Accepted Manuscript Title: Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: in vitro and in vivo evaluation Author: Joung-Pyo Nam Kyeong-Jae Lee Joung-Woo Choi Chae-OK Yun Jae-Woon Nah PII: DOI: Reference:
S0927-7765(15)00391-4 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.06.018 COLSUB 7148
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
31-3-2015 14-5-2015 8-6-2015
Please cite this article as: J.-P. Nam, K.-J. Lee, J.-W. Choi, C.-O.K. Yun, J.-W. Nah, Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: in vitro and in vivo evaluation, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.06.018 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.
Highlights We synthesized targeting ligand-modified multidrug grafted polymeric micelle (HPTOC-DOX). HPTOC-DOX formed micelles of approximately 151.9 – 311.2 nm in size in aqueous solution. Doxorubicin was released from HPTOC-DOX micelles at acidic pH 5.2 or 6.2. In vitro and in vivo anti-tumor assays showed higher anti-cancer activity than target peptide un-modified polymeric micelles.
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Targeting delivery of tocopherol and doxorubicin grafted-chitosan
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polymeric micelles for cancer therapy: in vitro and in vivo evaluation
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Joung-Pyo Nam1, Kyeong-Jae Lee1, Joung-Woo Choi2, Chae-OK Yun2, Jae-Woon Nah1,*
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University, 255 Jungang-ro, Suncheon, Jeollanam-do, Republic of Korea
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Republic of Korea
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Department of Polymer Science and Engineering, College of Engineering, Sunchon National
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Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791,
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* Corresponding authors: Department of Polymer Science and Engineering, Sunchon National
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University, Suncheon, Republic of Korea. Tel: 82 61 750 3566, Fax: 82 61 750 5423, Email:
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[email protected]
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Abstract In this study, we report the development of a novel, redox-sensitive chitosan-based targeted
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drug delivery system, containing two drugs. We determined whether the synthesized
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polymeric micelles (HPTOC-DOX) were suitable as a drug carrier. The formation of
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HPTOC-DOX micelles was confirmed by 1H-NMR. HPTOC-DOX formed micelles of
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approximately 151.9–311.2 nm in size in aqueous solution. Analysis of the drug release
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profile of HPTOC-DOX in different pH conditions (pH 5.2, 6.2, and 7.4) indicated that DOX
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was released from HPTOC-DOX micelles at acidic pH (5.2 or 6.2), while almost no DOX
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was released at pH 7.4. In vitro cell cytotoxicity and hemolysis assays indicated that HPTOC-
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DOX micelles safely deliver anti-cancer drugs and decrease the cytotoxicity of DOX. In vitro
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anti-cancer activity assays, confocal laser scanning microscopy analysis of SK-BR-3 cells,
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and in vivo anti-tumor activity in SK-BR-3-derived tumor-bearing mice were used to evaluate
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synergistic drug effects and the effect of the targeting peptide (anti-human epidermal growth
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factor receptor 2 [HER2] target peptide, epitope form; LTVSPWY) on receptor-mediated
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endocytosis.
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Keywords: chitosan, doxorubicin, pH-sensitive, targeted delivery
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1. Introduction In the past few decades, drug delivery systems (DDS) have been developed to effectively
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deliver drugs for cancer therapy. Several recent studies have described delivery carriers with
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redox-sensitive and site-specific targeting functions [1-4]. These carriers have advantages
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such as controlled release, decreased side effects, site-specific drug targeting, improved drug
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utilization, lower dose administration, and drug loss prevention [5]. Polymeric micelles that
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form a core-shell structure based on amphiphilic copolymers are ideal drug carriers because
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of their numerous advantages such as improved solubility and bioavailability of poorly
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soluble hydrophobic drugs in aqueous solutions [6, 7]. In addition, polymeric micelles
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effectively protect the encapsulated drug from fast degradation after intravenous injection.
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However, polymeric micelles have low cellular uptake because of the reticuloendothelial
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system [7, 8]. To overcome this problem, various targeting ligands such as transferrin [9],
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folic acid [10, 11], galactose [12], mannose [13], and an anti-human epidermal growth factor
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receptor 2 (HER2) antibody (trastuzumab) [14] have been used for drug or gene delivery.
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These targeting ligands enhance not only cellular uptake through receptor-mediated
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endocytosis, but also therapeutic efficacy.
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Chitosan is a linear polysaccharide with randomly distributed β-(1-4)-linked
D-
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glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is
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one of the major cationic polymers and has a structure similar to cellulose [15, 16]. Chitosan
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is obtained from chitin by alkaline deacetylation and inter- or intra-molecular hydrogen
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bonding between hydroxyl and amine groups [17]. Chitosan is widely used in DDS
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development as a backbone for polymeric micelles owing to its biocompatibility,
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biodegradability, high stability, and lack of toxicity. O-Carboxymethyl chitosan (OCMCh) is
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a derivative of chitosan with a CH2COOH modification at the C6 position of chitosan, which
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enhances its solubility in aqueous solutions. OCMCh has various advantages such as good 5
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bioactivity, solubility in aqueous solutions, antibacterial activity, and stability [18-20]. Doxorubicin (DOX), the model drug used in this study, is an anti-cancer drug also known
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as hydroxydaunorubicin, which is used in chemotherapy to treat cancers such as bladder,
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breast, stomach, ovarian, thyroid, and lung cancers, multiple myeloma, and soft tissue
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sarcoma [21]. The high antitumor activity of DOX is associated with multidrug resistance
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and acute cardiotoxicity [22, 23]. To solve these problems, several methods of DOX delivery
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were developed mainly using hydrophobic interactions with the polymeric micelles [24-26].
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Polymeric micelles with drugs loaded by hydrophobic interaction have some advantages,
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such as easy preparation and applicability for all hydrophobic drugs, but there are also some
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disadvantages associated with their use, such as an inconsistent drug release rate and
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modification However, polymeric micelles, with a drug covalently conjugated to the micelle
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backbone, have the potential to overcome these problems.
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α-Tocopherol (TP), also known as vitamin E, is a hydrophobic compound consisting of
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various methylated phenols. TP is not toxic to normal cells, but has anti-cancer activity
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against different cancer cell lines [27-30]. It can induce apoptosis and inhibit cell
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differentiation by blocking the cell cycle [27]. In addition, it can self-assemble into polymeric
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micelles.
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The aim of this study was to synthesize novel polymeric micelles consisting of TP, DOX,
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and OCMCh, to achieve a synergistic anti-cancer effect. We designed anti-cancer drug-
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micelle conjugates to overcome the limitations of micelles, in which drugs are loaded through
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hydrophobic interactions. DOX was conjugated to redox-sensitive dithiobis-succinimidyl
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propionate (DTSP) that is cleavable in the cytoplasm by an intracellular reducing agent such
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as glutathione (GSH, 0.5 – 10 mM) [1, 2, 31, 32]. Furthermore, a targeting ligand (anti-
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HER2/neu peptide-polyethylene glycol [PEG]; HP) was added to TP and DOX-conjugated
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polymeric micelles (TOC-DOX) to promote site-specific targeting. The synthesized targeting 6
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ligand-containing novel polymeric micelles (HPTOC-DOX) were expected to have the
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following effects: (1) synergistic effect of the conjugated TP and DOX, (2) redox-sensitive
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drug release in solid tumors owing to a disulfide bond, and (3) site-specific delivery of anti-
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cancer drugs such as TP and DOX. In this study, we performed in vitro and in vivo
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experiments to determine whether the novel polymeric HPTOC-DOX micelles can be used
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for cancer therapy.
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2. Materials and methods 2.1. Materials
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O-Carboxymethyl chitosan (OCMCh, molecular weight (MW) = 12,000 Da, deacetylation
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degree = 95%, and carboxymethylation degree = 83%) was prepared as previously described
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[33]. TP and DOX were purchased from Sigma-Aldrich Chemical Co. USA. The crosslinkers
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DTSP, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-
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hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Chemical Co. USA.
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HER2/neu-targeting peptides (epitope form, LTVSPWY-COOH) were synthesized with a
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Liberty microwave peptide synthesizer (CEM Co. Matthews, NC) using Fmoc solid-phase
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methods. NH2-PEG-COOH (MW = 3,400) was purchased from SunBio, Korea. Dulbecco’s
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Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium,
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and fetal bovine serum (FBS) were obtained from Gibco BRL (MD, USA). LysoTracker and
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Hoechst 33258 were purchased from Invitrogen. All other chemicals and solvents were of
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analytical or reagent grade and were used without further purification.
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2.2. Preparation of anti-cancer drug conjugated polymeric micelles
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The detailed method of TP conjugation to OCMCh (TOC), DOX conjugation to TOC (TOC-
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DOX), and targeting ligand addition to TOC-DOX (HPTOC-DOX) polymeric micelles is
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described in the Supplementary Information.
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2.3. Characterization of anti-cancer drug conjugated polymeric micelles
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The synthesized HPTOC-DOX micelles were characterized by 1H-NMR. The 1H-NMR
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spectra of micelles were measured in deuterium oxide (D2O) by using a 400 MHz NMR
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spectrometer (AVANCE 400FT-NMR 400 MHz, Bruker). Five milligrams of HPTOC-DOX 8
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were dissolved in 0.5 mL of D2O and analyzed with an NMR spectrometer. The products of
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intermediate structures, such as TOC, DTSP-modified DOX (DOX-DTSP), and TOC-DOX
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were also measured. The detailed method is described in the Supplementary Information. The average particle size and size distribution of TOC, TOC-DOX, and HPTOC-DOX
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micelles were determined using an ELS-8000 electrophoretic LS spectrophotometer
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(NICOMP 380 ZLS zeta potential/particle sizer; Otsuka Electronics INC., Japan), equipped
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with a He-Ne laser, at a wavelength of 632.8 nm at 25 °C (scattering angle of 90°). A micelle
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solution (10 µg/µL) was used for particle measurement (HE. 013.016-ALU cell adapter)
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without filtering.
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DOX concentrations and conjugation efficiency of TOC-DOX and HPTOC-DOX polymeric
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micelles were determined using high-performance liquid chromatography (HPLC) analysis.
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The polymeric micelles were dissolved in 50% DMSO (1 mg/mL) and then the solution was
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centrifuged. The supernatant was collected for analysis. The HPLC system consisted of a
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mobile phase delivery pump (LC-20AD HPLC pump, Shimadzu, Japan) and a UV detector
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(SPD-20A, UV/Vis detector, Shimadzu, Japan). A ZORBAX 300SB-C18 reverse-phase
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column (250 mm × 4.6 mm, 5 µm, Agilent Technologies Inc., USA) was used for separation
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of components. The mobile phase was composed of acetonitrile and water at a ratio of 40:60
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(v/v). The flow rate and column temperature were set at 1.0 mL/min and 30 °C, respectively.
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The UV absorbance at 280 nm was determined using an injection volume of 20 µL. DOX
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concentration and conjugation efficiency were calculated from standard curves. The curve
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was linear over the tested concentration range. The drug content (DC) and conjugation
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efficiency (CE) were calculated as follows:
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Weight of the drug in micelles
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DC% =
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Weight of the feeding polymer and drug
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CE% =
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Weight of the drug in micelles
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2.4. DOX release profile from micelles against different pH conditions
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The drug release profile of DOX from TOC-DOX or HPTOC-DOX was investigated by
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HPLC. One milligram of TOC-DOX or HPTOC-DOX was dissolved in 1 mL of phosphate-
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buffered saline (PBS) and then filtered using centrifugal filter devices (molecular weight cut-
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off [MWCO], 1 kDa; AMICON® Ultra-0.5, Millipore). The solution obtained by filtration
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and centrifugation was analyzed by HPLC. To confirm drug release from micelles in different
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pH conditions, we used acidic pH (pH 5.2 and 6.2) and neutral pH (7.4) with interval times
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from 30 min to 30 h. The pH conditions were stable at 7.4 from 30 min to 24 h and then
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decreased to 5.2.
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2.5. Cells and cell culture conditions
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Human breast cancer cells (SK-BR-3), human embryonic kidney cells (HEK 293), and
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human keratinocytes (HaCaT) were purchased from KCLB® (Seoul, Republic of Korea). The
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cells were cultured in RPMI-1640 or DMEM supplemented with 10% FBS and 1%
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penicillin/streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. The cells
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grown as a monolayer were harvested after incubation with trypsin-EDTA.
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2.6. Confocal laser scanning microscopy (CLSM) assay
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The localization of TOC-DOX or HPTOC-DOX in SK-BR-3 cells was determined with
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CLSM analysis. SK-BR-3 cells were seeded at a density of 1 × 105 cells/well in eight-well
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culture plates and cultured overnight. After 12 h, free DOX, TOC-DOX, and HPTOC-DOX
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(0.5 µg/mL equivalent concentration of DOX) were added to the SK-BR-3 cell culture
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medium. The cells were incubated for 1 h and then washed three times with PBS. The cells
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were pre-incubated with Hoechst 33258 (Dojindo Molecular Technologies), a nucleic acid
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stain, to determine the intracellular localization of micelles. In addition, the cells were labeled
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with the lysosome stain LysoTracker (Molecular Probes, OR, USA). The localization of
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TOC-DOX or HPTOC-DOX was determined using an inverted LSM510 laser scanning
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microscope (Carl Zeiss, Gottingen, Germany). The diode laser excitation wavelength of 405
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nm and helium neon laser excitation wavelengths of 511 and 543 nm were used to detect
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DOX-conjugated micelles, LysoTracker, and Hoechst 33258. Images were recorded digitally
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using a 512 × 512 pixel format.
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2.7. Cytotoxicity and anticancer activity assay in vitro
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The cytotoxicity and anti-cancer activity of OCMCh, TP, DOX, and polymeric micelles
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(TOC-DOX and HPTOC-DOX) in different cell lines (both normal and cancer cells) were
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evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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assay. The experiment was carried out as follows: HEK293, HaCaT, and SK-BR-3 cells were
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seeded into 96-well microplates at a density of 5 × 103 cells/well in 100 µL of medium. When
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cell confluence reached 80%, the cells were treated with OCMCh, TP, DOX, and polymeric
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micelles for 24 h at DOX equivalent concentrations ranging from 0.078125 to 10 µg/mL.
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After the incubation, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, and
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the plate was incubated for an additional 2 h at 37 °C. At the determined time, unreacted
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MTT was removed by aspiration. After removing the MTT-containing medium, the formazan
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crystals formed in live cells were dissolved with 100 µL of DMSO. Finally, the absorbance
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was measured at 570 nm (optical density) and 670 nm (background subtraction) with a
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VersaMax ELISA Microplate Reader (StakMax®, USA). The relative cell viability (%) was
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calculated according to the following equation: OD value = (OD560 - OD670) (OD sample - OD blank)
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Relative cell viability (%) = ————————————————— × 100% (OD control - OD blank)
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2.8. Hemolytic activity of TOC-DOX and HPTOC-DOX
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The hemolytic activity of TOC-DOX and HPTOC-DOX was evaluated in red blood cells
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(RBCs) from inbred rats (Orientbio Inc., Korea) by using a previously described method [34].
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Fresh rat blood was centrifuged at 800 × g and the collected RBCs were washed with pH 7.4
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PBS until the supernatant was clear. Polymeric micelles were dissolved in PBS at
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concentrations ranging from 0.625 to 10 µg/mL. Polymeric micelle solutions (equal volumes)
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were aliquoted in a 96-well plate and RBCs were then added to a final concentration of 8%
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(v/v). The samples were incubated with mild agitation for 1 h at 37 °C and then centrifuged at
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800 × g for 10 min. The absorbance of the supernatant was measured with a VersaMax
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ELISA Microplate Reader (StakMax®, USA) at 414 nm. Each measurement was made in
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triplicate, and the percentage of hemolysis was calculated using followed equation [35]:
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(A414 sample - A414 PBS)
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Hemolysis (%) =
× 100% (A414 0.1% Triton X-100 - A414 PBS) 12
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In addition, 100% hemolysis was defined as the absorbance of RBCs resuspended in 0.1%
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Triton X-100 and zero hemolysis as the absorbance of RBCs in PBS.
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2.9. In vivo anti-tumor effect of TOC-DOX and HPTOC-DOX and histological analysis
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To assess the anti-tumor effect of TOC-DOX and HPTOC-DOX, xenograft tumors of HER2
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overexpressing SK-BR-3 cells were established by injecting 1 × 106 cells into the lower
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abdomen of 6- to 8-week-old female athymic nude mice (Orientbio Inc.). Once tumor volume
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reached 100–120 mm3, the mice were randomized into three groups and injected
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intravenously with 200 µL of PBS, TOC-DOX, or HPTOC-DOX two times every other day
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(n = 5, 20 mg/kg). Anti-tumor efficacy was analyzed by measuring tumor size with a caliper
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three times a week until the end of study. The length (l) and width (w) of the tumor were
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measured and the tumor volume was calculated according to the following formula: tumor
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volume = 0.523 lw2.
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For histological analysis, tumor tissue was fixed in 4% formalin, embedded in paraffin, and
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cut into 5-μm sections by using a microtome (Wax-it, Vancouver, Canada). Representative
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sections were stained with hematoxylin and eosin (H&E), and then examined by light
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microscopy. For immunohistochemistry, slides were deparaffinized in xylene and then
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processed as previously described [36]. Immunohistochemical staining for proliferating cell
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nuclear antigen (PCNA) was performed on paraffin-embedded tissue sections by using an
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anti-PCNA antibody (Dako) as described previously [36]. Stained sections were
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counterstained with Mayer’s hematoxylin. All animal studies were approved by the Animal
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Care Committee of Sunchon National University (SCNU_IACUC-2013-6), and all animal
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care was in accordance with the guidelines of Korea Council on Animal Care.
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2.10. Statistical Analysis
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All data are expressed as mean ± standard error (SE) or standard deviation (SD).
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Statistically significant differences were determined using Stat View software (Abacus
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Concepts, Inc., Berkeley, CA) and the Mann-Whitney test (non-parametric rank sum test). P
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values less than 0.05 were considered statistically significant (*, P < 0.05; **, P < 0.01; ***,
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P < 0.001).
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3. Results 3.1. Characterization of TOC-DOX and HPTOC-DOX micelles
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In this study, we prepared novel polymeric micelles that have a redox-sensitive bond and
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contain anti-cancer drugs. The reaction scheme of the final product (HPTOC-DOX) and
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intermediate structures (TOC, DOX-DTSP, and TOC-DOX) is shown in Fig. S1.
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The 1H-NMR results of intermediate structures that were synthesized to prepare novel
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polymeric micelles are shown in Figs. S2–S4 (see Supplementary Information). The final
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product, which is a targeting ligand-modified TOC-DOX (HPTOC-DOX), was analyzed with 1
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H-NMR to confirm successful synthesis. The proton peaks of TP, DOX, OCMCh, PEG, and
anti-HER2 in HPTOC-DOX are classified in Fig. 1. Peak assignments are as follow: OCMCh,
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2.0 to 4.6 ppm; TP, 2.4 to 2.6 ppm; DOX, 1.1 to 1.4 ppm; PEG, 3.6 ppm; anti-HER2, 0.8 to 1
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ppm. These 1H-NMR results showed that targeting ligand- and anti-cancer drug-conjugated
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novel polymeric micelles were successfully synthesized.
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The particle size and distribution of TOC, TOC-DOX, and HPTOC-DOX micelles were
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measured by dynamic light scattering (DLS; Fig. 2). TOC, TOC-DOX, and HPTOC-DOX
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micelles had a unimodal particle size distribution (Fig. 2B, presented HPTOC-DOX). When
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TP increased the in the conjugation ratio of TOC, particle size increased from 1096.8 nm
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(TOC1) to 1347.1 nm (TOC3). The particle size significantly decreased when the
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hydrophobic drug was conjugated to TOC. In addition, targeting ligand-modified TOC-DOX
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had a larger particle size of approximately 151.9 nm (HPTOC1-DOX) and 311.2 nm
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(HPTOC3-DOX) compared to the unmodified TOC-DOX.
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3.2. In vitro DOX release profile of TOC-DOX and HPTOC-DOX micelles against different pH conditions
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The DOX release profile of TOC-DOX or HPTOC-DOX in different pH conditions (pH 5.2,
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6.2, and 7.4) was determined using HPLC to confirm drug release from the novel polymeric
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micelles. As shown Fig. 3, almost no DOX was released from any polymeric micelles
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(TOC1-DOX, TOC3-DOX, HPTOC1-DOX, and HPTOC3-DOX) at pH 7.4 (the pH was
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maintained in a time range from 30 min to 24 h). However, DOX was released when the pH
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was changed from 7.4 to 5.2 or 6.2. TOC3-DOX and HPTOC3-DOX micelles released less
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DOX compared to that released from TOC1-DOX and HPTOC1-DOX micelles at pH 6.2
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(Fig. 3A). More DOX was released from polymeric micelles at pH 5.2 than at pH 6.2. These
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results suggest that the novel polymeric micelles are maintained at neutral pH and start to
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release the drug in acidic pH conditions.
In addition, drug concentration (DC) and drug conjugation efficiency (CE) of polymeric
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micelles was investigated using HPLC (Table S1). DC did not change, but CE increased
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when the amount of TP, which was conjugated to OCMCh, increased. However, DC and CE
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decreased when a targeting ligand was added to TOC-DOX. This phenomenon can be
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explanined the accrued loss during the dialysis process and the targeting ligand modifying
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process.
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3.3. Localization assay of polymeric micelles in SK-Br3 cells using CLSM
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The polymeric micelles were taken up by cells and localized to the cytosol, endosomes,
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lysosomes, or nuclei. As shown Fig. 4, polymeric micelles were localized in lysosomes and
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nuclei, but free DOX localized only in nuclei. These results demonstrated that polymeric
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micelles were taken up by endocytosis. TOC1 and TOC3 had similar LysoTracker intensity.
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However, when the amount of conjugated TP increased in TOC1 to TOC3 (molar ratio), the
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fluorescence intensity of DOX also increased, indicating that more DOX was conjugated to
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TOC3 than to TOC1. This is consistent with the CE determined by HPLC. The merged 16
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images showed that targeting ligand-modified HPTOC-DOX micelles localized in cell nuclei,
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while unmodified TOC-DOX showed perinuclear localization. These results clearly
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demonstrated the effect of targeting ligand in HPTOC-DOX micelles. The targeting ligand
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(HER2/neu-targeting peptide [pepitope form, LTVSPWY]) induced HER2/neu receptor-
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mediated endocytosis in SK-BR-3 cells [37]. Therefore, the uptake of HPTOC-DOX by SK-
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BR-3 cells was more efficient than that of unmodified TOC-DOX.
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3.4. In vitro stability of polymeric micelles
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The results of the cell viability and hemolysis assays are shown in Fig. 5. The viability of
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HEK293 or HaCaT cells treated with intermediate structures, including OCMCh, TP, TOC1,
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and TOC3, is shown in Fig. S5 (see Supplementary Information). The cytotoxicity of DOX-
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conjugated polymeric micelles (TOC1-DOX, TOC3-DOX, HPTOC1-DOX, and HPTOC3-
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DOX) was lower than that of free DOX at equivalent DOX concentrations ranging from
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0.078125 to 10 µg/mL in both HEK293 and HaCaT cells (Fig. 5A and B). The viability of
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HEK293 cells treated with DOX-conjugated polymeric micelles at equivalent DOX
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concentrations ranging from 0.078125 to 5 µg/mL was higher than 85% (Fig. 5A). However,
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the viability of HaCaT cells treated with DOX-conjugated polymeric micelles at equivalent
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DOX concentrations ranging from 2.5 to 10 µg/mL was below 85% (Fig. 5B). The slight
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toxicity of polymeric micelles against normal cell lines is because of the toxicity of DOX.
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A hemolysis assay was performed to determine whether TOC-DOX and HPTOC-DOX
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micelles are safe for systemic administration. The results showed that TOC1-DOX, TOC3-
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DOX, HPTOC1-DOX, and HPTOC3-DOX did not induce hemolysis at DOX concentrations
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ranging from 0.625 to 1 µg/mL due to the low positive charge of OCMCh (Fig. 5C). These
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results demonstrated that the prepared polymeric micelles can be used for systemic
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administration.
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3.5. Anti-tumor effect of polymeric micelles in vitro
2
To investigate the anti-cancer effect of polymeric micelles we measured the viability of
3
HER2/neu-overexpressing SK-BR-3 cells treated with TOC-DOX and HPTOC-DOX
4
micelles with the MTT assay. Fig. 6 shows the anti-cancer effect of intermediate structures
5
(OCMCh, TP, TOC1, and TOC3) and final products (TOC-DOX and HPTOC-DOX) at
6
equivalent intermediate structure or DOX concentrations ranging from 0.078125 to 10 µg/mL.
7
The viability of TP-, TOC1-, and TOC3-treated cells decreased with increasing intermediate
8
structure concentration, but OCMCh-treated cells were 100% viable at all concentrations
9
tested (Fig. 6A). The 50% inhibitory concentration (IC50) of TP, TOC1, and TOC3 was above
10
0.625 µg/mL (Table S1). Treatment with the DOX-conjugated polymeric micelles TOC-
11
DOX and HPTOC-DOX decreased cell viability to below 60% at all equivalent DOX
12
concentrations (Fig. 6B). The IC50 of TOC-DOX and HPTOC-DOX was approximately
13
0.078125 µg/mL (Table S1). TOC-DOX and HPTOC-DOX decreased cell viability to a
14
similar extent as free DOX at concentrations ranging from 2.5 to 10 µg/mL. In addition,
15
treatment with targeting ligand-modified HPTOC-DOX micelles decreased cell viability to a
16
greater extent than unmodified TOC-DOX. However, the amount of TP did not have a
17
significant effect on cell viability. Therefore, to examine the anti-tumor effect of polymeric
18
micelles in vivo, we selected TOC3-DOX and HPTOC3-DOX.
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3.6. Anti-tumor effect and immunohistological analysis in HER2/neu-positive SK-Br3
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tumor-bearing mice
21
To evaluate the anti-tumor effect of TOC3-DOX or HPTOC3-DOX micelles, the tumor
22
growth rate was examined after systemic administration of polymeric micelles to HER2/neu-
23
positive SK-BR-3 tumor-bearing mice (Fig. 7A). Twenty days after the polymeric micelles
18
Page 17 of 36
were administered; the average tumor volume was 4092.3 mm3, 3142.6 mm3, and 1650.7
2
mm3 in PBS, TOC3-DOX, and HPTOC3-DOX-treated mice, respectively. The tumor volume
3
of polymeric micelle-treated group was decreased as compared with the PBS-treated group
4
due to the effect of DOX conjugated to the polymeric micelles. In particular, the targeting
5
ligand-modified HPTOC3-DOX showed a higher therapeutic efficacy than the other
6
treatments (P < 0.05 for TOC3-DOX; P < 0.001 for PBS). These results show that treatment
7
with the targeting ligand (anti-HER2/neu)-modified HPTOC-DOX micelles led to an
8
impressive anti-tumor effect through the specific receptor-mediated interaction between the
9
targeting ligand on the HPTOC-DOX micelle surface and the HER2/neu receptor on the SK-
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BR-3 cell surface.
To confirm the therapeutic efficacy of TOC3-DOX and HPTOC3-DOX in SK-BR-3 tumor-
12
bearing mice, histological observation of TOC3-DOX and HPTOC3-DOX micelle-treated
13
tumor tissues was carried out after H&E staining. Tumors of PBS-treated mice had a high cell
14
density. In contrast, the tumors of TOC3-DOX- and HPTOC3-DOX-treated mice contained
15
necrotic cells. Furthermore, the necrotic area in tumors of HPTOC3-DOX-treated mice
16
dramatically increased as compared to the control and TOC3-DOX-treated groups (Fig. 7B).
17
PCNA is a marker of proliferating cells and PCNA immunostaining was used to assess tumor
18
cell proliferation (Fig. 7B). Tumors in the PBS-treated group (control) were highly positive
19
for PCNA, whereas tumors in the TOC3-DOX- or HPTOC3-DOX- treated groups were not.
20
In particular, PCNA expression was considerably reduced in the HPTOC3-DOX-treated
21
group as compared with the control and TOC3-DOX groups. These in vivo results clearly
22
demonstrate that the polymeric micelles can suppress tumor growth. In addition, the targeting
23
ligand can enhance the ability of polymeric micelles to deliver anti-cancer drugs.
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1
4. Discussion In this study, we synthesized a novel polymeric micelle to develop a targeted and redox-
3
sensitive system for delivery of chemotherapeutic drugs such as TP and DOX. HPTOC-DOX
4
micelles were synthesized in three steps using chemical conjugation with a targeting ligand
5
(anti-HER2/neu targeting peptide), anti-cancer drugs (TP and DOX), and a redox-sensitive
6
bond (DTSP) to the OCMCh backbone (Fig. S1). This novel polymeric micelle has a
7
hydrophilic group and a hydrophobic group. Therefore, it can form micelles in aqueous
8
solution. Our results show that hydrophobic group-conjugated OCMCh formed particles. The
9
particle size of TOC1 and TOC3 was 1096.8 nm and 1347.1 nm, respectively. The
10
conjugation ratio of TP was increased 1% to 3% (molar ratio), but particle size increased
11
from 1096.8 nm to 1347.1 nm. The explanation for this phenomenon could be that TOC
12
formed micelles, but did not form strong hydrophobic interactions due to very low
13
hydrophobicity. We used OCMCh with an MW of 12,000 Da in this study. It consisted of
14
approximately 54.5 monomers (MW 220 Da). TP was conjugated to OCMCh monomers in
15
ratios of 0.5 (TOC1) and 1.6 (TOC3). Therefore, TOC has very low hydrophobicity and
16
relatively large particle sizes. The TOC micelles aggregated when TP was conjugated to
17
OCMCh with a molar ratio higher than 3%. However, when DOX was conjugated to TOC,
18
the particle size of TOC1-DOX and TOC3-DOX significantly decreased from 1096.8 nm to
19
127.4 and 1347.1 nm to 244.9 nm, respectively, because tight micelles were formed by strong
20
hydrophobic interactions. Several studies reported that the particle size decreased when
21
hydrophobic drugs were associated with polymeric micelles [38-40]. In addition, we
22
modified TOC-DOX micelles with a targeting ligand to allow site-specific delivery of anti-
23
cancer drugs. The particle size of HPTOC-DOX was slightly increased because of the
24
addition of the hydrophilic group (HER2/neu targeting peptide-PEG; HP).
25
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In this study, DTSP was used as a linker to conjugate DOX to the micelle and to confer 20
Page 19 of 36
redox-sensitivity. DTSP has a disulfide bond and DTSP-based drug carriers allow drugs to be
2
accumulated in target tissues at higher concentrations than they would with drug carriers
3
without disulfide bond-containing linkers [41]. As mention above, DTSP is is cleavable in the
4
cytoplasm by an intracellular reducing agent such as glutathione (GSH, 0.5 – 10 mM) [1, 2,
5
31, 32]. The disulfide bond in HPTOC-DOX micelles allows DOX release at the target site
6
due to the reducing agent after cellular uptake.
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The DOX release profile from polymeric micelles at different pH values indicated that the
8
synthesized polymeric micelles (TOC-DOX and HPTOC-DOX) released DOX at the tumor
9
site. TOC-DOX and HPTOC-DOX retained the conjugated DOX at pH 7.4. This means that
10
the TOC-DOX and HPTOC-DOX micelles are stable in blood plasma (pH 7.4). Drug carriers
11
consisting of polycationic polymers such as chitosan, polyethylenimine (PEI), and polylysine
12
(PLL) may not be considered as drug delivery systems for systemic administration due to
13
their positive charge. Positively charged polycationic polymers can interact with negatively
14
charged erythrocyte membranes and lead to hemolysis. Therefore, the stability of the
15
synthesized polymeric micelles is very important for RBC stability. Our results show that
16
TOC-DOX and HPTOC3-DOX micelles are stable in blood plasma and can be used as a
17
systemic drug delivery system.
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In general, targeted delivery systems are divided into two kinds: passive targeting systems
19
and systems with covalently conjugated targeting molecules on the surface of drug carriers.
20
The targeting ligand conjugated to drug carriers can bind to receptors expressed specifically
21
in cancer cells [11, 14]. The target receptor, human epidermal growth factor receptor 2
22
(HER2), which is also known as Neu, CD340, ErbB-2, or p185, is overexpressed in
23
approximately 30% of breast cancers and 20% of ovarian cancers, while its expression in
24
normal tissues is low [14, 42]. The HPTOC-DOX micelle, containing an anti-HER2 targeting
25
peptide, showed a better therapeutic effect better than TOC-DOX both in vitro in SK-BR-3 21
Page 20 of 36
cells and in vivo in SK-BR-3 tumor-bearing mice (Fig. 6B and Fig. 7). These results clearly
2
demonstrated that HPTOC-DOX cellular uptake was through receptor-mediated endocytosis
3
via an interaction between the anti-HER2 targeting peptide and the overexpressed HER2/neu
4
receptor on the SK-BR-3 cell surface. The receptor-mediated endocytosis enhanced cellular
5
uptake efficacy of micelles and led to increased therapeutic efficacy. In addition, we designed
6
multi-drug-conjugated polymeric micelles. TP was used to not only enhance hydrophobicity
7
but also as an anti-cancer drug. TP has anti-cancer activity against various cancer cells and
8
can induce apoptosis and diminish cell differentiation by blocking the cell cycle [27-30].
9
Therefore, TP-conjugated TOC micelles at concentrations exceeding 0.625 µg/mL
10
significantly decreased the viability of SK-BR-3 cells (Fig. 6A). The synergistic effect of TP
11
and DOX was evident from the low cell viability after treatment with a very low
12
concentration (0.078125 µg/mL) of TOC-DOX and HPTOC-DOX (Fig. 6B). Interestingly,
13
the viability of cells treated with polymeric micelles was higher than or similar to that of cells
14
treated with free DOX at concentrations exceeding 0.625 µg/mL. These results demonstrate
15
that that TP and DOX in multi-drug-conjugated TOC-DOX and HPTOC-DOX have a
16
synergistic effect in tumor therapy.
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In conclusion, we synthesized novel polymeric micelles with a targeting ligand, two anti-
18
cancer drugs (TP and DOX), and a redox-sensitive function. The synthesized TOC-DOX and
19
HPTOC-DOX formed polymeric micelles in aqueous solution through hydrophobic
20
interactions between the conjugated TP and DOX. The particle size of TOC-DOX and
21
HPTOC-DOX micelles was in the range of 124.7-244.9 nm and 151.9-311.2 nm, respectively.
22
The cumulative drug release of HPTOC-DOX of around 90% within 6 h after the pH changed
23
from 7.4 to 5.2. The anti-cancer drugs of TOC-DOX and HPTOC-DOX had a synergistic
24
effect against SK-BR-3 cells. The anti-HER2/neu targeting peptide (epitope form,
25
LTVSPWY) was used as the targeting moiety. It enhanced not only cellular uptake by 22
Page 21 of 36
HER2/neu overexpressing SK-BR-3 cells, but also therapeutic efficacy in SK-BR-3 tumor-
2
bearing mice. The targeting peptide-modified HPTOC-DOX had lower cytotoxicity than free
3
DOX and enhanced therapeutic efficacy as evidenced by results from both in vitro and in vivo
4
assays.
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Acknowledgements
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This work was supported by the National Research Foundation of Korea (NRF) grant funded
3
by the Ministry of Science, ICT & Future Planning (NRF-2014R1A2A1A10053027).
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Figure Legends
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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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Figure 3. DOX release profile of TOC-DOX and HPTOC-DOX micelles against various pH conditions. The pH conditions maintained at pH 7.4 during 24 h from 30 min and then decreased at pH 6.2 (A) or pH 5.2 (B) until 30 h. The results are presented as mean ± SD (n=3).
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Figure 4. Localization of various polymeric micelles in SK-Br3 cells. SK-Br3 cells were incubated with DOX, TOC-DOX, and HPTOC-DOX and then were observed in confocal laser scanning microscopy. SK-Br3 cells were pre incubated with LysoTracker and Hoechst 33258 to confirm intracellular localization.
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Figure 2. Particle size and distribution of various polymeric micelles. A) Particle size of TOC1, TOC1-DOX, HPTOC1-DOX, TOC3-DOX, TOC3-DOX, and HPTOC3-DOX. The results are presented as mean ± SD (n=3). B) Particle size distribution of HPTOC3-DOX.
Figure 5. A) In vitro cell cytotoxicity of DOX, TOC-DOX, and HPTOC-DOX against HEK 293 cells (a) or HaCa T cells (b). B) In vitro hemolytic assay of TOC-DOX and HPTOCDOX with different micelles concentration (0.625 to 5 µg/mL) against rat red blood cells (RBCs) from an inbred rat. D-PBS and 0.1% triton X were used the negative control and positive control. The results are presented as mean ± SD (n=5)
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Figure 1. 1H-NMR spectrum of HPTOC-DOX. Concentration of HPTOC-DOX is 5 mg/0.5 mL in D2O.
Figure 6. In vitro anti-tumor effect of intermediate structure or final products against SK-Br3 cells. The anti-tumor levels were expressed by percentage of cell viability. A) cell viability of OCMCh, TP, TOC1, and TOC3; B) cell viability of DOX, TOC1-DOX, HPTOC1-DOX, TOC3-DOX, HPTOC3-DOX. The results are presented as mean ± SD (n=5)
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Figure 7. Therapeutic efficacy and characterization of systemically injected TOC-DOX or HPTOC-DOX micelles in HER2 overexpressed SK-Br3 subcutaneous tumor models. A) Inhibition of tumor growth by intravenous injection of TOC-DOX or HPTOC-DOX micelles (20 mg/kg).*P
S0927-7765(15)00391-4 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.06.018 COLSUB 7148
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
31-3-2015 14-5-2015 8-6-2015
Please cite this article as: J.-P. Nam, K.-J. Lee, J.-W. Choi, C.-O.K. Yun, J.-W. Nah, Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: in vitro and in vivo evaluation, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.06.018 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.
Highlights We synthesized targeting ligand-modified multidrug grafted polymeric micelle (HPTOC-DOX). HPTOC-DOX formed micelles of approximately 151.9 – 311.2 nm in size in aqueous solution. Doxorubicin was released from HPTOC-DOX micelles at acidic pH 5.2 or 6.2. In vitro and in vivo anti-tumor assays showed higher anti-cancer activity than target peptide un-modified polymeric micelles.
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Targeting delivery of tocopherol and doxorubicin grafted-chitosan
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polymeric micelles for cancer therapy: in vitro and in vivo evaluation
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Joung-Pyo Nam1, Kyeong-Jae Lee1, Joung-Woo Choi2, Chae-OK Yun2, Jae-Woon Nah1,*
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University, 255 Jungang-ro, Suncheon, Jeollanam-do, Republic of Korea
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Republic of Korea
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Department of Polymer Science and Engineering, College of Engineering, Sunchon National
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Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791,
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* Corresponding authors: Department of Polymer Science and Engineering, Sunchon National
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University, Suncheon, Republic of Korea. Tel: 82 61 750 3566, Fax: 82 61 750 5423, Email:
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[email protected]
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Abstract In this study, we report the development of a novel, redox-sensitive chitosan-based targeted
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drug delivery system, containing two drugs. We determined whether the synthesized
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polymeric micelles (HPTOC-DOX) were suitable as a drug carrier. The formation of
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HPTOC-DOX micelles was confirmed by 1H-NMR. HPTOC-DOX formed micelles of
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approximately 151.9–311.2 nm in size in aqueous solution. Analysis of the drug release
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profile of HPTOC-DOX in different pH conditions (pH 5.2, 6.2, and 7.4) indicated that DOX
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was released from HPTOC-DOX micelles at acidic pH (5.2 or 6.2), while almost no DOX
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was released at pH 7.4. In vitro cell cytotoxicity and hemolysis assays indicated that HPTOC-
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DOX micelles safely deliver anti-cancer drugs and decrease the cytotoxicity of DOX. In vitro
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anti-cancer activity assays, confocal laser scanning microscopy analysis of SK-BR-3 cells,
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and in vivo anti-tumor activity in SK-BR-3-derived tumor-bearing mice were used to evaluate
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synergistic drug effects and the effect of the targeting peptide (anti-human epidermal growth
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factor receptor 2 [HER2] target peptide, epitope form; LTVSPWY) on receptor-mediated
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endocytosis.
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Keywords: chitosan, doxorubicin, pH-sensitive, targeted delivery
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1. Introduction In the past few decades, drug delivery systems (DDS) have been developed to effectively
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deliver drugs for cancer therapy. Several recent studies have described delivery carriers with
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redox-sensitive and site-specific targeting functions [1-4]. These carriers have advantages
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such as controlled release, decreased side effects, site-specific drug targeting, improved drug
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utilization, lower dose administration, and drug loss prevention [5]. Polymeric micelles that
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form a core-shell structure based on amphiphilic copolymers are ideal drug carriers because
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of their numerous advantages such as improved solubility and bioavailability of poorly
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soluble hydrophobic drugs in aqueous solutions [6, 7]. In addition, polymeric micelles
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effectively protect the encapsulated drug from fast degradation after intravenous injection.
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However, polymeric micelles have low cellular uptake because of the reticuloendothelial
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system [7, 8]. To overcome this problem, various targeting ligands such as transferrin [9],
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folic acid [10, 11], galactose [12], mannose [13], and an anti-human epidermal growth factor
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receptor 2 (HER2) antibody (trastuzumab) [14] have been used for drug or gene delivery.
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These targeting ligands enhance not only cellular uptake through receptor-mediated
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endocytosis, but also therapeutic efficacy.
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Chitosan is a linear polysaccharide with randomly distributed β-(1-4)-linked
D-
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glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is
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one of the major cationic polymers and has a structure similar to cellulose [15, 16]. Chitosan
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is obtained from chitin by alkaline deacetylation and inter- or intra-molecular hydrogen
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bonding between hydroxyl and amine groups [17]. Chitosan is widely used in DDS
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development as a backbone for polymeric micelles owing to its biocompatibility,
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biodegradability, high stability, and lack of toxicity. O-Carboxymethyl chitosan (OCMCh) is
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a derivative of chitosan with a CH2COOH modification at the C6 position of chitosan, which
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enhances its solubility in aqueous solutions. OCMCh has various advantages such as good 5
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bioactivity, solubility in aqueous solutions, antibacterial activity, and stability [18-20]. Doxorubicin (DOX), the model drug used in this study, is an anti-cancer drug also known
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as hydroxydaunorubicin, which is used in chemotherapy to treat cancers such as bladder,
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breast, stomach, ovarian, thyroid, and lung cancers, multiple myeloma, and soft tissue
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sarcoma [21]. The high antitumor activity of DOX is associated with multidrug resistance
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and acute cardiotoxicity [22, 23]. To solve these problems, several methods of DOX delivery
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were developed mainly using hydrophobic interactions with the polymeric micelles [24-26].
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Polymeric micelles with drugs loaded by hydrophobic interaction have some advantages,
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such as easy preparation and applicability for all hydrophobic drugs, but there are also some
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disadvantages associated with their use, such as an inconsistent drug release rate and
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modification However, polymeric micelles, with a drug covalently conjugated to the micelle
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backbone, have the potential to overcome these problems.
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α-Tocopherol (TP), also known as vitamin E, is a hydrophobic compound consisting of
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various methylated phenols. TP is not toxic to normal cells, but has anti-cancer activity
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against different cancer cell lines [27-30]. It can induce apoptosis and inhibit cell
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differentiation by blocking the cell cycle [27]. In addition, it can self-assemble into polymeric
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micelles.
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The aim of this study was to synthesize novel polymeric micelles consisting of TP, DOX,
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and OCMCh, to achieve a synergistic anti-cancer effect. We designed anti-cancer drug-
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micelle conjugates to overcome the limitations of micelles, in which drugs are loaded through
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hydrophobic interactions. DOX was conjugated to redox-sensitive dithiobis-succinimidyl
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propionate (DTSP) that is cleavable in the cytoplasm by an intracellular reducing agent such
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as glutathione (GSH, 0.5 – 10 mM) [1, 2, 31, 32]. Furthermore, a targeting ligand (anti-
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HER2/neu peptide-polyethylene glycol [PEG]; HP) was added to TP and DOX-conjugated
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polymeric micelles (TOC-DOX) to promote site-specific targeting. The synthesized targeting 6
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ligand-containing novel polymeric micelles (HPTOC-DOX) were expected to have the
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following effects: (1) synergistic effect of the conjugated TP and DOX, (2) redox-sensitive
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drug release in solid tumors owing to a disulfide bond, and (3) site-specific delivery of anti-
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cancer drugs such as TP and DOX. In this study, we performed in vitro and in vivo
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experiments to determine whether the novel polymeric HPTOC-DOX micelles can be used
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for cancer therapy.
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2. Materials and methods 2.1. Materials
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O-Carboxymethyl chitosan (OCMCh, molecular weight (MW) = 12,000 Da, deacetylation
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degree = 95%, and carboxymethylation degree = 83%) was prepared as previously described
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[33]. TP and DOX were purchased from Sigma-Aldrich Chemical Co. USA. The crosslinkers
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DTSP, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and N-
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hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich Chemical Co. USA.
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HER2/neu-targeting peptides (epitope form, LTVSPWY-COOH) were synthesized with a
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Liberty microwave peptide synthesizer (CEM Co. Matthews, NC) using Fmoc solid-phase
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methods. NH2-PEG-COOH (MW = 3,400) was purchased from SunBio, Korea. Dulbecco’s
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Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium,
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and fetal bovine serum (FBS) were obtained from Gibco BRL (MD, USA). LysoTracker and
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Hoechst 33258 were purchased from Invitrogen. All other chemicals and solvents were of
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analytical or reagent grade and were used without further purification.
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2.2. Preparation of anti-cancer drug conjugated polymeric micelles
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The detailed method of TP conjugation to OCMCh (TOC), DOX conjugation to TOC (TOC-
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DOX), and targeting ligand addition to TOC-DOX (HPTOC-DOX) polymeric micelles is
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described in the Supplementary Information.
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2.3. Characterization of anti-cancer drug conjugated polymeric micelles
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The synthesized HPTOC-DOX micelles were characterized by 1H-NMR. The 1H-NMR
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spectra of micelles were measured in deuterium oxide (D2O) by using a 400 MHz NMR
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spectrometer (AVANCE 400FT-NMR 400 MHz, Bruker). Five milligrams of HPTOC-DOX 8
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were dissolved in 0.5 mL of D2O and analyzed with an NMR spectrometer. The products of
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intermediate structures, such as TOC, DTSP-modified DOX (DOX-DTSP), and TOC-DOX
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were also measured. The detailed method is described in the Supplementary Information. The average particle size and size distribution of TOC, TOC-DOX, and HPTOC-DOX
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micelles were determined using an ELS-8000 electrophoretic LS spectrophotometer
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(NICOMP 380 ZLS zeta potential/particle sizer; Otsuka Electronics INC., Japan), equipped
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with a He-Ne laser, at a wavelength of 632.8 nm at 25 °C (scattering angle of 90°). A micelle
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solution (10 µg/µL) was used for particle measurement (HE. 013.016-ALU cell adapter)
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without filtering.
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DOX concentrations and conjugation efficiency of TOC-DOX and HPTOC-DOX polymeric
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micelles were determined using high-performance liquid chromatography (HPLC) analysis.
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The polymeric micelles were dissolved in 50% DMSO (1 mg/mL) and then the solution was
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centrifuged. The supernatant was collected for analysis. The HPLC system consisted of a
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mobile phase delivery pump (LC-20AD HPLC pump, Shimadzu, Japan) and a UV detector
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(SPD-20A, UV/Vis detector, Shimadzu, Japan). A ZORBAX 300SB-C18 reverse-phase
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column (250 mm × 4.6 mm, 5 µm, Agilent Technologies Inc., USA) was used for separation
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of components. The mobile phase was composed of acetonitrile and water at a ratio of 40:60
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(v/v). The flow rate and column temperature were set at 1.0 mL/min and 30 °C, respectively.
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The UV absorbance at 280 nm was determined using an injection volume of 20 µL. DOX
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concentration and conjugation efficiency were calculated from standard curves. The curve
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was linear over the tested concentration range. The drug content (DC) and conjugation
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efficiency (CE) were calculated as follows:
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Weight of the drug in micelles
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DC% =
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Weight of the feeding polymer and drug
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CE% =
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Weight of the drug in micelles
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2.4. DOX release profile from micelles against different pH conditions
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The drug release profile of DOX from TOC-DOX or HPTOC-DOX was investigated by
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HPLC. One milligram of TOC-DOX or HPTOC-DOX was dissolved in 1 mL of phosphate-
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buffered saline (PBS) and then filtered using centrifugal filter devices (molecular weight cut-
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off [MWCO], 1 kDa; AMICON® Ultra-0.5, Millipore). The solution obtained by filtration
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and centrifugation was analyzed by HPLC. To confirm drug release from micelles in different
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pH conditions, we used acidic pH (pH 5.2 and 6.2) and neutral pH (7.4) with interval times
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from 30 min to 30 h. The pH conditions were stable at 7.4 from 30 min to 24 h and then
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decreased to 5.2.
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2.5. Cells and cell culture conditions
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Human breast cancer cells (SK-BR-3), human embryonic kidney cells (HEK 293), and
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human keratinocytes (HaCaT) were purchased from KCLB® (Seoul, Republic of Korea). The
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cells were cultured in RPMI-1640 or DMEM supplemented with 10% FBS and 1%
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penicillin/streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. The cells
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grown as a monolayer were harvested after incubation with trypsin-EDTA.
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2.6. Confocal laser scanning microscopy (CLSM) assay
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The localization of TOC-DOX or HPTOC-DOX in SK-BR-3 cells was determined with
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CLSM analysis. SK-BR-3 cells were seeded at a density of 1 × 105 cells/well in eight-well
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culture plates and cultured overnight. After 12 h, free DOX, TOC-DOX, and HPTOC-DOX
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(0.5 µg/mL equivalent concentration of DOX) were added to the SK-BR-3 cell culture
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medium. The cells were incubated for 1 h and then washed three times with PBS. The cells
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were pre-incubated with Hoechst 33258 (Dojindo Molecular Technologies), a nucleic acid
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stain, to determine the intracellular localization of micelles. In addition, the cells were labeled
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with the lysosome stain LysoTracker (Molecular Probes, OR, USA). The localization of
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TOC-DOX or HPTOC-DOX was determined using an inverted LSM510 laser scanning
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microscope (Carl Zeiss, Gottingen, Germany). The diode laser excitation wavelength of 405
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nm and helium neon laser excitation wavelengths of 511 and 543 nm were used to detect
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DOX-conjugated micelles, LysoTracker, and Hoechst 33258. Images were recorded digitally
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using a 512 × 512 pixel format.
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2.7. Cytotoxicity and anticancer activity assay in vitro
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The cytotoxicity and anti-cancer activity of OCMCh, TP, DOX, and polymeric micelles
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(TOC-DOX and HPTOC-DOX) in different cell lines (both normal and cancer cells) were
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evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
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assay. The experiment was carried out as follows: HEK293, HaCaT, and SK-BR-3 cells were
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seeded into 96-well microplates at a density of 5 × 103 cells/well in 100 µL of medium. When
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cell confluence reached 80%, the cells were treated with OCMCh, TP, DOX, and polymeric
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micelles for 24 h at DOX equivalent concentrations ranging from 0.078125 to 10 µg/mL.
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After the incubation, 10 µL of MTT solution (5 mg/mL in PBS) was added to each well, and
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the plate was incubated for an additional 2 h at 37 °C. At the determined time, unreacted
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MTT was removed by aspiration. After removing the MTT-containing medium, the formazan
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crystals formed in live cells were dissolved with 100 µL of DMSO. Finally, the absorbance
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was measured at 570 nm (optical density) and 670 nm (background subtraction) with a
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VersaMax ELISA Microplate Reader (StakMax®, USA). The relative cell viability (%) was
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calculated according to the following equation: OD value = (OD560 - OD670) (OD sample - OD blank)
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Relative cell viability (%) = ————————————————— × 100% (OD control - OD blank)
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2.8. Hemolytic activity of TOC-DOX and HPTOC-DOX
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The hemolytic activity of TOC-DOX and HPTOC-DOX was evaluated in red blood cells
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(RBCs) from inbred rats (Orientbio Inc., Korea) by using a previously described method [34].
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Fresh rat blood was centrifuged at 800 × g and the collected RBCs were washed with pH 7.4
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PBS until the supernatant was clear. Polymeric micelles were dissolved in PBS at
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concentrations ranging from 0.625 to 10 µg/mL. Polymeric micelle solutions (equal volumes)
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were aliquoted in a 96-well plate and RBCs were then added to a final concentration of 8%
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(v/v). The samples were incubated with mild agitation for 1 h at 37 °C and then centrifuged at
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800 × g for 10 min. The absorbance of the supernatant was measured with a VersaMax
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ELISA Microplate Reader (StakMax®, USA) at 414 nm. Each measurement was made in
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triplicate, and the percentage of hemolysis was calculated using followed equation [35]:
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(A414 sample - A414 PBS)
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Hemolysis (%) =
× 100% (A414 0.1% Triton X-100 - A414 PBS) 12
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In addition, 100% hemolysis was defined as the absorbance of RBCs resuspended in 0.1%
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Triton X-100 and zero hemolysis as the absorbance of RBCs in PBS.
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2.9. In vivo anti-tumor effect of TOC-DOX and HPTOC-DOX and histological analysis
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To assess the anti-tumor effect of TOC-DOX and HPTOC-DOX, xenograft tumors of HER2
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overexpressing SK-BR-3 cells were established by injecting 1 × 106 cells into the lower
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abdomen of 6- to 8-week-old female athymic nude mice (Orientbio Inc.). Once tumor volume
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reached 100–120 mm3, the mice were randomized into three groups and injected
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intravenously with 200 µL of PBS, TOC-DOX, or HPTOC-DOX two times every other day
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(n = 5, 20 mg/kg). Anti-tumor efficacy was analyzed by measuring tumor size with a caliper
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three times a week until the end of study. The length (l) and width (w) of the tumor were
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measured and the tumor volume was calculated according to the following formula: tumor
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volume = 0.523 lw2.
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For histological analysis, tumor tissue was fixed in 4% formalin, embedded in paraffin, and
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cut into 5-μm sections by using a microtome (Wax-it, Vancouver, Canada). Representative
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sections were stained with hematoxylin and eosin (H&E), and then examined by light
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microscopy. For immunohistochemistry, slides were deparaffinized in xylene and then
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processed as previously described [36]. Immunohistochemical staining for proliferating cell
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nuclear antigen (PCNA) was performed on paraffin-embedded tissue sections by using an
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anti-PCNA antibody (Dako) as described previously [36]. Stained sections were
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counterstained with Mayer’s hematoxylin. All animal studies were approved by the Animal
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Care Committee of Sunchon National University (SCNU_IACUC-2013-6), and all animal
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care was in accordance with the guidelines of Korea Council on Animal Care.
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2.10. Statistical Analysis
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All data are expressed as mean ± standard error (SE) or standard deviation (SD).
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Statistically significant differences were determined using Stat View software (Abacus
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Concepts, Inc., Berkeley, CA) and the Mann-Whitney test (non-parametric rank sum test). P
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values less than 0.05 were considered statistically significant (*, P < 0.05; **, P < 0.01; ***,
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P < 0.001).
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3. Results 3.1. Characterization of TOC-DOX and HPTOC-DOX micelles
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In this study, we prepared novel polymeric micelles that have a redox-sensitive bond and
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contain anti-cancer drugs. The reaction scheme of the final product (HPTOC-DOX) and
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intermediate structures (TOC, DOX-DTSP, and TOC-DOX) is shown in Fig. S1.
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The 1H-NMR results of intermediate structures that were synthesized to prepare novel
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polymeric micelles are shown in Figs. S2–S4 (see Supplementary Information). The final
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product, which is a targeting ligand-modified TOC-DOX (HPTOC-DOX), was analyzed with 1
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H-NMR to confirm successful synthesis. The proton peaks of TP, DOX, OCMCh, PEG, and
anti-HER2 in HPTOC-DOX are classified in Fig. 1. Peak assignments are as follow: OCMCh,
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2.0 to 4.6 ppm; TP, 2.4 to 2.6 ppm; DOX, 1.1 to 1.4 ppm; PEG, 3.6 ppm; anti-HER2, 0.8 to 1
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ppm. These 1H-NMR results showed that targeting ligand- and anti-cancer drug-conjugated
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novel polymeric micelles were successfully synthesized.
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The particle size and distribution of TOC, TOC-DOX, and HPTOC-DOX micelles were
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measured by dynamic light scattering (DLS; Fig. 2). TOC, TOC-DOX, and HPTOC-DOX
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micelles had a unimodal particle size distribution (Fig. 2B, presented HPTOC-DOX). When
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TP increased the in the conjugation ratio of TOC, particle size increased from 1096.8 nm
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(TOC1) to 1347.1 nm (TOC3). The particle size significantly decreased when the
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hydrophobic drug was conjugated to TOC. In addition, targeting ligand-modified TOC-DOX
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had a larger particle size of approximately 151.9 nm (HPTOC1-DOX) and 311.2 nm
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(HPTOC3-DOX) compared to the unmodified TOC-DOX.
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3.2. In vitro DOX release profile of TOC-DOX and HPTOC-DOX micelles against different pH conditions
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The DOX release profile of TOC-DOX or HPTOC-DOX in different pH conditions (pH 5.2,
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6.2, and 7.4) was determined using HPLC to confirm drug release from the novel polymeric
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micelles. As shown Fig. 3, almost no DOX was released from any polymeric micelles
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(TOC1-DOX, TOC3-DOX, HPTOC1-DOX, and HPTOC3-DOX) at pH 7.4 (the pH was
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maintained in a time range from 30 min to 24 h). However, DOX was released when the pH
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was changed from 7.4 to 5.2 or 6.2. TOC3-DOX and HPTOC3-DOX micelles released less
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DOX compared to that released from TOC1-DOX and HPTOC1-DOX micelles at pH 6.2
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(Fig. 3A). More DOX was released from polymeric micelles at pH 5.2 than at pH 6.2. These
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results suggest that the novel polymeric micelles are maintained at neutral pH and start to
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release the drug in acidic pH conditions.
In addition, drug concentration (DC) and drug conjugation efficiency (CE) of polymeric
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micelles was investigated using HPLC (Table S1). DC did not change, but CE increased
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when the amount of TP, which was conjugated to OCMCh, increased. However, DC and CE
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decreased when a targeting ligand was added to TOC-DOX. This phenomenon can be
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explanined the accrued loss during the dialysis process and the targeting ligand modifying
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process.
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3.3. Localization assay of polymeric micelles in SK-Br3 cells using CLSM
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The polymeric micelles were taken up by cells and localized to the cytosol, endosomes,
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lysosomes, or nuclei. As shown Fig. 4, polymeric micelles were localized in lysosomes and
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nuclei, but free DOX localized only in nuclei. These results demonstrated that polymeric
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micelles were taken up by endocytosis. TOC1 and TOC3 had similar LysoTracker intensity.
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However, when the amount of conjugated TP increased in TOC1 to TOC3 (molar ratio), the
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fluorescence intensity of DOX also increased, indicating that more DOX was conjugated to
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TOC3 than to TOC1. This is consistent with the CE determined by HPLC. The merged 16
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images showed that targeting ligand-modified HPTOC-DOX micelles localized in cell nuclei,
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while unmodified TOC-DOX showed perinuclear localization. These results clearly
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demonstrated the effect of targeting ligand in HPTOC-DOX micelles. The targeting ligand
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(HER2/neu-targeting peptide [pepitope form, LTVSPWY]) induced HER2/neu receptor-
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mediated endocytosis in SK-BR-3 cells [37]. Therefore, the uptake of HPTOC-DOX by SK-
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BR-3 cells was more efficient than that of unmodified TOC-DOX.
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3.4. In vitro stability of polymeric micelles
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The results of the cell viability and hemolysis assays are shown in Fig. 5. The viability of
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HEK293 or HaCaT cells treated with intermediate structures, including OCMCh, TP, TOC1,
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and TOC3, is shown in Fig. S5 (see Supplementary Information). The cytotoxicity of DOX-
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conjugated polymeric micelles (TOC1-DOX, TOC3-DOX, HPTOC1-DOX, and HPTOC3-
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DOX) was lower than that of free DOX at equivalent DOX concentrations ranging from
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0.078125 to 10 µg/mL in both HEK293 and HaCaT cells (Fig. 5A and B). The viability of
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HEK293 cells treated with DOX-conjugated polymeric micelles at equivalent DOX
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concentrations ranging from 0.078125 to 5 µg/mL was higher than 85% (Fig. 5A). However,
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the viability of HaCaT cells treated with DOX-conjugated polymeric micelles at equivalent
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DOX concentrations ranging from 2.5 to 10 µg/mL was below 85% (Fig. 5B). The slight
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toxicity of polymeric micelles against normal cell lines is because of the toxicity of DOX.
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A hemolysis assay was performed to determine whether TOC-DOX and HPTOC-DOX
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micelles are safe for systemic administration. The results showed that TOC1-DOX, TOC3-
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DOX, HPTOC1-DOX, and HPTOC3-DOX did not induce hemolysis at DOX concentrations
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ranging from 0.625 to 1 µg/mL due to the low positive charge of OCMCh (Fig. 5C). These
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results demonstrated that the prepared polymeric micelles can be used for systemic
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administration.
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3.5. Anti-tumor effect of polymeric micelles in vitro
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To investigate the anti-cancer effect of polymeric micelles we measured the viability of
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HER2/neu-overexpressing SK-BR-3 cells treated with TOC-DOX and HPTOC-DOX
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micelles with the MTT assay. Fig. 6 shows the anti-cancer effect of intermediate structures
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(OCMCh, TP, TOC1, and TOC3) and final products (TOC-DOX and HPTOC-DOX) at
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equivalent intermediate structure or DOX concentrations ranging from 0.078125 to 10 µg/mL.
7
The viability of TP-, TOC1-, and TOC3-treated cells decreased with increasing intermediate
8
structure concentration, but OCMCh-treated cells were 100% viable at all concentrations
9
tested (Fig. 6A). The 50% inhibitory concentration (IC50) of TP, TOC1, and TOC3 was above
10
0.625 µg/mL (Table S1). Treatment with the DOX-conjugated polymeric micelles TOC-
11
DOX and HPTOC-DOX decreased cell viability to below 60% at all equivalent DOX
12
concentrations (Fig. 6B). The IC50 of TOC-DOX and HPTOC-DOX was approximately
13
0.078125 µg/mL (Table S1). TOC-DOX and HPTOC-DOX decreased cell viability to a
14
similar extent as free DOX at concentrations ranging from 2.5 to 10 µg/mL. In addition,
15
treatment with targeting ligand-modified HPTOC-DOX micelles decreased cell viability to a
16
greater extent than unmodified TOC-DOX. However, the amount of TP did not have a
17
significant effect on cell viability. Therefore, to examine the anti-tumor effect of polymeric
18
micelles in vivo, we selected TOC3-DOX and HPTOC3-DOX.
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3.6. Anti-tumor effect and immunohistological analysis in HER2/neu-positive SK-Br3
20
tumor-bearing mice
21
To evaluate the anti-tumor effect of TOC3-DOX or HPTOC3-DOX micelles, the tumor
22
growth rate was examined after systemic administration of polymeric micelles to HER2/neu-
23
positive SK-BR-3 tumor-bearing mice (Fig. 7A). Twenty days after the polymeric micelles
18
Page 17 of 36
were administered; the average tumor volume was 4092.3 mm3, 3142.6 mm3, and 1650.7
2
mm3 in PBS, TOC3-DOX, and HPTOC3-DOX-treated mice, respectively. The tumor volume
3
of polymeric micelle-treated group was decreased as compared with the PBS-treated group
4
due to the effect of DOX conjugated to the polymeric micelles. In particular, the targeting
5
ligand-modified HPTOC3-DOX showed a higher therapeutic efficacy than the other
6
treatments (P < 0.05 for TOC3-DOX; P < 0.001 for PBS). These results show that treatment
7
with the targeting ligand (anti-HER2/neu)-modified HPTOC-DOX micelles led to an
8
impressive anti-tumor effect through the specific receptor-mediated interaction between the
9
targeting ligand on the HPTOC-DOX micelle surface and the HER2/neu receptor on the SK-
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BR-3 cell surface.
To confirm the therapeutic efficacy of TOC3-DOX and HPTOC3-DOX in SK-BR-3 tumor-
12
bearing mice, histological observation of TOC3-DOX and HPTOC3-DOX micelle-treated
13
tumor tissues was carried out after H&E staining. Tumors of PBS-treated mice had a high cell
14
density. In contrast, the tumors of TOC3-DOX- and HPTOC3-DOX-treated mice contained
15
necrotic cells. Furthermore, the necrotic area in tumors of HPTOC3-DOX-treated mice
16
dramatically increased as compared to the control and TOC3-DOX-treated groups (Fig. 7B).
17
PCNA is a marker of proliferating cells and PCNA immunostaining was used to assess tumor
18
cell proliferation (Fig. 7B). Tumors in the PBS-treated group (control) were highly positive
19
for PCNA, whereas tumors in the TOC3-DOX- or HPTOC3-DOX- treated groups were not.
20
In particular, PCNA expression was considerably reduced in the HPTOC3-DOX-treated
21
group as compared with the control and TOC3-DOX groups. These in vivo results clearly
22
demonstrate that the polymeric micelles can suppress tumor growth. In addition, the targeting
23
ligand can enhance the ability of polymeric micelles to deliver anti-cancer drugs.
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1
4. Discussion In this study, we synthesized a novel polymeric micelle to develop a targeted and redox-
3
sensitive system for delivery of chemotherapeutic drugs such as TP and DOX. HPTOC-DOX
4
micelles were synthesized in three steps using chemical conjugation with a targeting ligand
5
(anti-HER2/neu targeting peptide), anti-cancer drugs (TP and DOX), and a redox-sensitive
6
bond (DTSP) to the OCMCh backbone (Fig. S1). This novel polymeric micelle has a
7
hydrophilic group and a hydrophobic group. Therefore, it can form micelles in aqueous
8
solution. Our results show that hydrophobic group-conjugated OCMCh formed particles. The
9
particle size of TOC1 and TOC3 was 1096.8 nm and 1347.1 nm, respectively. The
10
conjugation ratio of TP was increased 1% to 3% (molar ratio), but particle size increased
11
from 1096.8 nm to 1347.1 nm. The explanation for this phenomenon could be that TOC
12
formed micelles, but did not form strong hydrophobic interactions due to very low
13
hydrophobicity. We used OCMCh with an MW of 12,000 Da in this study. It consisted of
14
approximately 54.5 monomers (MW 220 Da). TP was conjugated to OCMCh monomers in
15
ratios of 0.5 (TOC1) and 1.6 (TOC3). Therefore, TOC has very low hydrophobicity and
16
relatively large particle sizes. The TOC micelles aggregated when TP was conjugated to
17
OCMCh with a molar ratio higher than 3%. However, when DOX was conjugated to TOC,
18
the particle size of TOC1-DOX and TOC3-DOX significantly decreased from 1096.8 nm to
19
127.4 and 1347.1 nm to 244.9 nm, respectively, because tight micelles were formed by strong
20
hydrophobic interactions. Several studies reported that the particle size decreased when
21
hydrophobic drugs were associated with polymeric micelles [38-40]. In addition, we
22
modified TOC-DOX micelles with a targeting ligand to allow site-specific delivery of anti-
23
cancer drugs. The particle size of HPTOC-DOX was slightly increased because of the
24
addition of the hydrophilic group (HER2/neu targeting peptide-PEG; HP).
25
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In this study, DTSP was used as a linker to conjugate DOX to the micelle and to confer 20
Page 19 of 36
redox-sensitivity. DTSP has a disulfide bond and DTSP-based drug carriers allow drugs to be
2
accumulated in target tissues at higher concentrations than they would with drug carriers
3
without disulfide bond-containing linkers [41]. As mention above, DTSP is is cleavable in the
4
cytoplasm by an intracellular reducing agent such as glutathione (GSH, 0.5 – 10 mM) [1, 2,
5
31, 32]. The disulfide bond in HPTOC-DOX micelles allows DOX release at the target site
6
due to the reducing agent after cellular uptake.
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The DOX release profile from polymeric micelles at different pH values indicated that the
8
synthesized polymeric micelles (TOC-DOX and HPTOC-DOX) released DOX at the tumor
9
site. TOC-DOX and HPTOC-DOX retained the conjugated DOX at pH 7.4. This means that
10
the TOC-DOX and HPTOC-DOX micelles are stable in blood plasma (pH 7.4). Drug carriers
11
consisting of polycationic polymers such as chitosan, polyethylenimine (PEI), and polylysine
12
(PLL) may not be considered as drug delivery systems for systemic administration due to
13
their positive charge. Positively charged polycationic polymers can interact with negatively
14
charged erythrocyte membranes and lead to hemolysis. Therefore, the stability of the
15
synthesized polymeric micelles is very important for RBC stability. Our results show that
16
TOC-DOX and HPTOC3-DOX micelles are stable in blood plasma and can be used as a
17
systemic drug delivery system.
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In general, targeted delivery systems are divided into two kinds: passive targeting systems
19
and systems with covalently conjugated targeting molecules on the surface of drug carriers.
20
The targeting ligand conjugated to drug carriers can bind to receptors expressed specifically
21
in cancer cells [11, 14]. The target receptor, human epidermal growth factor receptor 2
22
(HER2), which is also known as Neu, CD340, ErbB-2, or p185, is overexpressed in
23
approximately 30% of breast cancers and 20% of ovarian cancers, while its expression in
24
normal tissues is low [14, 42]. The HPTOC-DOX micelle, containing an anti-HER2 targeting
25
peptide, showed a better therapeutic effect better than TOC-DOX both in vitro in SK-BR-3 21
Page 20 of 36
cells and in vivo in SK-BR-3 tumor-bearing mice (Fig. 6B and Fig. 7). These results clearly
2
demonstrated that HPTOC-DOX cellular uptake was through receptor-mediated endocytosis
3
via an interaction between the anti-HER2 targeting peptide and the overexpressed HER2/neu
4
receptor on the SK-BR-3 cell surface. The receptor-mediated endocytosis enhanced cellular
5
uptake efficacy of micelles and led to increased therapeutic efficacy. In addition, we designed
6
multi-drug-conjugated polymeric micelles. TP was used to not only enhance hydrophobicity
7
but also as an anti-cancer drug. TP has anti-cancer activity against various cancer cells and
8
can induce apoptosis and diminish cell differentiation by blocking the cell cycle [27-30].
9
Therefore, TP-conjugated TOC micelles at concentrations exceeding 0.625 µg/mL
10
significantly decreased the viability of SK-BR-3 cells (Fig. 6A). The synergistic effect of TP
11
and DOX was evident from the low cell viability after treatment with a very low
12
concentration (0.078125 µg/mL) of TOC-DOX and HPTOC-DOX (Fig. 6B). Interestingly,
13
the viability of cells treated with polymeric micelles was higher than or similar to that of cells
14
treated with free DOX at concentrations exceeding 0.625 µg/mL. These results demonstrate
15
that that TP and DOX in multi-drug-conjugated TOC-DOX and HPTOC-DOX have a
16
synergistic effect in tumor therapy.
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In conclusion, we synthesized novel polymeric micelles with a targeting ligand, two anti-
18
cancer drugs (TP and DOX), and a redox-sensitive function. The synthesized TOC-DOX and
19
HPTOC-DOX formed polymeric micelles in aqueous solution through hydrophobic
20
interactions between the conjugated TP and DOX. The particle size of TOC-DOX and
21
HPTOC-DOX micelles was in the range of 124.7-244.9 nm and 151.9-311.2 nm, respectively.
22
The cumulative drug release of HPTOC-DOX of around 90% within 6 h after the pH changed
23
from 7.4 to 5.2. The anti-cancer drugs of TOC-DOX and HPTOC-DOX had a synergistic
24
effect against SK-BR-3 cells. The anti-HER2/neu targeting peptide (epitope form,
25
LTVSPWY) was used as the targeting moiety. It enhanced not only cellular uptake by 22
Page 21 of 36
HER2/neu overexpressing SK-BR-3 cells, but also therapeutic efficacy in SK-BR-3 tumor-
2
bearing mice. The targeting peptide-modified HPTOC-DOX had lower cytotoxicity than free
3
DOX and enhanced therapeutic efficacy as evidenced by results from both in vitro and in vivo
4
assays.
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Page 22 of 36
Acknowledgements
2
This work was supported by the National Research Foundation of Korea (NRF) grant funded
3
by the Ministry of Science, ICT & Future Planning (NRF-2014R1A2A1A10053027).
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Figure Legends
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10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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Figure 3. DOX release profile of TOC-DOX and HPTOC-DOX micelles against various pH conditions. The pH conditions maintained at pH 7.4 during 24 h from 30 min and then decreased at pH 6.2 (A) or pH 5.2 (B) until 30 h. The results are presented as mean ± SD (n=3).
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Figure 4. Localization of various polymeric micelles in SK-Br3 cells. SK-Br3 cells were incubated with DOX, TOC-DOX, and HPTOC-DOX and then were observed in confocal laser scanning microscopy. SK-Br3 cells were pre incubated with LysoTracker and Hoechst 33258 to confirm intracellular localization.
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Figure 2. Particle size and distribution of various polymeric micelles. A) Particle size of TOC1, TOC1-DOX, HPTOC1-DOX, TOC3-DOX, TOC3-DOX, and HPTOC3-DOX. The results are presented as mean ± SD (n=3). B) Particle size distribution of HPTOC3-DOX.
Figure 5. A) In vitro cell cytotoxicity of DOX, TOC-DOX, and HPTOC-DOX against HEK 293 cells (a) or HaCa T cells (b). B) In vitro hemolytic assay of TOC-DOX and HPTOCDOX with different micelles concentration (0.625 to 5 µg/mL) against rat red blood cells (RBCs) from an inbred rat. D-PBS and 0.1% triton X were used the negative control and positive control. The results are presented as mean ± SD (n=5)
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Figure 1. 1H-NMR spectrum of HPTOC-DOX. Concentration of HPTOC-DOX is 5 mg/0.5 mL in D2O.
Figure 6. In vitro anti-tumor effect of intermediate structure or final products against SK-Br3 cells. The anti-tumor levels were expressed by percentage of cell viability. A) cell viability of OCMCh, TP, TOC1, and TOC3; B) cell viability of DOX, TOC1-DOX, HPTOC1-DOX, TOC3-DOX, HPTOC3-DOX. The results are presented as mean ± SD (n=5)
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Figure 7. Therapeutic efficacy and characterization of systemically injected TOC-DOX or HPTOC-DOX micelles in HER2 overexpressed SK-Br3 subcutaneous tumor models. A) Inhibition of tumor growth by intravenous injection of TOC-DOX or HPTOC-DOX micelles (20 mg/kg).*P
