Self-assembly behaviors of thermal- and pH- sensitive magnetic nanocarriers for stimuli-triggered release
© Kuo et al.; licensee Springer. 2014
Received: 16 July 2014
Accepted: 7 September 2014
Published: 22 September 2014
In the present work, we prepare thermo- and pH-sensitive polymer-based nanoparticles incorporating with magnetic iron oxide as the remote-controlled, stimuli-response nanocarriers. Well-defined, dual functional tri-block copolymer poly[(acrylic acid)-block-(N-isopropylacrylamide)-block-(acrylic acid)], was synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization with S,S′-bis(α,α′-dimethyl-α″-acetic acid)trithiocarbonate (CMP) as a chain transfer agent (CTA). With the aid of using 3-aminopropyltriethoxysilane, the surface-modified iron oxides, Fe3O4-NH2, was then attached on the surface of self-assembled tri-block copolymer micelles via 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinamide (EDC/NHS) crosslinking method in order to furnish not only the magnetic resources for remote control but also the structure maintenance for spherical morphology of our nanocarriers. The nanocarrier was characterized by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FT-IR), and ultraviolet–visible (UV/Vis) spectral analysis. Rhodamine 6G (R6G), as the modeling drugs, was encapsulated into the magnetic nanocarriers by a simple swelling method for fluorescence-labeling and controlled release monitoring. Biocompatibility of the nanocarriers was studied via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed that neither the pristine nanocarrier nor the R6G-loaded nanocarriers were cytotoxic to the normal fibroblast cells (L-929 cells). The in vitro stimuli-triggered release measurement showed that the intelligent nanocarriers were highly sensitive to the change of pH value and temperature rising by the high-frequency magnetic field (HFMF) treatment, which provided the significant potential to apply this technology to biomedical therapy by stimuli-responsive controlled release.
Over the past decades, polymeric nanoparticles have received great interests in drug delivery systems owing to their drug loading capacity and biocompatibility for targeting therapy . As drug carriers, polymeric nanoparticles were able to load therapeutic agents, usually hydrophobic drugs, in the hydrophobic core region, while the hydrophilic shell region provided good dispersion in water and reduced the toxicity . Polymeric nanoparticles also have shown prolonged-circulation characteristics and great-accumulation properties in tumor tissue because of the inefficient drainage by the lymph system, the so-called enhanced permeation and retention (EPR) effect . Thus, efforts have been devoted to developing the stimuli-responsive polymers to be served as drug delivery vehicles, which could undergo a tremendous and reversible transition of morphology and dominate the precise controlled release, and work as a response to the various environmental stimuli, like temperature , pH , ionic strength , and magnetic field . Thermo-sensitive polymers were widely investigated since the temperature was easy to control and could provide several advantages for in vivo and in vitro treatments. Among these thermal-sensitive polymers, poly(N-isopropylacrylamide) (PNIPAAm) was the best-known one; it preserved the considerable phase transition in aqueous system from hydrophilic to hydrophobic form around 32°C, which is the lower critical solution temperature (LCST) .
Since the LCST was close to the human body temperature, hydrophilic-segments, like acrylic acid (AA), were introduced into PNIPAAm for LCST tuning by co-polymerization , surface modification, or physical blending in order to prepare hollow structured nanoparticles via thermo-triggered self-assembling. Actually, the spherical sub-micron particle with empty core region possessed many advantages, for instance, higher drug loading content . In spite of the benefits for drug delivery, the practical controlled release was still hindered with poor in vivo colloidal stability , which often caused low therapeutic efficiency and side effects.
On the other hand, magnetic materials were also widely used as nanocarriers' preparation because of the noncontact force which provided magnetically triggered release and remote treatment for drug delivery in therapy applications [12–17]. It was proved that the synergy-combined hyperthermia and chemotherapy was considerably effective for cancer therapy from the aid of tissue deoxygenating and cell-killing above 42°C . Although magnetic sub-micron particles were the outstanding source as magnetic agent in the application of magnetic resonance imaging (MRI) , bioseparation, specific cell-detection, and so on, the applications of pure iron oxide (Fe3O4) were still limited due to its poor water dispersion. Thus, the incorporation or the surface modification of magnetic iron oxide with polymers has been employed to promote the biocompatibility and the drug-delivery capability.
In order to obtain the well-defined molecular-weight-distribution polymers, dual functional tri-block copolymer, P(AA-b-NIPAAm-b-AA), was synthesized by reversible addition- fragmentation chain-transfer (RAFT) polymerization in this research. The temperature-inducing self-assembling characteristic of the tri-block copolymers provide the excellence in drug encapsulation, owing to the interaction between hydrophobic PNIPAAm and hydrophilic poly(acrylic acid) (PAA) segments. Surface-modified iron oxide (Fe3O4-NH2) was introduced into the system via N-hydroxysuccinamide (NHS) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) crosslinking method. Fe3O4-NH2 provided not only the stability of nanoparticles but also the ability of magnetic controlled release. After the encapsulated model drug, Rhodamine 6G (R6G), it gives the optical functionality via fluorescence labeling; the releasing behavior of R6G-loaded nanocarriers was studied under the condition of various temperatures and pH values. Furthermore, an external high-frequency magnetic field (HFMF) was applied for the remote control release of our intelligent magnetic nanocarriers.
N-Isopropylacrylamide (NIPAAm) was re-crystallized from n-hexane and acrylic acid (AA) was purified by vacuum distillation in order to remove the inhibitors before being used as the monomers. The RAFT chain-transfer agent, S,S′-bis(α,α′-dimethyl-α″-acetic acid)trithiocarbonate (CMP) was synthesized, following the protocol proposed by Lai and his coworkers . Azobisisobutyronitrile (AIBN) was re-crystallized from methanol and then used as the thermal initiator. Rhodamine 6G (R6G), the model drug was purchased from Sigma-Aldrich. Iron(II) chloride tetrahydrate (FeCl2 · 4H2O) and iron(III) chloride (FeCl3) were used as the precursors of magnetic iron oxide (Fe3O4) nanoparticles. Ammonia solution (33%) was used as the reducing agent. 3-Aminopropyl triethoxysilane (APTES), NHS and EDC were introduced for surface modification. Over 99% of HPLC methanol was purchased from Sigma-Aldrich. Deionized water (18.2 MΩ) was used throughout the work. All chemicals and solvents were purchased from Sigma-Aldrich (Taipei, Taiwan), Acros (Taipei, Taiwan), or Fluka (Taipei, Taiwan) and used as received except otherwise noted.
Synthesis of magnetic iron oxide nanoparticles (Fe3O4)
Magnetic nanoparticles, Fe3O4, were prepared by coprecipitation method as reported . In brief, a mixture of FeCl3 and FeCl2 · 4H2O with 2:1 molar ratio was dissolved in deionized water. After completely dissolving, ammonium hydroxide was added dropwise under mechanical stirring at room temperature for 30 min. Then, the magnetic iron oxides were collected by magnet and washed with water three times.
Surface-modification of magnetic iron oxide with amine groups (Fe3O4-NH2)
The sol–gel method was applied for the surface modification of Fe3O4 with amine groups. In short, Fe3O4 was dispersed with ammonia solution in an ethanol/water (9/1, v/v) solution at pH greater than 11 with sonication for 30 min. APTES was then added into the system and stirred for another 12 h at 25°C. Thereafter, the magnetic iron oxide nanoparticles were collected and re-dispersed in deionized water for further use.
RAFT polymerization of tri-block copolymers P(AA-b-NIPAAm-b-AA)
AA monomer (3.062 g) was dissolved in 20 ml of methanol containing 0.12 g of chain-transfer agent, CMP, and 0.017 g of initiator, AIBN. After the chemicals were totally dissolved, the reaction was preceded for 3 h at 70°C and purged with nitrogen, so that the conversion of AA monomers could reach 99.29%. NIPAAm monomer (7.21 g) was dissolved in 20 ml methanol then introduced into the system for another 14 h and the tri-block copolymer P(AA-b-NIPAAm-b-AA) solution was obtained. The solution was first dialyzed with methanol so as to remove unreacted monomers, and then vacuum-dried at room temperature. Therefore, the P(AA-b-NIPAAm-b-AA) copolymer, A100N150, was collected. AxNy represents the mole feeding ratio of x mole of AA and y mole of NIPAAm monomers to chain transfer agent (CTA) molecules.
Preparation of self-assembled magnetic nanocarriers (A100N150/Fe3O4-NH2)
Fe3O4-NH2 was covalently bonded with the tri-block copolymer, P(AA-bNIPAAm-bAA) via the 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinamide (EDC/NHS) method as described in the preparation of nanocarriers. A100N150 (0.25 g) was dissolved in 50 ml H2O, and the pH value was adjusted to 4.5 with the diluted NaOH solution. After the self-assembling process at 60°C for 2 h, 0.05 g of magnetic iron oxide, Fe3O4-NH2 was added into the system and then stirred for another 4 h at 60°C. EDC (0.2 g) and NHS (0.2 g) were then added into the solution for activation and chemical bonding at 60°C for another 24 h. The self-assembled magnetic nanocarriers, A100N150/Fe3O4-NH2, were centrifuged and washed with water three times and eventually collected as powder after lyophilization for further use.
Preparation of drug-loaded nanocarriers with high-concentration R6G solution
Controlled release behavior
Gel permeation chromatography (GPC)
The average molecular weight and molecular weight distribution of P(AA-b-NIPAAm-b-AA) via RAFT polymerization were determined by GPC, which was equipped with a solvent delivery system, two columns (Malvern Viscotek T-columns LT5000, Malvern Instruments, Ltd., Taipei, Taiwan) maintained at 40°C, and a differential refractometer (RI). Sodium sulfate aqueous solution (0.025 M) was applied as the eluent at a flow rate of 0.8 mL/min. Poly(methylacrylic acid) standards were used for calibration standard and the molecular weight was reported. A diluted sodium hydroxide solution (0.02 g) was also used to improve the solubility of tri-block copolymer in 4 ml of sodium sulfate aqueous solution.
Nuclear magnetic resonance spectroscopy (NMR)
NMR was applied to determine the ratio of two monomer units (AA and NIPAAm) in P(AA-b-NIPAAm-b-AA). Therefore, 1H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer (Bruker, Taipei, Taiwan), using D-methanol (D4) or D-chloroform (D1) as solvent.
Fourier transform infrared spectroscopy (FT-IR)
FT-IR absorption spectra were recorded by PerkinElmer Spectrum 100 spectrometer (PerkinElmer, Taipei, Taiwan) at frequencies ranging from 400 to 4,000 cm-1 with 4 cm-1 resolution. The samples were mixed with KBr and pressed into pellets.
LCST of tri-block copolymer, P(AA-b-NIPAAm-b-AA)
The cloud point or LCST of P(AA-b-NIPAAm-b-AA) was measured by using a UV/Vis spectrophotometer (Thermo Spectronic Gamma Series, Thermo Scientific, Taipei, Taiwan). Latex solutions were prepared by dissolving 120 mg of samples in 4 ml of different pH value solutions. Then, the transmittance of the solution at 480 nm was detected with the heating rate of 5°C/min at different temperatures.
Zeta potential measurement
The surface potential of self-assembled sub-micron particles was measured via zeta potential analyzer (Zetasizer Nano, Malvern Instruments Ltd., Taipei, Taiwan). The zeta potential data reported were the average values of triplicate samples with standard deviation.
The latex solutions were diluted with deionized water and dripped on a copper grid coated with a collodion. It was dried in air or in an oven with specific temperatures and observed by using JEOL JSM-1230 transmission electron microscope (TEM) (Jeol, Taipei, Taiwan).
Cell lines and cytotoxicity assay
L-929 fibroblast cells were purchased from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and was cultured using Dulbecco’s modified Eagle's medium (DMEM) composed of 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic antimycotic solution. The cell lines were incubated under saturated humid conditions at 37°C and 5% CO2, and the medium was changed every 1 to 2 days until confluence was reached at approximately 70% to 80% confluency.
Cytotoxicity analysis of the magnetic nanocarriers was determined via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay with L-929 fibroblasts cell as a model cell. L-929 cells were cultured (5,000) and placed in an incubator under saturated humid conditions at 37°C and 5% CO2 in each 24-well plates for 24 h cultivation. Our nanocarriers (A100N150/Fe3O4-NH2 or A100N150/Fe3O4-NH2/R6G) were mixed with PBS solution and sterilized through a 0.22 μm membrane filter. Thus, the sterilized solution was mixed with DMEM and denoted as the A100N150/Fe3O4-NH2-contained or A100N150/Fe3O4-NH2/R6G-contained medium. For toxicity studies, L-929 cells were incubated with those two mediums for 3 days. Then the control groups, pure DMEM (negative control) or 5% dimethyl sulfoxide (DMSO) in DMEM (positive control) were studied. After culturing, the resulting blue-purple crystals were dissolved in pure DMSO for 15 min, so that the cell number could be calculated by enzyme-linked immunosorbent assay (ELISA) (Sunrise, Tecan Group Ltd., Zürich, Switzerland).
Results and discussion
Synthesis and molecular characterization of P(AA-b-NIPAAm-b-AA)
Molecular characterization of P(AA-b-NIPAAm-AA)
LCST of P(AA-b-NIPAAm-b-AA)
The surface potential of sub-micron particles was determined as 0.002 via zeta potential analyzer which indicated that the tri-block copolymer, A100N150, was closed to the isoelectric point at pH = 4.5 and 60°C. Owing to the hydrophilic property of PAA segments, which provided the stability to the extreme hydrophobic PNIPAAm segments, the uncharged sub-micron particles could disperse well in an aqueous solution.
Preparation of R6G-loaded magnetic nanocarriers via swelling method
Cell line and cytotoxicity analysis
Controlled release with various pH values and temperatures
Polymer-framed, stimuli-response sub-micron particles were designed and prepared with magnetic iron oxides as intelligent multifunctional nanocarriers. RAFT polymerization was first employed for the P(AA-b-NIPAAm-b-AA) tri-block copolymer synthesis in order to obtain the well-defined self-assembling sub-micron particles. With EDC/NHS crosslinking method, Fe3O4-NH2 bonded with the tri-block copolymer micelle could not only provide the magnetically resource but also prevents the collapses of the spherical structure. The magnetic-triggered nanocarriers did not display cytotoxicity in L-929 fibroblasts, which provided the benefits for medical therapy like oral dosing. R6G was able to be encapsulated in the core as well as on the surface of nanocarriers due to the strong interaction between R6G and the polymers. The stimuli-response release behavior reveals that the intelligent multifunctional nanocarriers were highly pH and magnetic sensitive owing to the iron oxides and the functional groups (-COOH and -NH2) on its surface. Since the amount of release increased as the pH value increased, the nanocarrier has great potential for therapeutic application to intestinal illness with the demand of low leakage in stomach where the pH was in the range of 2 to 3. Moreover, the multifunctional nanocarriers exhibit three times of the release percentage under high-frequency magnetic field treatment in a short period (temperature above 50°C), which could acquire rapid and accurate therapy in practical applications. On the whole, there is a great potential of using the intelligent, multifunctional nanocarriers in biomedical targeting therapy especially for intestinal sickness.
CYK is a PhD students at National Taiwan University. AH is a PhD students at National Taiwan University of Science and Technology. CFL holds a professor position at Chia Nan University of Pharmacy and Science. MSW is a researcher at National Taipei University of Technology. TYL holds an assistant professor position at Ming Chi University of Technology. WYC holds a professor position at National Taiwan University.
This work was financially supported by the National Science Council of Taiwan (NSC 99-2221-E-002-019-MY3).
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