Doxorubicin-Loaded Unimolecular Micelle-Stabilized Gold

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Doxorubicin-Loaded Unimolecular Micelles-Stabilized Gold Nanoparticles as a Theranostic Nanoplatform for Tumor-Targeted Chemotherapy and CT Imaging Wenjing Lin, Xiaofang Zhang, Long Qian, Na Yao, Ya Pan, and Lijuan Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00810 • Publication Date (Web): 15 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Doxorubicin-Loaded Unimolecular Micelles-Stabilized Gold Nanoparticles as a Theranostic Nanoplatform for Tumor-Targeted Chemotherapy and CT Imaging

Wenjing Lin a,b, Xiaofang Zhang a, Long Qianc, Na Yao a, Ya Pan a, Lijuan Zhang a,*

a

School of Chemistry and Chemical Engineering, South China University of

Technology, Guangzhou 510640, P R China b

School of Chemical Engineering and Light Industry, Guangdong University of

Technology, Guangzhou 510006, PR China c

Department of Biology and Center for Genomics and Systems Biology, New York

University, NY 10003, USA

KEYWORDS: cancer theranostics, pH-sensitive, unimolecular micelles, gold nanoparticle, doxorubicin, DPD simulation

1 ACS Paragon Plus Environment

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ABSTRACT: Current researches are mainly trend to address the development of multi-functional nanocarriers which could precisely reach the disease sites, release drugs in a controlled-manner and act as an imaging agent for both diagnosis and targeted therapy. In this study, a pH-sensitive theranostic nanoplatform as a promising dual-functional nanovector for tumor therapy and CT imaging was developed. 21-arm star like triblock polymerof β-cyclodextrin-{poly(ε-caprolactone)-poly(2-aminoethyl methacrylate)-poly[poly(ethylene

glycol)

methyl

ether

methacrylate]}21

[β-CD-(PCL-PAEMA-PPEGMA)21] with stable unimolecular micelles formed in aqueous solution was firstly synthesized by combined ROP with ARGET ATRP techniques, and then were used as template for fabrication of gold nanoparticles (AuNPs) with uniform sizes and excellent colloidal stability in situ, followed by the encapsulation of doxorubicin (DOX) with maximum entrapment efficiency up to 60% to generate the final product β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX. Furthermore, dissipative particle dynamics (DPD) simulations revealed father details of the formation process of unimolecular micelles and the morphologies and distributions of AuNPs and DOX. Almost 80% of DOX was released in 120 h at acidic tumoral environment in in vitro drug release experiment; and both the experiments

of

in

vitro

and

in

vivo

demonstrated

the

fact

of

that

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX exhibited similar antitumor efficacy to free DOX and effective CT imaging performance. Therefore, we believe this structurally stable unimolecular micelle-based nanoplatform synergistically integrated with anticancer drug delivery and CT imaging capabilities hold a great promise for


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future cancer theranostics.

INTRODUCTION Cancer nanomedicine has received significant attention and has been extensively

exploited for applications in targeted therapy, cancer imaging and molecular diagnosis.1-3 An important prospect of cancer nanomedicine is to fabricate and realize multiple functionalities on one single nanoplatform e.g. for integrated cancer therapy and imaging. A few examples have been demonstrated, where inorganic or polymeric nanoparticles served as bi-functional theranostic platforms which outperformed traditional uni-functional systems.4-6 polymeric micelles self-assembled from amphiphilic polymers, as a new generation of anticancer drug delivery systems, have captured big interest among current smart-responsive nano-platforms because of the versatility of structure chemistry and ease of preparation.7-10 Micelles own unique amphiphilic structures where the inner core acts as a nanocontainer that accommodates hydrophobic drugs, while the outer hydrophilic shell maintains the stability of micelles and extends the in vivo circulation duration.11,12 However, previous work focused on multimolecular polymer micelles whose aggregation and thus in vivo stability is intrinsically sensitve to thermodynamic parameters such as concentrations of the block copolymers, temperature, pH, flow stress, and salt concentraitons.13,14 For example, when concentration in the blood stream fell below CMC, drug-loaded micelles could disassemble into unimers, leading to the premature burst-release of drugs. Such instability presents a hindrance to wide


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biomedical applications of multimolecular micelle-based nanoplatforms.15-17 In contrast, unimolecular micelles (micelles composed of a single copolymer) are relatively stable against changes in concentration, temperature, pH, etc, and they offer high drug loading capacities for cancer treatment and diagnosis.18-20 Unimolecular micelles based on hyperbranched polyesters (Boltorn Hx, x=30, or 40) inner core have been extensively exploited.21-24 For instance, Gong’s group reported unimolecular micelles based on a series of star polymers like H40-PCL-MPEG, H40-PLA-MPEG,

H40-PLA-PEG-OH/FA,

H40-PLA-PEG-Apt,

H40-BPLP-PEG-cRGD, and H40-PLA-PEG/OCT/TDP-A for enhanced therapeutic efficacy in dilute solution.25-30 Liu’s group developed multifunctional unimolecular micelles from a star polymer H40-PCL-P(OEGMA-Gd-FA) as a nanocarrier for synergistic targeted anticancer drug release and MR imaging.31 The adoption of H40 in many unimolecular micelle systems simplified the synthesis procedures, but the drawbacks like the irregularity of polymer structure, bad controllability, and wide molecular weight distributions are not conducive to the structural control of unimolecular micelles. Gold nanoparticles (AuNPs) possess good biocompatibility, controllable size and morphologies, desirable imaging sensitivity and can be easily synthesized for various functionalities. These features make it an attractive choice for CT contrast agent.32-36 For example, X-ray attenuation intensity of 30 nm AuNPs was 5.7-fold higher than iodine and thus produced more sensitive CT images. Shi’s group fabricated dendrimer-wrapped gold nanoparticles using the template G5.NH2-mPEG20 based on


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generation five amine-terminated poly(amidoamine) (PAMAM) dendrimers (G5.NH2) and used it in CT imaging application.37 They also developed Gd-Au-DENPs, produced from Gd modified G5.NH2-mPEG20, for CT/MR dual mode imaging applications.38 Despite these fascinating demonstrations, important issues pertaining to actual applicability are yet to be addressed. For instance, is the structure of the polymeric nanocarrier stable? How does micelle structure affect the distribution of AuNPs? What are the factors governing the size and morphology of AuNPs? Moreover, the idea of using unimolecualr micelles as a bi-functional template for dual loading of AuNPs and anticancer drugs has not been reported. To make a stable nanocarrier with well-controlled AuNP sizes and drug release properties, we designed and prepared a novel and stable pH-sensitive unimolecular micelle system as a potent dual-functional nanovector for tumor therapy and CT imaging, as illustrated in Scheme 1. The unimolecualr micelles were fabricated from the 21-arm star like polymer β-cyclodextrin-{poly(ε-caprolactone)-poly(2-aminoethyl methacrylate)-poly[poly(ethylene

glycol)

methyl

ether

methacrylate]}

[β-CD-(PCL-PAEMA-PPEGMA)21]. β-CD was chosen as the inner core by considerations of easy modification, good biocompatibility and nontoxicity. Biocompatible PCL comprises the hydrophobic micellar core that hosted anticancer drugs. The PAEMA mesosphere with amine groups had dual functions – it provided the coordination bonding for the in situ preparation of AuNPs, and at the same time acted as a pH-responsive block to trigger rapid drug release under weakly acidic tumoral conditions. Nonimmunogenic, nonantigenic and nontoxic PEGMA was


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utilized as the hydrophilic shell to offer a tight protective layer that sterically maintained the micelles during long biological circulation. DOX extensively utilized in the treatment of many cancers was employed as a model hydrophobic drug. In this study,

synthesis

of

β-CD-(PCL-PAEMA-PPEGMA)21,

preparation

and

characterization of unimolecular micelles, in situ AuNPs formation, DOX loading and release behavior, along with the antitumor activity and CT imaging efficacy of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX under in vitro and in vivo conditions were all explored. With the assistance of dissipative particle dynamics (DPD) simulation, we expected to gain a deeper understanding of the structure-property relationships from the polymer molecular structure to the micelle mesostructure and finally to macroscopic properties. This dual-functional nanovector provides a prototype and a design strategy for the development of specific and sensitive tools for early cancer treatment anddiagnosis.


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Scheme 1. Schematic representation of the fabrication of dual-functional β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX

nanoplatform

for

cancer

theranostics.

MATERIALS AND METHODS 2.1. Preparation of Unimolecular Micelles. For the preparation of blank micelles,

we applied the diafiltration method in view of our previous reports.39-41 Briefly, β-CD-(PCL-PAEMA-PPEGMA)21 (100 mg) was stirred 5 h in DMSO (20 mL); afterward, the mixed solution was dialyzed against 2 L of deionized water for 48 h using dialysis bag (MWCO=3.5 kDa) and have the deionized water refreshed for every 6 h; subsequently, the micelles received from dialysis were consequently filtered, then lyophilized and stored under condition of -20 °C. 2.2. Preparation of Unimolecular Micelle-Stabilized AuNPs. Briefly, 20 mL of 2.4 mM β-CD-(PCL-PAEMA-PPEGMA)21 ([AEMA]=4.8×10

-2

mmol) solution was

mixed with 200 µL, 400 µL or 2 mL of 24 mM HAuCl4 solution, respectively, to produce a mixture of [AEMA] and [HAuCl4] with molar ratios of 10, 5, or 1. After 30 min of this, NaBH4 in a molar ratio of 3 to HAuCl4 was injected dropwise to the mixture solution under stirring condition; then continuously stirred the reaction mixture at 25 oC in darkness for 24 h; afterward, the excess reagents were removed by centrifugation at the speed of 10,000 rpm for 10 min, and the resulting β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs

was

purified

using

three

wash-centrifugation cycles at speed of 10,000 rpm for 10 min and subsequently


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redispersed in water, and finally collected with freeze-drying technique. 2.3. Preparation of DOX-Loaded Micelles. We prepared the DOX-loaded micelles (β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX) and measured the LC and EE using UV-vis spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) according to our previous work.39-41 2.4. Measurements. The sizes and distributions (PDI) of blank micelles and β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX at 0.1 mg/mL concentration was measured bydynamic light scattering (DLS, Malvern Zetasizer Nano S, Malvern, WR, UK).

The

morphologies

of

blank

micelles

and

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs at 0.1 mg/mL concentration was observed by transmission electron microscopy (TEM, Hitachi H-7650) operating at 80 kV. Under Cu Kα (λ=1.5418 A°), as well as a current of 40 mA, an operation voltage of 40 kV, and the scanning range from 30° to 90°, x-ray diffraction spectrum (XRD, D8 ADVANCE, Bruker) was collected. 2.5. In Vitro DOX Release. Based on our previous report, We studied the release profiles of DOX from β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX in different media with pH of 5.0, 6.5 and 7.4, respectively, and then using a UV-vis spectrophotometer with wavelength of 480 nm to record the amounts of released DOX.39-41 2.6. Cytotoxicity Assay. The HepG2 cells were firstly cultured in a CO2 (5%) incubator for 3 days at 37 oC in DMEM with supplementary of 10% FBS, streptomycin (100 µg/mL), and penicillin (100 units/mL). For MTT assay, the cells at


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a density of 1 × 104 cells/well were separately incubated for 24 h and 48 h with serial dilutions of blank micelles, β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs, free DOX, and β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX solutions. Then the cells were incubated using fresh medium containing MTT solution (concentration of 5 mg/mL in PBS buffer solution) for 4 h. Afterwards, the internalized purple formazan crystals was dissolved with addition of DMSO. At last, the solution was gently agitated for 15 min and then recorded by a microplate reader at 490 nm (Multiskan Spectrum, Thermo Scientific, Vantaa, Finland). 2.7. CLSM Observation. Before the treatment, density of 4 × 105 HepG2 cells/well in DMEM (2 mL) were firstly seeded s on a 6-well plate and then cultured for 24 h under circumstances of 37°C and

5% CO2 atmosphere before treatment.

Then,

for

the

cells

were

incubated

1,

4

and

24

h

with

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX, and incubated with free DOX for 24 h at 37°C. At each pre-specified time point, the culture media were eliminated and theβ-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX that not ingested by the cells were removed by washing with PBS (3 × 1 min)Afterward, a paraformaldehyde aqueous solution with concentration of 4% (w/v) was used to have the cells fixedat ambient temperature for 30 minthen, it was

were stained with Hoechst 33324 (a PBS

solution of 5 mg/mL) for 15 min under temperature of 37 °C. Finally, the slides were thoroughly washed with PBS (3 × 2 min) and observed by the confocal laser scanning microscope (CLSM, Zeiss, LSM 510, Oberkochen, Germany). 2.8. In Vivo Antitumor Efficacy. All animal experiments have been approved by


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Institutional Animal Care and Use Committee of South China University of Technology and conducted in compliance to legal protocols. Nod-scid mice (13-16 g) in similar tumor size (180-230 mm3) supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd., were randomly separated into three groups (n=3) and fed under SPF conditions in GuangZhou Jennio Biotech Co., Ltd. The HepG2 tumor models were established by subcutaneous injection of 1×106 HepG2 cells into the right back of mouse. β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX and free DOX were administered by intravenous injection in 2 × 4 mg/kg equivalent DOX dosage with negative control of PBS. In the experiments, the mice were weighed for every 3 days, and have tumor volumes (V) calculated in view of the formula as V = 1/2 ab2, wherein the a and b means the longest diameter and shortest diameter of the tumor, respectively.42,43 2.9. Histological Examination. At day 18, tumor tissues of portions of mice were collected in 4% paraformaldehyde and embedded into paraffin. Then sections of tissue prepared and stained by hematoxylin and eosin (H&E, Beyotime Institute of Biotechnology, Jiangsu, China) were employed for histologic examination, by which the tissue sections wre examined with a fluorescence microscope (Carl Zeiss, Zeiss Axio Scope A1, Germany). 2.10. X-Ray Attenuation Measurements. The method as reported in the work by Shi’s team was applied.36,44 β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX or Omnipaque at a series of concentrations (0.01-0.08 M, and 0.1 M) were fabricated using Eppendorf tubes (0.5 mL) and then transferred to a self-designed scanning


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holder. Afterward the CT scans were conducted using an Inveon small-animal PET/computed tomography (CT) scanner (Siemens, Germany) with parameter of 500 mA, 100 kV, and a slice thickness of 0.08 mm. The contrast enhancement of which was

measured

in

hounsfield

units

(HU)

for

each

concentration

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX or Omnipaque was obtained with the digital CT images loaded in a standard display program choosing a uniform area of interest of each sample. 2.11. In Vitro CT Imaging of HepG2. HepG2 cells were seeded on 5-mL cell culture flask at a density of 1 × 106 cells/well and grew for 24 h. Then the old medium was

replaced

with

5

mL

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX

fresh or

one

Omnipaque

containing at

different

concentrations (0µM, 100µM, 400µM, and 800 µM). After 4 h of incubation, the cells were then sequentially trypsinized, centrifuged, resuspended in PBS (100 µL), and placed into Eppendorf tubes (1.5-mL) after that. At last, the cells were scanned by the same CT imaging system and procedures described in section of 2.10 . 2.12. In Vivo CT Imaging. Approximate 1 × 106 HepG2 cells were injected on the right back of each nod-scid mouse, then raised till tumor volume reached about 1.5 cm3 after 6-7 weeks. Each mouse was then anesthetized using 1% pelltobarbitalum natricum

and

intravenously

injected

with

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX or Omnipaque immersed in PBS ([Au] = 0.1 M, 100 µL). After that, the tumor-bearing mice were canned before injection and at 1, 2, and 4 h post-injection by the aforementioned CT imaging system


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in section 2.10. 2.13. DPD Simulation. DPD simulations based on coarse-graine models were applied to obtain a deeper exploration on the formation and microstructure of unimolecular micelles, and the detailed distributions and shapes of AuNPs and DOX on unimolecular micelles. As shown in Figure S7, the polymers of were seperated into six types as β-CD (β-cyclodextrin)-grey, CL-orange, MAA1-rose red, MAA2-light green, AE-pink and PEG-green. The molecular structure of DOX (Color: blue) was also seperated into three types of beads as D1, D2 and D3. As well, five water molecules, denoted by W (Color: black), were represented as one bead; the mass and radius of β-CD were 1135 amu and 4.36 Å, and 105 amu and 3.47 Å on average for other beads; a small cluster with unit cell crystal structure that contains four Au atoms, was considered as a gold bead (Color: wine). The crystal with a lattice length of 4.0783 Å possesses a space group of no. 225. The interaction parameters were computed in light of our previous method (Table S2).45,46 Then, within the Mesocite module of Materials Studio 5.5 we performed DPD simulations Furthermore, a cubic simulation box with size of 37×37×37 rc3 and periodic boundary conditions was applied in all directions, for which the total simulation steps were 100,000 andthe integration time step was 0.05 ns. 2.14. Statistical Analysis. A two-sample Student’s t-test with unequal variance was employed for the implementation statistical analysis of which, a p < 0.05 was defined as statistically significant.


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RESULTS AND DISCUSSION 3.1. Synthesis of β-CD-(PCL-PAEMA-PPEGMA)21 and Formation of

Unimolecular Micelles. In the current study, β-CD-(PCL-PAEMA-PPEGMA)21 (Mn,GPC=77284 g/mol, PDI=1.94, Mn,NMR=218002 g/mol) was generally synthesized via ring opening polymerization (ROP) along with activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) techniques, which was followed by hydrolysis of the tBoc groups. The routes and detailed methods of such synthesis were described in Supporting Information (Figure S3). The molecular weight, chemical structure, and composition of β-CD-(PCL-PAEMA-PPEGMA)21 and its precursors were fully investigated by GPC, FT-IR and 1H NMR spectra (Figure 1A,B, Figure S4 and Table S1). As measured by GPC, the molecular weight of the resultant polymers shifted rightward as blocks were successively added to the polymeric chain, and retracted slightly after hydrolysis (Figure 1A). And the FT-IR spectra showed characteristic vibrations of all polymer groups (Figure 1B), of which the most prominent were the stretching and the bending vibrations of -NH2 in β-CD-(PCL-PAEMA-PPEGMA)21 (at 3300~3500 cm-1 and 1625 cm-1, respectively), following by the removal of the tBoc group (1.46 ppm disappeared in Figuer S4E) which completed the synthesis process. All above measurements jointly confirmed that

well-defined

β-CD-(PCL-PAEMA-PPEGMA)21

with

well-defined

PCL/PAEMA/PPEGMA contents had been successfully synthesized. Dispalyed in Figure S5, as the increase of polymer concentration,the fluorescence emission intensity of Nile red increased even at extremely rather low polymer


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concentrations. We didn’t observe sudden changes in fluorescence intensity of the entirewhole range of polymer concentration, demonstrating the absence of a CMC in distilled water and the presence of unimolecular micelles. Compared to multimolecular micelles, such a structure is thermodynamically stable. The size of the unimolecular micelles of 10.5 nm and 18 nm, respectively, as determined by TEM and DLS measurements along with narrow distributions indicating uniformity (Figure 1C). The slightly lower values from TEM were due to the collapse of the micelle corona after sample drying, and however the slightly higher values from DLS were owing to scattering of the highly hydrated shell in micelles solution.47,48 These polymer micelles remained stable (18-19 nm) in water, PBS, and FBS solution without agglomeration for as long as one month (Figuer S6). Based on coarse-grain models, using the same concentration as in the actual experiment (polymer : water = 15.33 % : 84.67 %), DPD simulation was performed to simulate the process of micelle formation (Figure 1D, Figure S7 and Table S2). At the initial state, eight polymers were randomly placed in water (water molecules were hidden for clarity in Figure 1D), and eight discrete micelles were observed at the equilibrium state. With varying polymer concentrations from 10 % to 20 %, and the inital number of polymers being 6 or 20, the same numbers of micelles were observed at equilibrium, indicative of the formation of unimolecular micelles within a relatively wide range of reactant concentrations (Figure 1E&F). During the micelle formation process, the radius of gyration curve of PEGMA increased gradually before it plateaued, while the radius of gyration curve of the middle PAEMA layer remained


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unchanged (Figure 1D), suggesting that the micelles grew primarily by extension of hydrophilic PEGMA shell rather than PAEMA middle layer. Both experiment and DPD simulation revealed that in aqueous solution β-CD-(PCL-PAEMA-PPEGMA)21 existed as unimolecular micelles within a certain range of polymer concentrations.

Figure 1. (A) GPC traces and (B) FT IR spectra of β-CD-(PCL-OH)21 (a), β-CD-(PCL-Br)21 (b), β-CD-(PCL-PtBAM)21 (c), β-CD-(PCL-PtBAM-PPEGMA)21 (d), and β-CD-(PCL-PAEMA-PPEGMA)21 (e). (C) DLS plot and TEM image of β-CD-(PCL-PAEMA-PPEGMA)21 unimolecular micelles. (D) Radius of gyration of PEG and AE of β-CD-(PCL-PAEMA-PPEGMA)21 (polymer volume(%):15.33%) at 15 ACS Paragon Plus Environment

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different

simulation

timepoints.

(E&F)

Initial

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and

equilibrium

states

of

β-CD-(PCL-PAEMA-PPEGMA)21 at concentrations of 10%(E) and 20%(F).

3.2.

Preparation

and

Characterization

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs. Owing to complexation between Au3+ and the N atom, the amine group of PAEMA reduced Au3+ to zerovalent gold atom in situ by using reducing agents NaBH4. Subsequently, the gold atoms combined mutually to form stable AuNPs. The maximum wavelength (λmax) of AuNPs shown in UV-vis spectra increased with increasing concentrations of HAuCl4 (Figure 2A). This result is further confirmed by direct AuNP size measurements under TEM (mean sizes 2.2 nm, 3.5 nm and 5.7 nm at [AEMA]:[HAuCl4] with molar ratios of 10, 5, or 1, respectively (Figure 2B, and Figure S8). Using the same concentrations as in the actual experiment (volume fraction of unimolecular micelles 15.33 % and Au beads 0.23%, 0.45%, and 2.24%), the density profiles of β-CD-(PCL-PAEMA-PPEGMA)21 micelles were tracked in simulations, predicting that AuNPs tended to distribute in the PAEMA area. The peak values and area of the AuNP density profiles increased with an increasing number of feeding Au beads (Figure 2D). Typical simulated section views of the PAEMA area and the embedded growing AuNPs were given in Figure 2E. When the amount of HAuCl4 in aqueous solution increased, the distance between the Au beads decreased and more aggregated AuNPs appeared, which finallyresulted in the formation of large AuNPs. Herein, stability of AuNPs relies on the thermodynamic balance between the steric


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stabilization effect of the unimolecular micelles and the agglomeration effect of the AuNPs.49-51 Aggregation was well prevented at low HAuCl4 concentrations. At high concentrations of HAuCl4, steric stabilization of the nanocarrier micelles became insufficient to suppress the inter-collision of AuNPs, resulting in the formation of larger AuNPs. DPD simulation result also revealed that almost all the Au beads were entrapped into the micelles finally even at HAuCl4 concentration was highest (molar ratio of [AEMA]: [HAuCl4] = 1, Figure S9). As could be found in Figure 2C, the peaks of x-ray diffraction spectrum

at 38.5°,

44.8°, 64.2° and 78.0° corresponded to (111), (200), (220) and (311) crystal faces, respectively, which confirmed the face-centered-cubic (fcc) crystal structure of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs.38

Figure 2. (A) UV-vis absorption spectra of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs 17 ACS Paragon Plus Environment

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at different [AEMA]:[HAuCl4] molar ratios. (B) TEM image and (C) XRD plot of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs at [AEMA]:[HAuCl4] molar ratio of 5. (D) Density profiles of different beads and (E) cross-section views (The PEGMA shell and

water

molecules

were

hidden

for

clarity)

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs at different [AEMA]:[HAuCl4] molar ratios. 3.3. Preparation of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX and in Vitro Release of DOX. DOX, widely in the remedy of various cancers, was used in this

study

as

a

model

hydrophobic

drug

and

wrapped

into

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs micelles with suitable feeding weight ratios (DOX to micelles: 10-50 mg to 100 mg). With increasing amounts of the feeding DOX, DLS measurements in Figure 3A showed only a mild growth of micelle size. The size of the loaded micelles at the highest DOX feeding weight ratio was 20-35 nm with a narrow distribution (PDI 0.12-0.35). The growth in micelle size might be a result of the hydrophobic interactions between DOX and the PCL chains that promote the stretching of the otherwise collapsed PCL chains. Meanwhile, both DOX loading content (LC, 2.1-17.1 wt%) and entrapment efficiency (EE, 21.1-62.5%) grew with increasing amounts of feeding DOX. However, the LC growth was slowed down, and for the EE, it even decreased due to the limited capacity of solubilizing DOX in the miceller core, only except at 30% feeding weight ratio of DOX. The feeding weight ratio of 30/100, at where EE reached its maximum, was utilized in later in vitro and in vivo experiments.


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To gain further insight on the DOX loading properties, DPD simulation was carried out using the same experimental systems with DOX/micelle weight ratios of 10/100, 30/100, and 50/100, respectively. The volume fraction of the unimolecular micelles and Au beads were fixed at 15.33 % and 0.23%, and those of the DOX beads were 0.65%, 1.95%, and 3.26%, respectively. At increasing feeding DOX levels, both the peak value and the area of the DOX density profiles increased (Figure 3B & C). However, we observed a gradual shift of DOX distribution from the PAEMA area (at low feeding DOX) to the inner core of the micelles (at high feeding DOX). Although the interaction of DOX-CL is more favorable than DOX-AE, our findings suggests that DOX was first loaded in the mesosphere, and the inner miceller structures seemed to have imposed significant spatial resistance to slow down its diffusion into the inner core. In vitro drug release experiments were performed at 37 oC under physiological conditions of pH 7.4, pH 6.5 or 5.0, as the representative pH values in the intracellular medium, since anti-cancer drugs are most often delivered at where. We observed a sharp increase of DOX release rates through decreasing pH from 7.4 to 5.0 (Figure 3D). At pH 7.4, the release rate of DOX was at relatively low level – only 25% of DOX was released after 120 h; and its side chains were estimated to near 100% deprotonated given a pKa of 7.06 for copolymer (Figure S10). However, because of the protonation of the amino group of PAEMA and DOX at low pH, approximate 46% and 81% of DOX was released after 120 h at pH 6.5 and 5.0, respectively.52, Therefore,

it

is

concluded

that


the

53

pH-sensitive

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β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX could effectively reduce DOX leakage during long circulation in the blood stream (pH 7.4), and yet release a sufficient amount of DOX once the micelles are internalized into the acidic endosomes and lysosomes, to ensure cancer-targeted DOX delivery while shortening non-specific systemic toxicity.

Figure 3. (A) LC, EE, and sizes of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX as

a

function

of

feeding

weight

ratios

of

DOX

to

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs. (B) Density profiles of different beads and (C) cross-section views of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX micelles at

different

feeding

weight

ratios


of

DOX

to

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β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs. (D) In vitro drug release profiles of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX at pH 7.4, pH 6.5 or pH 5.0. 3.4. In Vitro Antitumor Efficacy. In an effort to test the antitumor efficacy of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX in vitro, cell viability experiment was conducted against HepG2 cells. No significant cytotoxicity and morphological changes were observed after 48 h of incubation with blank micelles or β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs, i.e. 90% viability even at the highest concentration (400 µg/mL), confirming that the blank micelles were biocompatible with the HepG2 cell line (Figure 4A, and Figure S11). On the contrary, cell viability was

dramatically

slowed

down

in

free

DOX

and

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX micelles solutions (Figure 4B). However, the cytotoxicity of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX was lower than that of free DOXbecause of the extra time needed for DOX-release from the micelles and a lower nuclear uptake rate at low DOX concentrations (1-15 µg/mL). Under a high concentration (20 µg/mL) after 48 h incubation, they both exhibited similar antitumor activities, with at least 77% reduction in cell viability. The fact that β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX induced substantial cytotoxicity proved that enough DOX was released (>70% after 48h) and had entered the nucleus even though the micelles enters cells relatively slowly through receptor-mediated endocytosis. 54,55 The IC50 values of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX and free DOX were 2.4 µg/mL and 1.7 µg/mL, respectively, indicating that β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX could kill cells efficiently, and the


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carrier micelles did not compromise DOX’s potency. CLSM was employed to track the location of DOX (red fluorescence) in cells whose nuclei were shown by Hoechst 33324 (blue fluorescence) in order to further prove the cellular uptake behavior of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX. Within

the

first

4

h

of

incubation,

the

red

fluorescence

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX was primarily observed in the cytoplasm with just a petty part in the nuclei. With a further 24 h of incubation, DOX fully localized in the nuclei, and showed a signal intensity comparable to free DOX (Figure 4C). Consistent with MTT assay, cells treated with free DOX showed a significantly

higher

cellular

uptake

rate

than

that

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX since free DOX enters cells through passive diffusion while β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX uptake relies on receptor-mediated endocytosis.54,55


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Biomacromolecules

Figure

4.

(A)

Cell

viability

of

blank

micelles

and

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs after 48 h incubation as a function of polymer concentrations measured by MTT assay against HepG2 cells (n=6). (B)Cell viability of free DOX and β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX after 24 h/48 h incubation as a function of concentrations of DOX measured by MTT assay against HepG2 cells (n=6). (C) CLSM images of HepG2 cells incubated with 23 ACS Paragon Plus Environment

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β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX at different time ((a) 1 h, (b) 4 h, (c) 24 h), and free DOX (d) for 24 h at 37 ℃ (Red: DOX; Blue: Hochest 33342, scale bar 25 µm, n=3). 3.5. In Vivo Antitumor Efficacy. To evaluate whether the decreased cell viability and enhanced biodistribution could result in the improvement of therapeutic efficacy, the antitumor efficacy was carried out in mice bearing HepG2 tumor. The body weight and tumor size of HepG2 xenografted mice were tracked for 18 days after treatment with β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX, free DOX or PBS as a control. All treatments were well tolerated as no body weight loss was observed for all animals (Figure 5A). The body weight of PBS-treated mice showed a relatively quick increase, which might be partially owing to the fast growth of tumor. A slight body weight increase of DOX-containing groups were also observed with time, indicating

the

drug

delivery

system

of

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX is safe to the body. The tumor volumes of mice in the PBS control group increased steadily throughout the experiment, while since the good antitumor efficacy of DOX, the tumor growth in the two DOX groups was suppressed (Figure 5B). By Day 18, the tumor size of PBS control, β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX and free DOX were 429 ± 67.6 mm3, 207 ± 56.7 mm3, and 204 ± 4.6 mm3, respectively. A significant (p < 0.05) decrease in tumor size was obtainved for the DOX groups in comparison with the PBS group, possibly due to the chemotherapeutic effect triggered by the multi-advantages including sustained and controlled release, higher cellular uptake


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along with enhanced tumor accumulation. The excised tumor tissues were examined histologically with hematoxylin-eosin staining (Figure 5C). Tumors treated with PBS typically were composed of some necrotic regions and densely packed tumor cells because of their rapid growth. In comparison, tumors treated with β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX or free DOX displayed extensive loss of cancer cells and condensation/fragmentation of the cellular nuclei, indicative of DOX-induced cellular apoptosis. To sum up, all these results suggest that β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX has great potential to serve as an effective antitumor drug nanocarrier with low systemic toxicity.

Figure 5. (A) Body weight and (B) tumor volumes of mice as at different time points (days after treatment). (C) Typical histologic images of tumor tissue sections (scale bar, 50 µm, n=3). *p < 0.05. 3.6. In Vitro and in Vivo CT Imaging Efficacy. Owing to its high electron density


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Page 26 of 39

andatomic number , gold has theoretically a higher coefficient of X-ray absorption than commercial contrast agent iodine that has been extensively utilized for the purposes of CT imaging. To test imaging capability of our nanocarrier, we investigated

the

X-ray

attenuation

property


of and

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX incubated with HepG2 cells, and the iodine-based Omnipaque was used as control (Figure 6A,B). The data generated revealed that the intensity of X-ray attenuation of all three systems increased with their concentration. However, at the same concentrations, AuNPs prepared by our group had higher X-ray attenuation intensity than Omnipaque, and as a result led to a higher CT contrast ability to produce higher sensitive CT imaging. Using a xenoplanted

HepG2

tumor

model,

the

possibility

to

employ

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX for CT imaging in vivo was then explored.


were

intravenously

injected to mice with HepG2 tumor implant. In CT images for these mice, high density bone tissues were observed to be white owing to stronger X-ray absorption, while soft tissue including blood vessels and muscles were dark orgray. As it was difficult to visually distinguish CT image brightness of the tumor site between β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX and Omnipaque (Figure 6C, Figure S12), the CT signal intensity was quantified with the manufacturer’s standard display program. In Figure 6D, tumor CT values gradually stepped up after injection, and the tumor CT values of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX were much


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higher than those of Omnipaque. When compared to pre injection, the average magnitude

of

CT

values

improvement

was

much

greater

in

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (32, 54, 65, or 71% increase after 0.5 1, 2 or 4 h post injection) than in Omnipaque systems (only 18, 26, 36, or 43% increase). The high-contrasting property of AuNPs, combined with the targeted delivery nanoplatform, makes our system particularly suitable for applications in disease site CT imaging.

Figure

6.

(A)

CT

values

of

Omnipaque

(1)

and

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (2) at different concentrations. (B) CT values of Omnipaque (1) and β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (2)


Biomacromolecules

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treated with the HepG2 cells at different concentrations (n=3). (C) CT images of the xenografted HepG2 tumor model before/after intravenous injection of Omnipaque (a) or β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (b) as a function of time. The stars indicate the tumor area (n=3). (D) CT values of the tumor region before/after intravenous

injection

of

Omnipaque

or

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (n=3). *p < 0.05.

CONCLUSIONS

We have developed the β-CD-(PCL-PAEMA-PPEGMA)21 unimolecular micelle system and demonstrated its promising potential as a theranostic nanovector. Our nanosystem could actively dual-load CT contrast agent AuNPs and the hydrophobic anticancer drug DOX, simultaneously achieving high CT imaging and antitumor efficacies

under

in

vitro

and

in

vivo

acidic

tumor

conditions.

The

β-CD-(PCL-PAEMA-PPEGMA)21 unimelecular micelles have the following distinct features: (i) The 21-arm star polymer β-CD-(PCL-PAEMA-PPEGMA)21 was readily synthesized and characterized via combined ROP and ARGET ATRP followed by hydrolysis. (ii) β-CD-(PCL-PAEMA-PPEGMA)21 formed well-defined and stable spherical unimolecular micelles in aqueous solution, which served as template for in situ fabrication of AuNPs with uniform sizes and excellent colloidal stability under steric stabilization effect. (iii) DPD simulations indicated that both AuNPs and DOX preferentially distributed in the PAEMA area, while at a high concentration and with time,

DOX

penetrated

to

the

inner

PCL

core.

(iv)

The

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs encapsulated DOX with an EE range of 21.1%-62.5% and rapidly released DOX (80% after 120 h) in acidic tumor conditions.


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(v)

Both

the

experiments

of

in

vitro

and

in

vivo

identified

that

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX exhibited similar antitumor activities to free DOX and had effective CT imaging properties. Herein, DPD simulations not only reproduced the experimental observations but also provided additional information on the intrinsic properties of the micelle system in terms of mesoscopic and microscopic scales. In conclusion, we hope that this unimolecular micelle-based dual-functional nanoplatform provides a promising tool for future cancer theranostic applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Detailed methods and additional results (Table S1, Figure S1-S4) on the synthesis and

characterization

of

β-CD-(PCL-PAEMA-PPEGMA)21,

Intensity

of

the

fluorescence emission of Nile red at 620 nm (Figure S5), DLS plots of unimolecular micelles after one month (Figure S6), coarse grain models and interaction parameters of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (Table S2, Figure S7), TEM images of β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs at [AEMA]:[HAuCl4] molar ratios of 10 and 1 (Figure S8), Morphologies of the formation of AuNPs at the [AEMA]: [HAuCl4] molar ratio of 1 (Figure S9), Titration curves (Figure S10), microscopic images of HepG2 cells (Figure S11), and CT images of the xenografted HepG2

tumor

model

of

Omnipaque

β-CD-(PCL-PAEMA-PPEGMA)21/AuNPs/DOX (b) (Figure S12) (PDF) 29 ACS Paragon Plus Environment

(a)

or

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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

AUTHOR INFORMATION Corresponding Author Li Juan Zhang, E-mail: [email protected]. Telephone/Fax: +86-20-87112046.

ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (No. 21776101 &No.91434125), Team Project of Natural Science Foundation of Guangdong Province, China (No.S2011030001366) were gratefully acknowledged. The authors would also like to thank Prof. Jufang Wang in School of Bioscience & Bioengineering of South China University of Technology for her help with the cell experiments. The authors would also like to thank Fenfen Guo in Sun Yat-sen University for her help with training course of animal experiments and Qingqiang Tu in Sun Yat-sen University for his help with CT measurements.

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for Table of Contents Only

Doxorubicin-Loaded Unimolecular Micelles-Stabilized Gold Nanoparticles as a Theranostic Nanoplatform for Tumor-Targeted Chemotherapy and CT Imaging

Wenjing Lin a,b, Xiaofang Zhang a, Long Qianc, Na Yao a, Ya Pan a, Lijuan Zhang a,*


Doxorubicin-Loaded Unimolecular Micelle-Stabilized Gold

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