Optimizing Hydrophobic Groups in Amphiphiles to Induce Gold

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Optimizing Hydrophobic Groups in Amphiphiles to Induce Gold Nanoparticle Complex Vesicles for Stability Regulation Jun Fu, and Liyan Qiu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02745 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 7, 2017

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Optimizing Hydrophobic Groups in Amphiphiles to

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Induce Gold Nanoparticle Complex Vesicles for

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Stability Regulation

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Jun Fu‡ and Liyan Qiu*,†

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†

Ministry of Education (MOE) Key Laboratory of Macromolecular Synthesis and

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Functionalization, Department of Polymer Science and Engineering, Zhejiang University, 38

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Zheda Road, Hangzhou 310027, China

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‡

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

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* Corresponding author. Tel.: +86 571 87952306 E-mail address: [email protected]

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ABSTRACT: Polymeric graft polyphosphazene containing 4-aminobenzoic acid

2

diethylaminoethyl ester (DEAAB) as hydrophobic side groups was rationally

3

designed and named PDEP. PDEP can self-assemble into nano-vesicle in water. More

4

importantly, when compared with the amphiphile PEP copolymer containing benzene

5

rings and the amphiphile PDP copolymer containing tertiary amino groups, the

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co-existence of benzene and tertiary amino groups in PDEP enabled it to effectively

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load water-soluble small molecule doxorubicin hydrochloride (DOX·HCl) into the

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vesicle and efficiently induce in situ transformation of gold tetrachloroaurate (HAuCl4)

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to gold nanoparticles (AuNPs) as both a reductant and a stabilizer. By optimizing the

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reduction conditions, such as the temperature, reaction time and hydrophobic group in

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polymer/HAuCl4 molar ratio, the AuNP complex PDEP vesicles significantly

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inhibited the DOX·HCl burst release at pH 7.4 while displaying a fast release

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responsive to pH 5.5.

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KEYWORDS: gold nanoparticles, vesicles, drug delivery, polyphosphazenes, stability

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Recently, nano-scaled polymersomes have become a novel research direction to load

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water-soluble substances, profiting from their characteristic core-shell architectures,

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which are similar to those of liposomes but exhibit better chemical stability and

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functionalization.1,

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easily they self-assemble into polymersomes just by regulating the ratio of the

7

hydrophilic chains to hydrophobic groups.2 However, the burst drug release is

8

difficult to avoid, especially at high levels of drug loading during the circulation

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period. For instance, Couffin-Hoarau, A. C. and his co-workers synthesized an

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amphiphilic poly(organophosphazene) vesicle to load trisodium 8-hydroxypyrene

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trisulfonate (HPTS).3 It was obvious that the HPTS release percent was approximately

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32 % in the first 30 min in phosphate buffer solution (PBS) (pH 7.4). Coincidentally,

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the study reported by Laurencin, C. T.4 addressed the same issue. They co-substituted

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the open-loop hexachlorocyclotriphosphazene by imidazole and methylphenoxy to

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load 14C-lableled bovine serum albumin (BSA). The BSA displayed a burst release of

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more than 25 % of the protein in the first 4 h. For the fellow-up developments of

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polyphosphazene vesicles, such formulations required adjustment to minimize the

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drug leakage at pH 7.4; otherwise, the drug would be stuck in the plasma before being

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delivered to the tissue target. In fact, burst drug release occurs frequently in many

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other vesicle systems, which would weaken the in vivo treatment effect.5-7

INTRODUCTION

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Notably, amphiphilic graft polyphosphazenes displayed how

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Over the last decade, metal nanoparticles have attracted much attention for use in

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the biomedical field because of their interesting shape- and size-dependent physical

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and chemical properties.8 Gold nanoparticle (AuNP) is undoubtedly one of the most

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remarkable members. They can be synthesized in many ways, but the most common

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method is the reduction of commercial gold tetrachloroaurate (HAuCl4) with

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appropriate reductants. To prevent AuNPs from aggregating, surfactant molecules are

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indispensable. For example, Leff, D. V. and his co-workers9 reduced HAuCl4 by

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NaBH4 in the presence of n-dodecyl mercaptan to achieve a stable colloid of

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thiol-capped AuNPs. Therefore, a system which can serve both as reductant and 3


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stabilizer to AuNPs is satisfactory to all researchers. In our recent work, we reported a

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new method to inhibit drug leakage before they arrived at tumor site by taking

3

advantage of AuNPs as inorganic crosslinkers to improve the compactness of

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hydrophobic membrane of polyphosphazene PNP vesicles.10 In the pre-formed blank

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or drug-loaded polymersome solution, HAuCl4 was reduced by NaBH4 to form

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AuNPs, which were trapped among hydrophobic lamella through the conjugation with

7

the tertiary amine groups of PNP. It should be noted that PNP we synthesized only

8

acted as a stabilizer to AuNPs while NaBH4 was a reductant in that case. Herein,

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however, we are pleasant to declare that HAuCl4 could be directly transferred to

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AuNPs after mixed with certain polymersome solution under the optimal conditions,

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that is, some amphiphilic polyphosphazenes with specific hydrophobic groups could

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play a role both as a reductant and as a stabilizing agent simultaneously in the process

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of in situ AuNPs formation after self-assembled into nano-scaled polymersomes. In

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addition, there was almost no aggregation of AuNPs beyond the polymersome

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membrane, indicating quite strong capability of these polymers to generate and

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stabilize AuNPs.

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Thereby, to illustrate the importance of screening hydrophobic groups, we

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constructed three different types of polyphosphazene vesicles with hydrophilic

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amino-terminal PEG2000 (NH2-PEG2000) groups and different hydrophobic groups of

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ethyl

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4-aminobenzoic acid diethylaminoethyl ester (DEAAB), namely PEP, PDP and

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PDEP, respectively. A minor but essential difference existed among the three

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hydrophobic groups. EAB contained a benzene ring but no tertiary amine group, DPA

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possessed tertiary amine group but no benzene ring, and both a benzene ring and

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tertiary amine group were present in DEAAB. We also investigated the factors that

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might affect the reactions between these hydrophobic groups in the polyphosphazenes

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and AuCl4-. The formation of polymer-AuNP complexes was evidenced by UV-Vis

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spectroscopy, transmission electron microscope (TEM), and X-ray diffraction (XRD).

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The mechanism between polymer and AuNPs capped in the hydrophobic membrane

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of vesicles was determined by fourier transform infrared spectroscopy (FTIR). The

p-aminobenzoate

(EAB),

N,N-diisopropylethylenediamine

(DPA)

and

4


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water-soluble

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(DOX·HCl) was used as a model drug, and the in vitro drug release was researched.

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Moreover, the drug loading capability of these three polyphosphazene vesicles was

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explored, which was a predominant issue for the drug delivery system and made the

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drug controlled release more significant. With this study, we expected to find some

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valuable strategies to improve the stability of polymersomes so the payload can be

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effectively maintained until it reaches the tumor tissues via the enhanced permeability

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and retention (EPR) effect. Based on this study, we expected to confirm some

9

valuable strategies to induce AuNPs complex vesicles for stability regulation.

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small-molecule

chemotherapeutics

doxorubicin

hydrochloride

MATERIALS AND METHODS

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Materials. Hexachlorocyclotriphosphazene (HCCP) (Acros Organics, Belgium)

12

was sublimated at 80-90 °C under a nitrogen atmosphere and ring-opening

13

polymerized at 250 °C to obtain poly(dichlorophosphazene).1,11-12 PEG2000 (Fluka,

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Steinheim, Germany) was azeotropically dehydrated with dry toluene and

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amino-terminal modified to NH2-PEG2000 according to our previous work.1 EAB and

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DPA were all purchased from Sigma-Aldrich (St. Louis, MO, USA). DEAAB was

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obtained from 4-aminobenzoic acid diethylaminoethyl ester hydrochloride (Acros

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Organics, Belgium) through desalination with trimethylamine (TEA). DOX·HCl was

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kindly supplied from Haikou Manfangyuan Chemical Company (Haikou, China).

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Chloroauric acid hydrate (HAuCl4·4H2O) and all other organic reagents were

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laboratory reagent grade and purchased from Sinopharm Chemical Reagent Co. Ltd

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(Shanghai, China), but again, the moisture in TEA and petroleum were removed by

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distillation and the water in NH2-PEG2000 was removed by toluene through azeotropic

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distillation because hexachlorocyclotriphosphazene was water-funk during the

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nucleophilic replacement process.

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Synthesis and Characterization of Polyphosphazenes. PEP was prepared under

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nitrogen conditions by a sequential nucleophilic substitution of NH2-PEG2000 and

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EAB onto a poly(dichlorophosphazene) backbone. Briefly, 1.60 g (0.8 mM)

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NH2-PEG2000 was dehydrated and dissolved in 30 mL dry toluene with equal molar 5


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dry TEA. Then, the solution was added dropwise into 20 mL toluene solution

2

containing 0.5 g poly(dichlorophosphazene) and stirred overnight at 30 °C. Morrow,

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to elevate the temperature of the reaction system to 50 °C and an excess of EAB (1.31

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g, 7.9 mM) in 30 mL dry toluene with equal molar TEA, was added slowly into the

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mixture. After 24 h stirring, the product was precipitated in cold diethyl ether and

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dialyzed against purified water for 24 h with frequent water changing. PDP and PDEP

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were achieved in the same way except for the different feed ratio of the hydrophilic

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chain NH2-PEG2000 and hydrophobic side groups DPA or DEAAB. For PDP, the

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ratios were 1.80 g (0.9 mM) NH2-PEG2000 and 1.13 g (7.8 mM) DPA. For PDEP, the

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reactants mass were 1.25 g (0.6 mM) NH2-PEG2000 and 1.59 g (8.1 mM) DEAAB.

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Copolymer chemical compositions were analyzed by FTIR (NICOLET 6700,

12

Nicolet, USA). 1H NMR (DMX-500, Bruker, Germany) in CDCl3 at 400 MHZ was

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carried out to back up the copolymers’ composition results by FTIR spectra, and the

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molar fraction of NH2-PEG2000 and EAB/DPA/DEAAB on each polymer unit was

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also evaluated by 1H NMR spectra. Furthermore, the molecular weights of PEP, PDP

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and PDEP and the polydispersity Mw/Mn of the copolymers were GPC (D40, TSK

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CO., Kyoto, Japan) with N,N-dimethylformamide (DMF) as an eluent with 1

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mL·min-1 flow rate at room temperature, relative to polystyrene standards.

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Preparation of Polyphosphazenes Particles and Morphological Examination.

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Polyphosphazene nanoparticles were prepared via a dialysis method. Briefly, PEP,

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PDP and PDEP polymers were separately dissolved in DMF at 95 mg·mL-1. Then,

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equal volumes of MilliQ water were added slowly with gentle stirring at room

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temperature. After 5 min, the mixture solutions were transferred into dialysis bags

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(8-14 kDa molecular weight cut-off (MWCO)) and dialyzed against pure water for 5 h

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with fresh water changing every hour. The obtained solutions were stored for

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morphological examination.

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TEM (JEM-1200EX, Japan) was applied to investigate the morphology of the

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polyphosphazene particles. Each sample was obtained through dipping a

29

carbon-coated copper grid into the particle solution diluted into 5 mg·mL-1 for 30 min

30

and dried at room temperature. 6


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The size and zeta potential of these polyphosphazene nanoparticles were analyzed

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by DLS (Malvern Nano-S90, UK). The concentrations of these nanoparticle solutions

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were diluted to 1 mg·mL-1 with distilled water for the measurements. The scattering

4

angle was fixed at 90o, and the detection temperature was maintained at a constant

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temperature of 25 °C.

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In-Situ Formulation and Characterizations of AuNPs by PEP, PDP and PDEP.

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AuNPs were generated by simply mixing an aqueous solution of HAuCl4·4H2O with

8

an aqueous solution of polyphosphazene vesicle and stirring for some time.

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Polymer-AuNP complex hydrosols were freeze-dried in a lyophilizer (FD-1A-50,

10

Beijing, China). Preparing KBr disks with 2 mg of each sample and collecting spectra

11

using a NICOLET 200SXV Infrared Spectrophotometer (Nicolet, USA) to confirm

12

the formation of AuNPs. Blank KBr disk was used as background spectra. XRD

13

patterns were obtained with a Bruker AXS D8 DISCOVER diffractometer using

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CuKα (0.1542 nm) radiation. UV-Vis measurements were performed with an

15

ultraviolet spectrophotometer (TU-1800PC, Beijing, China). The size and Zeta

16

potential of the obtained polymer-AuNP complexes were recorded on a Zetasizer

17

Nano ZS (Malvern, UK) equipped with a standard 633 nm laser to investigate the

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influence of AuNPs formation on the particle sizes and Zeta potentials. Moreover,

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TEM was carried on a JEOL JEM-1100 (Japan) microscope operating at 80 kV to

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observe the particle micromorphology. Samples for TEM were prepared by dipping a

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carbon-coated copper grid into mixture solution (5 mg·mL-1) for 30 min and air

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drying before measurements. We evaluated the influence of some factors, such as the

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type of reductant, temperature, reaction time and molar ratio of hydrophobic group in

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polymer to HAuCl4, on the size and reaction rate of particles generated. The molar

25

ratio of hydrophobic group in polymer to HAuCl4 ranged from 1:4 to 8:1 (gradually

26

reduction of HAuCl4). Different temperatures were considered for the generation of

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gold nanoparticles, i.e., 20 and 30 °C. During the experiments, we monitored the color

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change and gold nanoparticle generation as time progressed. In all cases, the reaction

29

mixtures were left at 4 °C for a minimum of one month to observe the gold

30

nanoparticles’ precipitation. 7


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In order to determine the loading content and conversion ratio of AuNP, we also

2

measured the concentration of Au in different polymersome samples at 4:1 molar ratio

3

of the hydrophobic group in polymers to HAuCl4 after dialysis against water to

4

remove the unreacted HAuCl4 by an inductively coupled plasma-atomic emission

5

spectrometer (ICP-AES, SPECTRO ARCOS, Spectro, Germany).

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Drug

Loaded

Polymer-AuNP

Complexes

Vesicles

Preparation

and

7

Characterizations. On the basis of the above experiments, we screened the desired

8

reductant, temperature and reaction time. Then, we prepared drug-loaded

9

polymer-AuNP complex vesicles at the optimal conditions. The specific steps were

10

similar to the above section, except 100 µL 5 mg·mL-1 DOX·HCl aqueous solution

11

took the place of the water solution, followed by the HAuCl4 liquor addition.

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The concentration of DOX·HCl in the obtained polymer-AuNP complexes vesicles

13

was determined by high-performance lipid chromatography (HPLC). The vesicles

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were destroyed by methyl alcohol to eject DOX·HCl from the vesicles’ hydrophilic

15

core. The resulting solutions were analyzed with a Prominence UFLC (Shimadzu

16

Corporation) equipped with a Kromasil 100-5C18 reverse-phase column and a

17

dual-wavelength UV-Vis detector operating at 233 nm wavelength and 1 mL·min-1

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flow rate. A 49:20:31 mixture (v/v) of pure HPLC-grade methanol, acetonitrile and

19

phosphate buffer solution (containing 25 mM·L-1 Na2HPO4 and 30 mM·L-1 KH2PO4

20

with the pH adjusted to 5.0) was used. DOX·HCl was eluted for 5 min at room

21

temperature. Eight DOX·HCl solutions with known concentrations ranging from 1 to

22

75 µg·mL-1 were prepared to construct a calibration curve. The loading content (LC)

23

and encapsulation efficiency (EE) were determined as:

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LC (%) = (weight of drug in drug-loaded vesicles / weight of drug-loaded vesicles) × 100%

25

(1)

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EE (%) = (weight of drug in drug-loaded vesicles / initial weight of drug-loaded vesicles) × 100%

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(2)

28 29

DLS experiments were conducted to measure the particle sizes of the drug-loaded vesicles, and TEM was carried out to verify their morphology. 8


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In Vitro Drug Release Behaviour. Specifically, 200 µL of drug-loaded vesicles

2

solution was transferred into a sealed dialysis bag (MWCO 8-14 kDa), dipped in 10

3

mL pH 7.4 phosphate buffered solution (PBS), and shaken at 100 rpm at 37 ºC in an

4

incubator. At the predetermined time points, 1 mL release media was withdrawn and

5

replaced by an equal volume of pre-warmed fresh PBS. The concentration of

6

DOX·HCl

7

spectrophotometer at a wavelength of 480 nm. All results were the mean of three test

8

runs. The release behaviors of drug-loaded vesicles in pH 5.5 were also studied

9

according to the above method.

in

the

collected

media

was

determined

using

an

ultraviolet

10

Statistical Analysis. The data are expressed as the means ± standard deviations of

11

the various measurements. The statistical significance of the intergroup differences

12

was analyzed using one-way analysis of variance (ANOVA) and the Duncan post hoc

13

test. Differences with a P value of less than 0.05 were considered statistically

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significant.

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RESULTS AND DISCUSSION

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Synthesis and Characterization. PEP, PDP and PDEP were prepared through

17

nucleophilic substitution by hydrophilic NH2-PEG2000 and hydrophobic EAB, DPA

18

and DEAAB, respectively. The detailed synthesis routes were illustrated in Scheme

19

S1. The spectra recorded by FTIR are shown in Figure 1A. The characteristic bands

20

of hydrophilic chain and polyphosphazene backbone were the same in PEP, PDP and

21

PDEP, that is, 1351 and 1021 cm-1 (-N=P- stretching vibration), 950 cm-1 (-N-P-

22

stretching vibration), 2872 cm-1 (-CH2- stretching vibration, NH2-PEG2000), 1467 cm-1

23

(-CH2- deformation vibration, NH2-PEG2000), and 1108 cm-1 (-C-O- stretching

24

vibration, NH2-PEG2000). Additionally, the peak assignments of the hydrophobic side

25

groups on the polyphosphazene backbone are as follows: PEP: 1710 cm-1 (-C=O-

26

stretching vibration, EAB) and 852 cm-1 (p-substituted benzene vibration, EAB).

27

PDP: 1525 and 3322 cm−1 (−NH− stretching vibration, DPA), 2969 cm−1 (−CH3

28

stretching vibration, DPA) and 2884 cm−1 (−CH2− stretching vibration, DPA). PDEP:

9


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852 cm-1 (p-substituted benzene vibration, DEAAB), and 1607 cm-1 (N-H

2

deformation vibration, DEAAB).

3

The common peak attributions of -NH-PEG2000 in these polymers’ 1H NMR spectra

4

(Figure 1B) were set as follows: 3.2 ppm (-NH-CH2-CH2-), 3.3 ppm (-OCH3,), 3.5

5

ppm (-NH-CH2-CH2-) and 3.6 (-O-CH2-CH2-). The differences among the spectra

6

were due to the different hydrophobic chains. PEP: 1.3 ppm (-CH3, EAB), 4.2 ppm

7

(-OCH2-, EAB), and 7.2-7.6 ppm (phenyl, EAB). PDP: 1.1 ppm (-CH3, DPA), 2.4

8

ppm (-CH2-, DPA), and 3.0 ppm (-CH- and –NH-CH2-, DPA). PDEP: 1.3 ppm

9

(-CH3, DEAAB), 2.5 ppm (--CH2-, DEAAB), 4.2 ppm (-OCH2-, DEAAB), and

10

7.2-7.6 ppm (phenyl, DEAAB). The molar ratios of the hydrophilic chain to the

11

hydrophobic chain in PEP, PDP and PDEP were calculated by the peak areas of the

12

chemical shifts at 3.3 and 1.3 ppm, 3.3 and 1.1 ppm, and 3.3 and 1.3 ppm,

13

respectively. Thus, the molar ratios of the hydrophilic chain/hydrophobic group of

14

three polymers were 0.5/1.5, 0.4/1.6 and 0.3/1.7, respectively. The number-averaged

15

molecular weight (Mn) and polydispersity index (PDI) of PEP, PDP and PDEP are

16

displayed in Table 1.

17

10


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Figure 1. Characterization of three different polyphosphazene polymers. (A) FTIR spectra and (B)

2

1

3

Table 1. Characteristics of the amphiphilic polyphosphazenes and their self-assembled vesicles.

H NMR spectra in CDCl3.

polymers

Mn

PDIa

fPEG

size (nm)(PDIb)

PEP

45950

1.89

0.77

94.37±4.50 (0.32)

PDP

22289

1.74

0.74

204.17±3.99 (0.31)

PDEP

53183

1.75

0.57

182.31±4.71 (0.22)

4

a

The polydispersity index of the copolymers’ number-average molecular weight.

5

b

The particle size polydispersity index of polyphosphazene vesicles.

6 7

Self-assemble Behavior of Polyphosphazene Particles. Amphiphilic polymers in

8

DMF solvent assembled gradually into a defined arrangement with the increase in

9

water content during the dialysis process. The configurations of polymers largely

10

depended on the chemical structure of the polymers.13 It was widely reported that the

11

PEG content (fPEG) in the copolymer controlled the architecture of polymers that

12

aggregation-shifted from micelles to vesicles with the decrease in the PEG level.14, 15

13

In our previous work, we prepared a series of amphiphilic polyphosphazenes with

14

various hydrophilic NH2-PEG2000/hydrophobic EAB molar ratios.2 The fPEG of

15

polyphosphazene vesicles was more than 0.5,1, 2 which was higher than that of the

16

amphiphilic diblock copolymer vesicles (0.20-0.42).16 According to the propositions,

17

we designed PEP, PDP and PDEP with comparatively low PEG content on the

18

premise of polymer water solubility. According to the ratio of hydrophilic

19

chain/hydrophobic groups, the fPEG of PEP, PDP and PDEP was calculated as 0.77,

20

0.74 and 0.57, respectively. The size of the aggregations were analyzed by DLS

21

(Table 1). All polymers had the tendency to form stable nanostructures with a narrow

22

size distribution. As shown in Figure 2A-C, the polymers all presented as vesicles in

23

aqueous solution with a hydrophobic polymer membrane and a hydrophilic inner

24

cavity, which further supported the above theory.

25

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Figure 2. TEM images of (A) PEP, (B) PDP, (C) PDEP, (D) PEP-AuNP complex, (E) PDP-AuNP

3

complex and (F) PDEP-AuNP complex with a hydrophobic group in polymer/ HAuCl4 molar ratio

4

of 4:1 after reacting for 24 h; the inset shows the magnified image of the polymers and

5

polymer-AuNP complexes. All scale bars are 0.2 µm.

6 7

Influence of Type of Hydrophobic Groups in Polymers. In addition to PEP,

8

there is a considerable quantity of tertiary amino groups in PDP and PDEP with

9

different chemical surroundings. We mixed three polyphosphazene vesicle solutions

10

with a HAuCl4 aqueous solution at a hydrophobic group in polymer/HAuCl4 molar

11

ratio of 4:1 and stirred the mixture for at least 24 h at 30 °C. We evaluated the AuNP

12

generation through the solutions’ color change and UV-Vis spectra. As shown in

13

Figure 3A, no color change was observed in the PEP-HAuCl4 mixture. On the

14

contrary, the PDP and PDEP mixture solutions exhibited a color change from yellow

15

to wine red. These phenomena indicated that the AuNPs were generated in-situ

16

through the reduction by PDP and PDEP, with the exception of PEP.17

17

The UV-Vis absorption spectroscopy studies shown in Figure 3B added credence

18

to these results. The inset displayed the magnified image range from 460 to 610 nm.

19

PDP-AuNP and PDEP-AuNP complex vesicle solutions presented a maximum

20

absorption at 535 nm and 525 nm, respectively, due to the surface plasmon resonance

21

(SPR) absorption of the AuNPs. According to the interpretation proposed by Mie,18

22

the SPR of the AuNPs was attributed to the free electrons’ collective oscillation on the 12


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surfaces of the AuNPs when exposed to light with a wavelength much larger than the

2

particles’ size. The SPR peak shift hinged on the size of the AuNPs to a great extent

3

and led to the gold hydrosol solution color change.19 Compared to the common SPR

4

peak shift at approximately 520 nm for the AuNPs, our results expressed a slight red

5

shift, and we proposed this phenomenon to account for the complexes generated

6

between the polymer and the AuNPs.

7 8

Figure 3. The characteristics of AuNP generation after mixing with different polyphosphazene

9

polymers at 30 °C. (A) The color change of these mixtures at 24 h, (B) the UV-Vis spectra of

10

polymers and polymer-AuNP complexes, (C, D) the UV-Vis spectra of PDP/PDEP-AuNP

11

complexes with different stirring time with hydrophobic group in polymer/HAuCl4 molar ratio

12

setting at 4:1 and (E, F) the UV-Vis spectra of PDP/PDEP-AuNP complexes with different

13

hydrophobic group in polymer/ HAuCl4 molar ratios. The inset shows the magnified image of the

14

polymer-AuNP complexes.

15 16

Then, XRD was employed to characterize the crystal texture of the AuNPs in

17

PDP-AuNP and PDEP-AuNP complexes. As shown in Figure S1, the four peaks that

18

appeared at 38.2, 44.4, 64.6 and 77.5° corresponded to the (111), (200), (220) and 13


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Page 14 of 24

1

(311) diffraction peaks of the gold face-center cubic (fcc), respectively, thus

2

demonstrating the pure crystalline nature of AuNPs reduced by PDP and PDEP.20

3

Furthermore, we quantitatively analyzed the AuNP concentration in each

4

complexes vesicles at a hydrophobic group in polymer/HAuCl4 molar ratio of 4:1

5

after dialysis against water to remove the unreacted HAuCl4 via ICP-AES. According

6

to the results, the conversions of the HAuCl4 to the AuNP for two amino groups

7

contained PDP and PEDP vesicles were 88.10% and 92.78%. And on the basis of the

8

amount of polymer adding, their loading content of Au were 5.22% and 6.89%,

9

respectively. In contrast, the AuNP loading content and conversion ratio of

10

PEP-AuNP complex vesicles was only 0.22% and 3.17%, respectively. In other words,

11

the vast majority of HAuCl4 added were unreacted with PEP, implying PEP almost

12

lacked the ability to reduce HAuCl4 due to the absence of tertiary amine groups.

13

The morphologies of the polymer-AuNP complexes were observed by TEM

14

(Figure 2D-F). And the actual mean particle sizes determined by DLS were 92.23 nm,

15

226.8 nm, 205.7 nm for PEP-AuNP, PDP-AuNP and PDEP-AuNP complex vesicles,

16

respectively. In a previous study by Richardson et al.,21 low molecular weight

17

compounds containing tertiary amino groups reacted promptly to generate AuNPs

18

within one hour, but aggregation occurred spontaneously. However, we successfully

19

avoided this phenomenon by substituting high molecular weight grafted

20

polyphosphazenes for low molecular weight compounds. From Figure 2E and F, the

21

coordination between the tertiary amino groups and generated AuNPs22-24 seemed to

22

effectively prevent the new-born AuNPs from aggregating and result in the

23

homogeneous distribution of AuNPs in the hydrophobic membranes of vesicles,

24

probably because of the considerable sequestration of tertiary amines to AuNPs and

25

the intertwined nature of the branched chains.25 When comparing Figure 2B and E or

26

Figure 2C and F, it is obvious that the introduction of the AuNPs into vesicles

27

thickened the hydrophobic membrane. This feature would be beneficial to the

28

compact nature of the vesicle membrane. However, Figure 2D illustrated the different

29

situation of PEP-AuNP vesicles that many irregular dark particles were scattered

30

outside the vesicles. Since we prepared the TEM samples without removing the 14


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unreacted HAuCl4 and the AuNP conversion ratio was quite low of only 0.22% as

2

discussed above, we speculated that these irregular dark particles were unreacted

3

HAuCl4 due to poor reactivity of PEP with HAuCl4.

4

Influence of Reaction Time and Temperature. As the above text suggested, PEP

5

did not possess the reduction capacity to HAuCl4; thus, the following research objects

6

were focused on PDP and PDEP vesicles. It has long been known that temperature is

7

a significant issue for chemical reactions.26, 27 Experiments were carried out at 20 and

8

30 ºC to seek the optimal temperature for the reaction at a constant copolymer

9

concentration of 2 mM and a hydrophobic group in polymer/HAuCl4 molar ratio

10

setting of 4:1. As shown in Figure S2A, after stirring for 24 h at 20 ºC, no color

11

changes were observed in the PDP and PDEP vesicles when mixing with the HAuCl4

12

aqueous solution. Conversely, AuNPs were achieved upon elevating the reaction

13

temperature to 30 ºC. The kinetics of the AuNP formation could explain the reason in

14

the framework of Marcus electron transfer theory.28-30 The increased temperature

15

provided more contacts between electron donors and receptors and cut down the free

16

energy of the reaction, leading to the spontaneous HAuCl4 reduction reaction.

17

A suite of studies concerning reaction time were supervised at 0.083, 0.17, 0.5, 1, 2,

18

4, 24 and 48 h at 30 ºC. The solution color and UV-Vis spectra are given in Figure

19

S2B and Figure 3C and D, respectively. PDP had a slightly higher reaction rate than

20

PDEP did, with color changes at 0.5 and 1 h, respectively, and reduced HAuCl4

21

completely without a significant color change after 4 h, which was shorter than found

22

in the previous work.21,

23

different reactivity occurred due to the combination of steric hindrance and electron

24

density of tertiary amino groups. The -C=O- group in PDEP acted as an electron

25

withdrawing role, decreasing the electron density of lone pair electrons on the tertiary

26

amino group. Furthermore, the ethyl group had a stronger steric effect than the methyl

27

group, thus lowering the reaction rate. In summary, PDEP displayed a lower reactivity

28

than PDP did, which was subjected to stronger steric hindrance and lower electron

29

density. Despite all this, the reactivity of PDEP was still considerable.

31

UV-Vis patterns revealed the same consequence. The

15


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1

Influence of Hydrophobic Group in Polymer/HAuCl4 Molar Ratio. High

2

hydrophobic group in polymer/HAuCl4 molar ratios from several to thousands were

3

necessary to guarantee the reaction completeness and the AuNP stability.21,

4

However, in our work, AuNPs formed even though the hydrophobic group in

5

polymer/HAuCl4 molar ratio was set to 0.25 (Figure 3E and F), which was probably

6

due to the unique structure of the polyphosphazenes. In the chain of

7

polyphosphazenes, each repeat unit contained nearly 2 tertiary amino groups, which

8

explained the outstanding reducing effect with a lower amount of reductant than that

9

used in other studies. We designed six molar ratios, 0.25, 0.5, 1, 2, 4 and 8, and found

10

that as the molar ratio decreased, the complex solutions were changed from pale

11

yellow to burgundy after 24 h (Figure S2C). Furthermore, we measured the size and

12

took TEM images of polymer-AuNP vesicles at various hydrophobic group in

13

polymer/HAuCl4 molar ratios (supporting information, Table S1 and Figure S3). The

14

results indicated that the particle sizes and the membrane thickness gradually

15

increased with the decrease of hydrophobic group in polymer/HAuCl4 molar ratio.

16

Based on the previous literatures,33-36 the amount of reductant affected the nucleation

17

rate of gold nanoparticles so that the size of AuNP increased as the reducant amount

18

decreased.

31, 32

19

Mechanism of AuNPs Generation. The remaining question was how gold cations

20

were reduced by PDP and PDEP. We assumed that tertiary amino groups in PDP and

21

PDEP took on the responsibility to reduce the gold ions, accompanied by

22

synchronously partially oxidized, which was supported by many previous studies. 37-42

23

They contended that the lone pair electron on the nitrogen of the unprotonated tertiary

24

amino groups was responsible for the effective reducing ability. The reducing process

25

is described by the following equation. 41, 42

26

2R-CH2-NR2 + AuCl4- → 2R-CH=N+R2 + Au0 + 2H + + 4Cl-

(3)

27

We verified this hypothesis by comparing the FTIR spectrograms of polymer and

28

polymer-AuNP complex (Figure 4). The expected concurrent peaks of new bands

29

related to the formation of imine appeared: -C=N- stretching vibration at

30

approximately 1650 cm-1 and 1645 cm-1 were observed in the PDP-AuNP and 16


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PDEP-AuNP complexes, respectively. Therefore, we can conclude that the tertiary

2

amino group played an active role in the reduction of HAuCl4.

3

4 5

Figure 4. The FTIR spectra of polymers and polymer-AuNP complexes. (A) PDP and PDP-AuNP

6

complex and (B) PDEP and PDEP-AuNP complex.

7 8

Drug Loading Behaviour. Then we encapsulated DOX·HCl into polyphosphazene

9

vesicles via a dialysis method. Surprising there was almost no DOX·HCl loaded into

10

PDP (loading content 0.23±0.03 %, encapsulation efficiency 2.2±0.06 %). Inversely,

11

PEP and PDEP displayed outstanding DOX·HCl loading capacity, with 4.87±0.12%

12

loading content, 97.31±2.18% encapsulation efficiency and 4.71±0.17% loading

13

content, and 93.74±4.91% encapsulation efficiency, respectively. And the in situ

14

reducing HAuCl4 by tertiary amino groups in PDEP had little effect on the drug

15

loading. For instance, when the hydrophobic group in polymer/HAuCl4 molar ratio

16

was 4:1, the drug loading content and encapsulation efficiency of PDEP-AuNP

17

complex system were 4.63±0.22% and 91.66±5.31%, respectively. The outstanding

18

drug-loading capacity was mainly due to two aspects. One was because of the strong

19

mutual attraction between the benzene ring in EAB or DEAAB and the anthracene

20

ring in DOX·HCl as we reported before.2 Therefore, the absence of a benzene ring in

21

DPA caused the loss of this strong action, following by poor drug encapsulation of 17


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Page 18 of 24

1

PDP vesicle. On the other hand it was probably attributed to electrostatic

2

incorporation. Namely, PDP possessed a strong positive charge with 21.45 mV zeta

3

potential and DOX·HCl was weakly electropositive43 so that the charge repulsion

4

between PDP and DOX·HCl inhibited the drug loading during the dialysis process.

5

Conversely, the zeta potentials of PEP and PDEP were -12.31 mV and -14.94 mV,

6

respectively, which facilitated the adsorption of DOX·HCl. Otherwise, adding

7

positively charged HAuCl4 partially decreased the potential of PDEP, but the whole

8

system was electronegative. Therefore, PEP, PDEP and PDEP-AuNP complex all

9

demonstrated better DOX·HCl loading capacity than did PDP and PDP-AuNP

10

complex. Satisfactory loading capability is an obligatory factor to construct a

11

nano-scaled drug delivery system; otherwise, there would be no sense in discussing

12

vesicle stability for controlled drug release. The particle sizes of the drug-loaded

13

polymer-AuNPs complexes were measured by DLS and were slightly enlarged

14

compared with blank polymer-AuNPs complexes (Table S2). TEM images indicated

15

the center cavity turned dark of PEP or PDP vesicles after DOX·HCl loaded (Figure

16

S4).

17

In Vitro Drug Release. On the basis of the above results, PDP exhibited a quite

18

poor loading content, which restricted its application for DOX·HCl delivery, although

19

its vesicle had enough dense shells after the AuNPs complexed. Thus, in this section,

20

we discuss the release behavior of the DOX·HCl-loaded PEP system (named PEP-D)

21

and DOX·HCl-loaded PDEP system (named PDEP-D). As shown in Figure 5A, the

22

burst drug release from PEP-D and PDEP-D was so serious that the accumulative

23

drug release ratios in the first 4 h were 47.21% and 44.77%, respectively. Since PEP

24

cannot induce AuNP formation, adding HAuCl4 into PEP vesicle system failed to

25

inhibit DOX·HCl burst release. However, it was clear that in situ reducing HAuCl4

26

into AuNPs by tertiary amino group of PDEP significantly alleviated the DOX·HCl

27

burst release, apart from the molar ratio setting at 8 (Figure 5B). The extent of the

28

slowdown in drug release increased with the decrease of tertiary amino group/HAuCl4

29

molar ratio. The most likely reason could be that the large molar ratio of hydrophobic

30

group in polymer to HAuCl4 resulted in the fast generation of Au nuclei, so the size of 18


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1

resultant AuNPs was relatively small. Therefore, the density of the vesicles’

2

membrane was relatively weak, which led to the fast drug release. Further, we

3

investigated the release profiles of PDEP-D and DOX·HCl-loaded PDEP-AuNP

4

complex system (named PEDP-AuNP-D) in different pH release media (Figure 5A).

5

Compared with the result obtained for pH 7.4 PBS, the lower pH obviously promoted

6

the drug release from PDEP-D and PEDP-AuNP-D. According to our previous

7

work,10 the zeta potential of PDEP vesicles in pH 7.4 and 5.5 were -14.9 mV and 2.0

8

mV, respectively, and the AuNP loading had no effect on their pH sensitivity. And the

9

corresponding sizes were 182.3 nm and 208.3 nm severally. The reason for this

10

phenomenon might be to the protonation of the tertiary amino groups in acidic

11

circumstances, leading to the decrease of the hydrophobicity of DEAAB in the PDEP,

12

which made the vesicles looser but maintained the core-shell structure, accompanied

13

by holes generated on the surface.1 Therefore, PDEP-AuNP-D at the optimal

14

hydrophobic group in polymer/HAuCl4 molar ratio achieved considerable inhibition

15

of drug leakage at pH7.4 and fast drug release responsive to lower pH.

16

17 18

Figure 5. The release profile of drug-loaded polymer systems. (A) PEP-D and PDEP-D in pH 7.4

19

PBS, PDEP-D and PDEP-AuNP-D (hydrophobic group in polymer/HAuCl4 molar ratio set at

20

4:1) in pH 5.5, (B) PDEP-AuNP-D with various hydrophobic group in polymer/HAuCl4

21

molar ratio in pH 7.4 PBS.

22 23

24

In summary, the presence of benzene rings and tertiary amino groups in PDEP

25

rendered the high DOX·HCl encapsulation and efficient in situ AuNP generation as

CONCLUSIONS

19


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Page 20 of 24

1

both a reductant and a stabilizer to improve the formulation stability under normal

2

physiological condition (pH7.4). Moreover, the protonation of the tertiary amine in

3

DEAAB of PEDP might contribute to the fast intracellular drug release. All these

4

properties of PDEP-AuNP-D would be expected to exert synergistic and positive

5

effect on final drug action.

6

ASSOCIATED CONTENT

7

Supporting Information

8

The Supporting Information is available free of charge on the ACS Publications

9

website.

10

Schematic illustration of the synthesis of polymers (Scheme S1)

11

The XRD results of PDP/PDEP-AuNP complexes (Figure S1)

12

The color change of PDP/PDEP and HAuCl4 mixture solutions at different

13

temperature, time and hydrophobic group in polymer/ HAuCl4 molar ratios (Figure

14

S2)

15

The TEM images of PDEP-AuNP complex vesicles at various hydrophobic group

16

in polymer/HAuCl4 molar ratios. (A) 0.25, (B) 0.5, (C) 1, (D) 2, (E) 4 and (F) 8

17

(Figure S3) The TEM images of (A) PEP-D, (B) PDEP-D and (C) PDEP-AuNP-D with

18 19

hydrophobic group in polymer/HAuCl4 molar ratio setting at 4:1 (Figure S4) The particle size of PDP-AuNP and PDEP-AuNP complexes vesicles with various

20 21

hydrophobic group in polymer/HAuCl4 molar ratios (Table S1) The particle size (nm) of PEP-D, PDEP-D and PDEP-AuNP-D with hydrophobic

22 23

group in polymer/HAuCl4 molar ratios setting at 4:1 (Table S2)

24

AUTHOR INFORMATION

25

Corresponding Author

26

*E-mail: [email protected]

27

Notes

28

The authors declare that there are no competing financial interests.

29

ACKNOWLEDGMENTS

20


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1

This work was supported by the National Natural Science Foundation of China

2

(81673384).

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

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Optimizing Hydrophobic Groups in Amphiphiles to Induce Gold

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