Facile construction of pH- and redox-responsive micelles from a biodegradable poly(β-hydroxyl amine) for drug delivery

Article abstract of DOI:10.1021/acs.biomac.5b01394

Here we demonstrate a type of pH and reduction dual-sensitive biodegradable micelles, which were self-assembled by a cationic polymer in an aqueous solution. Due to tumor cells or tissues showing low pH and high reduction concentration, these micelles possessed specific tumor targetability and maximal drug-release controllability inside tumor cells upon changes in physical and chemical environments, but presented good stability at physiological conditions. CCK-8 assay showed that the DOX-loaded micelles had a similar cytotoxicity for MCF-7 tumor cells as free DOX, and blank micelles had a very low cytotoxicity to the cells. Fluorescent microscopy observation revealed that the drug-loaded micelles could be quickly internalized by endosomes to inhibit cancer cell growth. These results indicated these biodegradable micelles, as a novel and effective pH- and redox-responsive nanocarrier, have a potential to improve drug delivery and enhance the antitumor efficacy.

Full text of DOI:10.1021/acs.biomac.5b01394

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Article
Facile Construction of pH- and Redox-Responsive Micelles
from a Biodegradable Poly(#-hydroxyl amine) for Drug Delivery
Dawei Li, Yazhong Bu, Lining Zhang, Xing Wang, Yanyu Yang,
Yaping zhuang, Fei Yang, Hong Shen, and De-Cheng Wu
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01394 • Publication Date (Web): 18 Dec 2015
Downloaded from http://pubs.acs.org on December 22, 2015
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Facile Construction of pHꢀ and Redoxꢀ
Responsive Micelles from a Biodegradable
Poly(βꢀhydroxyl amine) for Drug Delivery
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Dawei Li,†,‡,ǁ Yazhong Bu,†,ǁ Lining Zhang,§ Xing Wang,*,† Yanyu Yang,† Yaping Zhuang,† Fei
Yang,† Hong Shen† and Decheng Wu*,†
†Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics
& Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
‡Department of Orthopaedics, The 309th Hospital of the PLA, Beijing 100094, China
§Center of Rehabilitation, Chinese People’s Liberation Army General Hospital, Fuxing Road,
Beijing, 100853, China
KEYWORDS: disulfide, cationic polymer, micelles, dualꢀresponsive, drug delivery.
ABSTRACT: Here we demonstrate a type of pH and reduction dualꢀsensitive biodegradable
micelles, which were selfꢀassembled by a cationic polymer in an aqueous solution. Due to tumor
cells or tissues showing low pH and high reduction concentration, these micelles possessed
specific tumor targetability and maximal drugꢀrelease controllability inside tumor cells upon
changes in physical and chemical environments, but presented good stability at physiological
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conditions. CCKꢀ8 assay showed that the DOXꢀloaded micelles had a similar cytotoxicity for
MCFꢀ7 tumor cells as free DOX, and blank micelles had a very low cytotoxicity to the cells.
Fluorescent microscopy observation revealed that the drugꢀloaded micelles could be quickly
internalized by endosomes to inhibit cancer cell growth. These results indicated these
biodegradable micelles, as a novel and effective pHꢀ and redoxꢀresponsive nanocarrier, have a
potential to improve drug delivery and enhance the antitumor efficacy.
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INTRODUCTION
Biodegradable polymeric micelles in aqueous solutions have been extensively explored as a
most promising candidate in drug delivery systems for targeted cancer chemotherapy.1ꢀ4 To
overcome severe problems that are associated with traditional hydrophobic antitumor drugs,
polymeric micellar nanocarriers need to possess several unique advantages, including good water
solubility, long in vivo circulation times, efficient bioavailability, high preferential accumulation
at the tumor sites and low systemic side effects.5 Specially, stimuliꢀresponsive micelles were
furnished for specific tumor targetability and maximal drug release controllability inside the
target cells upon changes in physical and chemical environments, such as redox, pH,
temperature, magnetism, ultrasound, enzyme and glucose.6
The significantly chemical stimuli to trigger drug release were pH and redox. In general, pH
maintains about 7.4 in normal extracellular matrices and blood while tumors always presented a
weak acid in the intracellular compartments due to low oxygen in intercellular environments.7
When drug carriers were endocytosed into endosomes, the carriers firstly encountered a pH with
5.5ꢀ6.5 in the early endosomes, then pH declined into about 5.0 in the later endosomes, and
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finally lysosomes decrease even lower pH value. Accordingly, pHꢀresponsive polymeric micelles
have been widely developed for targeted tumor or tissue in biological systems. As one of
representative instances, tertiary amine group was generally introduced into polymers to control
assembled behaviors by protonation/deprotonation.8
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Redoxꢀresponsive micelles have own unique potential to facilitate drug release in tumor cells
or tissues, which is originated from varied concentrations of redox species in intracellular and
extracellular compartments.9 For example, glutathione (GSH) is a most abundant reductive
molecule with millimolar concentrations in the intracellular compartments (~10 mM in the
cytoplasm) and micromolar concentrations in the extracellular environment (~10 ꢁM in the
plasma). The disulfide bond is stable under physiological conditions during circulation as well as
in the extracellular tissues on account of a low concentration of GSH, but quickly cleaved upon
exposure to a highly reductive environment in cells.10 Therefore, utilizing disulfideꢀcontaining
redoxꢀresponsive micelles for drug carrier has been a biologically and friendly method for drug
delivery.
Cationization of polymers is another approach that can be applied for targeted drug
delivery.11,12 The cell surface membrane is negatively charged, so this charged surface provides
plentiful sites of interaction for cationic macromolecules. Therefore, cationic delivery systems
have been utilized for the delivery of drugs, proteins and genes to various tissues including liver,
brain and kidney.13 In recent years, amine−epoxy “click” reaction between diꢀamine and diꢀ
epoxy groups has been recognized as a facile access to prepare mainꢀchain cationic polymers.14
This method offers many natural advantages: 1) the polymerization proceeds at room
temperature without any catalyst; 2) the coupling reaction produces equivalent reactive hydroxyl
groups that are convenient for further functionalization; 3) many tertiary amines are generated
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upon the polymerization that can be transformed into positively charge quaternary structures by
protonation/alkylation. Therefore, a reactive polymer scaffold with several chemically distinct
functional sites can be created at ambient conditions in one simple step by amine−epoxy
reaction. This family of mainꢀchain cationic polymers is named as poly(βꢀhydroxyl amine)s
(PHA), which has a weakly basic characteristic on account of its tertiary amines with a pKb
value of about 6.5. Selfꢀassembly of the biodegradable poly(βꢀhydroxyl amine)s has been studied
in recent years, but due to poor hydrophilicity provided by hydroxyl groups and tertiary amines,
the mainꢀchain cationic polymers always were linked by hydrophilic segments or polymers to
build amphiphilic copolymers and corresponding stimuliꢀresponsive copolymeric micelles in
water for intracellular doxorubicin delivery.15
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Here, we reported a facile method to construct pHꢀ and redoxꢀresponsive micelles from a
novel reducible poly(βꢀhydroxyl amine)s (RPHA), in which a large number of hydroxyl groups
gave rise to good hydrophilicity to stable micelles in aqueous solutions, and the abundant
disulfide bonds embedded in the core and tertiary amines located in the shell provided the
stimulative responsibility for the micelles. The RPHA polymer and its micelles possessed many
unique advantages: 1) the RPHA polymer was easily prepared at ambient conditions in one
simple step by amine−epoxy “click” reaction; 2) a large number of disulfides in the inner
micelles maintain the compact coreꢀshell architectures in the normal physiological conditions,
but are high sensitive to reductive agents in a short time, which can facilitate intracellular rapid
release of the encapsulated drugs within the cells; 3) abundant tertiary amines endow the PRHA
polymer high buffer capacity in a certain pH range; 4) massive hydroxyl groups allow
convenient postꢀfunctionalization to yield multifunctional aggregations; 5) these particles would
be demicellized rapidly into waterꢀsoluble molecules at high levels of reducing agents and/or in
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an acidic environment, which was benefit for antitumor drug to release from the carriers when
the pH values and concentrations of reducing agents changed in intracellular and extracellular
compartments. Additionally, a few of hydroxyl groups were further modified by coumarins to
yield the RPHAꢀgꢀcoumarin polymer, and its corresponding spherical micelle exhibited high
fluorescence intensity in aqueous solutions.
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EXPERIMENTAL SECTION
Materials. Potassium tertꢀbutanolate (99%, J&K), 2,2'ꢀdithiodiethanol (90%, Energy Chemical),
epibromohydrin (97%, Energy Chemical), ethanolamine (99%, J&K), dithiothreitol (DTT, 99%,
J&K), 2ꢀHydroxybenzaldehyde (99%, J&K), diethyl malonate (99.5%, J&K), diethylamine (99%,
J&K), 4ꢀ(dimethylꢀamino)ꢀpyridine (DMAP, 99%, Aldrich), triethylamine (TEA, 99%, Beijing
Chemical Works), thionyl chloride (SOCl2, 99.5%, Energy Chemical), sodium hydroxide,
ethanol, acetic acid glacial, ethyl acetate, hydrochloric acid, acetone, isopropanol, hexane and
ether (reagent grade, Beijing Chemical Works), tetrahydrofuran (THF), N,Nꢀdimethyl formamide
(DMF), chloroform (CHCl3) and dichloromethane (CH2Cl2) were purified by stirring over
calcium hydride for 24 h followed by distillation.
Characterizations. 1H and 13C NMR spectra were obtained on a Bruker DRXꢀ400 spectrometer.
Gel permeation chromatography (GPC) measurement was carried out on a system comprised of a
Waters 515 HPLC pump, and a Waters 2414 RI detector equipped with four Waters Styragel
columns (HT 2, HT 3, HT 4, and HT 5). DMF with 0.01 M LiBr was used as the eluent at a flow
rate of 1.0 mL/min. Polystyrene standards were used for the calibration. Transmission electron
microscopy (TEM) images were obtained on a JEMꢀ2200FS microscope (JEOL, Japan). A 5 ꢁL
droplet of micellar solution was dropped onto a copper grid (300 mesh) coated with a carbon film,
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followed by drying at room temperature. The samples were sputterꢀcoated with a thin layer of Pt
for 120 s to make the samples conductive before testing. Dynamic light scattering (DLS) spectra
were obtained on a commercial laser light scattering spectrometer (ALV/DLS/SLSꢀ5022F)
equipped with a multiꢀτ digital time correlator (ALV5000) and a cylindrical 22 mW UNIPHASE
HeꢀNe laser (λ0=632.8 nm) was used. All data were averaged over three time measurements. The
laser light scattering cell is held in a thermostat index matching vat filled with purified and dustꢀ
free toluene with the temperature controlled to within 0.1 °C. UVꢀvis spectra of the samples were
measured on a Hitachi Uꢀ3010 spectrometer, and fluorescence measurements were carried out on
a Hitachi F4600 photoluminescence spectrometer with a xenon lamp as a light source. Confocal
laser scanning microscopy (CLSM, Zeiss LSM 510, Germany) was under excitation at 314 nm.
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Synthesis of 2,2'ꢀDithiodiethanol Diepoxypropyl Ether. In a 250 mL roundꢀbottomed flask
equipped with a magnetic stirring bar, potassium tertꢀbutanolate (4.48 g, 40 mmol) and 2,2'ꢀ
dithiodiethanol (3.08 g, 20 mmol) were dissolved in 50 mL of isopropanol, followed by addition
100 mL of isopropanol containing epibromohydrin (6.85 g, 50 mmol) to fully dissolve all the
solids. The reaction was allowed to proceed for 24 h at room temperature. After that, the mixture
was filtrated and the crude product was yielded after removing the solvent. Then the final
product was separated chromatographically on silica gel with ethyl acetate/hexane (v/v=2/1) to
afford the yellow viscous liquid with 33.5% yield.
Synthesis of Coumarinꢀ3ꢀcarboxylic Acid. In a 100 mL roundꢀbottomed flask equipped with a
magnetic stirring bar, 2ꢀhydroxybenzaldehyde (12.2 g, 0.1 mol), diethyl malonate (19.2 g, 0.12
mol), diethylamine (1.8 g, 0.025 mol) and acetic acid glacial (1 mL) were dissolved in 30 mL of
ethanol. After refluxing for 2 h, a mount of NaOH solution was added into the mixture, and then
kept refluxing for another 15 min. After cooling to the room temperature, a mount of
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concentrated hydrochloric acid was added to adjust pH = 2 to produce the solid precipitates in an
ice bath. The crude products were obtained after filtration, washing with DI water and drying.
Then recrystallization from ethanol afforded the white solid with 77.2% yield.
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Synthesis of Coumarinꢀ3ꢀacyl Chloride. Coumarinꢀ3ꢀcarboxylic acid (1.9 g, 10 mmol) and two
drops of DMF were added into 6 mL of SOCl2. The mixture was stirred at room temperature for
2 h and concentrated under reduced pressure to afford the faint yellow solid with 87.8% yield.
Synthesis of Reducible Poly(βꢀhydroxy amine)s (RPHA) Polymer. 2,2'ꢀdithiodiethanol
diepoxypropyl ether (267 mg, 1 mmol) and ethanolamine (61 mg, 1 mmol) were dissolved in 5
mL of methanol. After stirring for several days at room temperature, the mixture was precipitated
in a large excess amount of diethyl ether twice and dried in vacuum to afford the final product
with 85.4% yield.
Synthesis of RPHAꢀgꢀcoumarin Polymer. RPHA (240 mg, 0.02 mmol) and DMAP (5 mg, 0.04
mmol) were dissolved in 5 mL of anhydrous DMF. Under vigorous stirring at 0 °C, coumarinꢀ3ꢀ
acry chloride (34 mg, 0.16 mmol) was dropwise into the solution for 30 min. The solution was
stirred at room temperature for another 4 h, and precipitated in ether for three times to afford the
white solid with 84.7% yield.
Formation and Selfꢀassembly of the Amphiphilic RPHA Polymer. Polymeric micelles were
prepared by a dialysis method. A typical selfꢀassembly aggregate solution was prepared as
following: RPHA polymer (5 mg) was first dissolved in methanol (1 mL). Then deionized water
(4 mL) was added dropwise into the solution at the rate of 0.05 mL/min via a syringe pump. The
colloidal dispersion was further stirred for another 2 h and the temperature was fixed at 30 °C
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during the selfꢀassembling process. The organic solvent was removed by dialysis (MW cutoff, 4
kDa) against deionized water for 3 days.
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Determination of pH and ReductionꢀTriggered Destabilization of RPHA Micelles. The size
change of micelles in response to DTT in phosphate buffered saline (PBS, pH 7.4) or PBS (pH
5.0) was determined by DLS measurement. For determining the reduction sensitivity of micelles,
DTT (10 mM) was added into a 10 mL solution of RPHA micelles (1 mg/mL) in PBS (pH 7.4),
and then the solution was mildly stirring at 37 °C. After 8 h, the size was measured by DLS. For
determining the pH sensitivity of micelles, a certain amount of PBS (pH 5.0) was added into 10
mL solution of RPHA micelles (1 mg/mL), and then the solution was mildly stirring at 37 °C.
After 8 h, the size was measured by DLS.
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Formation and Selfꢀassembly of RPHAꢀgꢀcoumarin Polymer. Polymeric micelles were
prepared by a dialysis method. A typical selfꢀassembly aggregate solution was prepared as
following: RPHA polymer (5 mg) was first dissolved in methanol (1 mL), and then deionized
water (4 mL) was added dropwise into the solution at the rate of 0.05 mL/min via a syringe
pump. The colloidal dispersion was further stirred for another 2 h and the temperature was fixed
at 30 °C during the selfꢀassembling process. The organic solvent was removed by dialysis (MW
cutoff, 4 kDa) against deionized water for 3 days.
Preparation of DOXꢀLoaded RPHA Micelles. DOXꢀloaded micelles were prepared as follows:
20 mg RPHA was dissolved in 1 mL of DMF, followed by adding a predetermined amount of
DOXꢂHCl and 1.5 molar equiv of triethylamine and stirred at room temperature for 2 h. Then 4
mL of phosphate buffered saline (PBS, pH 7.4) was added dropwise into the solution at the rate
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of 0.05 mL/min via a syringe pump. Subsequently, the solution was dialyzed against deionized
water for 24 h (MW cutoff, 4 kDa) to remove free DOX and byproducts.
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In Vitro DOX Release from Polymeric Micelles. The pHꢀdependent DOX release
measurements were conducted as below: dispersed DOXꢀloaded polymeric micelles were added
into a dialysis membrane tube (MW cutoff, 4 kDa), which was then incubated in 30 mL of
phosphate buffer saline (PBS) at pH 5.0 or 7.4 at 37 °C in a shaking water bath at rate of 90 rpm.
The DTTꢀdependent DOX release measurements were conducted in the same manner as
described above. The incubation solutions were 30 mL of PBS (pH 7.4) with DTT concentrations
of 10 mM. At predetermined frequencies, 6 mL of incubated solution was taken out and 6 mL of
fresh PBS with 10 mM of DTT was added to refill the incubation solution to 30 mL. The pHꢀ
dependent DOX release profiles were determined by measuring the UVꢀvis absorbance of the
solutions at 480 nm. All DOXꢀrelease experiments were conducted in triplicate and the results
were expressed as the average data with standard deviations. To determine the total loading of
the drug, the DOXꢀloaded micellar suspensions were freezeꢀdried and then dissolved in DMF
again, and analyzed with fluorescence spectroscopy. A calibration curve was obtained using
DOX/DMF solutions with different DOX concentrations. For determining the amount of DOX
release, calibration curves were running with DOX/phosphate buffer solution (pH 7.4, 100 mM)
with different DOX concentrations. Release experiments were conducted in triplicate. The
results were presented as the average ± standard deviation.
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Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the
following formulas:
DLC (wt %) = (weight of loaded drug/weight of polymer) × 100%
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DLE (wt %) = (weight of loaded drug/weight of feeding drug) × 100%
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Fluorescent Microscope Observation of MCFꢀ7 cells Incubated with DOXꢀLoaded
Polymeric Micelles. MCFꢀ7 cells were plated on microscope slides in a 96ꢀwell plate (5 × 104
cells/well) using Dulbecco’s Modified Eagle medium (DMEM) medium containing 10% FBS.
After incubation for 24 h, the cells were incubated with prescribed amounts of DOXꢀloaded
RPHA micelles or free DOX at 37 °C and 5% CO2 for another 24 h. Then the culture medium
was removed and the cells on microscope plates were washed three times with PBS. After fixing
with 4% paraformaldehyde overnight, the cells were observed under a fluorescent microscope.
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CCKꢀ8 Assay. The cytotoxicity of RPHA micelles and DOXꢀloaded RPHA micelles was
studied by CCKꢀ8 assay using MCFꢀ7 cells. Cells were seeded onto a 96ꢀwell plate at a density
of 1 × 104 cells per well in 180 ꢁL of DMEM containing 10% fetal bovine serum (FBS) and
incubated for 24 h (37 °C, 5% CO2). The medium was replaced by 90 ꢁL of fresh DMEM
medium containing 10% FBS, and then 20 ꢁL samples of various concentrations (2ꢀ10 mg/mL)
of the micelle suspensions in PBS (pH 7.4) were added. The cells were incubated for another 24
h. After removal of the culture media from cell culture plates, 100 ꢁL fresh culture media and 10
ꢁL CCKꢀ8 kit solutions were immediately added and homogeneously mixed and then incubated
for 4 h in a CO2 incubator. Finally, 100 ꢁL reaction solutions were put into 96ꢀwell plate. The
optical density of each well at 450 nm was read by a microplate reader. Cells cultured in DMEM
medium containing 10% FBS (without exposure to micelles) were used as controls.
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Scheme 1. Synthetic Route of pHꢀ and RedoxꢀSensitive RPHA and RPHAꢀgꢀCoumarin
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Polymers
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RESULTS AND DISCUSSION
Synthesis and Characterization of the Reducible Poly(βꢀhydroxyl amine)s (RPHA).
Synthetic pathway of the RPHA polymer is summarized in Scheme 1. Firstly, the monomer of
2,2'ꢀdithiodiethanol diepoxypropyl ether (Figure 1A) was produced with high purity, and then
reacted with ethanolamine to yield cationic RPHA polymers by amine−epoxy reaction at room
temperature. Two polymers, RPHA1 and RPHA2, were produced after polymerization of 5 and 3
days. The every repetitive unit of this cationic polymer possessed a disulfide bond, a tertiary
amine and three hydroxyl groups. Figure 1B showed the clear assignment of the polymeric
framework. The typical epoxy proton resonances at 2.6, 2.8 and 3.1 ppm disappeared, and the
expected shifts in the proton resonances, such as methylene groups and hydroxyl groups,
1
appeared after polymerization in the H NMR spectra (Figure 1A and 1B). The chemical
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structures of the monomer and RPHA polymer were further clearly clarified in the C NMR
spectra of the monomer (Figure 1A) and RPHA polymer (Figure 1B). Figure 1C gave the
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molecule weights of two polymers were about 5700 and 12400, and the corresponding
polydispersity indices were 1.83 and 2.23, respectively. It is mentioned that the monomer
stoichiometry and the polymerization time are two critical factors to define Mn of RPHA
polymers in the stepꢀgrowth polymerization. These results manifested a simple synthetic
pathway for preparation of reducible poly(βꢀhydroxyl amine)s in a mild condition.
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Figure 1. H and C NMR spectra of (A) monomer and (B) RPHA1. (C) GPC curves of the
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RPHA polymers.
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Formation and pHꢀ and RedoxꢀTriggered Disruption of Micelles. Due to a large number of
hydrophilic hydroxyl groups and tertiary amines throughout the polymer backbone, these mainꢀ
chain cationic RPHA polymers were recognized as a novel amphiphilic polymer that can be selfꢀ
assembled into nanometre aggregations in aqueous solutions. We investigated selfꢀassembly
behaviours of the two polymers. The RPHA1 polymer could assemble into the stable micelles,
but the assemblies of the RPHA2 polymer quickly precipitated after dialysis with water. The poor
stability of the RPHA2ꢀbased assemblies in water may ascribe to that the hydrophobic terminal
epoxy groups have a certain effect on hydrophobicity /hydrophilicity balance when the repeat
units are not so high. So we adopted the RPHA1 polymer to systemically explore its selfꢀ
assembly and drugꢀrelease behaviours. The CMC of RPHA micelles was ca. 1.17 mg/L as
determined by fluorescence measurements using pyrene as a probe. Transmission electron
microscope (TEM) images in Figure 2A showed formation of the uniform spherical micelles, and
the average diameter is about 85 nm. Dynamic light scattering (DLS) revealed a large
hydrodynamic diameter of 185 nm and a PDI of 0.29 in Figure 2D. The bigger size was
attributed to the swollen molecules in water, indicating high hydrophilicity given by excess
pendant hydroxyl groups. This size of RPHA1 micelles is suitable for intracellular drug delivery,
because an appropriate diameter (less than 200 nm) of aggregations is favourable to keep the
lowered level of reticuloendothelial system (RES) uptake, minimal renal excretion and efficient
EPR effect for passive tumourꢀtargeting.16
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Figure 2. TEM images of the micelles before (A) and after treatment of (B) pH 5.0 and (C) 10
mM DTT for 8 h. (D) The size change of the micelles in response to 10 mM DTT in PBS (pH
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7.4 or 5.0) and at 37 C for 8 h determined by DLS measurement (micelles without any
treatments were used as the control).
A lot of disulfide bridge linkages, served as the hydrophobic moieties of micelles, were
throughout the whole polymeric main chain. Accordingly, the RPHA micelles can inevitably be
broken down in response to the reductive agents. Meanwhile, the same number of tertiary amines
in the architectural framework furnished the micelles breakable behaviour with pHꢀresponsive
property, because tertiary amines can be protonated to give cationic backbones that can be
dissolved in water. Therefore, these micelles possessed pH and reduction dualꢀresponsive
properties. To study this pHꢀ and redoxꢀtriggered disassembly responsiveness, the micelles were
treated with 10 mM DTT and/or in acidic conditions. The particle size changes of the micelles
were measured by DLS measurement. After adjusting pH value of the solution to 5.0, the
micelles were damaged as observed in Figure 2B corresponding to the diameters of about 0.3 and
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7 nm (Figure 2D). Similarly, for the reducible moiety, polymeric micelles were degraded in the
presence of a high level of DTT (10 mM) that triggered the disassembly of micelles in Figure 2C.
The corresponding sizes of DLS profile were ca. 1 and 10 nm in Figure 2D, demonstrating DTTꢀ
triggered cleavage of disulfide bonds and dissociation of the aggregation. As a control, the size
of micelles had no obvious alteration after 8 h immersion in PBS buffer (pH=7.4) without DTT.
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Figure 3. The size change of the micelles in response to (A) pH 5.0, (B) pH 7.4, (C) pH 10.0, (D)
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2 mM, (E) 5 mM and (F) 10 mM DTT in PBS (pH 7.4) at 37 C for various treated time
determined by DLS measurement.
We further quantitated the different pH (5.0, 7.4 and 10.0) and DTT concentration (2 mM, 5
mM and 10 mM) conditions at various treated time to study the biodegradation process of
particles by DLS measurement. By adjusting pH value of the solution to 5.0 for 0.5 h shown in
Figure 3A, the swelling and cracking of the polymeric micelles could be observed corresponding
to one peak with up to 450 nm and another peak with 80 nm. Then the size declined into 393 nm
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and 45 nm after 1 h. The incremental size ascribed to electrostatic repulsive force and the
micellar swelling originated from the protonated tertiary amines and improved hydrophilicity.
The shrunken size resulted from the partially destroyed micelles. After treatment for 2 h,
protonated tertiary amines markedly improved the solubility of RPHA polymers and the micelles
degraded into small particles, including the collapsed micelles and escaped polymeric chains
from the aggregates. With standing for the longer time, more and more polymeric chains were
released from the micelles. After 8 h, all the micelles decomposed into the protonated polymer
chains with the size of ca. 1ꢀ5 nm by DLS measurement. The nanoscale was in accordance with
the theoretic size (2.3 ± 0.2 nm) calculated by molecular dynamics (MD) simulation, proving
completely dissolution of micelles in pH 5.0 solution. In comparison, the micelles in pH 7.4 or
pH 10.0 can basically maintained the initial diameters (Figure 3B and 3C). As for DTTꢀtriggered
disassembly of the micelles, some disulfides were broken down in the first 1 h in the presence of
2 mM DTT (Figure 3D), and thus the micelles became slightly loose and swollen with the size
increasing into 355 nm. However, the majority of intact disulfides preserved the micellar
architectures even after 8 h, indicating that a low concentration of DTT can hardly destroy the
micelle completely. When the DTT concentration increased up to 5 and 10 mM, the disulfide
linkages were easily broken down, inducing the micelles rapidly disassembled in the first 2 h
(Figure 3E and 3F). After 8 h, the disulfide linkages were completely cleaved to degrade the
micelles into small waterꢀsoluble molecules with the diameter of ca. 1ꢀ10 nm. It is mentioned
that high level of DTT concentration and acidic condition can work in tandem to accelerate the
degradation of micelles into waterꢀsoluble protonated molecules with smaller diameters of ca.
0.3ꢀ7 nm (Figure 2D), which was consistent with the MD simulation result (1.1±0.1 nm). The
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degradation processes by pHꢀtriggered, DTTꢀtriggered and pHꢀ/DTTꢀtriggered disassembly of
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micelles were clearly illustrated in Scheme 2. These results further revealed these dualꢀsensitive
micelles were stable in the normal physiological conditions and degradable in response to high
concentration of reductive agents and/or acidic environment, which can be employed as an
intelligent responsive drug carrier.
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Scheme 2. Schematic of pH and/or DTTꢀTriggered the Demicellization of RPHA Micelles
Loading and In Vitro Release of DOX. DOX was loaded into the micelles by dialysis method.
The dialysate was analysed by fluorescence spectrum and the results showed the characteristic
absorbance at 554 nm for DOX was not detected, demonstrating the free DOX was removed
completely. The drug loading content in the micelles was 9.1%, and the corresponding drug
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loading efficiency was 36.6%. The in vitro release of DOX from the micelles was firstly
investigated at 37 °C under different conditions, i.e., (a) pH 7.4 in PBS, (b) pH 5.0 in PBS, (c) 10
mM DTT and pH 7.4 in PBS, and (d) 10 mM DTT and pH 5.0 in PBS. As shown in Figure 4A, a
slow release of below 20% of incorporated DOX within 24 h was observed in PBS at pH 7.4,
indicating that the DOXꢀloaded micelles can preserve the coreꢀshell architectures at
physiological pH and maintain a slow diffusion with a sustained release. However, when the
micelles were dispersed in an acidic environment (pH 5.0), the encapsulated DOX molecules
were rapidly released on account of pHꢀinduced quick demicellization, exhibiting much faster
release rate with 49.1% contents in the first 4 h and up to 74.7% in 24 h. This pH change from
extracellular environment (pH 7.4) to the endosomal compartments (pH 5.0) suggested that the
DOXꢀloaded micelles were suitable for intracellular drug delivery. It is noted that the positive
charged core of the micelles and its capacity of disrupting endosomes, due to the proton sponge
effects, may further promote the application of the micelles for cytoplasmic drug delivery. It is
well known that the great difference of GSH level from the circulation (10ꢀ100 ꢁM) to the tumor
cells (1ꢀ10 mM) can trigger cleavage of the disulfide bonds. Figure 4A showed that DOX release
burst from the micelles in the first 2 h (24.1%), and then sharply rose into 62.6 % after 24 h in
the presence of 10 mM DTT in PBS at pH 7.4. The fast release of DOX was attributed to a rapid
reductive degradation of the core by high DTT concentration. Notably, the fastest and almost
completely DOX release (96.9%) was observed at pH 5.0 in the presence of 10 mM DTT. These
results suggested that the micelles would significantly release their loading drugs in endosomal
environments.
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Figure 4. pH and/or redoxꢀtriggered release of DOX from RPHA micelles with various
treatment condition at 37 °C as a function of time.
In order to further mimic the intracellular trafficking pathway and clarify the mechanism that
accelerated the DOX release from the micelles, we performed the other tests including pH trend
experiments: pH 5.0, 7.4 and 10.0 with and without DTT, and DTT trend experiments: 2 mM, 5
mM and 10 mM at various pH values (5.0, 7.4 and 10.0). The results disclosed that both DTT
and pH worked in tandem to accelerate DOX release from the micelles. In the pHꢀinduced
degradation experiments (Figure 4B), less than 20% DOX was released from the micelles in pH
7.4 and pH 10.0 after 8 hours, but acidic environment quickly prompted DOX release (68% in
pH 5.0). In the DTTꢀtriggered degradation experiments (Figure 4C), high level of DTT
concentrations can quickly disrupt the micelles and promote DOX release (42% and 53% for 5
mM and 10 mM at 8 h), while 2 mM DTT cannot effectively disintegrate micelles and only 20%
DOX release was detected. After further testing with various pH and DTT concentration for 8 h
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(Figure 4DꢀF), the DOX release can quickly increase up to 88% in the presence of pH 5.0 and 10
mM DTT, testifying that the synergistic effects of pH and DTT worked in tandem to induce
demicellization and accelerate DOX release from the micelles. It was noted that DTT can
improve its capacity to break the disulfide linkages in alkaline environment, so the rate of DOX
release with pH 10.0 is quicker than that with pH 7.4 in the same DTT concentrations. Based on
the above analysis, it is reasonable that the micelles not only possessed good stability at
physiological conditions, but also had capacity to rapidly and thoroughly drug release by pH and
reduction dualꢀstimuli in the process of intracellular trafficking.
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Scheme 3. Activated Intracellular Drug Release from pH and GSH DualꢀSensitive
Biodegradable Micelles
Intracellular Drug Release and Antitumor Activity of DOXꢀLoaded RPHA Micelles. The
cellular uptake and intracellular drug release behaviours of DOXꢀloaded micelles (Scheme 3)
were investigated using fluorescent microscope in MCFꢀ7 cells. As displayed in Figure 5A, the
DOX molecules exhibited high fluorescence intensity in the cells, indicating the rapid DOX
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release from the micelles and the localization in the cells. The intracellular distribution of DOX
reflected that the synergistic effects of DTTꢀresponsive degradation of disulfide linkages and pHꢀ
induced quick demicellization accelerated the intracellular DOX release from the micelles. The
in vitro cytotoxicity to MCFꢀ7 cells of the micelles was determined by CCKꢀ8 assay. As shown
in Figure 5B, RPHA micelles were nontoxic to MCFꢀ7 cells due to their low charge density and
quick degradation capacity in the presence of high levels of reductive agents. The cell viability
was more than 95% until a tested concentration of 1.0 mg/mL, demonstrating excellent
biocompatibility of these biodegradable micelles. However, the DOXꢀloaded micelles displayed
high efficiency of antitumor activity towards MCFꢀ7 cells following 24 h of incubation. Figure
5C showed that the DOXꢀloaded micelles had a similar toxicity as free DOX in a low
concentration. In a high concentration, DOX molecules can diffuse into cells rapidly, whereas
the assembled micelles have to be endocytosed to enter the cells, which made free DOX
molecules quicker than the DOXꢀloaded micelles in internalization, thus presenting high
efficiency in cancer cell inhibition. The halfꢀmaximal inhibitory concentration (IC50) of the
DOXꢀloaded micelles was about 7.3 ꢁg/mL while that of free DOX was only 0.9 ꢁg/mL.
However, it should be noted that the antiꢀtumor activity of the micelles might be enhanced like
traditional carriers using targeting ligands, such as folic acid, peptide, aptamer, antibody or
antibody fragments, to facilitate efficient and specific cellular uptake of micelles.17 The results
revealed that the biodegradable micelles possessed an excellent biocompatibility and the DOXꢀ
loaded micelles showed a good antiꢀtumor capacity.
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Figure 5. (A) Fluorescent microscope images of MCFꢀ7 cells for cellular uptake. a: red
fluorescence image; b: a bright field image; c: overlap of fluorescence and bright field images.
(B) Cytotoxicity of RPHA micelles to MCFꢀ7 cells following 24 h incubation. (C) Viabilities of
MCFꢀ7 cells following 24 h incubation with DOXꢀloaded RPHA micelles and free DOX as a
function of DOX dosages. All the data are presented as the average ± standard deviation.
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Figure 6. (A) IR spectra of RPHA and RPHAꢀgꢀcoumarin polymers. (B) H NMR spectrum of
RPHAꢀgꢀcoumarin polymer. (C) TEM image and (D) DLS plot of RPHAꢀgꢀcoumarin micelles.
Coumarin and its derivatives type dyestuff, possessing high fluorescent efficiency, are used in
the fields of biology, medicine, perfumes and cosmetics, cigarettes, alcoholic beverages, laser
dyes and polymer science.18,19 On account of abundant hydroxyl groups that can be facilely
modified, a RPHAꢀgꢀcoumarin polymer was obtained by acylation reaction with a small amount
of coumarins anchoring on the side chain shown in Scheme 1. IR spectra gave an evidence to
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demonstrate the effective attachment on RPHA polymer with coumarins in Figure 6A. H NMR
spectrum showed the typical signals of coumarin groups (jꢀn) at 7.35, 7.52 and 8.86 ppm as well
as the new peaks of a' and d' at 4.25ꢀ4.28 ppm for the RPHAꢀgꢀcoumarin polymer in Figure 6B.
The integration ratio (Ij/Ig) was close to 1:60, indicating the grafting rate of the coumarin was
about 3.3%. Figure 6C disclosed the uniform sphere micelles with the size of 145 nm assembled
by the RPHAꢀgꢀcoumarin polymer, but the average diameter (195 nm) with a PDI of 0.379 from
the DLS result in Figure 6D was a little bigger than that of the RPHA micelles (185 nm, Figure
2D). The increase of the micellar size may be attributed to the faint increase of hydrophobicity.
Controlling low grafting rates of coumarins not only aimed to keep the RPHAꢀgꢀcoumarin
micelles stable in an aqueous solution, but also ensure the optimal size of the micelles for
intracellular drug delivery. Although the percentage of coumarin molecules was low, the RPHAꢀ
gꢀcoumarin polymer presented strong UV absorption and fluorescence intensity as shown in
Figure 7A and 7B. Confocal laser scanning microscopy (CLSM) in Figure 7C and 7D provided
intuitive structural insights to better observe the RPHAꢀgꢀcoumarin polymer and RPHAꢀgꢀ
coumarin micelles with high intense fluorescence. Importantly, compared to the encapsulated
dyes in the micelles, covalent conjugated coumarins are stable with a period of time in aqueous
solutions, which would be useful for application of fluorescence labelling. The further study of
coumarinꢀbased biodegradable micelles with multiple responses is an intriguing ongoing work in
our laboratory and will be discussed in future publications.
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The functional modification with excessive hydroxyl groups by acylation reaction is a general
strategy to produce smart multiꢀresponsive materials. Currently we have also synthesized a
paclitaxelꢀconjugated RPHA polymer, and obtained dualꢀdrugs loaded biodegradable micelles by
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manipulating selfꢀassembly condition to encapsulate DOX drugs, which presented the excellent
antitumor activity in targeted cells with pH and reduction dualꢀresponsiveness.
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Figure 7. (A) Absorption spectrum, (B) Emission spectrum (λex = 314 nm) and (C) CLSM image
of RPHAꢀgꢀcoumarin polymer in methanol. (D) CLSM image of RPHAꢀgꢀcoumarin micelles.
CONCLUSION
In summary, we synthesized a kind of mainꢀchain cationic polymer (RPHA) with a large number
of hydroxyl groups, disulfide linkages and tertiary amines. This polymer assembled into uniform
sphere micelles in aqueous solutions with pH and reduction dualꢀresponsive biodegradation. The
DOXꢀloaded micelles can preserve the coreꢀshell architectures in the normal physiological
conditions, and rapidly release the drug in the presence of high level of reductive agent and/or
acidic environment, which mimics the pH and reduction conditions in the endosomes.
Cytotoxicity assay of the DOXꢀloaded micelles indicated a fast internalization and a high cellular
proliferation inhibition to MCFꢀ7 cells. After further functionalization with excessive hydroxyl
groups by acylation reaction, the RPHA polymer was facilely modified into the multifunctional
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nanocarriers, paving a new pathway for acquiring intelligent biomaterials in the field of
fluorescent probe, bioimaging, drug delivery and biomedicine.
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AUTHOR INFORMATION
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Corresponding Author
*Eꢀmail: dcwu@iccas.ac.cn; wangxing@iccas.ac.cn
Author Contributions
ǁDawei Li and Yazhong Bu contributed equally to this work.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge MOST (2014CB932200 and 2014BAI11B04), ‘Young Thousand
Talents Program’ and NSFC (21504096, 21174147 and 21474115) for financial support.
REFERENCES
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