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Article
Temperature/pH/Enzyme Triple-Responsive Cationic Protein/
PAA-b-PNIPAAm Nanogels for Controlled Anticancer Drug and
Photosensitizer Delivery Against Multidrug Resistant Breast Cancer Cells
Trong-Ming Don, Kun-Ying Lu, Li-Jie Lin, Chun-Hua Hsu, Jui-Yu Wu, and Fwu-Long Mi
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00737 • Publication Date (Web): 23 Oct 2017
Downloaded from http://pubs.acs.org on October 25, 2017
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Molecular Pharmaceutics is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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Molecular Pharmaceutics
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Temperature/pH/Enzyme Triple-Responsive Cationic Protein/PAA-b-PNIPAAm
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Nanogels for Controlled Anticancer Drug and Photosensitizer Delivery
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Against Multidrug Resistant Breast Cancer Cells
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Trong-ming Don1‡, Kun-Ying Lu2,3‡, Li-Jie Lin1,
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Chun-Hua Hsu4, Jui-Yu Wu,3,5 Fwu-Long Mi3,5,6*
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1.
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Department of Chemical and Materials Engineering, Tamkang University, New
Taipei City 25137, Taiwan.
2.
Graduate Institute of Biomedical Materials and Tissue Engineering, College of
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Biomedical Engineering, Taipei Medical University, Taipei City 11031, Taiwan,
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R.O.C.
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3.
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University, Taipei 11031, Taiwan
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Department of Agricultural Chemistry, National Taiwan University, Taipei 10617,
Taiwan.
5.
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Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical
Department of Biochemistry and Molecular Cell Biology, School of medicine,
Taipei Medical University, Taipei 11031, Taiwan
6.
Graduate Institute of Nanomedicine and Medical Engineering, College of
Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan
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*Corresponding author
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Fwu-Long Mi, PhD
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Professor
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Department of Biochemistry and Molecular Cell Biology,
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School of medicine, Taipei Medical University
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Taipei City, Taiwan 110
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Fax: 886-2-2735-6689
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E-mail: flmi530326@tmu.edu.tw
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* To whom correspondence should be addressed: flmi530326@tmu.edu.tw (Dr. F. L. Mi)
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‡ These authors contributed equally to this work.
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ABSTRACT
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The tumor microenvironments are often acidic and overexpress specific enzymes. In
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this work, we synthesized a poly(AA-b-NIPAAm) copolymer (PAA-b-PNIPAAm)
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using a reversible addition-fragmentation chain transfer (RAFT) polymerization
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method. PAA-b-PNIPAAm and a cationic protein (protamine) were self-assembled
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into nanogels, which effectively reduced the cytotoxicity of protamine. The
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protamine/PAA-b-PNIPAAm nanogels were responsive to the stimuli including
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temperature, pH and enzyme due to disaggregation of PAA-b-PNIPAAm, change in
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random coil/α-helix conformation of protamine, and enzymatic hydrolysis of the
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protein. Changing the pH from 7.4 to a lowered pHe (6.5-5.0) resulted in an increase
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in mean particle size and smartly converted surface charge from negative to positive.
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The cationic nanogels easily passed through the cell membrane and enhanced
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intracellular localization and accumulation of doxorubicin-loaded nanogels in
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multidrug resistant MCF-7/ADR breast cancer cells. Cold shock treatment triggered
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rapid intracellular release of doxorubicin against P-glycoprotein (Pgp)-mediated drug
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efflux, showing significantly improved anticancer efficacy as compared with free
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DOX. Furthermore, the nanogels were able to carry a rose bengal photosensitizer and
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caused significant damage to the multidrug resistant cancer cells under irradiation.
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The cationic nanogels with stimuli-responsive properties show promise as drug carrier
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for chemotherapy and photodynamic therapy (PDT) against cancers.
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Keywords: N-isopropylacrylamide, protamine, nanogels, pH-responsive, thermo-responsive,
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enzymatic digestion
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■ INTRODUCTION
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Despite a variety of anticancer drugs have been developed, cancer therapy remains
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one of the greatest challenges in modern medicine because chemoresistance becomes
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a major problem that limits treatment with a success. Tumor-specific stimuli can
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trigger drug release from nanocarriers to overcome this limitation. The temperature of
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tumor microenvironment can be easily adjusted and controlled by applying an
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external heat source or cold shock while pH changes and enzymatic digestion can
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spontaneously occur in cancer cells or tumors.1-3 Polymeric micelles and nanoparticles
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are easily modified with stimuli-responsive properties, allowing drug release
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controlled by temperature, pH, enzymes and other biological active molecules.4-7
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Poly(N-isopropylacrylamide) (PNIPAAm) is a thermo-responsive polymer that
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exhibits a conformational change at a lower critical solution temperature (LCST)
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around 32°C. Especially, PNIPAAm-based drug delivery systems with thermo- and/or
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pH-responsive properties have been developed by different methods.8-13 Our
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previously prepared nanogels based on poly(AA-co-NIPAAm) copolymers have
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temperature and/or pH -responsive properties.14-16 However, the application of the
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poly(AA-co-NIPAAm) nanogels for anticancer drug delivery was limited due to its
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high solubility in physiological saline, leading to fast release of loaded drugs from the
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carriers during circulating in blood. Our previous study also prepared protamine-based
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nanoparticles that have enzyme-responsive and charge conversion properties.17
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Protamine is a cationic polypeptide which is rich in arginine and basic amino acids.
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The guanidino group of arginine residues in protamine has a pKa value near 13, thus
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protamine is usually positively charged at most pH levels. Protamine has been used as
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a cell-penetrating peptide which can effectively carry drugs, nucleic acids (DNA,
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siRNA and miRNA) and proteins into cells.18,19 It can be digested by a matrix serine
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protease (MSP) trypsin which is overexpressed in some cancer tissues, which has
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been found to be associated with tumor growth, invasion, and metastasis.20,21
In
this
work,
we
synthesized
a
poly(AA-b-NIPAAm)
copolymer
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(PAA-b-PNIPAAm), via a reversible addition-fragmentation chain transfer (RAFT)
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polymerization method. PAA-b-PNIPAAm greatly reduced the cytotoxicity of
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protamine after formation of protamine/PAA-b-PNIPAAm complex nanogels. The
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pH/thermo/enzyme-responsive properties were evaluated in vitro. The nanogels were
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negatively charged at pH 7.4 (blood circulation) and positively charged at 6.0 and 5.0
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(tumor microenvironments). The nanogel was also sensitive to trypsin, an enzyme
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expressed in several human cancer cells and tumors, and was responsive to cold shock
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treatments which could be used in cryotherapy to trigger doxorubicin (DOX) release
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in acid environments (endosomes/lysosomes) for intracellular anticancer drug
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delivery. Furthermore, the nanogels loaded with a photosensitizer for in vitro
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photodynamic therapy (PDT) against DOX-resistant MCF-7/ADR breast cancer cells
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were investigated.
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■ EXPERIMENTAL SECTION
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Materials and Reagents. N-isopropyl acrylamide (NIPAAm) and acrylic acid
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(AA) were purchased from Acros Organics, Belgium. NIPAAm was purified by its
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dissolution in hexane at 50 °C and then fitered to remove the impurity. It underwent
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recrystalization from the filtrate as placed in freezer, followed by filtration to remove
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the solvent. AA was purified by distilliation. Only the distillate in the middle stage
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was collected. 2,2′-Azobisisobutyronitrile (AIBN) was supplied from UniRegion
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Bio-Tech, Taiwan. It was purified by recrystallization in its methanol solution at -20
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°C.
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protamine, doxorubicin (DOX) and rose bengal (RB) were all purchased from
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Sigma–Aldrich (Louis, MO, USA). All other chemicals were at least analytical grade
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and used without further purfication.
2-(Dodecylsulfanylthiocarbonylsulfanyl)-2-methylpropionic
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(DMP),
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RAFT Polymerization of PAA-b-PNIPAAm Block Copolymer. A living free
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radical polymerization method, RAFT, was adopted to synthesize PAA-b-PNIPAAm
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block copolymer following previous studies with a slight modification (Schilli et al.,
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2004; Kulkarni et al., 2006). In the first stage, AA was dissolved in 20 mL methanol
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and placed in a reaction vessel equipped with a condenser and the addition funnel.
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The reactor was purged with nitrogen and heaed to 75 °C. The DMP as chain transfer
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agent for the RAFT polymerization and the AIBN as an initiator, both pre-dissolved
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in methanol, were added into the reactor to start polymerization. The molar feed ratio
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of AA: DMP: AIBN was 77.45: 1.0: 0.1. The reaction was continued for 1.5 h. To
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remove the un-reacted monomer and initiator, the reactor was cooled down by
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immersion in the ice-water bath. Cold ether was then added with 20-time volume of
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methanol to precipitate the reaction product, i.e. PAA-CTA, as the new macro-chain
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transfer agent. The precipitate was separated by centrifugation at 4 °C for 15 min. It
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was dissolved in methanol again and repeated the procedure of precipitation in ether
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and centrifugation. Finally, it was dried at 80 °C to obtain the yellowish PAA-CTA.
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In the second stage, NIPAAm was also dissolved in methanol and added with the
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PAA-CTA and AIBN followed by the same reaction procedure in the first-stage
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RAFT polymerization. Yet, the reaction time was increased to 3 h. The molar feed
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ratio of NIPAAm: PAA-CTA: AIBN was 160: 1: 0.1. The reaction is shown in the
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following Fig. 1A.
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Chemical structure of the synthesized PAA-b-PNIPAAm block copolymer was
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analyzed by Fourier transform infrared (FTIR) spectrometer (iS10, ThermoFisher,
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USA), nulcear magnetic resonance (NMR) spectrometer (DMX-600, Bruker,
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Germany) and gel permeation chromatography (GPC, Jasco PU-2080 plus pump and
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Shodex RI-101 detector, Japan). To obtain the FTIR spectrum, dried sample was
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ground with KBr powder and the mixture was then pressed into transparency disk. It
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was scanned 32 times from 4000 to 400 cm-1 with a resolution of 4 cm-1 in the IR
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transmittance mode. Characteristic absorption peaks for the PAA-b-PNIPAAm (cm-1):
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3435-2500 ((C=O)-OH), 3354 (NH), 2976 and 2877 (-CH3), 2937 and 1459 (>CH2),
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1732 (C=O), 1652 (C=O, amide I), 1548 (NH, amide II), 1388 and 1368 (CH3,
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isopropyl group), 1073 (C=S). For NMR analysis, PAA-b-PNIPAAm was dissolved
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in deuterated DMSO. Its NMR spectrum was then obtained for analysis.
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Characteristic resonance peaks (ppm): 0.95 (-CH3 in the terminal DMP), 1.10-1.45
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(>CH2 in the DMP, -CH3 in the isopropyl groups of both DMP and PNIPAAm),
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1.50-2.15 (>CH2 in the PAA and PNIPAAm blocks), 2.30-2.55 (>CH- in the PAA and
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PNIPAAm blocks), 3.85 (>CH- in the isopropyl groups of PNIPAAm).
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Preparation of Protamine/PAA-b-PNIPAAm Nanogel. PAA-b-PNIPAAm
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nanogels were prepared by directly heating the PAA-b-PNIPAAm aqueous solution (1
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mg/mL, 25 mL) at 37 °C above its LCST for 10 mins. On the other hand,
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protamine/PAA-b-PNIPAAm nanogels were prepared via a polyelectrolyte complex
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method by mixing the PAA-b-PNIPAAm(aq) (1 mg/mL) with the protamine(aq) (2
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mg/mL) at different weight ratios and at different temperatures (25 °C and 37 °C)
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using a pipette (Table 1). The surface morphology and particle size of nanogels in
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their dry state were observed by using a transmission electron microscope (TEM,
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H-7650, Hitachi, Japan). Dilute nanogel solutions were applied to carbon-coated
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copper grids and then dried. Measurements of particle size and surface charge of
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PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm nanogels in solutions were
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performed using a Zetasizer Nano (Malvern, UK). The FTIR spectra were also
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recorded to determine the interaction between protamine and PAA-b-PNIPAAm.
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LCST of PAA-b-PNIPAAm Solutions. A UV/Visible spectrophotometer
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(ThermoFisher, Helios α, USA) equipped with a temperature controller was used to
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investigate the lower critical solution temperature (LCST) of the nanogel solutions at
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different pH values. The copolymer solutions were prepared at a concentration of 1
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mg/mL and their transmittances were recorded at the wavelength of 500 nm at
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temperatures ranging from 10 °C to 60 °C. Values for the LCST were then determined
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at the differential peak temperatures of transmittance curves. The LCST values were
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also determined by differential scanning calorimeter (Diamond DSC, Perkin Elmer,
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USA) at a heating rate of 1 °C/min.
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Characterization of pH/Thermo/Enzyme-Responsive Properties. In order to
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investigate the pH/thermo-responsive properties of the PAA-b-PNIPAAm and
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protamine/PAA-b-PNIPAAm, both nanogels(aq) in distilled water were separately
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added into several dissolution mediums of pH 5.0 (acetate buffer), 6.0 (HCl +
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phosphate buffer) and 7.4 (phosphate-buffered saline, PBS) at different volume ratios.
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Two different temperatures at 4 °C and 37 °C were tested. A series of experiments to
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characterize the fluorescence recovery, optical transmittance and the change in
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particle size distribution were then performed at the selected pH values and
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temperatures. Procedures for measuring optical transmittance and particle size
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distribution were described in the previous section. For the experiment of
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fluorescence recovery, 5-aminofluorescein-labeled PAA-b-PNIPAAm was prepared
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as described in the following. 5-aminofluorescein (1.5 mg) was first dissolved in
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DMSO, which together with EDC(3.0 mg)/NHS(4.5 mg) were added into 5.0 mL of
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PAA-b-PNIPAAm solution (5.0 mg) at pH 4.8. The coupling reaction was allowed to
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continue for 8 h at 35 °C. The 5-aminofluorescein-labeled PAA-b-PNIPAAm was
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purified by dialysis against DI water in the dark. The fluorescence spectra of
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5-aminofluorescein-labeled PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm
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nanogels at different pH values and temperatures were recorded using a fluorescence
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spectrophotometer equipped with a temperature-controllable circulating water bath
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(Hitachi,
F-7000,
Japan)
to
examine
assembly-disassembly
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disassembly behavior, the nanogels were added into a phosphate buffer (pH 6.5)
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containing trypsin at a concentration of 0.2 mg/mL. At different time intervals, the
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optical transmittance was measured by using the above mentioned method.
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Doxorubicin
Loading
nanogels.
and
To
Page 8 of 37
Release.
investigate
Both
the
enzyme-triggered
PAA-b-PNIPAAm
and
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protamine/PAA-b-PNIPAAm nanogels loaded with doxorubicin (DOX) were
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prepared to determine their drug release behaviors. DOX (25 mg) was first dissolved
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in PAA-b-PNIPAAm aqueous solution (1 mg/mL, 25 mL) at 10 °C, and then the
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premixed DOX/PAA-b-PNIPAAm solutions were heated to 37 °C with and without
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the addition of protamine solution (protamine to PAA-b-PNIPAAm weight ratio= 2:9)
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to
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protamine/PAA-b-PNIPAAm. After centrifugation, drug loading efficiency of the
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nanogel was determined by measuring the optical transmittance at 480 nm in the
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supernatant using a Perkin Elmer EnSpire 2300 multimode plate reader (USA). The
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amount of free doxorubicin was thus determined from the calibration curve. To
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examine the drug release behavior, the DOX-loaded nanogel was then transferred to
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each of three dialysis tubes against different dialysis solutions (45 mL) of pH 5.0, 6.0
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and 7.4, which were placed in a beaker and shaken at 37 °C, 100 rpm. Drug release
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studies were also performed at pH 6.5 in the presence of enzyme (0.2 mg mL-1 trypsin)
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and its inhibitor. At specific time intervals, the dialysate (0.5 mL) was withdrawn and
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replaced with fresh dissolution medium, and the collected dialysate was used to
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determine the doxorubicin release percentage colorimetrically using the multimode
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plate reader.
obtain
the
DOX-loaded
nanogels
of
PAA-b-PNIPAAm
and
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In Vitro Cytotoxicity of DOX-loaded Nanogels. MCF-7 and MCF-7/ADR
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(doxorubicin-resistant) breast cancer cells were seeded in 96-well plates at a density
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of 1×104 cells per well. After 24 h of incubation, the cells were exposed to
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DOX-loaded protamine/PAA-b-PNIPAAm nanogels by replacing the cell culture
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medium with serum-free medium containing the nanogels. After 2 h of incubation at
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37 °C (pH 6.5), the cells were washed and treated with a low-temperature treatment
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for 15 min at 4 °C (a cold shock treatment). The cells were further cultured for 24 h
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and MTT assay was used to determine the dose-dependent cytotoxicity (0.9, 1.8, 9.0
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µg/mL doxorubicin equivalent) caused by the nanogels after reading the optical
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density at 570 nm on a microplate reader (Model 3550, Bio-Rad, USA).
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Cellular Uptake of Doxorubicin-Loaded Nanogels. To investigate cellar uptake
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of the nanogels, PAA-b-PNIPAAm was labeled with a green fluorescence
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5-aminofluorescein and the fluorescent protamine/ PAA-b-PNIPAAm nanogels were
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prepared according to the previously described procedure. The cells were then
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incubated with the fluorescent nanogels for 2 h at pH 6.5. After incubation, the cells
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were washed with PBS, and then DAPI staining and paraformaldehyde fixation were
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performed either immediately or after a cold shock treatment. The cells were
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visualized by a confocal microscope (TCS SP5, Leica Microsystems, Wetzlar,
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Germany). Densitometric analysis of fluorescence intensity was performed using the
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Image J software (National Institute of Health, Bethesda, MD, USA).
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Photodynamic Treatments. MCF-7 and MCF-7/ADR cells were cultured
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according to the above-mentioned procedure. Rose bengal-loaded nanogels were
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prepared by the method similar to that for preparation of DOX-loaded nanogels in
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section 2.7, except for dissolving a photosensitizer (2 mg/mL rose bengal) in the
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PAA-b-PNIPAAm aqueous solution. Rose bengal loading efficiency was determined
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by measuring the amount of free rose bengal in the supernatant at 546 nm in the
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supernatant using a Perkin Elmer EnSpire 2300 multimode plate reader (USA). The
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cells were incubated with the rose bengal-loaded nanogels (8.0 µg/mL rose bengal
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equivalent) for 4 h. After replacing the culture medium, the plates (w/ and w/o a cold
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shock treatments) were immediately exposed to a green light LED (550 nm, 50
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mW/cm2) for 8 min. The cells were cultured for an additional 24 h and the
242
photocytotoxicity was evaluated using the above-mentioned MTT assay.
243
Statistical Analysis. Statistical analyses were conducted using Student’s t-test with
244
replicate measurements for data (n= 5). Differences with a P value < 0.01 were regarded
245
to be significant.
246
RESULTS AND DISCUSSION
247
Characterization of PAA-b-PNIPAAm Block Copolymer. A living free radical
248
polymerization method, RAFT, was adopted to synthesize PAA-b-PNIPAAm block
249
copolymer following previous studies with a slight modification (Figure 1A).15
250
Because of the presence of both PAA block and PNIPAAm block in the same
251
polymer chain, this block copolymer is therefore sensitive to the environmental
252
changes of acidity and tmperature. After reaction of 1.5 h for synthesizing the PAA
253
block in the first stage and another 3 h for the further growth of PNIPAAm block,
254
NMR analysis was carried out not only to confirm the chemical structure bout also to
255
determine the chain length (degree of polymerization, DP) of each block. The most
256
distinct absorption peaks of the PAA-b-PNIPAAm block copolymer were caused by
257
the isopropyl group in the PNIPAAm block including -CH3 at 1.20 ppm and >CH- at
258
3.85 ppm. The broad absorption peaks of the >CH2 and >CH- groups in the main
259
chain were at 1.50-2.15 and 2.30-2.55 ppm, respectively. The individual chain lengths
260
of the PAA and PNIPAAm blocks were thus calculated from the respective peak area
261
ratios of the characteristic absorption peaks of PAA segment and PNIPAAm segment
262
to the terminal DMP group. The results indicated that the DP values of the PAA and
263
the PNIPAAm blocks were 28 and 106, respectively. The corresponding AA
264
conversion in the first stage was 36%, whereas the NIPAAm conversion in the second
265
stage was 66%. The molecular weight of the PAA-b-PNIPAAm was thus 11480 and
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the PDI value measured from GPC was 1.23. This narrow molecular-weight
267
distribution is agreed to the living characteristics of the RAFT polymerization.
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Chemical and Physical Properties of Nanogels. Fig. 1B shows the schematic
269
diagram for the preparation of protamine/PAA-b-PNIPAAm nanogels by mixing both
270
polymers that were dissolved in deionized (DI) water. The nanogels were prepared by
271
a method of thermo-induced aggregation in combination with polyelectrolyte complex.
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Protamine was largely assembled with PAA-b-PNIPAAm via polyelectrolyte complex
273
on the outer layer of thermally aggregated PAA-b-PNIPAAm nanogels (Figure 1B).
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The PAA-b-PNIPAAm aqueous solution (1 mg/mL) was transparent at room
275
temperature (25 °C), but formed thermally aggregated nanogels upon heating to 37 °C.
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Yet, at room temperature, upon the addition of a small amount of aqueous protamine
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into PAA-b-PNIPAAm solution (weight ratio of protamine to PAA-b-PNIPAAm=
278
1:45), protamine/PAA-b-PNIPAAm nanogels formed immediately. This is because of
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the electrostatic attraction between the negative-charged PAA block and the
280
positive-charged protamine and probably also due to their hydrogen bonding.
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However, the hydrodynamic particle size (volume-averaged) increased substantially
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from 92.5±3.5 nm to 601.1±23.2 nm when increasing the weight ratio from 1:45 to
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1:25. Moreover, the nanogels became unstable during 24 h of storage and then
284
precipitated, when they were prepared at weight ratios of protamine to
285
PAA-b-PNIPAAm higher than 1:25. As shown in Table 1, increasing the weight ratio
286
of protamine to PAA-b-PNIPAAm from 1:45 to 1:25 leads to the formation of nearly
287
neutral nanogels (–20.60±0.78 mV vs. –0.07±0.10 mV), which decreases the
288
repulsion among individual nanogels. The results indicate that the complex nanogels
289
of protamine/PAA-b-PNIPAAm prepared at room temperature can’t be used as a drug
290
delivery carrier.
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Table 1. Average size and zeta potential of protamine/PAA-b-PNIPAAm nanogels at
different weight ratios of protamine to PAA-b-PNIPAAm
Volume ratio1
Weight
ratio
Average size
2
PDI
Zeta potential
(nm)
PAA-b-PNIPAAm
25°C
37°C
(mV)
33.91±1.76
34.71±2.39
0.175
0.034
0.24±0.61
–0.13±0.36
protamine/PAA-b-PNIPAAm
25°C
1:90
1:50
1:45
1:25
92.52±3.46
601.10±23.20
0.110
0.116
–20.60±0.78
–0.07±0.10
1:9
2:9
3:9
2:9
4:9
6:9
142.23±0.71
137.27±1.05
201.57±3.00
0.103
0.020
0.046
9.97±0.96
–0.01±0.17
–0.14±0.17
37°C
294
295
1
The volume ratio of the added protamine(aq) solution (2 mg/mL) to the PAA-bPNIPAAm(aq) solution (1 mg/mL).
296
2
The weight ratio of the protamine to the PAA-b-PNIPAAm block copolymer.
297
298
Interestingly, when a protamine solution (2 mg/mL, 1 mL) was added into a
299
thermally aggregated PAA-b-PNIPAAm (1 mg/mL, 9 mL) at 37 °C, a highly cloudy
300
and stable suspension of protamine/PAA-b-PNIPAAm was obtained. The average
301
particle size and zeta potential of the nanogels with different compositions are
302
summarized
303
protamine-to-PAA-b-PNIPAAm weight ratio of 2:9, which have a zeta potential of
304
9.97±0.96 mV, revealing that the nanogels were covered with the positively charged
305
protamine. The nanogels were very stable and the average particle size wasn’t
306
obviously
307
PAA-b-PNIPAAm nanogels under heating at 37 °C (T > LCST), the positively
308
charged protamine are attracted to the negatively charged PAA block (Figure 1B),
309
leading to the formation of protamine/PAA-b-PNIPAAm polyelectrolyte complex
310
layer on the outer layer of PAA-b-PNIPAAm nanogels (Figure 1B).
in
Table
changed
1.
for
14
The
nanogels
days.
By
can
be
pre-forming
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(B)
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Figure 1. (A) Schematic diagram for the synthesis of PAA-b-PNIPAAm copolymer,
315
(B) schematic diagram for the preparation of protamine/PAA-b-PNIPAAm nanogels.
316
Protamine/PAA-b-PNIPAAm
317
aggregation/polyelectrolyte complex method. Changing the pH from systemic
318
circulation (pH 7.4) to tumor microenvironment (pH 6.5) resulted in smartly
319
converting surface charge from negative to positive. The cationic nanogels easily
320
passed through the cell membrane and enhanced intracellular accumulation of
321
DOX-loaded nanogels. Cold shock treatment triggered rapid intracellular release of a
nanogels
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large amount of doxorubicin or a photosensitizer rose bengal against multidrug
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resistant MCF-7/ADR breast cancer cells.
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TEM imagine shows the protamine/PAA-b-PNIPAAm nanogels that were prepared
325
by the thermal aggregation/polyelectrolyte complex method (Figure 2A-a). The
326
average size of the protamine/PAA-b-PNIPAAm nanogels is larger than that of
327
PAA-b-PNIPAAm nanogels (Figure 2A-b). The FT-IR spectra of protamine,
328
PAA-b-PNIPAAm, and protamine/PAA-b-PNIPAAm nanogels (weight ratio= 2:9)
329
are shown in Figure 2B. The bands at 1655 cm-1 and 1540 cm-1 are assigned to the
330
stretching of amide I (>C=O) and amide II (>N-H) of the peptide bonds in protamine,
331
respectively. The PAA-b-PNIPAAm copolymer shows several characteristic peaks
332
assigned to the absorption bands of carboxylic acid (1732 cm-1), amide group (1652
333
cm-1 and 1548 cm-1), and isopropyl group (1388 cm-1 and 1368 cm-1). Obviously, the
334
amide and guanidino absorption bands of protamine are overlapped with the
335
absorption of PAA-b-PNIPAAm. Still, the spectrum of protamine/PAA-b-PNIPAAm
336
nanogels shows a shoulder at about 1715 cm-1, revealing the formation of molecular
337
interactions between protamine and PAA-b-PNIPAAm.
(A)
(B)
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Figure 2. (A) TEM micrographs of PAA-b-PNIPAAm and protamine/PAA-b-
340
PNIPAAm nanogels, (B) FT-IR spectra of PAA-b-PNIPAAm, protamine, and
341
protamine/PAA-b-PNIPAAm nanogels,
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Thermo- and pH-Induced Assembly and Disassembly of Nanogels. The phase
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transition temperatures of PNIPAAm-based materials can be determined by DSC
344
analysis.15, 22 Figure 3A shows the DSC curves of the swollen PAA, PNIPAAm, and
345
PAA-b-PNIPAAm copolymer in the distilled water. The phase transition temperature
346
(LCST) of the empty PNIPAAm is found at 31.5 °C, which is agreed to the literature
347
value. After copolymerization with the PAA, the prepared PAA-b-PNIPAAm (about
348
pH 4.2 when dissolved in distilled water) has a lower LCST value than the empty
349
PNIPAAm. At this low pH value, most carboxylic acid groups are in their neutral
350
form (-COOH) since the pKa of the PAA is about 4.75. The result suggests that
351
hydrogen bonding might occur between the amide groups of PNIPAAm and the
352
carboxylic acid groups of PAA, thus decreasing the LCST value.
353
The transition behaviors of various nanogels were studied by the studies of
354
temperature-induced fluorescence quenching and protein conformational changes.
355
Figure 3B and 3C show the fluorescence quenching and recovery of fluorescein
356
amine-labeled PAA-b-PNIPAAm after assembly and disassembly of the nanogels.
357
Converting of PAA-b-PNIPAAm from solution into nanogels due to the increase of
358
temperature from 25 °C to 37 °C was monitored by the reduction in fluorescence due
359
to self-quenching of fluorescein amine conjugated with PAA-b-PNIPAAm (78.2% of
360
original intensity), indicating thermal-induced aggregation of the copolymer at a
361
temperature higher than its LCST. Adding protamine to the PAA-b-PNIPAAm
362
nanogels at a weight ratio of 2:9 further quenched the fluorescence (65.9% of original
363
intensity), suggesting that PAA-b-PNIPAAm was even more aggregated with the
364
assistance of protamine (Figure 3B). Unexpectedly, the quenching effect decreased
365
with increasing the protamine-to-PAA-b-PNIPAAm weight ratio from 2:9 to 4:9 and
366
6:9, resulting in the recovery of the fluorescence intensity from 65.9% to 82.7% and
367
84.5% of original intensity. The fluorescence change can be correlated to the decrease
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368
of zeta potential when increasing the weight ratio from 2:9 to 4:9 and even 6:9 (Table
369
1). This might be due to the decrease in thermal-induced aggregation of
370
PAA-b-PNIPAAm copolymer due to the protamine-involved electrostatic attraction
371
and possible hydrogen bonding as well. The existent ionized PAA block is assumed to
372
be fixed by the cationized protamine, thus the chain mobility of PAA-b-PNIPAAm
373
copolymer is further limited, leading to the unapparent fluorescence quenching. The
374
results suggest that the excessive increase in the number of protamine molecules
375
(positively
376
PAA-b-PNIPAAm, consequently leading to an increase of the fluorescence intensity
377
and decrease of the zeta potential. So, the optimal condition for preparing the complex
378
nanogels should be at a protamine-to-PAA-b-PNIPAAm weight ratio of 2:9.
379
Decreasing the temperature from 37 °C to 25 °C and 4 °C caused a fluorescence
380
recovery of the protamine/PAA-b-PNIPAAm nanogels (increases from 65.9% of
381
original intensity at 37 °C to 80.4% at 25 °C and 97.1% at 4 °C) (Figure 3C),
382
indicating the disassembly of the nanogels under a low-temperature condition.
charged)
may
disturb
the
thermal-induced
aggregation
of
383
Temperature-Induced Protein Conformational Changes. To evaluate if the
384
conformation of protamine could be affected by PAA-b-PNIPAAm after association
385
into
386
protamine/PAA-b-PNIPAAm
387
Formation of the protamine/PAA-b-PNIPAAm nanocomplex was indicated by the
388
shift of a negative band at 198 nm and the decrease of its ellipticity along with the
389
disappearance of a positive band near 217 nm.23 Figure 3D shows the CD spectra of
390
protamine and protamine/PAA-b-PNIPAAm nanogels at different temperatures. A
391
random coil-like conformation is characterized by a negative peak at 198 nm and a
392
positive band at 217 nm in the CD spectrum of only protamine molecules.24 The
393
conformation was found to change from a random coil to α-helix after the formation
polyelectrolyte
complex,
we
measured
the
CD
spectra
of
nanocomplex formed at different temperatures.
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of protamine/PAA-b-PNIPAAm nanocomplex. α-helix signal (a negative band at 208
395
nm and a positive band at 220 nm) was observed in the CD spectra for the complex
396
nanogel formed at 37 °C (Figure 3D). However, when decreasing the temperature
397
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398
nm and 205 nm, and a positive peak appeared at around 217 nm and 215 nm,
399
revealing that the conformation of protamine changed due to the disassembly of the
400
nanogels.
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(A)
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(B)
(C)
(D)
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Figure 3. (A) DSC curves of PAA, PNIPAAm, and PAA-b-PNIPAAm copolymer, (B)
405
fluorescence quenching of fluorescein amine-labeled PAA-b-PNIPAAm after addition
406
of protamine; the insert shows the fluorescence remaining and quenching ratios
407
calculated from the intensity of the fluorescein amine-labeled PAA-b-PNIPAAm and
408
the fluorescent nanogels self-assembled at protamine-to-PAA-b-PNIPAAm weight
409
ratio
of
2:9,
4:9
and
6:9,
(C)
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protamine/PAA-b-PNIPAAm nanogels at different temperatures; the insert shows the
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fluorescence remaing and quenching ratios calculated from the intensity of the
412
fluorescein amine-labeled PAA-b-PNIPAAm and the fluorescent nanogels at 37 °C,
413
25 °C and 4 °C, (D) CD spectra of protamine and the protamine/PAA-b-PNIPAAm
414
nanogels at different temperatures
415
Phase
Transition
Behavior.
Decrease
in
optical
transmittance
of
416
PAA-b-PNIPAAm solution caused by thermal and pH-induced aggregation was
417
investigated to observe its phase transition behavior. The temperature responsive
418
properties of PAA-b-PNIPAAm copolymer are easily affected by changes in the
419
solution pH value because the acid groups in the PAA block could undergo ionization
420
when the external pH is raised above its pKa (=4.75). The LCST of the
421
PAA-b-PNIPAAm copolymer solution was determined by optical transmittance in
422
various buffer solutions of pH 5.0, 6.0 and 7.4 (Figure 4A) and compared to the
423
protamine/PAA-b-PNIPAAm nanogels at the same pH values (Figure 4B).
424
PAA-b-PNIPAAm copolymer did not display LCST behavior in phosphate buffers at
425
pH 6.0 and 7.4 up to 60 °C. This is because the PAA block was highly ionized in
426
these buffers, leading to a great increase in electrostatic repulsion and hydrophilic
427
property for the copolymer. Yet, by decreasing the pH value to 5.0, close to the pKa
428
of PAA block, it was expected that the phase transition would become apparent and
429
the copolymer would self-assemble into nanogels, because the ionization extent of the
430
PAA block would be much lower. Indeed, as shown in Figure 4A, a rapid decline in
431
the optical transmittance upon heating was observed, and the LCST determined from
432
the differential peak temperature was 32.4 °C (Figure 4A). Interestingly, phase
433
transition could be observed even in the buffers of pH 6.0 and 7.4 when
434
PAA-b-PNIPAAm and protamine assembled together to form complex nanogels. The
435
LCST values of the protamine/PAA-b-PNIPAAm nanogels in pH 6.0 and 7.4 buffers
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were 22.8 and 25.4 °C, respectively (Figure 4B), which were lower than the LCST of
437
PNIPAAm. The protamine was bound with the ionized PAA-b-PNIPAAm in pH 6.0
438
and 7.4 buffers via the electrostatic interactions formed between the oppositely
439
charged protamine and PAA-b-PNIPAAm. Consequently, the phase transition
440
appeared in the transmittance-temperature curve with a lower LCST due to the
441
increased hydrophobicity and thermal-induced aggregation of PAA-b-PNIPAAm in
442
the complex nanogels (Figure 4B). In pH 5.0 buffer, the formation of
443
protamine/PAA-b-PNIPAAm
444
PAA-b-PNIPAAm
445
transformation of PNIPAAm segments. Therefore, the decline curves are not as sharp
446
as that of PAA-b-PNIPAAm copolymer (Figure 4A). It has to point out that after the
447
rapid decline of transmittance due to the aggregation, there was a slight increase in the
448
transmittance with temperature in the pH 5 solution. It is suspected that, in the pH 5.0
449
buffer, the PAA-b-PNIPAAm aggregates fixed by the protamine/PAA-b-PNIPAAm
450
complex layer that was formed on the outer layer of the nanogels was not as stable as
451
those in the pH 6.0 and 7.4 buffers at 37 °C, which is advantageous for pH-triggered
452
drug release in endosomes or lysosomes.
along
complex
with
the
reduced
decrease
the
of
chain
mobility
of
hydrophilic-to-hydrophobic
453
We next examined the pH- and temperature-dependent size changes of
454
protamine/PAA-b-PNIPAAm nanogels at various buffer solutions by dynamic light
455
scattering (DLS). As shown in Figure 4C, the hydrodynamic diameter of the nanogel
456
suspension would change accordingly with different temperature and pH values. At
457
37 °C; the PAA-b-PNIPAAm nanogel was very stable at pH 5.0 but was easily
458
dissolved at pH 6.0 and 7.4 as indicated by its much smaller particle sizes (less than 2
459
nm) (Figure 4C-a). On the other hand, the protamine/PAA-b-PNIPAAm complex
460
nanogel shows a completely different pH-responsive property compared with its
461
PAA-b-PNIPAAm counterpart. First, the protamine/PAA-b-PNIPAAm nanogels had
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larger particle size than the PAA-b-PNIPAAm alone at pH 5.0 and 37 °C. Moreover,
463
the protamine/PAA-b-PNIPAAm nanogels were very stable even at pH 6.0 and 7.4
464
(Figure 4C-b), in contrast to the rapid dissolution of PAA-b-PNIPAAm nanogels. As
465
explained previously, the hydrophilic portion of ionized PAA block can bind with
466
protamine to form electrostatic complex layers, and the PNIPAAm block can form
467
self-aggregated domain caused by raising the temperature above the LCST. In
468
addition, increasing the pH value resulted in a slight decrease in particle size of
469
protamine/PAA-b-PNIPAAm nanogels as shown in Figure 4C-b. This is because of
470
the higher ionization extents of the PAA block at higher pH values (such as pH 7.4),
471
leading to the stronger electrostatic interactions and thus the smaller particle sizes.
472
The protamine/PAA-b-PNIPAAm nanogels showed a pH-responsive size-tunable
473
property at 25 °C (Figure 4C-c) that were similar to those at 37 °C (Figure 4C-b). It
474
was worth noting that the zeta potential changed from -2.7 mV to 11.5 mV while the
475
average size change from 153.5 nm to 244.9 nm for pH decline 7.4 to 5.0 at 37 °C,
476
indicating that the nanogels may have pH-responsive and charge-conversion
477
properties (Figure 4D). These properties can be attributed to the conformational
478
change of protamine in the nanogels at different pH values, allowing the shielding and
479
stretching out the arginine-rich domain.17 As the temperature was decreased to 4 °C,
480
the nanogels completely dissolved in all pH buffer solutions (pH 5.0, 6.0 and 7.4),
481
thus the size distributions were not measurable (Figure 4C-d). Almost no signal
482
corresponding to the size distribution of the nanogels was detectable at this low
483
temperature. The hydrophilic effect was greatly increased on decreasing the
484
temperature to 4 °C, thus weakening the inter/intramolecular hydrogen bonding and
485
causing a disintegration in the aggregates of the PNIPAAm segments. The
486
protamine/PAA-b-PNIPAAm nanogels were disassembled and then were completely
487
dissolved in the medium at 4 °C regardless of pH values. Although the
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PAA-b-PNIPAAm was ionized at pH 7.4 to a large extent, the electrostatic force
489
between the PAA-b-PNIPAAm and protamine at such a low temperature (4 °C) did
490
not provide the nanogels with sufficient binding strength for self-aggregation,
491
consequently leading to the complete dissolution of the protamine/PAA-b-PNIPAAm
492
nanogels. Because a low temperature treatment at 4 °C caused a more pronounced
493
temperature-responsive behavior compared to the treatments at 25 °C, we selected 4
494
°C for the cold treatment in the following studies.
(A)
(B)
495
(C)
(a)
(b)
(d)
(c)
496
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(D)
497
498
Figure 4. Optical transmittance of PAA-b-PNIPAAm (A) and protamine/
499
PAA-b-PNIPAAm nanogels (B) at various buffer solutions of pH 5.0, 6.0 and 7.4, (C)
500
hydrodynamic diameters of PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm
501
nanogels at different temperatures (4 °C, 25 °C and 37 °C) and various buffer
502
solutions of pH 5.0, 6.0 and 7.4, (D) pH-dependent zeta potential change at 37 °C
503
Thermo- and pH-Responsive Drug Release. The doxorubicin encapsulation
504
efficiency of PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm nanogels were
505
42.4% and 54.9%, respectively. Figure 5A shows the pH-responsive drug release
506
behaviors
507
protamine/PAA-b-PNIPAAm nanogels. The nanogels prepared by self-aggregation of
508
PAA-b-PNIPAAm alone rapidly released doxorubicin in PBS (pH 7.4), revealing that
509
the nanogels were not able to prevent a quick release of the drug from the vesicles
510
under normal physiological conditions (normal tissues) or in blood circulation system.
511
By contrast, protamine/PAA-b-PNIPAAm nanogels demonstrated a distinct
512
pH-responsive property for controlled doxorubicin release. To simulate the conditions
513
of nanoparticles in blood circulation (pH 7.4) and subsequently transported to late
of
doxorubicin
from
the
PAA-b-PNIPAAm
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endosomes and lysosomes of cancer cells (pH 5.0) at different temperatures,
515
doxorubicin release was examined by changing the pH of the release medium. While
516
keeping the temperature at 37 °C, doxorubicin release from the nanogels was faster at
517
pH 5.0 (80% after 24 h of release), compared to the slower drug release at pH 7.4
518
(31.1%). As previously studied in Figure 4B, the nanogels has high stability at pH 7.4
519
and the release of doxorubicin was slow and consecutive, but the nanogels were
520
disassembled in acidic environment because the PAA blocks were at a lower level of
521
ionization, thus doxorubicin release from the nanogels was faster at pH 5.0 due to the
522
collapse of the nanogels and the weakened electrostatic forces between the positively
523
charged doxorubicin and protamine, with the negatively charged PAA-b-PNIPAAm.
524
To simulate the conditions of the low temperature treatment (cryotherapy),
525
doxorubicin release was examined by changing the temperature of the release medium
526
(Figure 5B). It was worth noting that a burst release of doxorubicin from the
527
protamine/PAA-b-PNIPAAm nanogels could be found when a low-temperature (cold
528
shock) treatment was performed. The cloudy nanogel suspension became clear when
529
the temperature drop to a low level at 4 °C. The accumulative release reached more
530
than 92.5% of the loaded doxorubicin from the nanogels placed in the medium with a
531
pH changing from 7.4 to 5.0, followed by a 15 min of cold shock treatment. On the
532
other hand, the nanogels released a smaller amount of doxorubicin during the same
533
time period if the low-temperature treatment was not performed. The total amount of
534
doxorubicin released from the nanogels was determined to be 57.6% upon changing
535
the pH from 7.4 to 5.0 w/o cold shock treatment, yet was still higher than the
536
doxorubicin release from the nanogels w/o changing the pH and temperature (31.2%
537
at pH 7.4, 37 °C). The PAA-b-PNIPAAm demonstrated greater hydrophilic properties
538
at
539
protamine/PAA-b-PNIPAAm nanogels were rapidly disassembled and became
4
°C
when
compared
to
that
at
37°C,
23
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the
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540
completely dissolved at 4 °C. The results suggest that the nanogels are indeed
541
pH-responsive and thermo-responsive at the same time. At pH 5.0 or at temperatures
542
below 4 °C, the nanogels were in a disassembled conformation, which obviously
543
accelerated doxorubicin release from the nanogels. These dual-responsive nanogels
544
thus will be able to deliver doxorubicin to cancer cells, in which the acidic
545
endosome/lysosome environment can trigger the drug release. Furthermore, this
546
nanogel is applicable to temperature-controlled drug release which can be associated
547
with cryotherapy.
548
Trypsin has also been confirmed to be expressed in various adenocarcinoma
549
tissues where pH levels are in the range of 6.5 to 6.8, which is able to digest
550
protamine via hydrolysis of peptides on the C-terminal side of arginine residues.25,26
551
Figure
552
protamine/PAA-b-PNIPAAm nanogels. Doxorubicin release in the medium w/ trypsin
553
was faster than that w/o trypsin at pH 6.5. Without digestion by trypsin, only 46.9%
554
doxorubicin was released within 24 h, however, the doxorubicin release percentage
555
considerably increased due to enzymatic digestion of protamine, and more than 88.6%
556
of DOX was released in the presence of trypsin. The enzyme-triggered drug release
557
could be inhibited by trypsin inhibitor (58.9% of DOX release), implying that the
558
trypsin-involved drug release could be due to enzymatic digestion-induced
559
disassembly of the nanogels. The transmittance of the protamine/PAA-b-PNIPAAm
560
nanogels with (w/) and without (w/o) trypsin was monitored to investigate the
561
enzyme-triggered disassembly of the protamine/PAA-b-PNIPAAm nanogels (Figure
562
5D). The nanogels have good stability in PBS w/o digestion by trypsin, showing no
563
appreciable change in size distribution; on the contrary, the optical + rapidly
564
increases under tryptic digestion, suggesting that the disassembly of nanogels was
565
caused by enzymatic digestion.
5C
shows
enzyme-responsive
release
of
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doxorubicin
from
the
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(C)
(D)
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568
Figure 5. (A) pH- (pH 5.0, 6.0 and 7.4) and (B) thermo-responsive (changed from 37
569
°C to 4°C) drug release behavior of doxorubicin from the PAA-b-PNIPAAm and
570
protamine/PAA-b-PNIPAAm
571
protamine/PAA-b-PNIPAAm nanogels in the medium w/ and w/o trypsin (0.2 mg/mL)
572
at pH 6.5, (D) optical transmittance of the protamine/ PAA-b-PNIPAAm nanogels w/
573
and w/o trypsin (0.2 mg/mL) at pH 6.5.
nanogels,
(C)
Doxorubicin
release
from
574
Intracellular Drug Delivery. Microenvironment of solid tumors is known to be
575
weakly acidic. Under hypoxic culture condition, the pH can even decrease to 6.1.27
576
Yang reported the charge conversion of photo- and pH-responsive polypeptides for
577
enhanced and targeted cancer therapy. The polypeptides were less positively charged
578
(1.2 mV) at physiological condition (pH 7.4) and became more positively charged
579
(5.5 mV) at the pH near tumor microenvironments (pH 6.0).28 As shown in Figure 4D,
580
the zeta potential was converted from -2.7 mV to 3.1 mV when pH changed from 7.4
581
to 6.5, revealing that the protamine/PAA-b-PNIPAAm nanogels became cationic
25
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acidic
conditions.
To
further
examine
intracellular
Page 26 of 37
582
under
distribution
of
583
doxorubicin-loaded nanogels, PAA-b-PNIPAAm was labeled with fluorescein amine
584
and then was used to prepare fluorescent protamine/PAA-b-PNIPAAm nanogels.
585
Figure 6A shows the cellular uptake and intracellular distribution of the fluorescein
586
amine-labeled and doxorubicin-loaded nanogels in MCF-7 breast cancer cells. Free
587
DOX penetrate rapidly into nuclei and the nuclei exhibited red fluorescence. The cells
588
incubated with protamine/PAA-b-PNIPAAm nanogels at pH 6.5 for 2 h showed a
589
green fluorescence in cytoplasm with their nuclei stained with doxorubicin (red
590
fluorescence) (Figure 6A), revealing that the nanogels were efficiently internalized by
591
MCF-7 cells and doxorubicin were delivered into nuclei. The effective uptake of the
592
nanogels by the cancer cells is possibly due to the reason that the nanogels are
593
positively charged at pH 6.5, which may improve endocytic uptakes of the nanogels
594
through binding to the negatively charged plasma membrane.
595
However, fluorescence of free DOX wasn’t observed in the multidrug resistant
596
MCF-7/ADR cells, indicating rapid DOX efflux from the drug-resistant cancer cells.
597
The cancer cells treated with DOX-loaded protamine/PAA-b-PNIPAAm nanogels
598
demonstrated weaker intracellular fluorescence intensity of DOX (red fluorescence)
599
because self-quenching of DOX fluorescence in the well-organized nanoparticles
600
(Figure 6B). Next, MCF-7 and MCF-7/ADR cells were treated by a cold shock at 4
601
°C. MCF-7 cells subjected to a cold-shock treatment demonstrated a similar
602
intracellular fluorescence intensity compared to the cells incubated at a normal
603
condition (Figure 6A). In contrast, MCF-7/ADR cells treated with DOX-loaded
604
protamine/PAA-b-PNIPAAm nanogels demonstrated much stronger intracellular
605
DOX fluorescence intensity than their free DOX-treated counterparts (Figure 6B).
606
The relative DOX fluorescence intensity in the nanogels-treated MCF-7/ADR cells
607
was shown to be much stronger than their counterparts treated with free DOX,
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revealing that the nanogels can overcome the cellular barrier and doxorubicin can
609
enter into the cancer cells by the nanogel delivery system (Figure 6C). These results
610
revealed that the nanogels may be internalized by the cells and then releasing
611
doxorubicin into cytosol. The nanogels tend to escape from endosome for releasing
612
doxorubicin in cytoplasm, causing a stronger red fluorescence in the cells under cold
613
shock treatment.
(A)
614
615
(B)
616
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(C)
617
618
Figure 6. Cellular uptake and intracellular distribution of the fluorescein
619
amine-labeled and DOX-loaded protamine/PAA-b-PNIPAAm nanogels in MCF-7 (A)
620
and MCF-7/ADR (B) breast cancer cells, (C) the relative DOX fluorescence intensity
621
in MCF-7/ADR breast cancer cells treated with free DOX and DOX-loaded
622
protamine/PAA-b-PNIPAAm nanogels w/ and w/o cold treatment at 4 °C for 15
623
minutes. Each data is represented as mean ± SD (n = 3); * indicates p < 0.01.
624
Cytotoxicity. Figure S1 shows that PAA-b-PNIPAAm significantly reduces the
625
cytotoxicity of protamine. The empty nanogels didn’t cause noticeable cellular death
626
in MCF-7 cells while free doxorubicin and the doxorubicin-loaded nanogels caused
627
remarkable cytotoxicity towards the cancer cells. As shown in Figure 7A, free DOX
628
exhibited greater cytotoxicity in MCF-7 cells than DOX-loaded nanogels (5.1% vs.
629
24.3% cell viability and 39.5% vs. 65.8% cell viability for the cells treated with 10.0
630
µg/mL and 1.0 µg/mL equivalent free RB and RB-loaded nanogels) (p < 0.01), which
631
is consistent with the higher cellular uptake of free DOX (Figure 6A). Free DOX
632
rapidly diffused into nuclei and effectively inhibited the replication of DNA. However,
633
doxorubicin was gradually released from the protamine/PAA-b-PNIPAAm nanogels
634
that were taken up by MCF-7 cells via endocytosis, causing a lower cytotoxicity in the
28
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cancer cells than free DOX. Because PAA-b-PNIPAAm nanogels readily dissolved in
636
culture medium (pH near 7.4) before cellular uptake and then released DOX quickly
637
(Figure 5A), its cytotoxicity is similar to that of free DOX.
638
In contrast, the DOX-loaded protamine/PAA-b-PNIPAAm nanogels demonstrated
639
significant inhibitory effect on MCF-7/ADR cells compared to its free doxorubicin
640
counterpart (Figure 7B). At the same doxorubicin equivalent, the cytotoxicity of
641
doxorubicin was improved by loading it in the complex nanogels (92.1% vs. 82.8%
642
cell viability for the cells treated with 10.0 µg/mL equivalent free RB and RB-loaded
643
nanogels) (p < 0.1). Doxorubicin is an anthracycline that poorly accumulates in
644
MCF-7/ADR cells because of the over-expression of multidrug efflux pumps in the
645
doxorubicin-resistant cells. Protamine is arginine-rich peptide, which can enhance the
646
cellular uptake and further endosomal escaping of nucleic acid. Doxorubicin-loaded
647
protamine/PAA-b-PNIPAAm nanogels exhibited superior accumulation of the drug in
648
MCF-7/ADR cells compared to free doxorubicin (Figure 6B), consequently may
649
enhance the cytotoxicity against the cancer cells.
650
Cell viability of MCF-7 and MCF-7/ADR cells treated with DOX-loaded
651
protamine/PAA-b-PNIPAAm nanogels followed by treatment of the cells at different
652
temperatures (4-37 °C) for 15 minutes are shown in Figure 7C. The chemotherapy
653
using DOX-loaded nanogels associated with a cold shock treatment (4 °C) induced a
654
higher cytotoxicity in MCF-7/ADR cells compared with isothermal (37 °C) treated
655
groups (63.4% vs. 82.3% and 81.8% cell viability for the cells treated with
656
DOX-loaded nanogels at 4 °C vs. at 25 °C and 37 °C) (p < 0.01). The higher
657
cytotoxicity
658
protamine/PAA-b-PNIPAAm nanogels rapidly disintegrate at 4 °C after endocytosis,
659
facilitating a rapid release of a large amount of anticancer drugs.29 The excess
660
doxorubicin released in cytoplasm that was not readily effluxed from the
in
the
cells
under
a
cold
shock
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661
drug-resistant cancer cells caused more notable cellular death.
662
We further examine photocytotoxicity of rose bengal (RB)-loaded nanogels
663
against MCF-7/ADR cells when it was combined with cold shock treatment. To
664
evaluate whether the RB-loaded nanogels can affect the proliferation of MCF-7/ADR
665
cells upon green light excitation, the cancer cells internalized with the empty and
666
RB-loaded nanogels were photo-irradiated using green LED light (550 nm),
667
respectively (Figure 7D). The MCF-7/ADR cells internalized with free RB or empty
668
nanogels did not show noticeable cellular death upon photo irradiation while the
669
RB-loaded nanogels caused obvious phototoxicity towards the cancer cells.
(A)
(B)
670
671 (C)
(D)
672
673
Figure 7. (A) Dose-dependent inhibition of cell proliferation in MCF-7 (A) and
674
MCF-7/ADR (B) breast cancer cells by DOX-loaded protamine/PAA-b-PNIPAAm
675
nanogels (w/o cold shock treatment), (C) inhibition of cell proliferation in
676
MCF-7/ADR breast cancer cells by DOX-loaded protamine/PAA-b-PNIPAAm
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677
nanogels (10.0 µg/mL DOX equivalent; w/ cold shock treatment at different
678
temperatures), (D) the photodynamic treatments of MCF-7/ADR cells with rose
679
bengal (RB)-loaded protamine/PAA-b-PNIPAAm nanogels (8.0 µg/mL RB
680
equivalent; w/ cold shock treatment at 4 °C) under irradiated with 550 nm using a
681
high-power green light-emitting diode (LED) array. The data are shown as mean ± SD
682
(n = 5); * indicates P < 0.05 and ** indicates P < 0.01.
683
The RB encapsulation efficiency of protamine/PAA-b-PNIPAAm nanogels was
684
52.3%. Free RB induced lower cytotoxicity in MCF-7/ADR cells upon photo
685
irradiation as compared with the RB-loaded nanogels. Under exposure to green LED
686
light, the phototoxicity of RB was improved by loading it into the nanogels (82.3% vs.
687
39.3% cell viability for photodynamic treatment with 8.0 µg/mL equivalent free RB
688
and RB-loaded nanogels) (p < 0.01) (Figure 7D). Rose bengal is a photosensitizer that
689
poorly accumulates in cancer cells because it is difficult to penetrate through cell
690
membranes. Furthermore, RB can be effluxed from MCF-7/ADR cells, resulting in a
691
reduced photocytotoxicity. Rose bengal loaded in protamine/PAA-b-PNIPAAm
692
nanogels exhibited superior accumulation in MCF-7/ADR cells compared to its free
693
form, consequently the cytotoxicity against the cancer cells was enhanced upon photo
694
irradiation. However, without photo irradiation, free RB and the RB-loaded nanogels
695
exhibited negligible cytotoxicity in MCF-7/ADR cells, indicating that, without the
696
photodynamic treatment, the empty and rose bengal-loaded nanogels were safe and
697
did not show phototoxicity.
698
■ CONCLUSION
699
The triple-stimuli-responsive nanogels were successfully prepared via electrostatic
700
interactions
701
protamine/PAA-b-PNIPAAm
702
PAA-b-PNIPAAm aggregates remarkably increased the stability of the nanogels that
between
PAA-b-PNIPAAm
complex
and
layer
protamine.
on
the
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thermally
of
induced
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703
were well dispersed in PBS. The nanogels were disassembled and then released
704
doxorubicin quickly by decreasing temperature, reducing pH, and treating with
705
enzyme. After loading doxorubicin or rose bengal in the nanogels, an improvement in
706
inhibition efficiencies, caused by chemical therapeutic or photodynamic effects, was
707
observed in doxorubicin-resistant MCF-7/ADR cancer cells.
708
■ ACKNOWLEDGMENTS
709
The authors gratefully acknowledge the financial support provided by the Ministry of
710
Science and Technology, Taiwan, ROC (MOST 101-2221-E-038-016-MY3).
711
SUPPORTING INFORMATION
712
Cell viability: protamine and empty nanogels
713
714
■ REFERENCE
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Table of Contents (TOC)
+
H 2N
C-terminal domain
(arginine-rich)
NH
N
H
m
O
O
O
2
11 O
PAA-b-PNIPAAm copolymer
PAA-b-PNIPAAm nanogel
PAA block
N
H
Systemic circulation
(pH 7.4)
OH
polyelectrolyte
complex (PEC)
37°C
-
O
cationic protein (protamine)
thermo-induced
aggregation
HN
O
H
N
N
H
2
2 O
NH2
NH
O
H
N
n
NH2
NH
O
+
H 2N
+
H 2N
NH2
+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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42
43
44
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46
47
Molecular Pharmaceutics
doxorubicin
Protamine/PAA-b-PNIPAAm nanogel
PNIPAAm block
conformational change
Tumor environment
(pH 6.3)
Cold shock (4°C)
endocytosis
photodynamic
therapy
I
O
chemotherapy
Burst intracellular release
I
O
O
O
I
O
OH
OH
I
Cl
COO
Cl
Cl
charge conversion
OH
ROS and 1O2
O
O
OH
doxorubicin
H
O
O
Cl
rose bengal
+
N H3
Overcome drug resistance
ACS Paragon Plus Environment
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