Mo, R.; Xue, J.; Zhang, L.; Zhao, Z.; Xue, L.; Ping, Q.; Zhang, C. Angew. Chem. Int. Ed. Engl. 2014, 53, 6253−6258. (13) Mura, S.; Nicolas, J.; Couvreur, P. Nat. Mater. 2013, 12, 991–1003. (14) Li, H. J.; Du, J. Z.; Du, X. J.; Xu, C. F.; Sun, C. Y.; Wang, H. X.; Cao, Z. T.; Yang, X. Z.; Zhu, Y. H.; Nie, S.; Wang, J. Proc. Natl. Acad. Sci. U S A 2016, 113, 4164–4169. (15) Wang, Y.; Deng, Y.; Luo, H.; Zhu, A.; Ke, H.; Yang, H.; Chen, H. ACS Nano 2017, 11, 12134–12144. (16) Luo, H.; Wang, Q.; Deng, Y.; Yang, T.; Ke, H.; Yang, H.; He, H.; Guo, Z.; Yu, D.; Wu, H.; Chen, H. Adv. Funct. Mater. 2017, 27, 1702834. (17) Minchinton, A. I.; Tannock, I. F. Nat. Rev. Cancer 2006, 6, 583–592. (18) Chauhan, V. P.; Stylianopoulos, T.; Boucher, Y.; Jain, R. K. Annu. Rev. Chem. Biomol. Eng. 2011, 2, 281–298. (19) Ernsting, M. J.; Murakami, M.; Roy, A.; Li, S. D. J. Control. Release 2013, 172, 782– 794. (20) Zuo, Z. Q.; Chen, K. G.; Yu, X. Y.; Zhao, G.; Shen, S.; Cao, Z. T.; Luo, Y. L.; Wang, Y. C.; Wang, J. Biomaterials 2016, 82, 48–59. 16 ACS Paragon Plus Environment

Page 16 of 27

Page 17 of 27 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

Nano Letters

(21) Axelson, H.; Fredlund, E.; Ovenberger, M.; Landberg, G.; Pahlman, S. Semin. Cell Dev. Biol. 2005, 16, 554–563. (22) Li, Z.; Bao, S.; Wu, Q.; Wang, H.; Eyler, C.; Sathornsumetee, S.; Shi, Q.; Cao, Y.; Lathia, J.; McLendon, R. E.; Hjelmeland, A. B.; Rich, J. N. Cancer Cell 2009, 15, 501–513. (23) Wang, J.; Mao, W.; Lock, L. L.; Tang, J.; Sui, M.; Sun, W.; Cui, H.; Xu, D.; Shen, Y. ACS Nano 2015, 9, 7195–7206. (24) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.;Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Nat. Nanotechnol. 2011, 6, 815–823. (25) Tang, L.; Yang, X.; Yin, Q.; Cai, K.; Wang, H.; Chaudhury, I.; Yao, C.; Zhou, Q.; Kwon, M.; Hartman, J. A.; Dobrucki, I. T.; Dobrucki, L. W.; Borst, L. B.; Lezmi, S.; Helferich, W. G.; Ferguson, A. L.; Fan, T. M.; Cheng, J. Proc. Natl. Acad. Sci. U S A 2014, 11, 15344– 15349. (26) Wang, H. X.; Zuo, Z. Q.; Du, J. Z.;Wang, Y. C.; Sun, R.; Cao, Z. T.; Ye, X. D.; Wang, J. L.; Leong, K. W.; Wang, J. Nano Today 2016, 11, 133–144. (27) Yuan, Y. Y.; Mao, C. Q.; Du, X. J.; Du, J. Z.; Wang, F.; Wang, J. Adv. Mater. 2012, 24, 5476–5480. (28) Zhou, M.; Zhang, X.; Yu, C.; Nan, X.; Chen, X.; Zhang, X. Nanomedicine 2016, 12, 181–189. (29) Alexander, J. F.; Kozlovskaya, V.; Chen, J.; Kuncewicz, T.; Kharlampieva, E.; Godin, B. Adv. Healthc. Mater. 2015, 4, 2657–2666. (30) Du, X. J.; Wang, J. L.; Liu, W. W.; Yang, J. X.; Sun, C. Y.; Sun, R.; Li, H. J.; Shen, S.; Luo, Y. L.; Ye, X. D.; Zhu, Y. H.; Yang, X. Z.; Wang, J. Biomaterials 2015, 69, 1–11. (31) Jiang, L.; Zhou, Q.; Mu, K.; Xie, H.; Zhu, Y.; Zhu, W.; Zhao, Y.; Xu, H.; Yang, X. Biomaterials 2013, 34, 7418–7428. (32) Yang, H.; Wang, Q.; Chen, W.; Zhao, Y.; Yong, T.; Gan, L.; Xu, H.; Yang, X. Mol. 17 ACS Paragon Plus Environment

Nano Letters 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

Pharm. 2015, 12, 1636–1647. (33) Li, H. J.; Du, J. Z.; Liu, J.; Du, X. J.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. ACS Nano 2016, 10, 6753–6761. (34) Sun, C. Y.; Shen, S.; Xu, C. F.; Li, H. J.; Liu, Y.; Cao, Z. T.; Yang, X. Z.; Xia, J. X.; Wang, J. J. Am. Chem. Soc. 2015, 137, 15217–15224. (35) Hatakeyama, H.; Akita, H.; Ito, E.; Hayashi, Y.; Oishi, M.; Nagasaki, Y.; Danev, R.; Nagayama, K.; Kaji, N.; Kikuchi, H.; Baba, Y.; Harashima, H. Biomaterials 2011, 32, 4306– 4316. (36) Hatakeyama, H.; Akita, H.; Harashima, H. Adv. Drug Deliv. Rev. 2011, 63, 152–160. (37) Xu, C. F.; Zhang, H. B.; Sun, C. Y.; Liu, Y.; Shen, S.; Yang, X. Z.; Zhu, Y. H.; Wang, J. Biomaterials 2016, 88, 48–59. (38) Sun, C. Y.; Liu, Y.; Du, J. Z.; Cao, Z. T.; Xu, C. F.; Wang, J. Angew. Chem. Int. Ed. Engl. 2016, 55, 1010–1014. (39) Zhang, L.; Cao, Z. Q.; Li, Y. T.; Ella-Menye, J. R.; Bai, T.; Jiang, S. Y. ACS Nano 2012, 6, 6681–6686. (40) H.K.Hall, Jr. J. Am. Chem. Soc. 1957, 79, 5441–5444. (41) Dai, Y.; Xu, C.; Sun, X.; Chen, X. Chem. Soc. Rev. 2017, 46, 3830–3852. (42) Wang, X.; Low, X. C.; Hou, W.; Abdullah, L. N.; Toh, T. B.; Mohd Abdul Rashid, M.; Ho, D.; Chow, E. K. ACS Nano 2014, 8, 12151–12166. (43) Pelton, R. H.; Pelton, H. M.; Morphesis, A.; Rowell, R. L. Langmuir 1989, 5, 816–818. (44) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1–33. (45) Hoare, T.; Pelton, R. Curr. Opin. Colloid Interface Sci. 2008, 13, 413–428. (46) Lyon, L. A.; Fernandez-Nieves, A. Annu. Rev. Phys. Chem. 2012, 63, 25–43. (47) Liu, J.; Tan, Y.; Zhang, H.; Zhang, Y.; Xu, P.; Chen, J.; Poh, Y. C.; Tang, K.; Wang, N.; Huang, B. Nat. Mater. 2012, 11, 734–741. (48) Ma, J.; Zhang, Y.; Tang, K.; Zhang, H.; Yin, X.; Li, Y.; Xu, P.; Sun, Y.; Ma, R.; Ji, T.; 18 ACS Paragon Plus Environment

Page 18 of 27

Page 19 of 27 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

Nano Letters

Chen, J.; Zhang, S.; Zhang, T.; Luo, S.; Jin, Y.; Luo, X.; Li, C.; Gong, H.; Long, Z.; Lu, J.; Hu, Z.; Cao, X.; Wang, N.; Yang, X.; Huang, B. Cell Res. 2016, 26, 713–7727. (49) Webb, B. A.; Chimenti, M.; Jacobson, M. P.; Barber, D. L. Nat. Rev. Cancer 2011, 11, 671–677. (50) Yong, T.; Hu, J.; Zhang, X.; Li, F.; Yang, H.; Gan, L.; Yang, X. ACS Appl. Mater. Interfaces 2016, 8, 27611−27621.

19 ACS Paragon Plus Environment

Nano Letters 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

Figure 1. Schematic illustration of hydrophobicity-adaptive nanogels for programmed anticancer drug delivery. (A) Construction of the nanogels. (B) Characterization of the nanogels in response to the tumor microenvironment. (C) Schematic illustration of the in vivo transport process of the nanogels during anticancer drug delivery.

20 ACS Paragon Plus Environment

Page 20 of 27

Page 21 of 27 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

Nano Letters

Figure 2. Characterization of SNG. (A) Diameters of SNG and DOX@SNG at pH 7.4 as determined by DLS. (B) TEM images of SNG and DOX@SNG (scale bar: 100 nm). (C) Transmittance of SNG at 600 nm after incubation in PBS containing 10% FBS at pH 7.4, 6.5 or 4.5 at the indicated temperatures for 10 min. The insets are pictures of DOX@SNG incubated in PBS containing 10% FBS at pH 7.4 (right), 6.5 (left) or 4.5 (middle) at 37 °C. (D) Diameters of SNG after incubation in PBS at pH 7.4, 6.5 or 4.5 at the indicated temperatures for 5 min. (E) Transmittance of SNG at 600 nm after incubation in PBS containing 10% FBS at pH 7.4, 6.5 or 4.5 at 37 °C for the indicated time intervals. The data are presented as the mean ± SD (n=3).

21 ACS Paragon Plus Environment

Nano Letters 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

Figure 3. Blood circulation and internalization of DOX@SNG into bulk tumor cells and CSCs in vitro and in vivo. (A) Plasma DOX concentrations versus time curves after intravenous injection with free DOX, DOX@ING or DOX@SNG at DOX dose of 4 mg/kg. The inset is the pharmacokinetic parameters of DOX, DOX@ING and DOX@SNG. The data are presented as the mean ± SD (n=3). *P