A Trackable Mitochondria-Targeting Nanomicellar ... - ACS Publications

Subscriber access provided by UNIV OF NEWCASTLE

Article

A Trackable Mitochondria-Targeting Nanomicellar Loaded with Doxorubicin for Overcoming Drug Resistance Ye Zhang, Congjun Zhang, Jing Chen, Li Liu, Mengyue Hu, Jun Li, and Hong Bi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07219 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 12, 2017

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 free 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 accessible to all readers and 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.

ACS Applied Materials & Interfaces 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 54

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

ACS Applied Materials & Interfaces

A Trackable Mitochondria-Targeting Nanomicellar Loaded with Doxorubicin for Overcoming Drug Resistance Ye Zhang, † Congjun Zhang, ‡ Jing Chen, † Li Liu, † Mengyue Hu, † Jun Li, † Hong Bi†,* †

College of Chemistry and Chemical Engineering, Anhui University, Hefei 230601,

China ‡

Department of Oncology, First Affiliated Hospital, Anhui Medical University, Hefei

230022, China KEYWORDS: nanomicelles, mitochondria targeting, multidrug resistance, carbon quantum dots, D-α-tocopheryl polyethylene glycol succinate

ABSTRACT Multidrug resistance (MDR) has been recognized as a major obstacle to successful chemotherapy for cancer in the clinic. In recent years, more and more nanoscaled drug delivery systems (DDS) are constructed to modulate drug efflux protein (P-gp) and deliver chemotherapeutic drugs for overcoming MDR. Among them, D-α-tocopheryl polyethylene glycol succinate (TPGS) has been widely used as a drug carrier due to its capability of inhibiting overexpression of P-gp and good amphiphilicity favorable for improving permeation and long-circulation property of DDS. In the present work, a novel kind of mitochondria-targeting nanomicelles-based DDS is developed to integrate chemotherapeutics delivery with fluorescence imaging functionalities on a comprehensive nanoplatform. The mitochondria-targeting

ACS Paragon Plus Environment


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

nanomicelles are prepared by self-assembly of triphenylphosphine (TPP)-modified TPGS and fluorescent carbon quantum dots (CQDs) in an n-hexane/H2O mixed solution, and named as CQDs-TPGS-TPP. Notably, although the drug loading content of doxorubicin (DOX) in the as-prepared nanomicelles is as low as 3.4%, the calculated resistant index (RI) is greatly decreased from 66.23 of free DOX to 7.16 of DOX-loaded nanomicelles while both treating parental MCF-7 cells and drug-resistant MCF-7/ADR cells. Compared with free DOX, the penetration efficiency of DOX-loaded nanomicelles in three-dimensional multicellular spheroids (MCs) of MCF-7/ADR is obviously increased. Moreover, the released DOX from the nanomicelles can cause much more damage to cells of drug-resistant MCs. These results demonstrate that our constructed mitochondria-targeting nanomicelles-based DDS has potential application in overcoming MDR of cancer cells as well as their MCs that mimic in vivo tumor tissues. The MDR-reversal mechanism of the DOX-loaded CQDs-TPGS-TPP nanomicelles is also discussed.

INTRODUCTION Chemotherapy is one of the mostly adopted clinical therapeutic strategies for cancer treatment. However, its therapeutic efficiency is severely restricted by multidrug resistance (MDR) of cancer cells1,2. The major mechanisms of MDR are rather complicated, often including intrinsic resistance before chemotherapy and acquired resistance via exposure to chemotherapeutics or substrates3. The intrinsic MDR originating from genetic and epigenetic changes in cancer cells through altered


Page 2 of 54

Page 3 of 54

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


apoptosis signaling pathways4. Different from the intrinsic MDR, the acquired MDR is mainly caused by overexpression of ATP-binding cassette (ABC) transporters, especially for P-glycoprotein (P-gp)5. As a result, P-gp will capture and pump anticancer drugs from cytoplasm out towards the extracellular space, which can reduce drugs accumulation within cells and induce a lower therapeutic effect. Both the intrinsic MDR and the acquired MDR are closely related to mitochondrion that plays a key role in cell apoptosis and energy production6,7. In the past decades, mitochondrion has been demonstrated to be an alternative target to overcome MDR by suppressing the mitochondrion’s function of producing ATP and subsequently inhibiting the drug efflux function of ABC protein8,9. In order to surmount the MDR, many small molecular efflux inhibitors, e. g., verapamil, have been employed to combine with chemotherapeutics in cancer clinic therapy10. However, undesirable pharmacokinetics, severe dose-limiting toxicities and low selectivity have obstructed their therapeutic effects11. In recent years, more and more considerations have been focused on constructing new nanoscaled drug delivery systems (DDS) to modulate drug efflux proteins and deliver chemotherapeutic drugs. Various kinds of new nanostructures, such as drug conjugates nanoparticles12, liposomes13, polymeric nanogels14, inorganic nanoparticles15 as well as self-assembled micelles from amphiphilic copolymers16 and surfactants17, have been widely used in DDS to overcome MDR. Among them, D-α-tocopheryl polyethylene glycol succinate (TPGS), as a derivative of Vitamin E and a sort of natural surfactants, has attracted much attention especially for constructing biofunctional nanomicelles. TPGS is



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

believed to be able to inhibit overexpression of P-gp, enhance encapsulation efficiency and oral bioavailability of anticancer drugs, in the meanwhile improve permeation and long-circulation property of DDS18-20. Compared with those small molecular inhibitors, these nanostructures-based DDS could passively target to tumor tissue via the enhanced permeability and retention (EPR) effect. Unfortunately, this single passive targeting strategy cannot achieve neither an ideal cellular uptake efficiency nor an effective inhibition of P-gp in MDR cancer cells. On the other hand, targeted delivery of chemotherapeutics into specific organelles in cancer cells, e.g., mitochondria, has been proved as an efficient way to evade the efflux of transporters21,22. Triphenylphosphine (TPP) and its derivatives belong to lipophilic cationic species, which can transport across mitochondrial membranes and accumulate into highly negatively charged mitochondria23. Recent studies reported by several groups, including ours, have demonstrated that TPP modified nanoparticles would selectively target to mitochondria24,25. In particular, TPP-conjugated TPGS has been incorporated onto the surface of paclitaxel liposomes for treating resistant lung cancer26. It is undoubted that TPGS-based DDS have positive therapeutic effects on MDR cancer models, but the fate of these DDS in cancer cells or tissues is hardly to be traced. In the literature, organic dyes, semiconductor quantum dots and up-conversion nanoparticles etc. have been employed as fluorescent indicators in the TPGS-based DDS27,28, however, the compositions of these materials may arouse potentially complex toxicity in their biomedical applications29,30. In the present study, we design a new trackable, mitochondria-targeting DDS based


Page 4 of 54

Page 5 of 54

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


on the self-assembly of TPP-conjugated TPGS nanomicelles and fluorescent carbon quantum dots (CQDs), as illustrated in Scheme 1. Being a new and promising family member of carbon nanomaterials, CQDs have been successfully served as fluorescent probes in bioimaging, biolabeling and biosensing etc. due to their strong and stable fluorescence, excellent biocompatibility and extremely low cytotoxicity31,32. Herein, CQDs are used as a fluorescent indicator to enable real-time monitoring of the as-prepared TPP-TPGS nanomicelles internalization and localization inside cancer cells, with regard to mitochondria targeting effect. Further, Doxorubicin (DOX) is selected as a model chemotherapeutic drug, and then drug release behavior as well as resistant index (RI) of the DOX-loaded nanomicelles are investigated by co-incubating with parental human breast cancer cells (MCF-7), drug-resistant MCF-7 cells (MCF-7/ADR), and their three-dimensional (3D) multicellular spheroids (MCs), respectively. Our findings demonstrate that the as-constructed mitochondria-targeting nanomicelles-based DDS could overcome MDR of cancer cells as well as their MCs that mimic in vivo tumor tissues. MATERIALS AND METHODS Materials.

The

citric

acid

anhydrate

(CA),

1-Hexadecylamine

(HDA),

1-Octadecene (ODE), (4-Carboxybutyl) triphenylphosphonium bromide (CTPB), N,N’-Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP) and pyrene

were

purchased

poly(2-hydroxyethyl

from

Aladdin

methacrylate)

Industrial

Corporation.

(poly-HEMA)

TPGS, and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained



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

from Sigma-Aldrich Chemicals. Acetone, n-hexane, diethyl ether and ethanol were purchased from Sinopharm Chemical Reagent Co. and used without further purification. Toluene were obtained from Sinopharm Chemical Reagent Co. Ltd. and distilled under reduced pressure before use. Doxorubicin hydrochloride was purchased from Beijing HVSF United Chemical Materials Co. Ltd. Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 Medium, fetal bovine serum (FBS), penicillin/streptomycin and tyrisin were purchased from Hyclone. Mitotracker Red and Lysotracker Red were obtained from Invitrogen. Tetramethylrhodamine ethyl ester (TMRE) mitochondrial membrane potential (MMP) assay kit was purchased from Abcam. Synthesis of HDA-capped CQDs. In a typical process of one-step rapid pyrolysis33, 1 g of HDA and 10 mL of ODE were added into a three-neck flask and then heated to 300 oC under nitrogen atmosphere. When temperature stabilized at 300 o

C for 10 min, 0.67 g of CA was quickly injected into the reaction flask. Then the

resulting solution kept at this temperature for 3 hours. After cooling down to room temperature, the final products were purified by precipitating with acetone three times. Synthesis of TPGS-TPP conjugate. The DCC/DMAP method was used for coupling of TPP to TPGS26. Briefly, 0.202 g of TPGS was dissolved in 6 mL of DMSO and then 0.293 g of CTPB, 0.165 g of DCC and 0.0013 g of DMAP were added into the brown bottle under magnetic stirring. After stirring at room temperature under nitrogen atmosphere for 24 h, the crude product was filtered to


Page 6 of 54

Page 7 of 54

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


remove N, N-dicyclohexylurea (DCU). And then, the filtrate was dialyzing in regenerated cellulose dialysis tubing with MWCO 3500 for 48 h to remove unreacted DCC, DMAP and CTPB. The final product was obtained by freeze-drying. Self-assembly of CQDs-TPGS-TPP. An emulsion and solvent-evaporation method was employed to prepare CQDs-TPGS-TPP nanomicelles. First, 150 mg of TPGS-TPP and 20 mg of HDA-capped CQDs were dispersed in 10 mL of n-hexane under 10 min sonication, then 20 mL of ultrapure water was added into the flask and the mixture was sonicated for additional 20 mins. The flask was transferred into 70 oC water bath under magnetic stirring to evaporate n-hexane after sonication ended. Afterwards, the resultant solution was dialyzed in a dialysis bag with MWCO 3500 for 24 h and then the nanomicelles were freeze-dried. When adding excessive TPGS during the synthesis process, the CQDs-TPGS-TPP nanomicelles would further self-assemble into large-sized nested vesicles. Regarding that only small-sized nanomicelles are appropriate for cellular uptake, so the larger-sized nested vesicles have to be filtered out through a millipore filter membrane of 0.22 µm. In the meantime, TPGS and the same HDA-capped CQDs were used to prepare non-targeting CQDs-TPGS nanomicelles as a control. Characterization. The morphology and structure of CQDs and nanomicelles were observed by a transmission electron microscope (TEM, JEM 2100, JEOL Ltd., Japan) with an accelerated voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) studies were carried out with a spectrophotometer (ESCALAB250). The ζ-potentials and hydrodynamic diameters of the samples were measured using a Malvern Zetasizer



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 ZS90 analyzer (Worcestershire, UK). The Fourier Transform Infrared (FT-IR) and 1H NMR spectra of samples were recorded using a NEXUS-870 spectrometer and a 400 MHz Bruker NMR spectrometer, respectively. The ultraviolet-visible (UV-Vis) and photoluminescence (PL) measurements of samples were performed using an UV759 spectrophotometer and a Hitachi F-4500 fluorescence spectrophotometer, respectively. Drug loading and releasing behavior. 300 mM of DOX aqueous solution was mixed with 10 mg of CQDs-TPGS-TPP nanomicelles in a 20 mL brown vial. The mixture was stirred in the dark for 24 h under room temperature. Thereafter, the DOX-loaded nanomicelles were collected by centrifugation (14000 rpm/min), washed with PBS (pH7.4) for several times until the supernatant became free of reddish color (corresponding to free DOX). The DOX releasing investigation was performed by adding CQDs-TPGS-TPP/DOX in the dialysis membrane tubing (MWCO 1000) into PBS medium (pH 7.4 or 5.0) at 37 oC under continuous stirring. At certain time intervals, 1 mL of the release medium was taken out for UV-Vis measurement to determine DOX release efficiency. The loading content and released content of DOX were determined by the absorbance at 480 nm for DOX, according to the standard calibration curve of free DOX. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated using the following formulae: amount of DOX loaded to micelles ×100% total amount of micelles add DOX amount of DOX loaded to micelles DLE ሺ%ሻ= ×100% total amount of DOX in the system DLCሺ%ሻ=

Cell culture. MCF-7 (DOX-sensitive) and P-gp overexpressing MCF-7/ADR


Page 8 of 54

Page 9 of 54

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


(DOX-resistant) cells were cultured in DMEM and 1640 medium both containing 10% FBS and 100 U/mL penicillin/streptomycin, respectively. The cells were maintained in a humidified 5% CO2 incubator at 37 °C. MCF-7/ADR cells were maintained with free DOX at 1 µg/mL. The cells were routinely harvested by treatment with a trypsin-EDTA solution (0.25%). Cytotoxicity assay. The cytotoxicity of mitochondria targeted CQDs-TPGS-TPP and non-targeted CQDs-TPGS nanomicelles were evaluated using MTT assay with MCF-7 and MCF-7/ADR cells. Typically, cells were initially seeded in a 96-well cell culture plate at a density of 1 × 104 cells per well for 12 h before a total of 100 mL of planed materials (CQDs-TPGS-TPP, DOX-loaded CQDs-TPGS/DOX, DOX-loaded CQDs-TPGS-TPP/DOX, or free DOX) with various concentrations in medium were added into each well. After further co-incubation for 24 h, 10 µL of MTT (5 mg/mL in PBS solution) was added into each well. After 4 h of incubation, culture supernatants were aspirated and purple formazan crystals were dissolved into 150 mL of DMSO for an additional incubation of 15 min. The absorbance was measured at a reference wavelength of 630 nm while using a test wavelength of 570 nm employing a microplate reader (318C, INESA Instrument, China). Cell viability was normalized to that of MCF-7 or MCF-7/ADR cells cultured in complete culture media. Mitochondrial Membrane Potential (MMP) Measurement. MMP was determined by using a TMRE MMP assay kit. Firstly, MCF-7 and MCF-7/ADR cells were plated onto a 96-well plate with a density of 1 × 104 cells per well. After 24 h incubation, the cells were treated with equivalent free DOX, CQDs-TPGS/DOX and



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 10 of 54

CQDs-TPGS-TPP/DOX nanomicelles. At the end of 24 h treatment, the cells were washed with PBS, and TMRE with a final concentration of 200 nM were added into MCF-7 and MCF-7/ADR cells. Then returned the cells to incubator and cultured for an additional 20 min. The cells were then washed once with PBS/0.2% BSA, and the flourescence intensity of TMRE was measured by using a microplate reader (spectramax M2, molecular devices) at the wavelength of Ex/Em = 549/575 nm. Cell culture medium treated cells were used as control groups, and 20 µM carbonyl cyaninde 4-(trifluoromethoxy) phenylhydrazone) (FCCP) pre-treated cells were used as positive control. Confocal

laser

scanning

microscopy

(CLSM).

The

cell

imaging

of

CQDs-TPGS-TPP nanomicelles was observed under the ZEISS 710 CLSM with 488 nm excitation. MCF-7 cells were seeded on a Φ35 mm confocal laser dish at a density of 5 × 104 cells per dish and the medium was changed to the CQDs-TPGS-TPP micelles (150 µg/mL) next day. After further co-incubation for 12 h, the cells were washed with PBS three times and fixed with 4% paraformaldehyde. For the energy-dependent effect on cellular uptake of CQDs-TPGS-TPP nanomicelles, MCF-7 and MCF-7/ADR cells were seeded on confocal laser dishes and pre-incubated for 30 min at either 37 oC or 4 oC before exposure to nanomicelles. After further co-incubation with 150 µg/mL of CQDs-TPGS-TPP for 2 h at 37 oC and 4 oC, respectively, both kinds of cells were washed three times with PBS and fixed with 4% paraformaldehyde. The treated cells were observed under the Leica DMi8 CLSM.


Page 11 of 54

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


Mitochondria co-localization of CQDs-TPGS-TPP nanomicelles in MCF-7 and MCF-7/ADR cells also observed by CLSM. After co-incubated with 150 µg/mL of CQDs-TPGS or CQDs-TPGS-TPP for 12 h, MCF-7 and MCF-7/ADR cells were washed with PBS and further stained by Mitotracker Red (150 nM). The cells were then washed twice with PBS, and differential interference contrast (DIC) and fluorescent images were taken with Leica DMi8 CLSM. In the merged pictures, yellow spots arising from overlap of red and green signals denote the co-localization of the nanomicelles within mitochondrial compartments. Co-localization studies with lysosomes in MCF-7 and MCF-7/ADR cells were also performed. Cells were pre-incubated for 24 h prior to addition of nanomicelles, after reaching 70% confluence, cells were treated with 150 µg/mL of CQDs-TPGS-TPP for 12 h, followed by staining with Lysotracker Red (250 nM) for 30 min. Cells were washed twice with PBS to remove free dyes and then visualized using the Leica DMi8 CLSM. The Pearson’s correlation coefficient (R) of mitochondria or lysosomes with nanomicelles were calculated by ImageJ software. Formation of MCF-7 and MCF-7/ADR MCs. A layer of Poly HEMA thin film was coated on the bottom of cell culture flask to produce MCs. First, 450 mg of Poly HEMA was dissolved into 30 mL of 95% ethanol solution and the mixture was kept stirring at 37 oC for 24 h. Second, every 5 mL of completely dissolved Poly HEMA mixture was added into each cell culture flask. Third, the flasks were transferred to oven to dry for at least 24 h at 37 oC. Finally, coated flasks should be exposed under



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 12 of 54

ultraviolet light overnight to sterile before use. The monolayer cultured MCF-7 and MCF-7/ADR cells were trypsinized to obtain single-cell suspension and counted using the hemocytometer. 5× 105 of MCF-7 or 7 × 105 of MCF-7/ADR cells incubated with 5 mL of fresh DMEM or 1640 medium were placed in cell culture flasks coated with Poly HEMA. The cells were maintained in a humidified 5% CO2 incubator at 37 °C and the culture medium was replaced every other day. MCF-7 MCs and MCD-7/ADR MCs with a mean diameter of 300 - 400 µm formed spontaneously in 7 days. Penetration in MCs. The distributions of CQDs-TPGS-TPP/DOX nanomicelles and free DOX within MCs were determined by Olympus FV1000 CLSM. For each experiment, 15-20 of MCF-7 MCs or MCD-7/ADR MCs with diameters of 300-400 µm were handpicked and transferred into a 24-well cell culture plate with Poly HEMA coating severally. Appropriate concentrations of CQDs-TPGS-TPP/DOX nanomicelles and free DOX were added into each well, respectively. After co-incubation for 2, 6 and 12 h, MCs were transferred to a 1.5 mL Eppendorf tube and washed with PBS for three times before CLSM observation. The semi-quantitative analysis of mean fluorescence intensity of CQDs-TPGS-TPP/DOX micelles and free DOX were calculated by ImageJ software. Growth

inhibition

study

in

MCs.

The

cytotoxicity

of

free

DOX,

CQDs-TPGS-TPP/DOX, CQDs-TPGS-TPP, CQDs-TPGS/DOX and CQDs-TPGS nanomicelles to MCs were investigated through a growth inhibition assay. MCF-7/ADR MCs were incubated with free DOX, CQDs-TPGS-TPP/DOX,


Page 13 of 54

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


CQDs-TPGS-TPP, CQDs-TPGS/DOX and CQDs-TPGS nanomicelles for 7 days, respectively. As an indication of MCs proliferation, the morphology and diameters of spheroid were measured every other day after images were taken from an Olympus IX51 fluorescence microscope. RESULTS AND DISCUSSION

Scheme

1.

Illustrations

of

synthesis

procedure

of

drug-loaded

CQDs-TPGS-TPP/DOX nanomicelles. Preparation and characterization of CQDs-TPGS-TPP nanomicelles. As depicted in Scheme 1, oil-soluble CQDs was firstly synthesized by one-step rapid pyrolysis of citric acid in hot 1-octadecene33. The morphology and average diameter analysis of CQDs are shown in Figure 1A and supplementary Figure S1, respectively. The quasi-spherical CQDs with an average diameter of 2.68 nm are capped with HDA and monodispersed in n-hexane. High-resolution TEM (HRTEM) images of CQDs show that the distances between lattice fringes are 0.227 nm and 0.263 nm which correspond to the (100) and (020) facets of graphitic carbon, respectively34. In order to determine the chemical compositions and structures of CQDs, XPS, 1H NMR and FT-IR spectroscopy studies have been carried out. XPS survey scan of CQDs (Figure S2) displays the presence of carbon (C 1s, 284.8 eV), nitrogen (N 1s, 400.5 eV), and oxygen (O 1s, 532.2 eV) elements in CQDs. The C 1s spectrum (Figure S2B) shows three distinct carbon states of sp2C–sp2C at 284.6 eV, C-OR at 285.1 eV and C=O at



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

288.4 eV35. The O 1s spectrum (Figure S2C) exhibits the oxygen signals of C=O and O=C-OH at 531.9 and 532.4 eV, respectively. N 1s spectrum (Figure S2D) can be resolved into two different peaks located at 400.2 and 400.8 eV, which corresponds to quaternary-N (N–C3) and N-H36. The relative contents of C, N and O are calculated to be 84.67, 3.31 and 12.02 wt%, respectively. As shown in 1H NMR spectrum of HDA-capped CQDs (Figure S3A), the chemical shift at 0.88 ppm can be assigned to -CH3 group whilst another at 1.26 ppm assigned to -CH2- group in HDA37. Moreover, the corresponding

13

C NMR spectrum (Figure S3B) also shows -CH3 and -CH2-

carbons of HDA located within 20-35 ppm, and C=C carbons of CQDs within 100-140 ppm38. FT-IR spectrum (Figure S4) further confirms the HDA capping of CQDs, in which two sharp peaks at 1702 and 1643 cm-1 reveal the appearance of ν(C=O) and ν(CONR) vibrations, suggesting that carbonyl group should have converted into amide group. The UV-Vis absorption and photoluminescence emission spectra of CQDs are recorded to investigate the optical properties of oil-soluble CQDs. The insets of Figure S5 show that CQDs disperse well in n-hexane and exhibit a yellow fluorescence under an UV lamp of 365 nm. In the UV-Vis spectrum of CQDs, the absorption peak at 240 nm is attributed to the π-π* electron transition within the carbon framework. Under an excitation of 470 nm, the CQDs exhibit a strong PL emission centered at 545 nm with a Stokes shift of 75 nm. The mitochondria-targeting TPGS-TPP conjugate was synthesized by a DCC/DMAP method, which coupled the carboxylic group of CTPB to hydroxyl group of TPGS in DMSO (Figure S6)26. 1H-NMR spectra (Figure S7) are used to


Page 14 of 54

Page 15 of 54

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


determine the detailed structure. The characteristic signals of phenyl groups at 7.70-7.78 ppm appear in both spectra of CTPB (blue line) and TPGS-TPP (red line), as while the signals of phenyl groups at 3.65 ppm are also present in the spectra of TPGS (green red) and TPGS-TPP (red line). FT-IR spectra of TPGS, CTPB and TPGS-TPP are shown in supplementary Figure S8, the signal of ν(C=O) vibrations at 1720 cm-1 in CTPB (red line) disappears after conjugated to TPGS (TPGS-TPP, green line), however, the signal of ν(C-O-C) vibrations at 1246 cm-1 of TPGS (black line) and characteristic signal of phenyl groups of CTPB (red line) still emerge in that of TPGS-TPP (green line). The above-mentioned results indicate that TPGS-TPP conjugate has been successful synthesized. In addition, the hydrophilic-lipophilic balance (HLB) values of TPGS and TPGS-TPP are evaluated from their 1H-NMR spectra39. According to the respective integrals of lipophilic groups (δ < 2.5 ppm) and hydrophilic groups (δ > 2.5 ppm), the HLB value of TPGS is calculated to be 14.36 that is close to the previously reported value of 13.240. As expected, the HLB value of TPGS-TPP decreases to 12.38 when amphiphilic TPGS conjugated with a lipophilic TPP moiety. Next, CMC value of TPGS-TPP was measured again by a fluorescence spectrometer using pyrene as a probe41. In a mixture of micelles/pyrene, the fluorescence intensity of pyrene increases remarkably when the concentration of micelles exceeds their CMC42. The CMC value of TPGS-TPP is determined to be 0.008 mg/mL according to the intersecting point of two plots of fluorescence intensity ratio (I338/I334) verses log(concentration of TPGS-TPP), as shown in Figure S9. This value is far lower than that of TPGS (0.2 mg/mL) as previously reported43, which



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

means that the formed TPGS-TPP micelles would have a better dilution stability than TPGS17. Subsequently, we prepared mitochondria-targeting nanomicelles by self-assembly of TPGS-TPP conjugate and HDA-capped CQDs through an emulsification and solvent-evaporation method. Figure 1B shows a typical TEM image of the CQDs-TPGS-TPP nanomicelles, where the dotted CQDs (black spots) are encapsulated by TPGS-TPP and self-assemble into nanometer-sized micelles. The average diameter of CQDs-TPGS-TPP nanomicelles is around 76.63 nm, based on the manual statistics from more than 100 nanomicelles (Figure S1B). The CQDs distributing mainly on the periphery of nanomicelles contain not only their quasi-spherical shape but also their crystalline structure (the inset shows a HRTEM image with the lattice fringes of CQDs individually). Figure 1C shows a typical TEM image of the self-assembled CQDs-TPGS nanomicelles, which acting as a non-targeting control sample. Both amphiphilic TPGS and TPGS-TPP have lipophilic D-α-tocopheryl acid succinate heads as well as hydrophilic PEG tails, in which the lipophilic heads have a high affinity to oil-soluble HDA-capped CQDs, and then aggregate into inner core of the nanomicelles, while the hydrophilic PEG tails extend out to the surrounding aqueous solution. According to the previous reports, TPGS-based micelles were used to load therapeutic drugs as well as other functional nanoparticles for diagnosis, in which these inorganic nanoparticles usually distributed randomly, and the formed micelles always exhibited an elliptical or spherical morphology20. However, in our CQDs-TPGS-TPP nanomicelles, the CQDs majorly


Page 16 of 54

Page 17 of 54

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


locate on the periphery of nanomicelles (Figure 1B), showing a more regular distribution way. Very differently, the CQDs show a random distribution in the control sample of CQDs-TPGS nanomicelles (Figure 1C). In the present case, it is supposed that TPP modification at the end of TPGS may have a positive influence on the formation of more organized structure such as the nanomicelles. In addition, Figure 1D shows the UV-Vis spectra of CQDs, CQDs-TPGS and CQDs-TPGS-TPP, respectively. It can be seen that new absorption peaks appear at 286 nm in both CQDs-TPGS and CQDs-TPGS-TPP, which can be attributed to a characteristic absorption of TPGS (as arrowed in Figure 1D). In addition, the digital photographs in the left column insets of Figure 1D demonstrate that the HAD-capped CQDs originally disperse well in oil phase (n-hexane), and then either CQDs-TPGS or CQDs-TPGS-TPP cannot stay in oil phase (upper layer) any longer but completely dissolve in water phase (down layer). Therefore, the oil phase containing HAD-capped CQDs as well as water phase containing CQDs-TPGS or CQDs-TPGS-TPP all can emit blue fluorescence under an UV lamp of 365 nm, as exhibited in the right column of insets of Figure 1D. Regarding the mitochondrial targeting effect of TPP-conjugated TPGS, it is known that optimum size and positive zeta potential of nanoparticles/nanomicelles are required for efficient cellular uptake and mitochondrial targeting24. In Shanta Dhar’s previous study, they demonstrated that TPP containing positively charged nanoparticles of suitable size (< 160 nm) have the ability to enter the mitochondria of cells, whereas negatively charged nanoparticles that are not highly positively charged



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

or are larger in size, are mostly distributed in the cytosol44,45. We have measured the hydrodynamic diameters and ξ potential of CQDs-TPGS and CQDs-TPGS-TPP by DLS, as shown in Figures 1E and 1F, respectively. Obviously, non-targeting CQDs-TPGS nanomicelles have a mean hydrodynamic diameter of 87.64 nm with a ξ potential of -10.86 mV, however, the hydrodynamic size of CQDs-TPGS-TPP nanomicelles increases to 101.40 nm and ξ potential changes from the negative charge to a positive charge of +21.04 mV. These results have suggested that CQDs-TPGS-TPP nanomicelles with positive potential and appropriate size should have mitochondria-targeting functionality.

Figure 1 Typical TEM and HRTEM (insets) images of (A) CQDs, (B) CQDs-TPGS-TPP and (C) CQDs-TPGS nanomicelles. (D) UV-Vis spectra of CQDs, CQDs-TPGS and CQDs-TPGS-TPP (inset: the photographs of CQDs, CQDs-TPGS and CQDs-TPGS-TPP dissolved in a mixed solution of n-hexane and water (Vhex:Vwat = 1:1) under visible light and an UV lamp of 365 nm, respectively). (E) The hydrodynamic diameters distribution of CQDs-TPGS and CQDs-TPGS-TPP. (F) ζ-potentials of TPGS, CTPB, TPGS-TPP, CQDs-TPGS and CQDs-TPGS-TPP.


Page 18 of 54

Page 19 of 54

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


Cytotoxicity, cell imaging and mitochondria targeting studies of nanomicelles. CQDs have already emerged as universal fluorophores due to their tunable fluorescence properties, high photostablity and excellent biocompatibility in bioimaging and drug or gene delivery carriers46. From the cytotoxicity and in vivo toxicity evaluations, it has been confirmed that CQDs are nontoxic and well biocompatible to exhibit competitive in pre-clinical and clinical imaging applications to that of commercially semiconductor CdSe/ZnS quantum dots and fluorescent dyes47,48. Before CQDs-TPGS-TPP nanomicelles can be ready for loading anticancer drugs and further overcoming MDR, the cytotoxicity, cell imaging and mitochondria targeting ability of CQDs-TPGS-TPP nanomicelles are evaluated in MCF-7 and adriamycin-resistant MCF-7/ADR cells. In this study, MCF-7/ADR cell line is derived from the parental MCF-7 with increasing concentration of the anthracycline antibiotic ADR treatment (the final treatment concentration of DOX is 1 µg/mL)49,50. Figure S10 shows the MTT result of MCF-7 and MCF-7/ADR cells treated with different concentrations of CQDs-TPGS-TPP nanomicelles for 24 h. The cell viabilities of both MCF-7 and MCF-7/ADR cells can keep more than 80% when the concentration of CQDs-TPGS-TPP nanomicelles increases up to 200 µg/mL. However, MCF-7/ADR cells show a little higher cell viability than MCF-7 cells while both treated with CQDs-TPGS-TPP nanomicelles at the same concentration. The possible reason for this difference is that TPGS itself has been reported also possessing anticancer activity due to its increased ability of inducing apoptosis51, which means TPGS-based nanomicelles at high concentration (> 150 µg/mL) will



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 20 of 54

cause more toxicity to drug-sensitive MCF-7 cells. Next, MCF-7 cells co-incubated with the CQDs-TPGS-TPP nanomicelles (150 µg/mL) for 12 h and then were observed via a CLSM. Figure S11 shows a merged CLSM image, in which the internalized CQDs-TPGS-TPP nanomicelles give off green fluorescence in cytoplasm of MCF-7 cells due to fluorescent CQDs under excitation of 488 nm laser. To further investigate CQDs-TPGS-TPP nanomicelles internalization pathway, cells were pre-treated at low temperature (4 oC) before adding nanomicelles. As shown in Figure S12, lowering the temperature resulted in obvious reduction on the cell uptake level of nanomicelles both in MCF-7 and MCF-7/ADR cells, implying that majority of CQDs-TPGS-TPP nanomicelles could be

internalized

through

energy-dependent

endocytosis.

As

we

known,

energy-dependent clathrin-mediated endocytosis is probably the primary characterized mechanism for the cellular uptake of nanoparticles with usual diameter < 100 nm52. Moreover, in contrast to neutral or negatively charged nanoparticles, the positively charged nanoparticles has a better internalization ability via clathrin-mediated endocytosis53. We can conclude from the temperature inhibition of cellular uptake that CQDs-TPGS-TPP nanomicelles are internalized by MCF-7 and MCF-7/ADR cells via an endocytosis pathway, and clathrin-mediated endocytosis might be one of the major endocytosis pathways. Further, mitochondria or lysosomes in MCF-7 and MCF-7/ADR cells were stained with commercial dyes, Mitotracker Red or Lysotracker Red to evaluate mitochondria targeting ability of TPP-modified nanomicelles. Figure 2 shows the CLSM images of


Page 21 of 54

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


MCF-7/ADR cells co-incubated with CQDs-TPGS-TPP nanomicelles and the control CQDs-TPGS nanomicelles. In the merged images, the yellow fluorescence is overlaid by green fluorescence of CQDs-TPGS-TPP and red fluorescence of Mitotracker Red or Lysotracker Red. As shown in the upper panel of Figure 2, the insignificant co-localization of lysosomes and nanomicelles with Pearson’s correlation coefficient (R) of 0.26 can be observed, which indicates that most CQDs-TPGS-TPP nanomicelles can escape from endosomes or lysosomes after 12 h treatment. On the contrary, much more yellow dots can be found in the middle panel of Figure 2, CQDs-TPGS-TPP nanomicelles can evidently co-localize with mitochondria in MCF-7/ADR cells with R value of 0.66. Compared with CQDs-TPGS-TPP, non-targeting CQDs-TPGS nanomicelles show a much lower R value of 0.19 (lower panel of Figure 2). In addition, the same protocols were also performed in MCF-7 cells, the co-localization profile of the CQDs-TPGS-TPP nanomicelles is shown in Figure S13. The R value of lysosomes with CQDs-TPGS-TPP, mitochondria with CQDs-TPGS-TPP, as well as mitochondria with CQDs-TPGS are 0.30, 0.61 and 0.18, respectively. It should be pointed out that herein all of the correlation coefficients of CQDs-TPGS-TPP

are

much

lower

than

other

previously

reported

mitochondria-targeting agents54, probably because the lipophilic ending group of TPP will approach to lipophilic D-α-tocopheryl acid succinate head of TPGS during the self-assembly process, and then partially be encapsulated into the interior of nanomicelles.



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 22 of 54

Figure 2. CLSM images of MCF-7/ADR cells treated with mitochondria-targeting CQDs-TPGS-TPP or non-targeting CQDs-TPGS nanomicelles for 12 h and stained with Lysotracker Red or Mitotracker Red, respectively. Scale bar = 15 µm. Drug loading and release behavior. A commonly used anticancer drug, DOX, can be easily loaded on the surface of sp2-bonded carbon nanostructures or conjugated polymers via hydrophobic interaction and π–π stacking55.56. In a typical experiment, CQDs-TPGS-TPP nanomicelles are impregnated with DOX solution under magnetic stirring for 24 h to load DOX into the hydrophobic core of CQDs-TPGS-TPP to obtain CQDs-TPGS-TPP/DOX nanomicelles, and then unattached free DOX drugs are

removed

through

centrifugation.

The

optical

characterization

of

CQDs-TPGS-TPP/DOX nanomicelles is shown in Figure 3A. It is well-known that DOX

has

a

characteristic

absorption

peak

at

480

nm,

whereas

CQDs-TPGS-TPP/DOX also shows an absorption around 500 nm in the UV-Vis spectrum. The redshift in absorption of CQDs-TPGS-TPP/DOX means that DOX has been successfully loaded into nanomicelles and further interact with lipophilic D-α-tocopheryl acid succinate heads of TPGS. The DLC and DLE of


Page 23 of 54

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


CQDs-TPGS-TPP/DOX nanomicelles are calculated to be 3.4% and 48.85%, respectively. To investigate the pH-triggered release of CQDs-TPGS-TPP/DOX nanomicelles, the same quantities of nanomicelles are dispersed in PBS buffer at different pH values (pH = 7.4 and pH = 5.0). The drug release profiles of DOX from nanomicelles are shown in Figure 3B, in which the released amount of DOX is estimated via a standard curve of DOX at 480 nm measured by using an UV-Vis spectrophotometer. The release rate of DOX at pH 5.0 is significantly higher than that at pH 7.4. It is found that DOX release efficiency increases to ~65% in acidic conditions, but it in the neutral buffer is only as low as ~18%. Importantly, the release rate of DOX in PBS buffer at pH 5.0 and pH 7.4 are both less than 30% in the initial 4 h, showing a delayed drug releasing behavior. It will benefit for preventing most anticancer drugs inside nanomicelles from early leaking before the nanomicelles reach the target sites.

Figure 3. Drug loading and release behavior of CQDs-TPGS-TPP nanomicelles. (A) UV-Vis

absorption

spectra

of

free

DOX,

CQDs-TPGS-TPP

and

CQDs-TPGS-TPP/DOX. (B) Release behavior of DOX from CQDs-TPGS-TPP at pH 5.0 and 7.4, respectively. Killing effect and intracellular accumulation of DOX-loaded Nanomicelles.



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 24 of 54

Compared with parental MCF-7 cells, MCF-7/ADR cells from DOX continuous treatment have been found to overexpress of P-gp-encoding MDR1 gene and P-gp protein50. Any kind of P-gp substrates, e.g. DOX, interacts with the P-gp binding-site will inhibit another P-gp substrate, which causes complex resistant interactions in the situation of polypharmacy to cancer patients57. As TPGS is a typical P-gp inhibitor, it is assumed that TPGS-based nanomicelles could evade the efflux pump and increase the

accumulation

of

DOX

in

MDR

cancer

cells.

The

ability

of

CQDs-TPGS-TPP/DOX nanomicelles overcoming MDR in MCF-7/ADR cells is investigated via MTT assay. As shown in Figures 4A and B, free DOX appears obviously increasing cytotoxicity to MCF-7 cells with the increased dosage of DOX, but it has very limited inhibition effect on MCF-7/ADR cells even at a highest concentration

of

10

µM

DOX.

In

contrast,

CQDs-TPGS/DOX

and

CQDs-TPGS-TPP/DOX nanomicelles display higher therapeutic efficiency against drug resistant MCF-7/ADR cells than equivalent free DOX, although they show a lower cytotoxicity in drug-sensitive MCF-7 cells than free DOX, which have a similar killing effect as other TPGS-modified DOX-loaded nanoparticles in previous report28. The mitochondria targeting ability of CQDs-TPGS-TPP/DOX nanomicelles could increase the cellular uptake and mitochondria accumulation of nanomiclles, which result in more DOX delivered into MCF-7 and MCF-7/ADR cells to induce better cancer cell killing efficiency than non-targeting CQDs-TPGS/DOX nanomicelles. Basically, IC50 value as well as drug resistant index (RI) can reflect the chemoresistance level of MDR cells in comparison to its parental sensitive cells58.


Page 25 of 54

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


IC50 and RI of free DOX and CQDs-TPGS-TPP/DOX in MCF-7 and MCF-7/ADR cells are calculated from MTT results, as shown in Table S1. According to literature, MDR cells can be classified into highly drug-resistant with RI > 10, moderately drug resistant with 2 < RI < 10, and drug-sensitive with 0 < RI < 259. It can be seen clearly that MCF-7/ADR cells in the present study are highly drug-resistant to DOX with RI as high as 66.23. When MCF-7/ADR cells are treated with CQDs-TPGS-TPP/DOX, the IC50 value reduces remarkably from 49.67 µg/mL to 8.45 µg/mL with a much lower RI of 7.16. It illustrates that CQDs-TPGS-TPP/DOX treatment has successfully turned highly drug-resistant MCF-7/ADR cells into moderately drug resistant cancer cells. Mitochondria play key roles in controlling the activation of apoptosis and emerged as targets for overcoming MDR in cancer therapy6,7. The mitochondrial membrane potential (∆Ψm) is highly interlinked to many mitochondrial processes, and the loss of ∆Ψm is an early event in mitochondria triggered apoptosis8,60. Subsequently, we measured intracellular ∆Ψm changes caused by free DOX, CQDs-TPGS/DOX and CQDs-TPGS-TPP/DOX nanomicelles via a TMRE MMP assay kit. TMRE is a positively-charged, red-orange fluorescent dye that can be readily taken up by active mitochondria into the negatively charged mitochondrial matrix. FCCP is a ionophore uncoupler of oxidative phosphorylation that usually used as a positive control. Different levels of decrease in ∆Ψm of MCF-7 and MCF-7/ADR cells are shown in Figure 4C and 4D, respectively. Compared with free DOX and non-targeting CQDs-TPGS/DOX treated groups, more significant decrease in ∆Ψm can be observed



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

in CQDs-TPGS-TPP/DOX treated MCF-7 and MCF-7/ADR cells. Elimination of ∆Ψm could reflect the alterations of mitochondria DNA (mtDNA) caused by DOX delivered from mitochondria targeting nanocarriers61. Thereby, the targeting property of CQDs-TPGS-TPP/DOX nanomicelles to mitochondria is promising for inducing apoptosis and damaging mtDNA in resistant MCF-7/ADR cells.

Figure 4. Cell viabilities of (A) MCF-7 and (B) MCF-7/ADR cells treated with free DOX, CQDs-TPGS/DOX and CQDs-TPGS-TPP/DOX with various equivalent DOX concentrations, respectively. Mitochondrial membrane potential of (C) MCF-7 and (D) MCF-7/ADR cells treated with 0.5 µg/mL and 5 µg/mL equivalent free DOX, CQDs-TPGS/DOX and CQDs-TPGS-TPP/DOX nanomicelles, respectively. CLSM images of (E) MCF-7 and (F) MCF-7/ADR cells treated with free DOX and CQDs-TPGS-TPP/DOX. Large-scaled intracellular DOX accumulation is marked out with white arrows. Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. Next, Figures 4E and F show the CLSM images of MCF-7 and MCF-7/ADR cells treated with free DOX and CQDs-TPGS-TPP/DOX for 24 h, respectively. As for the parental MCF-7 cells, free DOX (red fluorescence) localized inside the nucleus while


Page 26 of 54

Page 27 of 54

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


CQDs-TPGS-TPP/DOX distributed in the cytoplasm as arrowed in merged image of Figure 4E, since free DOX can target to DNA in the nucleus after a passive diffusion process crossing cell membrane62. However, in respect to the drug-resistant MCF-7/ADR cells, free DOX merely localized in the cytoplasm due to overexpression of P-gp in drug-resistant MCF-7/ADR cells (Figure 4F). In the meanwhile, the majority of CQDs-TPGS-TPP/CQDs remained in the cytoplasm, albeit some DOX released from CQDs-TPGS-TPP/CQDs nanomicelles penetrated into the nucleus of MCF-7/ADR cells, as arrowed in Figure 4F, which indicates that CQDs-TPGS-TPP nanomicelles are helpful for overcoming MDR in MCF-7/ADR cells. Penetration in 3D multicellular spheroids (MCs). Compared with in vitro monolayer-cultured cells, their 3D MCs could be able to mimic in vivo tumor tissues more approximately, which has much easier controlled environment in systematic evaluating tumor penetration and therapeutic effect of DDS63. MCs have a complex network of cell-cell interactions and extracellular matrix (ECM), as well as pH, oxygen,

glucose,

metabolic

and

proliferative

gradients

similar

to

the

microenvironment in poorly vascularized and avascular regions of solid tumors64,65. More importantly, cells in MCs are more resistant than monolayers to a variety of chemotherapeutic agents66. In the literature, 3D culture could decrease the proliferative rate of cells in MCs, but it did not affect the resistance of MCF-7/ADR MCs67. In this work, 3D cultured MCs of parental MCF-7 and drug resistant MCF-7/ADR

cells

are

used

to

test

the

uptake


and

penetration

of


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 28 of 54

CQDs-TPGS-TPP/DOX nanomicelles, respectively. The morphology of MCF-7 MCs and MCF-7/ADR MCs are shown in Figures S14 and S15. After 7 days’ culture, MCF-7 and MCF-7/ADR-derived spheroids show symmetrical shape and similar average diameters of 300 - 400 µm in cell medium. Notably, these MCs have strong interactions of cell-cell and cell-ECM in this time. Figure

5A

shows

a

time-dependent

penetration

of

free

DOX

and

CQDs-TPGS-TPP/DOX into MCF-7 MCs, respectively. At first 2 h, the red fluorescence arising from DOX only appears around the outer layers of MCs for both free DOX and CQDs-TPGS-TPP/DOX treated MCs. As co-incubation time elapses, CQDs-TPGS-TPP/DOX treated MCs exhibit higher fluorescence spreading from the periphery to the core of spheroids than free DOX alone treated MCs. Figure 5B shows the semi-quantitative analysis of mean fluorescence intensity of red fluorescent signals from DOX in both MCs. It also presents a time-dependent penetration behavior, and the mean intensity in CQDs-TPGS-TPP/DOX treated groups is higher than that in free DOX treated MCs. Furthermore, the section images of MCs from the top to the core after 12 h treatment are obtained through Z-stacked method of CLSM. As shown in Figure 5C, the red fluorescence from CQDs-TPGS-TPP/DOX can be observed from the periphery to the inner of MCs with a maximum depth of ~120 µm, which exhibits a deeper penetration ability than free DOX with a maximum depth of ~80 µm. Similar phenomenon can be found in free DOX alone and CQDs-TPGS-TPP/DOX treated MCF-7/ADR MCs, as shown in Figure S16. These results reveal that CQDs-TPGS-TPP/DOX nanomicelles can improve the DOX


Page 29 of 54

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


penetration efficiency in both MCF-7 MCs and MCF-7/ADR MCs, leading to more DOX accumulation in CQDs-TPGS-TPP/DOX treated MCs than free DOX treated MCs within the same co-incubation time.

Figure 5. (A) CLSM images of MCF-7 MCs co-incubated with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 2, 6 and 12 h, respectively. (B) Semi-quantitative mean fluorescence intensity in MCF-7 MCs cultured with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 2, 6 and 12 h, respectively. (C) Representative Z-stack images of tumor spheroids after treatment with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 12 h are obtained from starting at the top of the spheroid in 20 µm intervals. Scale bars = 200 µm. Growth inhibition of multicellular spheroids (MCs). The MCF-7/ADR MCs are further used to screen the anticancer effect as well as MDR reversal of CQDs-TPGS-TPP/DOX nanomicelles. As shown in Figure 6 A, the optical images in left three columns represent for 1640 medium treated MCF-7/ADR MCs (as a control), free DOX and CQDs-TPGS-TPP/DOX treated MCF-7/ADR MCs at equivalent



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

concentration of DOX, respectively. More and more obvious differences in volume and morphology can be seen in free DOX and CQDs-TPGS-TPP/DOX treated MCF-7/ADR MCs as co-incubation time continues to one week. In particular, at 7 days, CQDs-TPGS-TPP/DOX treated MCs appear not only a remarkably reduced volume, but also a great change in morphology compared with free DOX treated MCs. Figure 6C displays the fluorescent image of CQDs-TPGS-TPP/DOX treated MCs at 7 days, which indicates clearly that the outer layer of MCs has been completely disrupted and divided into small fragments, accompanied by many dead cells peeling off from the spheroids and distributing randomly in 1640 medium. On the contrary, free DOX treated MCF-7/ADR MCs still keep their spherical morphology although their volume size has reduced a little which is comparable to the MCs treated with non-drug loaded CQDs-TPGS-TPP (Figure 6 A). This result confirms the drug resistant character of MCF-7/ADR MCs but a certain of MDR reversal effect of CQDs-TPGS-TPP/DOX occurred as while. We next examined whether the mitochondria targeting moiety could have a help for this MDR reversal behavior or not. For comparison, MCF-7/ADR MCs were treated with non-targeting CQDs-TPGS/DOX and CQDs-TPGS in same culture protocol for 1, 3, 5, and 7 days, respectively (shown in right side of Figure 6 A). It can be seen that the volume of either


Page 30 of 54

Page 31 of 54

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 6. (A) Growth inhibition assay in MCF-7/ADR MCs. Representative optical images of MCs treated with culture medium, free DOX, CQDs-TPGS-TPP/DOX, CQDs-TPGS-TPP, CQDs-TPGS/DOX and CQDs-TPGS nanomicelles. Scale bars = 100 µm. (B) Growth curves of MCs statistically obtained from growth inhibition assay. **P < 0.01 (versus 1640 medium group from the 7th day). (C) Representative fluorescent images of MCs treated with free DOX and CQDs-TPGS-TPP/DOX for 7 days, respectively.



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

CQDs-TPGS/DOX or CQDs-TPGS treated MCs decrease gradually in response to increasing cultivation time, but both of the MCs still hold their original spherical shape at 7 days while display a bigger volume size than CQDs-TPGS-TPP/DOX treated MCs. Furthermore, the statistical quantiﬁcation of MCs volume varying with treatment time for all of six specimens is summarized in Figure 6B, the results of mean diameters confirm again that CQDs-TPGS-TPP/DOX treated MCs possess the smallest volume in whole duration of one week. Finally, at 7 days, the mean diameter of CQDs-TPGS-TPP/DOX treated MCs has reduced 29.88 % and 20.70% respectively compared to control group and the free DOX treated MCs. Taken together, it can be considered that mitochondria-targeting modified nanomicelles indeed have help for overcoming MDR compared to non-targeting nanomicelles. We seek to identify the mechanism of why CQDs-TPGS-TPP/DOX nanomicelles have an enhanced MDR reversal effect in both MCF-7/ADR cells and MCF-/ADR MCs, compared with CQDs-TPGS/DOX and free DOX. Careful inspections of the morphology change of MCF-7/ADR MCs and intracellular distribution of CQDs-TPGS-TPP/DOX in MCF-7/ADR cells, suggest a possible MDR-reversal mechanism, involving overexpressed P-gp inhibition, DNA damage as well as mitotoxic effect. At study termination (7 days), statistically signiﬁcant reductions in CQDs-TPGS-TPP/DOX treated volumes are found relative to CQDs-TPGS/DOX treated volumes (Figures 6A and B). It has been reported that DOX not only inhibits topoisomerase II (Topo II) in the nucleus, but also damages mtDNA to generate mitotoxic effects21,68. We therefore infer that TPP, as a mitochondria targeting group,


Page 32 of 54

Page 33 of 54

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


may help CQDs-TPGS-TPP/DOX nanomicelles lead to more ∆Ψm decrease and mtDNA damage both in MCF-7/ADR cells and their MCs than those non-targeting nanomicelles. On the other hand, in the TPGS-based CQDs-TPGS-TPP/DOX nanomicelles, the overexpressed P-gp in cell membrane will be inhibited so as to increase the accumulation of released-DOX in cytoplasm and the interaction with the nucleus of MCF-7/ADR cells, resulting in a higher cytotoxicity than free DOX (Figures 4B, 4D and 4F). Therefore, both nuclear DNA as well as mtDNA have become the target for CQDs-TPGS-TPP/DOX. Moreover, with co-incubation time increasing, DOX penetration efficiency will be improved via CQDs-TPGS-TPP/DOX nanomicelles, inducing more DOX accumulation in MCF-7/ADR MCs to inhibit the growth of MCs (Figure 6). Finally, in view of above-mentioned killing effect, CQDs-TPGS-TPP/DOX nanomicelles have an enhanced MDR reversal influence on drug-resistant cancer cells and their MCs. CONCLUSION In summary, a mitochondria-targeting group, TPP, has been modified onto TPGS through a DCC/DMAP coupling method. Subsequently, the TPGS-TPP conjugate and fluorescent, oil-soluble CQDs can self-assemble into CQDs-TPGS-TPP nanomicelles. In vitro monolayer cell experiment results indicate that the fluorescent CQDs-TPGS-TPP nanomicelles are suitable for cell imaging especially with mitochondria targeting functionality. Furthermore, the in vitro DOX loading experiments show that CQDs-TPGS-TPP nanomicelles have a DLC of 3.4%, and the release of DOX from nanomicelles can be accelerated at lower pH value. Compared



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

with equivalent CQDs-TPGS/DOX and free DOX, CQDs-TPGS-TPP/DOX nanomicelles cause more cytotoxicity to MCF-7/ADR cells owing to the synergistic effect from P-gp inhibition of TPGS, nucleus interaction and mtDNA damage from DOX. Using 3D cultured MCF-7 and MCF-7/ADR MCs as mimicking models, the experimental results reveal that CQDs-TPGS-TPP/DOX nanomicelles can improve the DOX penetration efficiency in both MCF-7 MCs and MCF-7/ADR MCs, and show an enhanced MDR reversal effect in MCF-/ADR MCs, compared with CQDs-TPGS/DOX and free DOX. It is considered that mitochondria-targeting modified nanomicelles own advantages in overcoming MDR compared to non-targeting nanomicelles. This work opens up a new strategy in designing, constructing and testing chemotherapeutics-loaded nanomicellar DDS for overcoming MDR via self-assembly as well as modification with mitochondria-targeting groups, and studying them in 3D MCs model before in vivo test. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: Particles size distribution of CQDs and CQDs-TPGS-TPP nanomicelles. XPS survey spectrum, 1H and

13

C NMR, FT-IR spectrum, UV-Vis and PL emission

spectrum of CQDs. Synthetic route, 1H NMR, FT-IR spectra and CMC measurements of TPGS-TPP. MTT results, CLSM images and mitochondria co-localization results of CQDs-TPGS-TPP in MCF-7 cells. IC50 values and RI of free DOX and CQDs-TPGS-TPP/DOX nanomicelles. Optical microscope images of MCF-7 MCs


Page 34 of 54

Page 35 of 54

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


and MCF-7/ADR MCs. Penetration of CQDs-TPGS-TPP/DOX in MCF-7/ADR MCs. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financed by the 211 project of Anhui University, the National Natural Science Foundation of China (grant No. 51272002) and the 2016 Foundation for Imported Leading Talent Teams in Universities and Colleges of Anhui Province. We thank the Key Laboratory of Environment-Friendly Polymer Materials of Anhui Province, and Collaborative Innovation Center of Modern Biomanufacture, Anhui University. REFERENCES (1) Szakács, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.; Gottesman, M. M. Targeting Multidrug Resistance in Cancer. Nat. Rev. Drug Discov. 2006, 5, 219-234. (2) Holohan, C.; Schaeybroeck, S. V.; Longley, D. B.; Johnston, P. G. Cancer Drug Resistance: An Evolving Paradigm. Nat. Rev. Cancer 2013, 13, 714-726. (3) Bagrodia, S.; Smeal, T.; Abraham, R. T. Mechanisms of Intrinsic and Acquired Resistance to Kinase-Targeted Therapies. Pigment Cell Melanoma Res. 2012, 25, 819-831.



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

(4) Tredan, O.; Galmarini, C. M.; Patel, K.; Tannock, I. F. Drug Resistance and the Solid Tumor Microenvironment. J. Natl. Cancer Inst. 2007, 99, 1441-1454. (5) Turner, N. C.; Reis-Filho, J. S. Genetic Heterogeneity and Cancer Drug Resistance. Lancet Oncol. 2012, 13, e178-185. (6) McBride, H. M.; Neuspiel, M.; Wasiak, S. Mitochondria: More Than Just a Powerhouse. Curr. Biol. 2006, 16, 551-660. (7) Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting Mitochondria for Cancer Therapy. Nat. Rev. Drug Discovery 2010, 9, 447-464. (8) Zhang, Z. W.; Liu, Z. Y.; Ma, L.; Jiang, S. J.; Wang, Y. X.; Yu, H. J.; Yin, Q.; Cui, J. B.; Li, Y. P. Reversal of Multidrug Resistance by Mitochondrial Targeted Self-Assembled Nanocarrier Based on Stearylamine. Mol. Pharmaceutics 2013, 10, 2426-34. (9) Yu, P. C.; Yu, H. J.; Guo, C. Y.; Cui, Z. R.; Chen, X. Z.; Yin, Q.; Zhang, P. C.; Yang, X. L.; Cui, H. G.; Li, Y. P. Reversal of Doxorubicin Resistance in Breast Cancer by Mitochondria-Targeted pH-Responsive Micelles. Acta biomater. 2015, 14, 115-124. (10) Krishna, R.; Mayer, L. D. Multidrug Resistance (MDR) in Cancer. Mechanisms, Reversal Using Modulators of MDR and the Role of MDR Modulators in Influencing the Pharmacokinetics of Anticancer Drugs. Eur. J. Pharm. Sci. 2000, 11, 265-283. (11) Kunjachan, S.; Rychlik, B.; Storm, G.; Kiessling, F., Lammers, T. Multidrug Resistance: Physiological Principles and Nanomedical Solutions. Adv. Drug Deliver. Rev. 2013, 65, 1852-1865.


Page 36 of 54

Page 37 of 54

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


(12) Mallick, A.; More, P.; Ghosh, S.; Chippalkatti, R.; Chopade, B. A.; Lahiri, M.; Basu, S. Dual Drug Conjugated Nanoparticle for Simultaneous Targeting of Mitochondria and Nucleus in Cancer Cells. ACS Appl. Mater. Interfaces 2015, 7, 7584-7598. (13) Wang, X. X.; Li, Y. B.; Yao, H. J.; Ju, R. J.; Zhang, Y.; Li, R. J.; Yu, Y.; Zhang, L.; Lu, W. L. The Use of Mitochondrial Targeting Resveratrol Liposomes Modified with a Dequalinium Polyethylene Glycol-Distearoylphosphatidyl Ethanolamine Conjugate to Induce Apoptosis in Resistant Lung Cancer Cells. Biomaterials, 2011, 32, 5673-5687. (14) Wu, H. Q.; Jin, H. J.; Wang, C.; Zhang, Z. H.; Ruan, H. Y.; Sun, L. Y.; Yang, C.; Li, Y. J.; Qin, W. X.; Wang, C. C. Synergistic Cisplatin/Doxorubicin Combination Chemotherapy for Multidrug-Resistant Cancer via Polymeric Nanogels Targeting Delivery. ACS Appl. Mater. Interfaces 2017, 9, 9426-9436. (15) Chen, Y.; Chen, H. R.; Shi, J. L. Inorganic Nanoparticle-Based Drug Codelivery Nanosystems to Overcome the Multidrug Resistance of Cancer Cells. Mol. Pharmaceutics 2014, 11, 2495-2510. (16) Zhao, S.; Tan, S. W.; Guo, Y. Y.; Huang, J.; Chu, M.; Liu, H. D.; Zhang, Z. P. pH-Sensitive

Docetaxel-Loaded

D-α-Tocopheryl

Polyethylene

Glycol

Succinate-Poly(β-amino ester) Copolymer Nanoparticles for Overcoming Multidrug Resistance. Biomacromolecules 2013, 14, 2636-2646. (17) Hao, T. N.; Chen, D. W.; Liu, K. X.; Qi, Y.; Tian, Y.; Sun, P. Y.; Liu, Y. H.; Li, Z. Micelles of d‑α-Tocopheryl Polyethylene Glycol 2000 Succinate (TPGS2K) for



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

Doxorubicin Delivery with Reversal of Multidrug Resistance. ACS Appl. Mater. Interfaces 2015, 7, 18064-18075. (18) Dintaman, J. M.; Silverman, J. A. Inhibition of P-glycoprotein by D-α-tocopheryl Polyethylene Glycol 1000 succinate (TPGS). Pharm. Res. 1999, 16, 1550-1556. (19) Varma, M. V. S.; Panchagnula, R. Enhanced Oral Paclitaxel Absorption with Vitamin E-TPGS: Effect on Solubility and Permeability in Vitro, in Situ and in Vivo. Eur. J. Pharm. Sci. 2005, 25, 445-453. (20) Zhang, Z.; Tan, S.; Feng, S. S. Vitamin E TPGS as a Molecular Biomaterial for Drug Delivery. Biomaterials 2012, 33, 4889-4906. (21) Chamberlain, G. R.; Tulumello, D. V.; Kelley, S. O. Targeted Delivery of Doxorubicin to Mitochondria. ACS Chem. Biol. 2013, 8, 1389-1395. (22) Jean, S. R.; Pereira, M. P.; Kelley, S. O. Structural Modifications of Mitochondria-Targeted Chlorambucil Alter Cell Death Mechanism but Preserve MDR Evasion. Mol. Pharmaceutics 2014, 11, 2675-2682. (23) Modica-Napolitano, J. S.; Weissig, V. Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents. Int. J. Mol. Sci. 2015, 16, 17394-17421. (24) Pathak, R. K.; Kolishetti, N.; Dhar, S. Targeted Nanoparticles in Mitochondrial Medicine. WIREs Nanomed. Nanobiotechnol. 2015, 7, 315-329. (25) Zhang, Y.; Shen, Y. J.; Teng, X. Y.; Yan, M. Q.; Bi, H.; Morais, P. C. Mitochondria-Targeting Nanoplatform with Fluorescent Carbon Dots for Long Time Imaging and Magnetic Field-Enhanced Cellular Uptake. ACS Appl. Mater. Interfaces


Page 38 of 54

Page 39 of 54

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


2015, 7, 10201-10212. (26) Zhou, J.; Zhao, W. Y.; Ma, X.; Ju, R. J.; Li, X. Y.; Li, N.; Sun, M. G.; Shi, J. F.; Zhang, C. X.; Lu, W. L. The Anticancer Efficacy of Paclitaxel Liposomes Modified with Mitochondrial Targeting Conjugate in Resistant Lung Cancer. Biomaterials 2013, 34, 3626-3638. (27) Tan, Y. F.; Chandrasekharan, P.; Maity, D.; Yong, C. X.; Chuang, K. H.; Zhao, Y.; Wang, S.; Ding, J.; Feng, S. S. Multimodal Tumor Imaging by Iron Oxides and Quantum Dots Formulated in Poly(lactic acid)-d-alpha-tocopheryl Polyethylene Glycol 1000 Succinate Nanoparticles. Biomaterials 2011, 32, 2969-2978. (28) Tian, G.; Zheng, X. P.; Zhang, X.; Yin, W. Y.; Yu, J.; Wang, D. L.; Zhang, Z. P.; Yang, X. L.; Gu, Z. J.; Zhao. Y. L. TPGS-Stabilized NaYbF4:Er Upconversion Nanoparticles for Dual-Modal Fluorescent/CT Imaging and Anticancer Drug Delivery to Overcome Multi-Drug Resistance. Biomaterials 2015, 40, 107-116. (29) Chen, N.; He, Y.; Su, Y. Y.; Li, Y. M.; Huang, Q.; Wang, H. F.; Zhang, X. Z.; Tai, R. Z.; Fan, C. H. The Cytotoxicity of Cadmium Based Quantum Dots. Biomaterials 2012, 33, 1238-1244. (30) Guller, A. E.; Generalova, A. N.; Petersen, E. V.; Nechaev, A. V.; Trusova, I. A.; Landyshev, N. N.; Nadort, A.; Grebenik. E. A.; Deyev, S. M.; Shekhter, A. B.; Zvyagin, A. V. Cytotoxicity and Non-specific Cellular Uptake of Bare and Surface-Modified Upconversion Nanoparticles in Human Skin Cells. Nano Res. 2015, 8, 1546-1562. (31) Lim, S. Y.; Shen, W. Gao, Z. P. Carbon Quantum Dots and Their Applications.



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

Chem. Soc. Rev. 2015, 44, 362-381. (32) Wang, J.; Qiu, J. A Review of Carbon Dots in Biological Applications. J. Mater. Sci. 2016, 51, 4728-4738. (33) Wang, F.; Pang, S. P.; Wang, L.; Li, Q.; Kreiter, M.; Liu, C. Y. One-Step Synthesis of Highly Luminescent Carbon Dots in Noncoordinating Solvents. Chem. Mater. 2010, 22, 4528-4530. (34) Nie, H.; Li, M. J.; Li, Q. S.; Liang, S. J.; Tan, Y. Y.; Sheng, L.; Shi, W.; Zhang, S. X-A. Carbon Dots with Continuously Tunable Full-Color Emission and Their Application in Ratiometric pH Sensing. Chem. Mater. 2014, 26, 3104−3112. (35) Kang, Y. F.; Fang, Y. W.; Li, T. H.; Li, W.; Yin, X. B. Nucleus-Staining with Biomolecule-Mimicking Nitrogen-Doped Carbon Dots Prepared by a Fast Neutralization Heat Strategy. Chem. Commun. 2015, 51, 16956-16959. (36) Liu, X. J.; Hu, X. J.; Xie, Z.; Chen, P.; Sun, X. M.; Yan, J.; Zhou, S. Y. In Situ Bifunctionalized Carbon Dots with Boronic Acid and Amino Groups for Ultrasensitive Dopamine Detection. Anal. Methods 2016, 8, 3236-3241. (37) Yang, L.; Jiang, W. H.; Qiu, L. P.; Jiang, X. W.; Zuo, D. Y.; Wang, D. K.; Yang, L. One Pot Synthesis of Highly Luminescent Polyethylene Glycol Anchored Carbon Dots Functionalized with a Nuclear Localization Signal Peptide for Cell Nucleus Imaging. Nanoscale 2015, 7, 6104-6113. (38) Wang, J.; Wang, C. F.; Chen, S. Amphiphilic Egg-Derived Carbon Dots: Rapid Plasma Fabrication, Pyrolysis Process, and Multicolor Printing Patterns. Angew. Chem. Int. Ed. 2012, 51, 9297-9301.


Page 40 of 54

Page 41 of 54

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


(39) Ben-Et, G.; Tatarsky, D. Application of NMR for the Determination of HLB Values of Nonionic Surfactants. J. Am. Oil. Chem. Soc. 1972, 9, 499-500. (40) Zhang, Z. P.; Tan, S. W.; Feng, S. S. Vitamin E TPGS as a Molecular Biomaterial for Drug Delivery. Biomaterials 2012, 33, 4889-4906. (41) Lu, J. Q.; Huang, Y. X.; Zhao, W. C.; Chen, Y. C.; Li, J.; Gao, X.; Venkataramanan, R.; Li, S. Design and Characterization of PEG-Derivatized Vitamin E as a Nanomicellar Formulation for Delivery of Paclitaxel. Mol. Pharmaceutics 2013, 10, 2880-2890. (42) Kabanov, A. V.; Nazarova, I. R.; Astafieva, I. V.; Batrakova, E. V.; Alakhov, V. Y.; Yaroslavov, A. A.; Kabanov, V. A. Micelle Formation and Solubilization of Fluorescent Probes in Poly (oxyethylene-b-oxypropylene-b-oxyethylene) Solutions. Macromolecules 1995, 28, 2303-2314. (43) Yu, L.; Bridgers, A.; Polli, J.; Vickers, A.; Long, S.; Roy, A.; Winnike, R.; Coffin, M. Vitamin E-TPGS Increases Absorption Flux of an HIV Protease Inhibitor by Enhancing Its Solubility and Permeability1. Pharm. Res. 1999, 16, 1812-1817. (44) Marrache, S.; Dhar, S. Engineering of Blended Nanoparticle Platform for Delivery of Mitochondria-Acting Therapeutics. Proc. Natl. Acad. Sci. USA 2012, 109,16288-16293. (45) Marrache, S.; Dhar, S. The Energy Blocker Inside the Power House: Mitochondria Targeted Delivery of 3-bromopyruvate. Chem. Sci. 2015, 6, 1832-1845. (46) Ostadhossein, F.; Pan, D. Functional Carbon Nanodots for Multiscale Imaging and Therapy. WIREs Nanomed Nanobiotechnol. 2017, 9, e1436.



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

(47) Yang, S. T.; Wang, X.; Wang, H. F.; Lu, F. S.; Luo, P. G.; Cao, Li.; Meziani, M. J.; Liu, J. H.; Liu, Y. F.; Chen, M.; Huang, Y. P.; Sun, Y. P. Carbon Dots as Nontoxic and High-Performance Fluorescence Imaging Agents[J]. J. Phys. Chem. C 2009, 113, 18110–18114. (48) Hola, K.; Zhang, Y.; Wang, Y.; Giannelis, E.P.; Zboril, R.; Rogach, A. L. Carbon Dots-Emerging Light Emitters for Bioimaging, Cancer Therapy and Optoelectronics. Nano Today 2014, 9, 590-603. (49) Batist, G.; Tulpule, A.; Sinha, B. K.; Katki, A. G.; Myers, C. E.; Cowan, K. H. Overexpression of a Novel Anionic Glutathione Transferase in Multidrug-Resistant Human Breast Cancer Cells. J. Biol. Chem. 1986, 261, 15544-15549. (50) Wang, F.; Wang, Y. C.; Dou, S.; Xiong, M. H.; Sun, T. M; Wang, J. Doxorubicin-Tethered Responsive Gold Nanoparticles Facilitate Intracellular Drug Delivery for Overcoming Multidrug Resistance in Cancer Cells. ACS Nano 2011, 5, 3679-3692. (51) Youk, H. J.; Lee, E.; Choi, M. K.; Lee, Y. J.; Chung, J. H.; Kim, S. H.; Lee, C. H.; Lim, S. J. Enhanced Anticancer Efficacy of α-tocopheryl Succinate by Conjugation with Polyethylene Glycol. J. Control. Release 2005, 107, 43-52. (52) Rappoport, J. Z. Focusing on Clathrin-Mediated Endocytosis. Biochem. J. 2008, 412, 415-423. (53) Zhao, F.; Zhao, Y.; Liu, Y.; Chang, X. L.; Chen, C. Y.; Zhao, Y. L. Cellular Uptake, Intracellular Trafficking, and Cytotoxicity of Nanomaterials. Small 2011, 7, 1322-1327.


Page 42 of 54

Page 43 of 54

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


(54) Boddapati, S. V.; D’Souza, G. G. M.; Erdogan, S.; Torchilin, V. P.; Weissig, V. Organelle-Targeted Nanocarriers: Specific Delivery of Liposomal Ceramide to Mitochondria Enhances Its Cytotoxicity in Vitro and in Vivo. Nano Lett. 2008, 8, 2559-2563. (55) Zhong, X.; Yang, K.; Dong, Z.; Yi, X.; Wang, Y.; Ge, C.; Zhao, Y.; Liu, Z. Polydopamine as a Biocompatible Multifunctional Nanocarrier for Combined Radioisotope Therapy and Chemotherapy of Cancer. Adv. Funct. Mater. 2015, 25, 7327−7336. (56) Zhang, P.; Li, J.; Ghazwani, M.; Zhao, W. C.; Huang, Y. X.; Zhang, X. L.; Venkataramanan, R.; Li, S. Effective Co-delivery of Doxorubicin and Dasatinib Using a PEG-Fmoc Nanocarrier for Combination Cancer Chemotherapy. Biomaterials 2015, 67,104-114. (57) Sobot, D.; Mura, S.; Couvreur, P. How Can Nanomedicines Overcome Cellular-Based Anticancer Drug Resistance? J. Mater. Chem. B 2016, 4, 5078-5100. (58) Wu, Q.; Yang, Z. P.; Nie, Y. Z.; Shi, Y. Q.; Fan, D. M. Multi-Drug Resistance in Cancer Chemotherapeutics: Mechanisms and Lab Approaches. Cancer Lett. 2014, 347, 159-166. (59) Wang, R. H.; Bai, J.; Deng, J.; Fang, C. J.; Chen, X. Y. TAT-Modified Gold Nanoparticle Carrier with Enhanced Anticancer Activity and Size Effect on Overcoming Multidrug Resistance. ACS Appl. Mater. Interfaces 2017, 9, 5828−5837. (60) Li, W. Q.; Wang, Z. G.; Hao, S. J.; He, H. Z.; Wan, Y.; Zhu, C. D.; Sun, L. P.; Cheng, G. Zheng, S. Y. Mitochondria-Targeting Polydopamine Nanoparticles to



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 44 of 54

Deliver Doxorubicin for Overcoming Drug Resistance. ACS Appl. Mater. Interfaces 2017, 9, 16793−16802. (61) Qu, Q. Y.; Ma, X.; Zhao, Y. L. Targeted Delivery of Doxorubicin to Mitochondria Using

Mesoporous

Silica

Nanoparticle

Nanocarriers.

Nanoscale

2015, 7,

16677-16686. (62) Kunjachan, S.; Błauż, A.; Möckel, D.; Theeka, B.; Kiesslinga, F.; Etrychc, T.; Ulbricha, K.; Blooisd, L.; Stormd, G.; Bartoszb, G.; Rychlik, B. Overcoming Cellular Multidrug Resistance Using Classical Nanomedicine Formulations. Eur. J. Pharm. Sci. 2012, 45, 421-428. (63) Wang, X.; Yang, C. C.; Zhang, Y. J.; Zhen, X.; Wu, W.; Jiang, X. Q. Delivery of Platinum (IV) Drug to Subcutaneous Tumor and Lung Metastasis Using Bradykinin-Potentiating Peptide-Decorated Chitosan Nanoparticles. Biomaterials 2014, 35, 6439-6453. (64) Hirschhaeuser, F.; Menne, H.; Dittfeld, C.; Westb, J.; Mueller-Kliesera, W.; Kunz-Schughart, Leoni. A. Multicellular Tumor Spheroids: An Underestimated Tool is Catching Up Again. J. Biotechnol. 2010, 148, 3-15. (65) Eetezadi, S.; Ekdawi, S. N.; Allen, C. The Challenges Facing Block Copolymer Micelles for Cancer Therapy: in Vivo Barriers and Clinical Translation. Adv. Drug Deliver. Rev. 2015, 91, 7-22. (66) Olive, P. L.; Durand, R. E. Drug and Radiation Resistance in Spheroids: Cell Contact and Kinetics. Cancer Metast. Rev. 1994, 13, 121-138. (67) Faute, M. A.; Laurent, L.; Ploton, D.; Poupon, M. F.; Jardillier, J. C.; Bobichon H.


Page 45 of 54

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


Distinctive Alterations of Invasiveness, Drug Resistance and Cell-Cell Organization in 3D-Cultures of MCF-7, a Human Breast Cancer Cell Line, and Its Multidrug Resistant Variant. Clin. Exp. Metastas. 2002, 19, 161-168. (68) Han, M.; Vakili, M. R.; Abyaneh, H. S.; Molavi, O.; Lai, R.; Lavasanifar, A. Mitochondrial Delivery of Doxorubicin via Triphenylphosphine Modification for Overcoming Drug Resistance in MDA-MB-435/DOX Cells. Mol. Pharmaceutics 2014, 11, 2640–2649.



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

TOC


Page 46 of 54

Page 47 of 54

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


Scheme 1. Illustrations of synthesis procedure of drug-loaded CQDs-TPGS-TPP/DOX nanomicelles. 46x7mm (600 x 600 DPI)



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 Typical TEM and HRTEM (insets) images of (A) CQDs, (B) CQDs-TPGS-TPP and (C) CQDs-TPGS nanomicelles. (D) UV-Vis spectra of CQDs, CQDs-TPGS and CQDs-TPGS-TPP (inset: the photographs of CQDs, CQDs-TPGS and CQDs-TPGS-TPP dissolved in a mixed solution of n-hexane and water (Vhex:Vwat = 1:1) under visible light and an UV lamp of 365 nm, respectively). (E) The hydrodynamic diameters distribution of CQDs-TPGS and CQDs-TPGS-TPP. (F) ζ-potentials of TPGS, CTPB, TPGS-TPP, CQDs-TPGS and CQDs-TPGS-TPP. 58x27mm (300 x 300 DPI)


Page 48 of 54

Page 49 of 54

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 2. CLSM images of MCF-7/ADR cells treated with mitochondria-targeting CQDs-TPGS-TPP or nontargeting CQDs-TPGS nanomicelles for 12 h and stained with Lysotracker Red or Mitotracker Red, respectively. Scale bar = 15 µm. 99x77mm (300 x 300 DPI)



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. Drug loading and release behavior of CQDs-TPGS-TPP nanomicelles. (A) UV-Vis absorption spectra of free DOX, CQDs-TPGS-TPP and CQDs-TPGS-TPP/DOX. (B) Release behavior of DOX from CQDs-TPGS-TPP at pH 5.0 and 7.4, respectively. 58x19mm (300 x 300 DPI)


Page 50 of 54

Page 51 of 54

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 4. Cell viabilities of (A) MCF-7 and (B) MCF-7/ADR cells treated with free DOX, CQDs-TPGS/DOX and CQDs-TPGS-TPP/DOX with various equivalent DOX concentrations, respectively. Mitochondrial membrane potential of (C) MCF-7 and (D) MCF-7/ADR cells treated with 0.5 µg/mL and 5 µg/mL equivalent free DOX, CQDs-TPGS/DOX and CQDs-TPGS-TPP/DOX nanomicelles, respectively. CLSM images of (E) MCF-7 and (F) MCF-7/ADR cells treated with free DOX and CQDs-TPGS-TPP/DOX. Large-scaled intracellular DOX accumulation is marked out with white arrows. Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01. 62x30mm (300 x 300 DPI)



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 5. (A) CLSM images of MCF-7 MCs co-incubated with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 2, 6 and 12 h, respectively. (B) Semi-quantitative mean fluorescence intensity in MCF-7 MCs cultured with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 2, 6 and 12 h, respectively. (C) Representative Z-stack images of tumor spheroids after treatment with free DOX and CQDs-TPGS-TPP/DOX nanomicelles for 12 h are obtained from starting at the top of the spheroid in 20 µm intervals. Scale bars = 200 µm. 100x59mm (300 x 300 DPI)


Page 52 of 54

Page 53 of 54

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 6. (A) Growth inhibition assay in MCF-7/ADR MCs. Representative optical images of MCs treated with culture medium, free DOX, CQDs-TPGS-TPP/DOX, CQDs-TPGS-TPP, CQDs-TPGS/DOX and CQDs-TPGS nanomicelles. Scale bars = 100 µm. (B) Growth curves of MCs statistically obtained from growth inhibition assay. **P < 0.01 (versus 1640 medium group from the 7th day). (C) Representative fluorescent images of MCs treated with free DOX and CQDs-TPGS-TPP/DOX for 7 days, respectively. 210x260mm (300 x 300 DPI)



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

TOC 61x47mm (300 x 300 DPI)


Page 54 of 54

A Trackable Mitochondria-Targeting Nanomicellar ... - ACS Publications

Recommend Documents