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Preparation of reduction-triggered degradable microcapsules for intracellular delivery of anti-cancer drug and gene
Accepted Manuscript Preparation of Reduction-triggered Degradable Microcapsules for Intracellular Delivery of anti-Cancer Drug and Gene Fuli Feng, Rongrong Li, Qingyun Zhang, Yinsong Wang, Xiaoying Yang, Hongquan Duan, Xinlin Yang PII:
S0032-3861(13)01086-0
DOI:
10.1016/j.polymer.2013.11.035
Reference:
JPOL 16623
To appear in:
Polymer
Received Date: 8 August 2013 Revised Date:
25 October 2013
Accepted Date: 23 November 2013
Please cite this article as: Feng F, Li R, Zhang Q, Wang Y, Yang X, Duan H, Yang X, Preparation of Reduction-triggered Degradable Microcapsules for Intracellular Delivery of anti-Cancer Drug and Gene, Polymer (2013), doi: 10.1016/j.polymer.2013.11.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preparation of reduction triggered P(MAA-co-BAC)/PEI microcapsules for delivery of anti-cancer drug and gene.
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Preparation
of
Reduction-triggered
Degradable
Microcapsules for Intracellular Delivery of anti-Cancer
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Drug and Gene
Fuli Feng,[a] Rongrong Li,[a] Qingyun Zhang,[b] Yinsong Wang,[a] Xiaoying Yang,*[a]
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Hongquan Duan,[a] and Xinlin Yang*[c]
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[a] Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), Basic Medical Research Center, School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China [b] Department of Chemistry, Logistics University of Chinese People’s Armed Police,
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Tianjin 300162, PR China
[c] Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, PR China
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* Corresponding authors.
Phone: +86 (22) 23502023. Fax: +86 (22) 23503510.
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E-mail: [email protected] (X. Y. Yang); [email protected] (X. L. Yang).
1
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Abstract:
The
reduction-triggered
degradable
acid-co-N,N-bis(acryloyl)cystamine)/polyethyleneimine
poly(methacrylic
(P(MAA-co-BAC)/PEI)
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microcapsules were prepared by distillation–precipitation polymerization for delivery of anti-cancer drug and gene. N,N-bis(acryloyl)cystamine (BAC) as a crosslinker containing a disulfide bond can be triggered by reductive agents, such as glutathione
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(GSH) and dithiothreitol (DTT), to endow the functional microcapsules with reduction-triggered drug release. The P(MAA-co-BAC)/PEI microcapsules were
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characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectra (FT-IR), laser particle size analyzer and elemental analysis. The degradable behavior of microcapsules was investigated by analysis of UV-vis spectroscopy. The controlled drug release behavior for P(MAA-co-BAC)/PEI microcapsules was
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strongly dependent on the absence/presence of GSH and the pH values with doxorubicin hydrochloride (DOX) as a model drug molecule. The in vitro gene transfection ability was evaluated by Hela cells with the transfection of plasmid DNA
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(pDNA) encoded with green fluorescent protein (GFP) and the transfection efficiency was determined by confocal fluorescence microscopy. Furthermore, the cytotoxicities
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of (P(MAA-co-BAC)/PEI) microcapsules before and after loading of DOX were assessed via WST-1 assay. The P(MAA-co-BAC)/PEI microcapsules provide the potential novel vectors for delivery of drugs and genes, promising for future applications in anticancer drug and gene combined therapy. Keywords: Reduction triggered degradable microcapsules; Distillation-precipitation polymerization; Drug delivery; Gene delivery. 2
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Introduction Combination therapy has shown several potential advantages (e.g., synergistic
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effects and reversal of drug resistance) and may prove more effective than single drug therapy.[1] It is essential for the sequential delivery of drugs and nucleic acids, which
can enhance their therapeutic effects.[2] For example, the combination of
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chemotherapy and RNAi therapy by sequential delivery of anticancer drug and siRNA can reverse the tumor multidrug resistance. A variety of organic/inorganic
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nanomaterials have been used as delivery vehicles to develop effective therapeutic modalities. A recent advance in nanomedicine-mediated cancer therapy is the development of multifunctional carriers for jointly delivering a chemotherapeutic drug and a genetic material such as pDNA or siRNA. Zhong et al prepared the
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biodegradable cationic micelles from PDMAEMA–PCL–PDMAEMA triblock copolymers and applied for the delivery of MDR-1-targeted siRNA and paclitaxel into cancer cells.[3] Li group reported the folate-modified multifunctional
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nano-assembling lipid for co-delivery of antitumor agent docetaxel and iSur-pDNA, a suppressor of metastatic and resistance-related protein survival.[4] Many nano-carriers
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have great potential to enhance the efficacy of chemotherapy via co-delivery of doxorubicin (DOX) and siRNA or anti-sense oligonucleotide targeting B-cell leukemia/lymphoma 2 (Bcl-2) protein. These nano-carrier systems include biodegradable nanoparticles formed via hierarchical assemblies of diblock copolymers
(PEI-PCL),[5]
cationic
liposomes,[6]
mesoporous
silica
based
nanoparticles,[7] etc. These results indicate that synergistic effect has been achieved 3
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during the treatment of cancer via combination of antitumor drug and gene therapeutics.
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The nanocarriers will have many advantages for delivery of drugs and genes as well as constructing intelligent controlled release systems based on their multi-stimuli
properties, including redox, pH, temperature, light, enzyme, magnetic field, and
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electric current. Among them, the redox-responsive carriers for delivery of drug and gene have been exclusively investigated due to the large difference of GSH
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concentration in intracellular compartments (3-10 mM) and extracellular plasma (∼2.8 µM). [8,9]
Distillation–precipitation polymerization can be considered as a novel technique to synthesize environmentally responsive polymer microspheres for various applications.
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Especially in the past a few years, there have been several researches according to their applications as carriers for drugs or genes delivery because of their uniform size and the shape in absence of any either surfactant or additive required for the
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stabilization of the polymer particles.[10-15] However, this method has only been used for the synthesis of polymer microspheres via free radical polymerization and the
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resultant products from this process are usually non-degradable. This may lead to an accumulative toxicity to human body, which limits their further applications in the biomedical field. The recent works resolve this problem via utilization of a reduction triggered crosslinker. For examples, Wang group prepared the degradable reduction/pH nanohydrogels
dual
stimuli-responsive via
poly(methacrylic
distillation-precipitation 4
acid)
(PMAA)-based
polymerization
using
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N,N-bis(acryloyl)cystamine (BAC) as crosslinkers.[16] The nanohydrogels could be facilely degraded into individual linear short chains (Mn ≈1200) in the presence of 10
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mM dithiothreitol (DTT) or glutathione (GSH). Bilalis et al reported the preparation of magnetic, pH and redox sensitive microcontainers with an efficient magnetic response and investigated their pH responsiveness, gradual and controlled collapse
In
this
paper,
poly(methacrylic
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under a highly reducing environment. [17]
acid-co-N,N-bis(acryloyl)cystamine)
non-crosslinked
poly(methacrylic
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(P(MAA-co-BAC)) microcapsules were prepared via the selective removal of the acid)
(PMAA)
core
from
the
PMAA/
P(MAA-co-BAC) core-shell microspheres, which were synthesized by a two-stage distillation precipitation polymerization. Since polyethyleneimine (PEI) has been
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considered as the most effective carrier, it provides stable pDNA or siRNA complexation and exhibits a unique ‘proton sponge effect’ for endosomal release of the nano-complexes into cytosol.[18] The (P(MAA-co-BAC) microcapsules were then
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modified with PEI on their surface through hydrogen-bonding as well as electrostatic interactions between the amino groups of the PEI chains and the carboxyl groups on
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the surface of the P(MAA-co-BAC) microcapsules. As a result, the PEI-modified (P(MAA-co-BAC) microcapsules had a large space inside and a lot of positive charges on the surface, which would enable them as efficient drug carriers for loading drug and gene transfer vectors via strong electrostatic interaction with negatively charged genes. Scheme 1 illustrates the whole procedure for the preparation of P(MAA-co-BAC) microcapsules and the further surface functionalization with PEI as 5
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well as the application for delivery of anti-cancer drug and gene.
Scheme 1 Preparation of reduction-triggered degradable P(MAA-co-BAC)/PEI
Experimental Section
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microcapsules for delivery of anti-cancer drug and gene
Materials: Acetonitrile (analytical grade, Tianjin Chemical Reagents II Co.) was
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dried over calcium hydride and purified by distillation before use. Methacrylic acid (MAA) was purchased from Tianjin Chemical Reagent II Co. and purified by vacuum
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distillation. 2, 2’-Azobisisobutyronitrile (AIBN) was provided by Chemical Factory of Nankai University and recrystallized from ethanol. Glutathione (GSH) was purchased from Shanghai Aladdin Chemistry Co. Ltd. Doxorubicin hydrochloride (DOX) was obtained from Beijing Huafeng United Technology Company. N,N-Bis(acryloyl)cystamine (BAC) was purchased from Alfa Aesar. Branched polyethyleneimine (PEI) was purchased from Aldrich with molecular mass of 10 K. 6
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Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Co.
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Preparation of PMAA/P(MAA-co-BAC) core-shell microspheres via a two-stage distillation precipitation polymerization: A typical procedure for the distillation
precipitation polymerization to afford PMAA microspheres: MAA (2.02 g, 23.5 mmol)
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and AIBN (40.0 mg, 0.244 mmol) were dissolved in 80 mL of acetonitrile in a
single-necked flask attaching with a fractionating column, Liebig condenser and a
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receiver. The flask was submerged in a heating mantle and the reaction system was heated from ambient temperature to boiling state within 20 min. Then the solvent started to be distilled and the reaction mixture became milky white after keeping boiling for 10 min. After 40 mL of acetonitrile was distilled out of the reaction system
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within 70 min, the reaction was ended. The resultant PMAA microspheres were purified by ultracentrifugation and washed three times with acetonitrile. The PMAA microspheres were dried in a vacuum oven at 50 oC till constant weight.
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The synthesis of PMAA/P(MAA-co-BAC) core-shell microspheres was performed via distillation precipitation polymerization in acetonitrile with AIBN as initiator in
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presence of PMAA microspheres as templates. A typical procedure for such polymerization is as follows: 0.703 g of PMAA microspheres were suspended in 80 mL of acetonitrile as seeds of the second-stage polymerization. Then BAC (0.176 g, 0.677 mmol), MAA (0.377 g, 4.39 mmol) and AIBN (11.5 mg, 0.070 mmol) were dissolved in the suspension. The flask was submerged in a heating mantle and the reaction mixture was heated from room temperature to the boiling state within 20 min. 7
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After the reaction system keeping the boiling state for 15 min, the acetonitrile began to be distilled off. When 40 mL of acetonitrile was distilled out of the reaction system
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within 60 min, the reaction was ended. The resultant PMAA/P(MAA-co-BAC) core-shell microspheres were purified by repeating centrifugation, decantation and resuspension in acetonitrile for three times.
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Preparation of P(MAA-co-BAC) microcapsules: To get the P(MAA-co-BAC)
microcapsules, 0.820 g of PMAA/P(MAA-co-BAC) core-shell microspheres were
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dispersed in ethanol with ultrasonic irradiation and the linear PMAA cores were selectively removed after stirring for 3 days. Then the resultant P(MAA-co-BAC) microcapsules were re-dispersed in ethanol, followed by centrifugation and ultra-sonication for three times to remove the residual linear PMAA.
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Preparation of P(MAA-co-BAC)/PEI microcapsules: 0.120 g of P(MAA-co-BAC) microcapsules and 0.700 g of PEI were dispersed in water by ultrasonication for 15 min. After the reaction system was stirred for 4 h, the resultant P(MAA-co-BAC)/PEI
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microcapsules were washed for fifteen times with deionized water by centrifugation and ultrasonication till free PEI in supernatant cannot be detected by UV-visible
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spectroscopy.
Characterization of the polymer microspheres: The morphology of the resultant polymer microspheres was determined by transmission electron microscopy (TEM) using a Hitachi, HT7700 microscope. The size and size distribution reflect the average of about 100 particles, which are calculated according to the following formulae: U = DW / Dn
k
k
D n = ∑ n i Di / ∑ n i i =1
i =1
8
k
k
i =1
i =1
Dw = ∑ ni Di4 / ∑ ni Di3
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where, U is the polydispersity index, Dn is the number-average diameter, Dw is the weight-average diameter, Di is the particle diameter of the determined microparticles.
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The dynamic size, size distribution in water and the zeta potential of the polymer microcapsules were measured by a Zeta Pals (Brookhaven Instrument Co., US) to
determine the electrophoretic mobility of the nanoparticles using deionized water as
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the solvent.
Fourier transform infrared spectra (FT-IR) were determined on a Bruker Tensor 27
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FT-IR spectrometer over potassium bromide pellet and the diffuse reflectance spectra were scanned over the range of 400-4000 cm-1.
Elemental analysis (EA) was performed on a Perkin Elmer 2400 to determine the nitrogen contents of the resultant polymer particles.
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Reduction-triggered disassembly of P(MAA-co-BAC)/PEI microcapsules was characterized via the turbidity change of P(MAA-co-BAC)/PEI microcapsules. The turbidity change of P(MAA-co-BAC)/PEI microcapsules was monitored by a
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UV-visible spectroscopy (JASCO, V570) at the wavelength of 630 nm. Briefly, 2 mg P(MAA-co-BAC)/PEI microcapsules was dispersed into 1.5 mL of phosphate buffer
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(10 mM, pH 7.4) solution in absence or presence of GSH (10 mM). The solution was kept under room temperature and the turbidity was determined by UV-vis spectroscopy at different intervals of time. The molecular weight of the degraded polymers from the microspheres in the presence of 10 mM GSH was measured by mass (MS) spectrum (Thermo Fisher, LTQ-Orbitrap Discovery).
Loading and release of DOX from P(MAA-co-BAC)/PEI microcapsules: DOX 9
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was chosen for the investigation of the drug loading and controlled release behavior of
the
P(MAA-co-BAC)/PEI
microcapsules.
Typically,
12
mg
of
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P(MAA-co-BAC)/PEI microcapsules (8 mg mL-1) and 4 mg of DOX (4 mg mL-1) were mixed together (pH value of the mixture is 6.86) and stirred for 24 h in dark at
room temperature. The DOX loaded P(MAA-co-BAC)/PEI microcapsules was
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separated from the dispersion via ultra-centrifugation. The DOX amount loaded into P(MAA-co-BAC)/PEI microcapsules was calculated by subtracting the weight of
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DOX in the supernatant from the total amount of the drug in the initial solution by a UV-vis absorption spectroscopy at 480 nm. The DOX loading capacity and encapsulation efficiency of the P(MAA-co-BAC)/PEI microcapsules were calculated according to the following equations:
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Drug loading capacity=(Wadministered dose-Wresidual dose in solution)/Wmicrocapsules *100% Encapsulation efficiency=(Wadministered dose-Wresidual dose in solution)/Wadministered dose*100% where, Wadministered
dose
is the weight of drug for loading, Wresidual dose in solution is the
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weight of residual drug in solution after loading into polymer microcapsules, and Wmicrocapsules is the weight of P(MAA-co-BAC)/PEI microcapsules for loading.
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The release behavior of DOX-loaded P(MAA-co-BAC)/PEI microcapsules was
investigated under phosphate buffer (pH 5.5 or 7.4) and in presence or absence of concentration of GSH (10 mM) at room temperature. At given time intervals, 2 mL of aliquots were withdrawn and centrifuged. The DOX concentration was determined in the supernatant by measuring the absorbance at 480 nm. For keeping a constant volume,
2
mL of fresh
buffer medium 10
together with
the
recoverable
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P(MAA-co-BAC)/PEI microcapsules after the centrifugation were added back to the reservoir after each sampling.
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In vitro transfection study: Hela cells were seeded in 24-well plates, in which a disinfected glass slide is contained at a density of 5.0×104 cells/well and cultivated with DMEM media plus 10% FBS, 100 units mL-1 penicillin and 100 µg mL-1
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streptomycin at 37 oC in a humidified atmosphere containing 5% CO2. Plasmid DNA
(0.5 µg/well) was complexed with P(MAA-co-BAC)/PEI microcapsules at different
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weight ratios of N and P and incubated for 1 h. After the cells were treated with the plasmid loaded P(MAA-co-BAC)/PEI microcapsules for 6 h in DMEM (without FBS), the medium was exchanged with DMEM with 10% FBS and the cells were further incubated for 24 h before analysis. Confocal fluorescence microscopy (Olympus,
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FV1000) were used to detect the intracellular transfection ability of green fluorescence protein (GFP) gene by P(MAA-co-BAC)/PEI microcapsules in Hela cells.
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Cytotoxicity assay: The cytotoxicities of P(MAA-co-BAC)/PEI microcapsules, PEI, free DOX and DOX loaded P(MAA-co-BAC)/PEI microcapsules against HeLa cells
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were determined by standard water-soluble tetrazolium (WST) assay using the WST-1 cell proliferation and cytotoxicity assay kit. In brief, HeLa cells were seeded in 96-well plates (5000 cells/well) using DMEM (200 µL) and incubated at 37 °C for 24 h. The medium in each well was then replaced with culture medium (200 µL) containing treatments of P(MAA-co-BAC)/PEI microcapsules, PEI, free DOX and DOX
loaded
P(MAA-co-BAC)/PEI
microcapsules. 11
The
concentration
of
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P(MAA-co-BAC)/PEI microcapsules was changed from 1 to 2000 mg L-1 and the concentration of PEI was varied from 0.01 to 100 mg L-1 as a control. The con-
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centration of free DOX as diluted with culture medium to obtain a concentration range of 0.01-100 mg L-1. The concentration of DOX loaded P(MAA-co-BAC)/PEI
microcapsules was diluted with culture medium to obtain a DOX concentration range
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of 0.01-200 mg L-1. After the incubation for 48 h, the medium in each well was
replaced with fresh medium (100 µL) and WST-1 solution (10 µL). The plate was
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incubated further for 1 h at 37 °C, which allowed viable cells to reduce WST-1 into the orange formazan crystal. The plate was read at 450 nm on a Bio-Rad microplate reader. Data are presented as the average (SD, n = 3).
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Results and discussion
Preparation of P(MAA-co-BAC)/PEI microcapsules and their structure characterizations: The PMAA microspheres were first prepared by the first-stage
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distillation precipitation polymerization according to our previous report11 and the corresponding transmission electron microscopy (TEM) image was shown in Figure
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1a. For the second-stage polymerization, the MAA monomers were strongly absorbed onto the surface of PMAA template to introduce the surface vinyl group via the efficient hydrogen-bonding interaction between the carboxyl groups on the surface of PMAA template and those on MAA comonomers as well as the amide group of BAC. The mechanism of the growth for PMAA microspheres with the aid of an efficient hydrogen-bonding interaction has been investigated in detail by distillation 12
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precipitation
polymerization
in
our
previous
work.11
The
formation
of
P(MAA-co-BAC) shell with 15 wt% of BAC crosslinking degree was designed for second-stage
copolymerization.
Figure
1b
indicated
that
all
the
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the
PMAA/P(MAA-co-BAC) core–shell microspheres had narrow size distribution without formation of secondary-initiated small particles during the second-stage
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copolymerization. In our experiments, it was found that the uniform P(MAA-co-BAC) microcapsules cannot be obtained with 10 mass% or lower BAC crosslinking degree,
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during which the too low level of the crosslinker cannot provide efficient stability for formation of the resultant microcapsules with uniform shape and structure. The driving force for removal of PMAA cores to afford the P(MAA-co-BAC) microcapsules were based on the solubility of the non-crosslinked polymer core in
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ethanol at room temperature, which was much mild and environmentally friendly. The TEM micrograph in Figure 1c indicated that P(MAA-co-BAC) microcapsules was spherical with an integrity structure. This implied that the P(MAA-co-BAC) shell
of
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layer with 26.5 nm was thick enough to support the cavity after the selective removal non-crosslinked
core
from
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microspheres.
PMAA
13
PMAA/P(MAA-co-BAC)
core-shell
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Figure 1. TEM images: a) PMAA microspheres; b) PMAA/P(MAA-co-BAC) core-shell microspheres; c) P(MAA-co-BAC) microspheres; d) P(MAA-co-BAC)/PEI
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microcapsules; e) The degrading P(MAA-co-BAC)/PEI microcapsules after addition of GSH for 5 min; f) The size and size distribution of P(MAA-co-BAC)/PEI microcapsules and g) the size and size distribution of P(MAA-co-BAC)/PEI
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microcapsules complexes with pDNA (GFP) with N/P ratio of 48:1 in water by a laser
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particle size analyzer.
The size, size distribution, the zeta potentials and elemental analysis of the resultant
polymer
microspheres
were
summarized
in
Table
1.
The
size
of
PMAA/P(MAA-co-BAC) core–shell microspheres with 222 nm was larger than that of the PMAA cores (about 169 nm), suggesting the successful formation of the P(MAA-co-BAC) shell layer. The size of the P(MAA-co-BAC) microcapsules with 14
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the shell thickness of 26.5 nm was decreased from 222 to 201 nm, which was originated from a certain extent collapse of the resultant P(MAA-co-BAC) shell layer
PMAA/P(MAA-co-BAC)
core-shell
microspheres,
and
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after the selective removal of PMAA cores. The polydispersity indexes (U) of PMAA, P(MAA-co-BAC)
microcapsules from TEM characterization were all less than 1.05 as summarized in
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Table 1, which implied that all the polymer microspheres remained narrow dispersion during the whole preparation.
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The size of P(MAA-co-BAC)/PEI microspheres was remarkably increased to 339 nm with a monodisperse index of 1.0283 after the branched PEI modification on the surface of the P(MAA-co-BAC) microcapsules, as shown by TEM image in Figure 1d. The dynamic average diameter of the resultant P(MAA-co-BAC)/PEI microcapsules
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in water was 704.8 nm determined by a laser particle analyzer, which was significantly larger than that (339 nm) from TEM characterization. This implied that the P(MAA-co-BAC)/PEI microcapsules were highly hydrophilic, as the dynamic
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diameter was determined in aqueous suspension as a fully swollen state. The dynamic average size distribution of P(MAA-co-BAC)/PEI microcapsules was narrow with a
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PDI of 0.141 with a single peak as shown in Figure 1f, which implied that the PEI-functionalized nanoparticles did not aggregate in the distilled water. The successful formation of P(MAA-co-BAC)/PEI microcapsules were
confirmed further by the elemental analysis as shown in Table 1. The trace nitrogen content of PMAA cores was 0.63%, which was originated from the residual cyano group from the AIBN initiator during the distillation precipitation polymerization. 15
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The nitrogen content of PMAA/P(MAA-co-BAC) core–shell microspheres was considerably increased to 1.76%. After removing the PMAA cores, the nitrogen
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contents of P(MAA-co-BAC) microcapsules was increased further to 5.45%. The nitrogen content increase of the particles containing BAC component was mainly originated from the imide groups in cross-linking agent. After the PEI-modification,
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the nitrogen content of P(MAA-co-BAC)/PEI microcapsules was significantly
increased to 18.86%, which confirmed the successful PEI modification onto the
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surface of polymer microspheres. All these results implied that the PEI contents in P(MAA-co-BAC)/PEI microcapsules was around 37.84%, as the theoretical nitrogen content of PEI was calculated as 35.44%.
The Zeta-potential of the P(MAA-co-BAC) microspheres was –29.22 mV as
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summarized in Table 1. The positive ion polymer PEI were then modified on the surface of these negatively charged P(MAA-co-BAC) microcapsules via the electrostatic interactions. During the PEI functionalization, the residual PEI chains
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were cleaned away from the system after 15 times of centrifugation and decantation, which was confirmed by absence of absorption at 230 nm of UV-vis spectrum for the
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decanted solution corresponding to the typical peak of PEI component. As a result, the zeta-potentials of P(MAA-co-BAC)/PEI microcapsules were +31.09 mV after surface modification. As a result, a hydrophilic polymer (PEI) with the positive charges on surface of the particles not only provides the ability for carrying and disassembling gene molecules but also improves their stability in water. After P(MAA-co-BAC)/PEI microcapsules carrying with pDNA, the size of the resultant 16
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polyplexes was 715.8 nm with a PDI of 0.260, as shown in Figure 1g, which was kept as a stable dispersion even for 72 h. These implied that all the polymer microcapsules
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were physically stable, which were similar to the results reported by Chen et al [19] and Hao et al. [20]
microspheres Dn (nm)
169
PMAA/P(MAA-co-BAC)
222
U
(nm)
Shell
Zeta
N
thickness
potential
Content
(nm)
(mV)
(%)
171
1.0077
-
0.63
224
1.0090
-
1.76
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PMAA
Dw
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Entry
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Table 1 The size, size distribution, Zeta-potential and elemental analysis of polymer
201
211
1.0510
26.5
–29.22
5.45
P(MAA-co-BAC)/PEI microcapsules
339
348
1.0283
-
+31.09
18.86
EP
P(MAA-co-BAC) microcapsules
The chemical structure of polymer microspheres formed after each step was
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characterized by FT-IR spectroscopy. The FT-IR spectrum of PMAA had a strong peak at 1707 cm-1 assigning to the vibration of the carbonyl group in PMAA segment. The typical amide I and II bands of BAC could be observed at 1642 and 1544 cm-1 in the spectrum of PMAA/P(MAA-co-BAC) core–shell microspheres. After the linear PMAA cores were selectively removed, the amide I and II bands of BAC in P(MAA-co-BAC) microcapsules obviously increased and slightly shifted to 1646 and 17
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1545 cm-1, respectively. The peak at 1710 cm-1 attributing to the carbonyl group of PMAA segment in P(MAA-co-BAC) microcapsules disappeared after modification of
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PEI. A strong peak at 3286 cm-1 assigning to the vibration of the imine group and the peaks at 2988 and 2833 cm-1 corresponding to the stretching modes of methylene
group in PEI segment existed in the spectrum of P(MAA-co-BAC)/PEI microcapsules.
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This suggested that P(MAA-co-BAC) microcapsules were formed and the positive
PEI had been modified on the surface of them, which provided a further opportunity
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for the utilization of such PEI-modified microspheres as gene transfer vectors.
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Figure 2 FT-IR spectra of polymer microspheres: a) PMAA seeds; b) PMAA/P(MAA-co-BAC)
core–shell
microspheres;
c)
P(MAA-co-BAC)
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microcapsules; d) P(MAA-co-BAC)/PEI microcapsules.
Reduction-triggered disassembly of P(MAA-co-BAC)/PEI microcapsules: The UV-vis spectroscopy was used to monitor the degradation behavior of the hollow P(MAA-co-BAC)/PEI microspheres. The white color of the microspheres solution was quickly changed to lighter and became a clear solution within 20 min when 10 18
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mM GSH (Figure 3a) was added in the suspension as a reducing agent. The degradability of the hollow P(MAA-co-BAC)/PEI microspheres were clearly reflected
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via combination of the decreased turbidity of the microspheres solution as illustrated in Figure 3 together with the degraded P(MAA-co-BAC)/PEI species as shown by TEM micrograph in Figure 1e. Upon the addition of the PBS buffer containing GSH,
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the P(MAA-co-BAC)/PEI microcapsules were degraded gradually with a much lower rate within about 48 h (Figure 3b), which may be due to the salting out effect of the
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functionalized microspheres to form some aggregates in PBS buffer solution. The much higher turbidity of the suspension of P(MAA-co-BAC)/PEI microcapsules in BSF buffer (Figure 3b) than that in distilled water (Figure 3a) implied that it may be more difficult for GSH molecule to contact the surface of P(MAA-co-BAC)/PEI in
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the former case. However, the P(MAA-co-BAC)/PEI microcapsules were very stable in the solution in absence of GSH, which were proven by a stable turbidity with a straight line as shown in Figures 3a,b. Obviously, the decomposition of BAC
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crosslinker with presence of GSH as a reductant was the main driving force for the degradation of hollow P(MAA-co-BAC)/PEI microspheres in these cases. Figure 3c
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indicated that the molecular weight of the most reduction triggered degradable P(MAA-co-BAC) species was below 800 with a wide and random distribution ranging from tens to around 800 measured by MS spectrum. All these data demonstrated
that
the
P(MAA-co-BAC)
microcapsules
from
the
distillation-precipitation polymerization possessed the uniform polymer structure and the
BAC crosslinkers were randomly and homogeneously distributed in 19
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P(MAA-co-BAC) network. As a result, the oligomers with low molecular weight were afforded after the complete decomposition of the P(MAA-co-BAC)
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microcapsules with GSH as a reducatant. This would provide the possible utilization of these nanocapsules as degradable carriers to be facilely discharged from the body
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in the form of oligomers with low and random distribution of molecular weight.
c
Figure 3 Investigations on the degradation of P(MAA-co-BAC)/PEI microcapsules via a reduction-responsive process (10 mM GSH) by turbidity measurements in different media: a) Distilled water; b) PBS butter (pH 7.4); c) The MS spectrum of the degraded P(MAA-co-BAC) microcapsules, in which the peak at about 307 is corresponding to GSH. 20
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Loading and release of DOX from P(MAA-co-BAC)/PEI microcapsules: To evaluate the potential application of P(MAA-co-BAC)/PEI microcapsules as a drug
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carrier, DOX was used as the model molecule to perform the loading and releasing test. The loading capacity of DOX incorporated into the P(MBAAm-co-MAA)
microcapsules was calculated from the difference between the residual DOX
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concentration remaining in the solution during the loading process and the initial concentrations. As a result, the DOX loading content of P(MAA-co-BAC)/PEI
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microcapsules was 31.1% and the encapsulation efficiency was 93% when an initial concentration of DOX was 1.6 mg mL-1 used for such a loading process. The controlled release property of drug from P(MAA-co-BAC)/PEI microcapsules was investigated under different pH values in the presence or absence of a reducing
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agent, GSH. On the one hand, DOX cumulative release from P(MAA-co-BAC)/PEI microcapsules was about 12 wt% at pH 7.4 after 25 h in absence of GSH as shown in Figure 4, which confirmed the stability of the P(MAA-co-BAC)/PEI microcapsules
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with a low premature drug release in a physiological pH condition. As a comparison, the cumulative release of DOX was considerably increased to 48 wt% in presence of
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10 mM GSH under a physiologic pH at the same time scale. The significant increase of
DOX
release
rate
was
mainly
attributed
to
the
decomposition
of
P(MAA-co-BAC)/PEI microcapsules in presence of GSH. Since the cell nucleus contained a high concentration of GSH, the controlled release
of the
P(MAA-co-BAC)/PEI microcapsules with GSH-trigger has an advantage of drug release in the cell. On the other hand, the DOX drug release behavior was faster in an 21
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acidic pH condition (pH 5.0) than that in a physiological pH. For example, in absence of GSH, DOX released from the P(MAA-co-BAC)/PEI microcapsules in pH 5.0 was
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about 17 wt% in 25 h, which was higher than that (12 wt%) under the neutral condition within the same period. The re-protonation of the amino group in DOX at
lower pH environments may be the driving force for a slightly faster release than that
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at pH 7. This was much similar to the loading and releasing behavior of DOX with pH-responsive microcapsules in our previous work11 and DOX-loaded systems with
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the pH-responsive polymer micelle as a carrier. 21
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Figure 4 The release of DOX from P(MAA-co-BAC)/PEI microcapsules at different pH values and presence or absence of 10 mM GSH
In vitro gene transfection study: P(MAA-co-BAC)/PEI microcapsules would find the potential application as gene transfer vectors via the effective interaction between pDNA encoded with GFP gene and P(MAA-co-BAC)/PEI microcapsules, which was 22
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investigated by the electrophoresis assays. As shown in Figure 5, when the N/P ratio of polymer microspheres to pDNA reached 12, nearly all the pDNA chains were
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retained in the gel well as observed in column 3, indicating all pDNA chains were bound to P(MAA-co-BAC)/PEI microcapsules in this circumstance. These results demonstrated that there was a strong and efficient interaction between pDNA chains
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and P(MAA-co-BAC)/PEI microcapsules.
5.
Agarose
gel
electrophoresis
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Figure
of
bare
pDNA,
mixtures
of
P(MAA-co-BAC)/PEI microcapsules and pDNA (GFP) at different N/P ratios. 1)
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30:1; 2) 22:1; 3) 12:1; 4) 3:1; 5) 0.3:1; 6) 0.03:1; 7) 0. Here, atoms of N in N/P refers to N content in PEI modified on the surface of P(MAA-co-BAC) microcapsules result
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from elemental analysis. Each sample was incubated at room temperature for 1 h before electrophoresis.
The
transfection
ability
of
the
reduction-triggered
degradable
P(MAA-co-BAC)/PEI microcapsules was compared to that of PEI as a control in HeLa cells. Figure 6 showed the confocal fluorescence images of the natural cultured Hela cells in the presence of GFP loaded P(MAA-co-BAC)/PEI microcapsules 23
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(Figure 6b-e) or GFP loaded PEI (Figure 6f) at 37 oC for 24 h, in which the green luminescence in some cases of transfecting by P(MAA-co-BAC)/PEI microcapsules
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were obviously stronger than that by PEI. The transfection experiments were carried out at a series of weight ratios of N and P to determine the optimal weight ratio of
P(MAA-co-BAC)/PEI microcapsules for gene delivery as illustrated in Figures 6b-e.
Almost
no
green
luminescence
can
be
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The remarkable difference of fluorescence level in all cases was clearly observed. observed
in
the
absence
of
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P(MAA-co-BAC)/PEI microcapsules. While P(MAA-co-BAC)/PEI microcapsules were demonstrated good transfection efficiency above a N/P ratio of 168:1 as shown in Figure 6e, for which the final concentration of P(MAA-co-BAC)/PEI microcapsules to Hela cells was 140 mg L-1. This was likely due to the ability of
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P(MAA-co-BAC)/PEI microcapsules and GFP to form more compact polyplexes and more residual positive charges on the polylexes to bind the negatively charged proteoglycans on the outer face of the cell membrane. These results indicate that the
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pDNA.
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P(MAA-co-BAC)/PEI microcapsules greatly enhance the transfection efficiency of
24
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Figure 6 Confocal fluorescence images of GFP transfected Hela cells using P(MAA-co-BAC) /PEI microcapsules (a-e) and using PEI with weight ratio of N and P 1:1 as a control. Plasmid DNA (0.5 µg/well) was complexed with P(MAA-co-BAC)/PEI microcapsules at different weight ratios of N and P: a) Without P(MAA-co-BAC)/PEI microspheres as control; b) N/P=24:1; c) N/P=72:1; d) N/P=120:1; e) N/P=168:1. The scale bar is 50 µm. 25
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Cytotoxicity assay: The optimal polycationic polymer for gene delivery carrier should combine high transfection efficiency with low cytotoxicity. Meanwhile, its
in biomedical fields. Therefore, the cytotoxicity of
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potential toxicity as a drug carrier material also should be concerned for its further use P(MAA-co-BAC)/PEI
microcapsules themselves was evaluated at first. From the result of WST assay, the
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P(MAA-co-BAC)/PEI microcapsules didn’t show the obvious toxicity even at high concentration of 1000 mg L-1 as shown in Figure 7a, suggesting their low levels of
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cytotoxicity. While for PEI, a synthetic cationic polymer used as gene carrier, it showed very high cytotoxicity even at a low concentration of 100 mg mL-1 as shown in Figure 7b. The IC50 values for the P(MAA-co-BAC)/PEI microcapsules and PEI were 1709.5 µg/mL and 80.4 µg/mL, respectively. As a result, the cytotoxicity of PEI
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was significantly decreased after incorporation onto the P(MAA-co-BAC) microcapsules.
In order to evaluate the cytotoxicity of DOX loaded P(MAA-co-BAC)/PEI
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microcapsules against tumor cells, DOX was loaded into P(MAA-co-BAC)/PEI microcapsules with a capacity of 311 µg mg-1. The relative viability of HeLa cells
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treated with free DOX was used as a control. From the cytotoxicity assay results, the IC50 values for DOX loaded P(MAA-co-BAC)/PEI microcapsules and free DOX were 77.1 µg/mL and 16.8 µg/mL, respectively. As shown in Figures 7c-d, free DOX behaves the higher toxicity than DOX loaded P(MAA-co-BAC)/PEI microcapsules to Hela cells at the DOX concentration higher than 10 mg L-1, which may be due to the partially inefficient release of DOX from P(MAA-co-BAC)/PEI microcapsules. DOX 26
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loaded P(MAA-co-BAC)/PEI microcapsules can kill nearly about 80% Hela cells at the DOX concentration of 200 mg L-1. The concentration of P(MAA-co-BAC)/PEI
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microcapsules under this case is 643 mg L-1, much lower than their cytotoxic dose.
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Figure 7 Relative cellular viability: a) P(MAA-co-BAC)/PEI microcapsules; b) Free
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PEI; c) Free DOX; d) DOX loaded P(MAA-co-BAC)/PEI microcapsules.
Conclusions
In summary, the P(MAAm-co-BAC)/PEI microcapsules with average size of 339 nm were
prepared
by
the
modification
of
PEI
on
the
surface
of
the
P(MAAm-co-BAC)/PEI microcapsules. The structure of P(MAAm-co-BAC)/PEI microcapsules were confirmed by the results from TEM observation, FT-IR spectra, 27
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laser particle size analyzer and elemental analysis. The investigation of the in-vitro loading
and
release
behavior
demonstrated
that
P(MAAm-co-BAC)/PEI
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microcapsules toward DOX drug possessed good loading capacity (as high as 311 µg mg-1). The release of DOX from P(MAAm-co-BAC)/PEI microcapsule as a reservoir
was highly dependent on presence/absence of GSH and the pH values in the
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environment. In vitro gene transfection study indicate that the P(MAA-co-BAC)/PEI
microcapsules can transfect pDNA into Hela cells with high efficiency. The
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cotytoxicity assays results indicated that the P(MAA-co-BAC)/PEI microcapsules had no obvious toxicity even at the high concentration of 1000 mg L-1 to the incubated cells and their toxicity remarkably lower than that of PEI. These reduction-triggered degradable P(MAA-co-BAC)/PEI microcapsules are promising in combination cancer
Acknowledgments
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therapy with therapeutic siRNA and chemotherapeutics.
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This work was supported by the NSFC (Grant Nos. 51103106, 21174065, 21374049), Tianjin Science Technology Research Funds of China (11JCYBJC02100) and
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PCSIRT (IRT1257).
28
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References [1] Greco F, Vicent MJ. Adv Drug Delivery Rev 2009;61:1203–1213. [2] Wang Y, Gao S, Ye WH, Yoon HS, Yang YY. Nat Mater 2006;5:791–796.
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[3] Zhu CH, Jung S, Luo SB, Meng FH, Zhu XL, Park TG, Zhong ZY. Biomater 2010;31:2408–2416.
[4] Xu ZH, Zhang ZW, Chen Y, Chen LL, Lin LP, Li YP. Biomater 2010;31:916–922.
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[5] Cao N, Cheng D, Zou SY, Ai H, Gao JM, Shuai XT. Biomater 2011;32:2222–2232.
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[6] Saad M, Garbuzenko O, Minko T. Nanomedicine 2008;3:761–776.
[7] Chen AM, Zhang M, Wei DG, Stueber D, Taratula O, Minko T, He HX. Small 2009;5:2673–2677.
[8] Ottaviano FG, Handy DE, Loscalzo J. Circ J 2008;72:1–16. [9] Saito G, Swanson JA, Lee KD. Adv Drug Delivery Rev 2003;55:199–20 5.
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[10] Li GL, Yang XY, Wang B, Wang JY, Yang XL. Polymer 2008;49:3436–3443. [11] Yang XY, Chen LT, Huang B, Bai F, Yang XL. Polymer 2009;50:3556–3563. [12] Yang XY, Chen L, Han B, Yang XL, Duan HQ. Polymer 2010;51:2533–2539.
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[13] Ma WF, Xu SA, Li JM, Guo J, Lin Y, Wang CC. J Polym Sci Part A Polym Chem 2011;49:2725–2733.
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[14] Bilalis P, Efthimiadou EK, Chatzipavlidis A, Boukos N, Kordas G. Polym Int 2012;61:888–894.
[15] Gao CY, Zhang H, Wu M, Liu Y, Wu YP, Yang XL, Feng XZ. Polym Chem 2012;3:1168–1173.
[16] Pan YJ, Chen YY, Wang DR, Wei C, Guo J, Lu DR, Chu CC, Wang CC. Biomater 2012;33:6570–6579. [17] Bilalis P, Chatzipavlidis A, Tziveleka LA, Boukos N, Kordas G. J Mater Chem 29
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2012;22:13451–13454. [18] Boussif O, Lezoualch F, Zanta MA, Mergny MD, Scherman D, Demeneix B,
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Behr JP. Proc Natl Acad Sci USA 1995;92:7297–7301. [19] Tian HY, Chen XS, Lin H, Deng C, Zhang PB, Wei Y, Jing XB. Chem Eur J 2006;12:4305–4312.
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[20] Wei GC, Yan MM, Dong RH, Wang D, Zhou XZ, Chen JF, Hao JC. Chem Eur J 2012;18:14708–14716.
[21] Shuai XT, Ai H, Nasongkla N, Kim S, Gao JM. J Control Release
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S0032-3861(13)01086-0
DOI:
10.1016/j.polymer.2013.11.035
Reference:
JPOL 16623
To appear in:
Polymer
Received Date: 8 August 2013 Revised Date:
25 October 2013
Accepted Date: 23 November 2013
Please cite this article as: Feng F, Li R, Zhang Q, Wang Y, Yang X, Duan H, Yang X, Preparation of Reduction-triggered Degradable Microcapsules for Intracellular Delivery of anti-Cancer Drug and Gene, Polymer (2013), doi: 10.1016/j.polymer.2013.11.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Preparation of reduction triggered P(MAA-co-BAC)/PEI microcapsules for delivery of anti-cancer drug and gene.
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Preparation
of
Reduction-triggered
Degradable
Microcapsules for Intracellular Delivery of anti-Cancer
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Drug and Gene
Fuli Feng,[a] Rongrong Li,[a] Qingyun Zhang,[b] Yinsong Wang,[a] Xiaoying Yang,*[a]
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Hongquan Duan,[a] and Xinlin Yang*[c]
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[a] Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), Basic Medical Research Center, School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China [b] Department of Chemistry, Logistics University of Chinese People’s Armed Police,
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Tianjin 300162, PR China
[c] Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, Nankai University, Tianjin 300071, PR China
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* Corresponding authors.
Phone: +86 (22) 23502023. Fax: +86 (22) 23503510.
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E-mail: [email protected] (X. Y. Yang); [email protected] (X. L. Yang).
1
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Abstract:
The
reduction-triggered
degradable
acid-co-N,N-bis(acryloyl)cystamine)/polyethyleneimine
poly(methacrylic
(P(MAA-co-BAC)/PEI)
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microcapsules were prepared by distillation–precipitation polymerization for delivery of anti-cancer drug and gene. N,N-bis(acryloyl)cystamine (BAC) as a crosslinker containing a disulfide bond can be triggered by reductive agents, such as glutathione
SC
(GSH) and dithiothreitol (DTT), to endow the functional microcapsules with reduction-triggered drug release. The P(MAA-co-BAC)/PEI microcapsules were
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characterized by transmission electron microscopy (TEM), Fourier-transform infrared spectra (FT-IR), laser particle size analyzer and elemental analysis. The degradable behavior of microcapsules was investigated by analysis of UV-vis spectroscopy. The controlled drug release behavior for P(MAA-co-BAC)/PEI microcapsules was
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strongly dependent on the absence/presence of GSH and the pH values with doxorubicin hydrochloride (DOX) as a model drug molecule. The in vitro gene transfection ability was evaluated by Hela cells with the transfection of plasmid DNA
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(pDNA) encoded with green fluorescent protein (GFP) and the transfection efficiency was determined by confocal fluorescence microscopy. Furthermore, the cytotoxicities
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of (P(MAA-co-BAC)/PEI) microcapsules before and after loading of DOX were assessed via WST-1 assay. The P(MAA-co-BAC)/PEI microcapsules provide the potential novel vectors for delivery of drugs and genes, promising for future applications in anticancer drug and gene combined therapy. Keywords: Reduction triggered degradable microcapsules; Distillation-precipitation polymerization; Drug delivery; Gene delivery. 2
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Introduction Combination therapy has shown several potential advantages (e.g., synergistic
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effects and reversal of drug resistance) and may prove more effective than single drug therapy.[1] It is essential for the sequential delivery of drugs and nucleic acids, which
can enhance their therapeutic effects.[2] For example, the combination of
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chemotherapy and RNAi therapy by sequential delivery of anticancer drug and siRNA can reverse the tumor multidrug resistance. A variety of organic/inorganic
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nanomaterials have been used as delivery vehicles to develop effective therapeutic modalities. A recent advance in nanomedicine-mediated cancer therapy is the development of multifunctional carriers for jointly delivering a chemotherapeutic drug and a genetic material such as pDNA or siRNA. Zhong et al prepared the
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biodegradable cationic micelles from PDMAEMA–PCL–PDMAEMA triblock copolymers and applied for the delivery of MDR-1-targeted siRNA and paclitaxel into cancer cells.[3] Li group reported the folate-modified multifunctional
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nano-assembling lipid for co-delivery of antitumor agent docetaxel and iSur-pDNA, a suppressor of metastatic and resistance-related protein survival.[4] Many nano-carriers
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have great potential to enhance the efficacy of chemotherapy via co-delivery of doxorubicin (DOX) and siRNA or anti-sense oligonucleotide targeting B-cell leukemia/lymphoma 2 (Bcl-2) protein. These nano-carrier systems include biodegradable nanoparticles formed via hierarchical assemblies of diblock copolymers
(PEI-PCL),[5]
cationic
liposomes,[6]
mesoporous
silica
based
nanoparticles,[7] etc. These results indicate that synergistic effect has been achieved 3
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during the treatment of cancer via combination of antitumor drug and gene therapeutics.
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The nanocarriers will have many advantages for delivery of drugs and genes as well as constructing intelligent controlled release systems based on their multi-stimuli
properties, including redox, pH, temperature, light, enzyme, magnetic field, and
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electric current. Among them, the redox-responsive carriers for delivery of drug and gene have been exclusively investigated due to the large difference of GSH
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concentration in intracellular compartments (3-10 mM) and extracellular plasma (∼2.8 µM). [8,9]
Distillation–precipitation polymerization can be considered as a novel technique to synthesize environmentally responsive polymer microspheres for various applications.
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Especially in the past a few years, there have been several researches according to their applications as carriers for drugs or genes delivery because of their uniform size and the shape in absence of any either surfactant or additive required for the
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stabilization of the polymer particles.[10-15] However, this method has only been used for the synthesis of polymer microspheres via free radical polymerization and the
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resultant products from this process are usually non-degradable. This may lead to an accumulative toxicity to human body, which limits their further applications in the biomedical field. The recent works resolve this problem via utilization of a reduction triggered crosslinker. For examples, Wang group prepared the degradable reduction/pH nanohydrogels
dual
stimuli-responsive via
poly(methacrylic
distillation-precipitation 4
acid)
(PMAA)-based
polymerization
using
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N,N-bis(acryloyl)cystamine (BAC) as crosslinkers.[16] The nanohydrogels could be facilely degraded into individual linear short chains (Mn ≈1200) in the presence of 10
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mM dithiothreitol (DTT) or glutathione (GSH). Bilalis et al reported the preparation of magnetic, pH and redox sensitive microcontainers with an efficient magnetic response and investigated their pH responsiveness, gradual and controlled collapse
In
this
paper,
poly(methacrylic
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under a highly reducing environment. [17]
acid-co-N,N-bis(acryloyl)cystamine)
non-crosslinked
poly(methacrylic
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(P(MAA-co-BAC)) microcapsules were prepared via the selective removal of the acid)
(PMAA)
core
from
the
PMAA/
P(MAA-co-BAC) core-shell microspheres, which were synthesized by a two-stage distillation precipitation polymerization. Since polyethyleneimine (PEI) has been
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considered as the most effective carrier, it provides stable pDNA or siRNA complexation and exhibits a unique ‘proton sponge effect’ for endosomal release of the nano-complexes into cytosol.[18] The (P(MAA-co-BAC) microcapsules were then
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modified with PEI on their surface through hydrogen-bonding as well as electrostatic interactions between the amino groups of the PEI chains and the carboxyl groups on
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the surface of the P(MAA-co-BAC) microcapsules. As a result, the PEI-modified (P(MAA-co-BAC) microcapsules had a large space inside and a lot of positive charges on the surface, which would enable them as efficient drug carriers for loading drug and gene transfer vectors via strong electrostatic interaction with negatively charged genes. Scheme 1 illustrates the whole procedure for the preparation of P(MAA-co-BAC) microcapsules and the further surface functionalization with PEI as 5
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well as the application for delivery of anti-cancer drug and gene.
Scheme 1 Preparation of reduction-triggered degradable P(MAA-co-BAC)/PEI
Experimental Section
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microcapsules for delivery of anti-cancer drug and gene
Materials: Acetonitrile (analytical grade, Tianjin Chemical Reagents II Co.) was
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dried over calcium hydride and purified by distillation before use. Methacrylic acid (MAA) was purchased from Tianjin Chemical Reagent II Co. and purified by vacuum
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distillation. 2, 2’-Azobisisobutyronitrile (AIBN) was provided by Chemical Factory of Nankai University and recrystallized from ethanol. Glutathione (GSH) was purchased from Shanghai Aladdin Chemistry Co. Ltd. Doxorubicin hydrochloride (DOX) was obtained from Beijing Huafeng United Technology Company. N,N-Bis(acryloyl)cystamine (BAC) was purchased from Alfa Aesar. Branched polyethyleneimine (PEI) was purchased from Aldrich with molecular mass of 10 K. 6
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Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Co.
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Preparation of PMAA/P(MAA-co-BAC) core-shell microspheres via a two-stage distillation precipitation polymerization: A typical procedure for the distillation
precipitation polymerization to afford PMAA microspheres: MAA (2.02 g, 23.5 mmol)
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and AIBN (40.0 mg, 0.244 mmol) were dissolved in 80 mL of acetonitrile in a
single-necked flask attaching with a fractionating column, Liebig condenser and a
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receiver. The flask was submerged in a heating mantle and the reaction system was heated from ambient temperature to boiling state within 20 min. Then the solvent started to be distilled and the reaction mixture became milky white after keeping boiling for 10 min. After 40 mL of acetonitrile was distilled out of the reaction system
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within 70 min, the reaction was ended. The resultant PMAA microspheres were purified by ultracentrifugation and washed three times with acetonitrile. The PMAA microspheres were dried in a vacuum oven at 50 oC till constant weight.
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The synthesis of PMAA/P(MAA-co-BAC) core-shell microspheres was performed via distillation precipitation polymerization in acetonitrile with AIBN as initiator in
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presence of PMAA microspheres as templates. A typical procedure for such polymerization is as follows: 0.703 g of PMAA microspheres were suspended in 80 mL of acetonitrile as seeds of the second-stage polymerization. Then BAC (0.176 g, 0.677 mmol), MAA (0.377 g, 4.39 mmol) and AIBN (11.5 mg, 0.070 mmol) were dissolved in the suspension. The flask was submerged in a heating mantle and the reaction mixture was heated from room temperature to the boiling state within 20 min. 7
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After the reaction system keeping the boiling state for 15 min, the acetonitrile began to be distilled off. When 40 mL of acetonitrile was distilled out of the reaction system
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within 60 min, the reaction was ended. The resultant PMAA/P(MAA-co-BAC) core-shell microspheres were purified by repeating centrifugation, decantation and resuspension in acetonitrile for three times.
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Preparation of P(MAA-co-BAC) microcapsules: To get the P(MAA-co-BAC)
microcapsules, 0.820 g of PMAA/P(MAA-co-BAC) core-shell microspheres were
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dispersed in ethanol with ultrasonic irradiation and the linear PMAA cores were selectively removed after stirring for 3 days. Then the resultant P(MAA-co-BAC) microcapsules were re-dispersed in ethanol, followed by centrifugation and ultra-sonication for three times to remove the residual linear PMAA.
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Preparation of P(MAA-co-BAC)/PEI microcapsules: 0.120 g of P(MAA-co-BAC) microcapsules and 0.700 g of PEI were dispersed in water by ultrasonication for 15 min. After the reaction system was stirred for 4 h, the resultant P(MAA-co-BAC)/PEI
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microcapsules were washed for fifteen times with deionized water by centrifugation and ultrasonication till free PEI in supernatant cannot be detected by UV-visible
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spectroscopy.
Characterization of the polymer microspheres: The morphology of the resultant polymer microspheres was determined by transmission electron microscopy (TEM) using a Hitachi, HT7700 microscope. The size and size distribution reflect the average of about 100 particles, which are calculated according to the following formulae: U = DW / Dn
k
k
D n = ∑ n i Di / ∑ n i i =1
i =1
8
k
k
i =1
i =1
Dw = ∑ ni Di4 / ∑ ni Di3
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where, U is the polydispersity index, Dn is the number-average diameter, Dw is the weight-average diameter, Di is the particle diameter of the determined microparticles.
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The dynamic size, size distribution in water and the zeta potential of the polymer microcapsules were measured by a Zeta Pals (Brookhaven Instrument Co., US) to
determine the electrophoretic mobility of the nanoparticles using deionized water as
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the solvent.
Fourier transform infrared spectra (FT-IR) were determined on a Bruker Tensor 27
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FT-IR spectrometer over potassium bromide pellet and the diffuse reflectance spectra were scanned over the range of 400-4000 cm-1.
Elemental analysis (EA) was performed on a Perkin Elmer 2400 to determine the nitrogen contents of the resultant polymer particles.
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Reduction-triggered disassembly of P(MAA-co-BAC)/PEI microcapsules was characterized via the turbidity change of P(MAA-co-BAC)/PEI microcapsules. The turbidity change of P(MAA-co-BAC)/PEI microcapsules was monitored by a
EP
UV-visible spectroscopy (JASCO, V570) at the wavelength of 630 nm. Briefly, 2 mg P(MAA-co-BAC)/PEI microcapsules was dispersed into 1.5 mL of phosphate buffer
AC C
(10 mM, pH 7.4) solution in absence or presence of GSH (10 mM). The solution was kept under room temperature and the turbidity was determined by UV-vis spectroscopy at different intervals of time. The molecular weight of the degraded polymers from the microspheres in the presence of 10 mM GSH was measured by mass (MS) spectrum (Thermo Fisher, LTQ-Orbitrap Discovery).
Loading and release of DOX from P(MAA-co-BAC)/PEI microcapsules: DOX 9
ACCEPTED MANUSCRIPT
was chosen for the investigation of the drug loading and controlled release behavior of
the
P(MAA-co-BAC)/PEI
microcapsules.
Typically,
12
mg
of
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P(MAA-co-BAC)/PEI microcapsules (8 mg mL-1) and 4 mg of DOX (4 mg mL-1) were mixed together (pH value of the mixture is 6.86) and stirred for 24 h in dark at
room temperature. The DOX loaded P(MAA-co-BAC)/PEI microcapsules was
SC
separated from the dispersion via ultra-centrifugation. The DOX amount loaded into P(MAA-co-BAC)/PEI microcapsules was calculated by subtracting the weight of
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DOX in the supernatant from the total amount of the drug in the initial solution by a UV-vis absorption spectroscopy at 480 nm. The DOX loading capacity and encapsulation efficiency of the P(MAA-co-BAC)/PEI microcapsules were calculated according to the following equations:
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Drug loading capacity=(Wadministered dose-Wresidual dose in solution)/Wmicrocapsules *100% Encapsulation efficiency=(Wadministered dose-Wresidual dose in solution)/Wadministered dose*100% where, Wadministered
dose
is the weight of drug for loading, Wresidual dose in solution is the
EP
weight of residual drug in solution after loading into polymer microcapsules, and Wmicrocapsules is the weight of P(MAA-co-BAC)/PEI microcapsules for loading.
AC C
The release behavior of DOX-loaded P(MAA-co-BAC)/PEI microcapsules was
investigated under phosphate buffer (pH 5.5 or 7.4) and in presence or absence of concentration of GSH (10 mM) at room temperature. At given time intervals, 2 mL of aliquots were withdrawn and centrifuged. The DOX concentration was determined in the supernatant by measuring the absorbance at 480 nm. For keeping a constant volume,
2
mL of fresh
buffer medium 10
together with
the
recoverable
ACCEPTED MANUSCRIPT
P(MAA-co-BAC)/PEI microcapsules after the centrifugation were added back to the reservoir after each sampling.
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In vitro transfection study: Hela cells were seeded in 24-well plates, in which a disinfected glass slide is contained at a density of 5.0×104 cells/well and cultivated with DMEM media plus 10% FBS, 100 units mL-1 penicillin and 100 µg mL-1
SC
streptomycin at 37 oC in a humidified atmosphere containing 5% CO2. Plasmid DNA
(0.5 µg/well) was complexed with P(MAA-co-BAC)/PEI microcapsules at different
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weight ratios of N and P and incubated for 1 h. After the cells were treated with the plasmid loaded P(MAA-co-BAC)/PEI microcapsules for 6 h in DMEM (without FBS), the medium was exchanged with DMEM with 10% FBS and the cells were further incubated for 24 h before analysis. Confocal fluorescence microscopy (Olympus,
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FV1000) were used to detect the intracellular transfection ability of green fluorescence protein (GFP) gene by P(MAA-co-BAC)/PEI microcapsules in Hela cells.
EP
Cytotoxicity assay: The cytotoxicities of P(MAA-co-BAC)/PEI microcapsules, PEI, free DOX and DOX loaded P(MAA-co-BAC)/PEI microcapsules against HeLa cells
AC C
were determined by standard water-soluble tetrazolium (WST) assay using the WST-1 cell proliferation and cytotoxicity assay kit. In brief, HeLa cells were seeded in 96-well plates (5000 cells/well) using DMEM (200 µL) and incubated at 37 °C for 24 h. The medium in each well was then replaced with culture medium (200 µL) containing treatments of P(MAA-co-BAC)/PEI microcapsules, PEI, free DOX and DOX
loaded
P(MAA-co-BAC)/PEI
microcapsules. 11
The
concentration
of
ACCEPTED MANUSCRIPT
P(MAA-co-BAC)/PEI microcapsules was changed from 1 to 2000 mg L-1 and the concentration of PEI was varied from 0.01 to 100 mg L-1 as a control. The con-
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centration of free DOX as diluted with culture medium to obtain a concentration range of 0.01-100 mg L-1. The concentration of DOX loaded P(MAA-co-BAC)/PEI
microcapsules was diluted with culture medium to obtain a DOX concentration range
SC
of 0.01-200 mg L-1. After the incubation for 48 h, the medium in each well was
replaced with fresh medium (100 µL) and WST-1 solution (10 µL). The plate was
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incubated further for 1 h at 37 °C, which allowed viable cells to reduce WST-1 into the orange formazan crystal. The plate was read at 450 nm on a Bio-Rad microplate reader. Data are presented as the average (SD, n = 3).
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Results and discussion
Preparation of P(MAA-co-BAC)/PEI microcapsules and their structure characterizations: The PMAA microspheres were first prepared by the first-stage
EP
distillation precipitation polymerization according to our previous report11 and the corresponding transmission electron microscopy (TEM) image was shown in Figure
AC C
1a. For the second-stage polymerization, the MAA monomers were strongly absorbed onto the surface of PMAA template to introduce the surface vinyl group via the efficient hydrogen-bonding interaction between the carboxyl groups on the surface of PMAA template and those on MAA comonomers as well as the amide group of BAC. The mechanism of the growth for PMAA microspheres with the aid of an efficient hydrogen-bonding interaction has been investigated in detail by distillation 12
ACCEPTED MANUSCRIPT
precipitation
polymerization
in
our
previous
work.11
The
formation
of
P(MAA-co-BAC) shell with 15 wt% of BAC crosslinking degree was designed for second-stage
copolymerization.
Figure
1b
indicated
that
all
the
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the
PMAA/P(MAA-co-BAC) core–shell microspheres had narrow size distribution without formation of secondary-initiated small particles during the second-stage
SC
copolymerization. In our experiments, it was found that the uniform P(MAA-co-BAC) microcapsules cannot be obtained with 10 mass% or lower BAC crosslinking degree,
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during which the too low level of the crosslinker cannot provide efficient stability for formation of the resultant microcapsules with uniform shape and structure. The driving force for removal of PMAA cores to afford the P(MAA-co-BAC) microcapsules were based on the solubility of the non-crosslinked polymer core in
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ethanol at room temperature, which was much mild and environmentally friendly. The TEM micrograph in Figure 1c indicated that P(MAA-co-BAC) microcapsules was spherical with an integrity structure. This implied that the P(MAA-co-BAC) shell
of
EP
layer with 26.5 nm was thick enough to support the cavity after the selective removal non-crosslinked
core
from
AC C
microspheres.
PMAA
13
PMAA/P(MAA-co-BAC)
core-shell
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SC
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ACCEPTED MANUSCRIPT
Figure 1. TEM images: a) PMAA microspheres; b) PMAA/P(MAA-co-BAC) core-shell microspheres; c) P(MAA-co-BAC) microspheres; d) P(MAA-co-BAC)/PEI
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microcapsules; e) The degrading P(MAA-co-BAC)/PEI microcapsules after addition of GSH for 5 min; f) The size and size distribution of P(MAA-co-BAC)/PEI microcapsules and g) the size and size distribution of P(MAA-co-BAC)/PEI
EP
microcapsules complexes with pDNA (GFP) with N/P ratio of 48:1 in water by a laser
AC C
particle size analyzer.
The size, size distribution, the zeta potentials and elemental analysis of the resultant
polymer
microspheres
were
summarized
in
Table
1.
The
size
of
PMAA/P(MAA-co-BAC) core–shell microspheres with 222 nm was larger than that of the PMAA cores (about 169 nm), suggesting the successful formation of the P(MAA-co-BAC) shell layer. The size of the P(MAA-co-BAC) microcapsules with 14
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the shell thickness of 26.5 nm was decreased from 222 to 201 nm, which was originated from a certain extent collapse of the resultant P(MAA-co-BAC) shell layer
PMAA/P(MAA-co-BAC)
core-shell
microspheres,
and
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after the selective removal of PMAA cores. The polydispersity indexes (U) of PMAA, P(MAA-co-BAC)
microcapsules from TEM characterization were all less than 1.05 as summarized in
SC
Table 1, which implied that all the polymer microspheres remained narrow dispersion during the whole preparation.
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The size of P(MAA-co-BAC)/PEI microspheres was remarkably increased to 339 nm with a monodisperse index of 1.0283 after the branched PEI modification on the surface of the P(MAA-co-BAC) microcapsules, as shown by TEM image in Figure 1d. The dynamic average diameter of the resultant P(MAA-co-BAC)/PEI microcapsules
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in water was 704.8 nm determined by a laser particle analyzer, which was significantly larger than that (339 nm) from TEM characterization. This implied that the P(MAA-co-BAC)/PEI microcapsules were highly hydrophilic, as the dynamic
EP
diameter was determined in aqueous suspension as a fully swollen state. The dynamic average size distribution of P(MAA-co-BAC)/PEI microcapsules was narrow with a
AC C
PDI of 0.141 with a single peak as shown in Figure 1f, which implied that the PEI-functionalized nanoparticles did not aggregate in the distilled water. The successful formation of P(MAA-co-BAC)/PEI microcapsules were
confirmed further by the elemental analysis as shown in Table 1. The trace nitrogen content of PMAA cores was 0.63%, which was originated from the residual cyano group from the AIBN initiator during the distillation precipitation polymerization. 15
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The nitrogen content of PMAA/P(MAA-co-BAC) core–shell microspheres was considerably increased to 1.76%. After removing the PMAA cores, the nitrogen
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contents of P(MAA-co-BAC) microcapsules was increased further to 5.45%. The nitrogen content increase of the particles containing BAC component was mainly originated from the imide groups in cross-linking agent. After the PEI-modification,
SC
the nitrogen content of P(MAA-co-BAC)/PEI microcapsules was significantly
increased to 18.86%, which confirmed the successful PEI modification onto the
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surface of polymer microspheres. All these results implied that the PEI contents in P(MAA-co-BAC)/PEI microcapsules was around 37.84%, as the theoretical nitrogen content of PEI was calculated as 35.44%.
The Zeta-potential of the P(MAA-co-BAC) microspheres was –29.22 mV as
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summarized in Table 1. The positive ion polymer PEI were then modified on the surface of these negatively charged P(MAA-co-BAC) microcapsules via the electrostatic interactions. During the PEI functionalization, the residual PEI chains
EP
were cleaned away from the system after 15 times of centrifugation and decantation, which was confirmed by absence of absorption at 230 nm of UV-vis spectrum for the
AC C
decanted solution corresponding to the typical peak of PEI component. As a result, the zeta-potentials of P(MAA-co-BAC)/PEI microcapsules were +31.09 mV after surface modification. As a result, a hydrophilic polymer (PEI) with the positive charges on surface of the particles not only provides the ability for carrying and disassembling gene molecules but also improves their stability in water. After P(MAA-co-BAC)/PEI microcapsules carrying with pDNA, the size of the resultant 16
ACCEPTED MANUSCRIPT
polyplexes was 715.8 nm with a PDI of 0.260, as shown in Figure 1g, which was kept as a stable dispersion even for 72 h. These implied that all the polymer microcapsules
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were physically stable, which were similar to the results reported by Chen et al [19] and Hao et al. [20]
microspheres Dn (nm)
169
PMAA/P(MAA-co-BAC)
222
U
(nm)
Shell
Zeta
N
thickness
potential
Content
(nm)
(mV)
(%)
171
1.0077
-
0.63
224
1.0090
-
1.76
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PMAA
Dw
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Entry
SC
Table 1 The size, size distribution, Zeta-potential and elemental analysis of polymer
201
211
1.0510
26.5
–29.22
5.45
P(MAA-co-BAC)/PEI microcapsules
339
348
1.0283
-
+31.09
18.86
EP
P(MAA-co-BAC) microcapsules
The chemical structure of polymer microspheres formed after each step was
AC C
characterized by FT-IR spectroscopy. The FT-IR spectrum of PMAA had a strong peak at 1707 cm-1 assigning to the vibration of the carbonyl group in PMAA segment. The typical amide I and II bands of BAC could be observed at 1642 and 1544 cm-1 in the spectrum of PMAA/P(MAA-co-BAC) core–shell microspheres. After the linear PMAA cores were selectively removed, the amide I and II bands of BAC in P(MAA-co-BAC) microcapsules obviously increased and slightly shifted to 1646 and 17
ACCEPTED MANUSCRIPT
1545 cm-1, respectively. The peak at 1710 cm-1 attributing to the carbonyl group of PMAA segment in P(MAA-co-BAC) microcapsules disappeared after modification of
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PEI. A strong peak at 3286 cm-1 assigning to the vibration of the imine group and the peaks at 2988 and 2833 cm-1 corresponding to the stretching modes of methylene
group in PEI segment existed in the spectrum of P(MAA-co-BAC)/PEI microcapsules.
SC
This suggested that P(MAA-co-BAC) microcapsules were formed and the positive
PEI had been modified on the surface of them, which provided a further opportunity
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for the utilization of such PEI-modified microspheres as gene transfer vectors.
EP
Figure 2 FT-IR spectra of polymer microspheres: a) PMAA seeds; b) PMAA/P(MAA-co-BAC)
core–shell
microspheres;
c)
P(MAA-co-BAC)
AC C
microcapsules; d) P(MAA-co-BAC)/PEI microcapsules.
Reduction-triggered disassembly of P(MAA-co-BAC)/PEI microcapsules: The UV-vis spectroscopy was used to monitor the degradation behavior of the hollow P(MAA-co-BAC)/PEI microspheres. The white color of the microspheres solution was quickly changed to lighter and became a clear solution within 20 min when 10 18
ACCEPTED MANUSCRIPT
mM GSH (Figure 3a) was added in the suspension as a reducing agent. The degradability of the hollow P(MAA-co-BAC)/PEI microspheres were clearly reflected
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via combination of the decreased turbidity of the microspheres solution as illustrated in Figure 3 together with the degraded P(MAA-co-BAC)/PEI species as shown by TEM micrograph in Figure 1e. Upon the addition of the PBS buffer containing GSH,
SC
the P(MAA-co-BAC)/PEI microcapsules were degraded gradually with a much lower rate within about 48 h (Figure 3b), which may be due to the salting out effect of the
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functionalized microspheres to form some aggregates in PBS buffer solution. The much higher turbidity of the suspension of P(MAA-co-BAC)/PEI microcapsules in BSF buffer (Figure 3b) than that in distilled water (Figure 3a) implied that it may be more difficult for GSH molecule to contact the surface of P(MAA-co-BAC)/PEI in
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the former case. However, the P(MAA-co-BAC)/PEI microcapsules were very stable in the solution in absence of GSH, which were proven by a stable turbidity with a straight line as shown in Figures 3a,b. Obviously, the decomposition of BAC
EP
crosslinker with presence of GSH as a reductant was the main driving force for the degradation of hollow P(MAA-co-BAC)/PEI microspheres in these cases. Figure 3c
AC C
indicated that the molecular weight of the most reduction triggered degradable P(MAA-co-BAC) species was below 800 with a wide and random distribution ranging from tens to around 800 measured by MS spectrum. All these data demonstrated
that
the
P(MAA-co-BAC)
microcapsules
from
the
distillation-precipitation polymerization possessed the uniform polymer structure and the
BAC crosslinkers were randomly and homogeneously distributed in 19
ACCEPTED MANUSCRIPT
P(MAA-co-BAC) network. As a result, the oligomers with low molecular weight were afforded after the complete decomposition of the P(MAA-co-BAC)
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microcapsules with GSH as a reducatant. This would provide the possible utilization of these nanocapsules as degradable carriers to be facilely discharged from the body
AC C
EP
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SC
in the form of oligomers with low and random distribution of molecular weight.
c
Figure 3 Investigations on the degradation of P(MAA-co-BAC)/PEI microcapsules via a reduction-responsive process (10 mM GSH) by turbidity measurements in different media: a) Distilled water; b) PBS butter (pH 7.4); c) The MS spectrum of the degraded P(MAA-co-BAC) microcapsules, in which the peak at about 307 is corresponding to GSH. 20
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Loading and release of DOX from P(MAA-co-BAC)/PEI microcapsules: To evaluate the potential application of P(MAA-co-BAC)/PEI microcapsules as a drug
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carrier, DOX was used as the model molecule to perform the loading and releasing test. The loading capacity of DOX incorporated into the P(MBAAm-co-MAA)
microcapsules was calculated from the difference between the residual DOX
SC
concentration remaining in the solution during the loading process and the initial concentrations. As a result, the DOX loading content of P(MAA-co-BAC)/PEI
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microcapsules was 31.1% and the encapsulation efficiency was 93% when an initial concentration of DOX was 1.6 mg mL-1 used for such a loading process. The controlled release property of drug from P(MAA-co-BAC)/PEI microcapsules was investigated under different pH values in the presence or absence of a reducing
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agent, GSH. On the one hand, DOX cumulative release from P(MAA-co-BAC)/PEI microcapsules was about 12 wt% at pH 7.4 after 25 h in absence of GSH as shown in Figure 4, which confirmed the stability of the P(MAA-co-BAC)/PEI microcapsules
EP
with a low premature drug release in a physiological pH condition. As a comparison, the cumulative release of DOX was considerably increased to 48 wt% in presence of
AC C
10 mM GSH under a physiologic pH at the same time scale. The significant increase of
DOX
release
rate
was
mainly
attributed
to
the
decomposition
of
P(MAA-co-BAC)/PEI microcapsules in presence of GSH. Since the cell nucleus contained a high concentration of GSH, the controlled release
of the
P(MAA-co-BAC)/PEI microcapsules with GSH-trigger has an advantage of drug release in the cell. On the other hand, the DOX drug release behavior was faster in an 21
ACCEPTED MANUSCRIPT
acidic pH condition (pH 5.0) than that in a physiological pH. For example, in absence of GSH, DOX released from the P(MAA-co-BAC)/PEI microcapsules in pH 5.0 was
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about 17 wt% in 25 h, which was higher than that (12 wt%) under the neutral condition within the same period. The re-protonation of the amino group in DOX at
lower pH environments may be the driving force for a slightly faster release than that
SC
at pH 7. This was much similar to the loading and releasing behavior of DOX with pH-responsive microcapsules in our previous work11 and DOX-loaded systems with
EP
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the pH-responsive polymer micelle as a carrier. 21
AC C
Figure 4 The release of DOX from P(MAA-co-BAC)/PEI microcapsules at different pH values and presence or absence of 10 mM GSH
In vitro gene transfection study: P(MAA-co-BAC)/PEI microcapsules would find the potential application as gene transfer vectors via the effective interaction between pDNA encoded with GFP gene and P(MAA-co-BAC)/PEI microcapsules, which was 22
ACCEPTED MANUSCRIPT
investigated by the electrophoresis assays. As shown in Figure 5, when the N/P ratio of polymer microspheres to pDNA reached 12, nearly all the pDNA chains were
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retained in the gel well as observed in column 3, indicating all pDNA chains were bound to P(MAA-co-BAC)/PEI microcapsules in this circumstance. These results demonstrated that there was a strong and efficient interaction between pDNA chains
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and P(MAA-co-BAC)/PEI microcapsules.
5.
Agarose
gel
electrophoresis
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Figure
of
bare
pDNA,
mixtures
of
P(MAA-co-BAC)/PEI microcapsules and pDNA (GFP) at different N/P ratios. 1)
EP
30:1; 2) 22:1; 3) 12:1; 4) 3:1; 5) 0.3:1; 6) 0.03:1; 7) 0. Here, atoms of N in N/P refers to N content in PEI modified on the surface of P(MAA-co-BAC) microcapsules result
AC C
from elemental analysis. Each sample was incubated at room temperature for 1 h before electrophoresis.
The
transfection
ability
of
the
reduction-triggered
degradable
P(MAA-co-BAC)/PEI microcapsules was compared to that of PEI as a control in HeLa cells. Figure 6 showed the confocal fluorescence images of the natural cultured Hela cells in the presence of GFP loaded P(MAA-co-BAC)/PEI microcapsules 23
ACCEPTED MANUSCRIPT
(Figure 6b-e) or GFP loaded PEI (Figure 6f) at 37 oC for 24 h, in which the green luminescence in some cases of transfecting by P(MAA-co-BAC)/PEI microcapsules
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were obviously stronger than that by PEI. The transfection experiments were carried out at a series of weight ratios of N and P to determine the optimal weight ratio of
P(MAA-co-BAC)/PEI microcapsules for gene delivery as illustrated in Figures 6b-e.
Almost
no
green
luminescence
can
be
SC
The remarkable difference of fluorescence level in all cases was clearly observed. observed
in
the
absence
of
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P(MAA-co-BAC)/PEI microcapsules. While P(MAA-co-BAC)/PEI microcapsules were demonstrated good transfection efficiency above a N/P ratio of 168:1 as shown in Figure 6e, for which the final concentration of P(MAA-co-BAC)/PEI microcapsules to Hela cells was 140 mg L-1. This was likely due to the ability of
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P(MAA-co-BAC)/PEI microcapsules and GFP to form more compact polyplexes and more residual positive charges on the polylexes to bind the negatively charged proteoglycans on the outer face of the cell membrane. These results indicate that the
AC C
pDNA.
EP
P(MAA-co-BAC)/PEI microcapsules greatly enhance the transfection efficiency of
24
EP
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ACCEPTED MANUSCRIPT
AC C
Figure 6 Confocal fluorescence images of GFP transfected Hela cells using P(MAA-co-BAC) /PEI microcapsules (a-e) and using PEI with weight ratio of N and P 1:1 as a control. Plasmid DNA (0.5 µg/well) was complexed with P(MAA-co-BAC)/PEI microcapsules at different weight ratios of N and P: a) Without P(MAA-co-BAC)/PEI microspheres as control; b) N/P=24:1; c) N/P=72:1; d) N/P=120:1; e) N/P=168:1. The scale bar is 50 µm. 25
ACCEPTED MANUSCRIPT
Cytotoxicity assay: The optimal polycationic polymer for gene delivery carrier should combine high transfection efficiency with low cytotoxicity. Meanwhile, its
in biomedical fields. Therefore, the cytotoxicity of
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potential toxicity as a drug carrier material also should be concerned for its further use P(MAA-co-BAC)/PEI
microcapsules themselves was evaluated at first. From the result of WST assay, the
SC
P(MAA-co-BAC)/PEI microcapsules didn’t show the obvious toxicity even at high concentration of 1000 mg L-1 as shown in Figure 7a, suggesting their low levels of
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cytotoxicity. While for PEI, a synthetic cationic polymer used as gene carrier, it showed very high cytotoxicity even at a low concentration of 100 mg mL-1 as shown in Figure 7b. The IC50 values for the P(MAA-co-BAC)/PEI microcapsules and PEI were 1709.5 µg/mL and 80.4 µg/mL, respectively. As a result, the cytotoxicity of PEI
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was significantly decreased after incorporation onto the P(MAA-co-BAC) microcapsules.
In order to evaluate the cytotoxicity of DOX loaded P(MAA-co-BAC)/PEI
EP
microcapsules against tumor cells, DOX was loaded into P(MAA-co-BAC)/PEI microcapsules with a capacity of 311 µg mg-1. The relative viability of HeLa cells
AC C
treated with free DOX was used as a control. From the cytotoxicity assay results, the IC50 values for DOX loaded P(MAA-co-BAC)/PEI microcapsules and free DOX were 77.1 µg/mL and 16.8 µg/mL, respectively. As shown in Figures 7c-d, free DOX behaves the higher toxicity than DOX loaded P(MAA-co-BAC)/PEI microcapsules to Hela cells at the DOX concentration higher than 10 mg L-1, which may be due to the partially inefficient release of DOX from P(MAA-co-BAC)/PEI microcapsules. DOX 26
ACCEPTED MANUSCRIPT
loaded P(MAA-co-BAC)/PEI microcapsules can kill nearly about 80% Hela cells at the DOX concentration of 200 mg L-1. The concentration of P(MAA-co-BAC)/PEI
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microcapsules under this case is 643 mg L-1, much lower than their cytotoxic dose.
EP
Figure 7 Relative cellular viability: a) P(MAA-co-BAC)/PEI microcapsules; b) Free
AC C
PEI; c) Free DOX; d) DOX loaded P(MAA-co-BAC)/PEI microcapsules.
Conclusions
In summary, the P(MAAm-co-BAC)/PEI microcapsules with average size of 339 nm were
prepared
by
the
modification
of
PEI
on
the
surface
of
the
P(MAAm-co-BAC)/PEI microcapsules. The structure of P(MAAm-co-BAC)/PEI microcapsules were confirmed by the results from TEM observation, FT-IR spectra, 27
ACCEPTED MANUSCRIPT
laser particle size analyzer and elemental analysis. The investigation of the in-vitro loading
and
release
behavior
demonstrated
that
P(MAAm-co-BAC)/PEI
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microcapsules toward DOX drug possessed good loading capacity (as high as 311 µg mg-1). The release of DOX from P(MAAm-co-BAC)/PEI microcapsule as a reservoir
was highly dependent on presence/absence of GSH and the pH values in the
SC
environment. In vitro gene transfection study indicate that the P(MAA-co-BAC)/PEI
microcapsules can transfect pDNA into Hela cells with high efficiency. The
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cotytoxicity assays results indicated that the P(MAA-co-BAC)/PEI microcapsules had no obvious toxicity even at the high concentration of 1000 mg L-1 to the incubated cells and their toxicity remarkably lower than that of PEI. These reduction-triggered degradable P(MAA-co-BAC)/PEI microcapsules are promising in combination cancer
Acknowledgments
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therapy with therapeutic siRNA and chemotherapeutics.
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This work was supported by the NSFC (Grant Nos. 51103106, 21174065, 21374049), Tianjin Science Technology Research Funds of China (11JCYBJC02100) and
AC C
PCSIRT (IRT1257).
28
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References [1] Greco F, Vicent MJ. Adv Drug Delivery Rev 2009;61:1203–1213. [2] Wang Y, Gao S, Ye WH, Yoon HS, Yang YY. Nat Mater 2006;5:791–796.
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[3] Zhu CH, Jung S, Luo SB, Meng FH, Zhu XL, Park TG, Zhong ZY. Biomater 2010;31:2408–2416.
[4] Xu ZH, Zhang ZW, Chen Y, Chen LL, Lin LP, Li YP. Biomater 2010;31:916–922.
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[5] Cao N, Cheng D, Zou SY, Ai H, Gao JM, Shuai XT. Biomater 2011;32:2222–2232.
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