Synthesis and characterization of NIR-responsive Aurod@pNIPAAm-PEGMA nanogels as vehicles for delivery of photodynamic therapy agents
© Shang et al.; licensee Springer. 2013
Received: 19 November 2012
Accepted: 11 December 2012
Published: 2 January 2013
A near-infrared (NIR)-responsive Aurod@pNIPAAm-PEGMA nanogel was synthesized in two steps, growing a PEGMA monolayer on the surface of gold nanorods (AuNRs), followed by in situ polymerization and cross-linking of N-iso-propylacrylamide (NIPAAm) and poly-(ethylene glycol)-methacrylate (PEGMA). The AuNRs and Aurod@pNIPAAm-PEGMA nanogel were characterized by UV–vis spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy, respectively. The lower critical solution temperature of the Aurod@pNIPAAm-PEGMA nanogel could be tuned by changing the molar ratio of NIPAAm/PEGMA. The NIR-mediated drug release behavior of the Aurod@pNIPAAm-PEGMA nanogel was studied with zinc phthalocyanines (ZnPc4) as a drug model. It was also demonstrated that the loaded ZnPc4 could keep the capability of generating singlet oxygen, and the in vitro study showed a great photodynamic therapy (PDT) effect on Hela cells. It thus indicated the potential of this Aurod@pNIPAAm-PEGMA nanogel for application as a drug carrier in PDT, which might make contributions to oncotherapy.
Gold nanoparticles including nanoshells, nanocages, and nanorods have drawn increasing attention in photodynamic therapy (PDT), drug delivery, and diagnostic imaging field in recent years [1–5]. Among them, gold nanorods (AuNRs) are of particular interest due to their unique optical properties. With the different aspect ratios and the resulting longitudinal surface plasmon resonance (SPR), AuNRs exhibit an absorption band in the near-infrared (NIR) region , which conduces to higher photothermal conversion and also shows significant biomedical application in view of the penetration of NIR light into biological tissues [7, 8].
Poly(N-isopropylacrylamide) (pNIPAAm) gel, as one of the most widely studied temperature-responsive polymers [9–11], undergoes phase transition in water when the temperature increases or decreases beyond its lower critical solution temperature (LCST; approximately 32°C) [12, 13]. Besides, its LCST can be tuned by the addition of a comonomer during polymerization [14, 15]. Combining this temperature-responsive gel and gold nanoparticles together, the temperature-responsive gel can be induced to collapse by the photothermal conversion of gold nanoparticles, which gives rise to much possibility for the application of the temperature-responsive gel in drug delivery. The composites of Au@pNIPAAm have been synthesized and studied in many works [16–18]. However, the combination mostly through the physical embedding effect or electrostatic interaction between gold nanoparticles and pNIPAAm may make the composites lack long-term stability, especially in the biological environment. Our previous work has reported the synthesis of a core-shell structured multifunctional hybrid Au@IPN-pNIPAAm nanogel in which the hydrogel could be chemically grafted onto a single gold nanoparticle .
Herein, we developed a new way to immobilize pNIPAAm combined with poly-(ethylene glycol)-methacrylate (PEGMA) on the surface of AuNRs through chemical grafting to obtain NIR-responsive Aurod@pNIPAAm-PEGMA nanogel. ZnPc4, a photosensitizer, was used as drug model to investigate the drug loading and release properties of the Aurod@pNIPAAm-PEGMA nanogel. The capacity of generating singlet oxygen of ZnPc4 after being loaded in the Aurod@pNIPAAm-PEGMA nanogel was measured, and the in vitro PDT was also studied. Our current results suggested the potential of Aurod@pNIPAAm-PEGMA nanogel as a carrier in PDT.
Synthesis of PEGMA-SH compound
Concentrations of 1.0 mmol 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and 2.0 mmol dicyclohexylcarbodiimide (DCC) were dissolved in 50 mL of dichlormethane, followed by the addition of 2.2 mmol 4-dimethylaminopyridine (DMAP) and 2.0 mmol PEGMA. The mixture was degassed with nitrogen and then stirred for 48 h at room temperature. After filtration, the filtrate was washed sequentially with water, 5% acetic acid, and water. Then, the organic phase was dried over magnesium sulfate, filtered, and evaporated to dryness. The product was dissolved in 100 mL of water/ethanol (V/V, 4/1) with the addition of 2 mL of 1 M sodium borohydride (NaBH4) and stirred for 2 h, and was used without further purification.
Synthesis of Aurod@pNIPAAm-PEGMA nanogel
AuNRs with a length of 50 nm were synthesized using the seed-mediated growth method as reported previously . Subsequently, 0.1 mmol PEGMA-SH was added to 25 mL of the as-prepared AuNRs suspension (1.6 × 10−6 μmol) and continuously stirred for 5 h at room temperature. Aurod@PEGMA was collected by centrifugation at 9,500 rpm for 12 min and then re-dispersed in 15 mL of the deionized water, followed by the addition of 1.8 mmol NIPAAm, 0.2 mmol PEGMA, 86.69 μmol sodium dodecyl sulfate (SDS), and 12.97 μmol N,N-methylenebisacrylamide (BIS). The mixture was heated to 75°C with stirring and maintained in vacuum. After equilibration for 1 h, the polymerization was initiated by adding 109.6 μmol ammonium persulfate (APS). The reaction was allowed to proceed for 4 h at 75°C and terminated by opening the system to air. The resulting Aurod@pNIPAAm-PEGMA nanogels were purified by repeated centrifugation (9,000 rpm for 12 min) and subsequently lyophilized for further use.
The optical properties of AuNRs and Aurod@pNIPAAm-PEGMA nanogels were characterized by an UV–vis spectrophotometer (DUTM800, Beckman Coulter, Brea, CA, USA) with a scanning speed of 1,200 nm/min from 400 to 1,000 nm. The transmission electron microscopy (TEM) images were obtained from a JEM 2100 microscope (JEOL Ltd., Tokyo, Japan) operating at an acceleration voltage of 200 kV. Raman spectra were performed on an UV-1000x instrument (Renishaw, Wotton-under-Edge, UK) (path length = 200 nm) using a red light-emitting diode laser (λ = 785 nm, 0.5 mW). A Fourier transform interferometer (AVATAR360, Nicolet Instrument Corporation, Madison, WI, USA) was used to record the absorption spectra of AuNRs and Aurod@pNIPAAm-PEGMA nanogels between 400 and 4,000 cm−1 at a spectral resolution of 4 cm−1.
LCST measurement of Aurod@pNIPAAm-PEGMA nanogel
In order to investigate the thermal property of the Aurod@pNIPAAm-PEGMA nanogel, nanogels with different molar ratios of NIPAAm/PEGMA (1:0, 18:1, 12:1, 9:1, 6:1, 4.5:1) were synthesized. LCSTs of nanogels were measured through turbidimetric measurement. The concentration for each Aurod@pNIPAAm-PEGMA nanogel in the deionized water was maintained at 1 mg/mL. The light transmittances at 600 nm were then measured by an UV–vis spectrophotometer (TU-1901, Beijing Purkinje General Instrument Co. Ltd, Beijing, China) equipped with a temperature-controlled sample holder, and the heating rate was set at 0.1°C/min. The LCST was defined as the initial break point in the resulting transmittance versus temperature curves.
ZnPc4 loading and NIR-mediated ZnPc4 release
where Wt represents the total amount of ZnPc4 and W0 represents the unloaded amount of ZnPc4.
where Wr represents the released amount of ZnPc4 and Wl represents the loaded amount of ZnPc4.
Singlet oxygen detection
The generation of singlet oxygen (1O2) from ZnPc4 loaded in the Aurod@pNIPAAm-PEGMA nanogel was determined by the transformation of 9,10-dimethylanthracene (DMA) which exhibits a maximum absorption at 377 nm . The DMA can react irreversibly with 1O2 to yield an endoperoxide. The reaction could be monitored by recording the decrease in the absorption at 377 nm. In a typical experiment, 0.105 mg of the Aurod@pNIPAAm-PEGMA nanogel loaded with 0.0135 μmol ZnPc4 was dispersed in 3 mL of DMF, and then, 0.45 μmol DMA was added. Pure ZnPc4 (0.0135 μmol) was used as a control. The solutions were then irradiated with a LED lamp (680 nm, 10 mW/cm2) or a NIR laser (808 nm, 400 mW/cm2). The absorption measurements followed by irradiation were carried out every 5 min.
Light-induced in vitro PDT effect
Hela cells were seeded into 24-well cell culture plates (1 × 105 cells/well) and incubated for 24 h. After being treated with ZnPc4-loaded Aurod@pNIPAAm-PEGMA nanogels (300 μg/mL) in serum-free medium at 37°C for 22 h, chloroquine (10 mg/mL) was added into every well for another 2 h to promote endosomal escape . Then, Hela cells were washed with PBS and incubated in a nanogel-free medium and treated with an 808-nm laser at 400 mW/cm2 for 15 min and a 680-nm LED lamp at 10 mW/cm2 for 40 min. For cell survival test, the irradiated plates were returned to the incubator, and cell viability was colorimetrically measured 48 h later with MTT assay .
Results and discussion
Synthesis of Aurod@pNIPAAm-PEGMA nanogel
Thermosensitive property of Aurod@pNIPAAm-PEGMA nanogel
The LCSTs of Au rod @pNIPAAm-PEGMA nanogels with different molar ratios of NIPAAm/PEGMA
NIR-mediated ZnPc4 release
The effect of laser power density on drug release was studied (Figure 8B). Exposure of Aurod@pNIPAAm-PEGMA nanogels to an 808-nm laser with the power of 100 mW/ cm2 for 15 min caused 20% of the loaded ZnPc4 released. More loaded ZnPc4 (43.7%) in Aurod@pNIPAAm-PEGMA nanogels could be released upon the irradiation power of 800 mW/ cm2. This is because when irradiated with a low-power NIR laser, small shrinkage of nanogels occurred, whereas a laser at high power might make nanogels shrink considerably and rapidly , consequently more ZnPc4 could be released. It is thus speculated that the NIR-responsive Aurod@pNIPAAm-PEGMA nanogel, acting as drug delivery carriers, could offer specific drug delivery to the targeted site, such as a tumor zone.
Singlet oxygen detection
In PDT, the photosensitizing drugs should preferentially accumulate in target tissues and subsequently be activated by light with a matching wavelength to generate reactive singlet oxygen . The singlet oxygen will cause the destruction of target cells by a complex cascade of chemical, biological, and physiological reactions . The Aurod@pNIPAAm-PEGMA nanogels served as ZnPc4 carrier in PDT; besides the excellent properties of drug loading and release, its effect on the capability of loaded ZnPc4 to generate singlet oxygen was also investigated.
In vitro PDT study on Hela cells
A facile approach to prepare near-infrared-responsive Aurod@pNIPAAm-PEGMA nanogels was described. The LCSTs of these Aurod@pNIPAAm-PEGMA nanogels could be tuned by changing the molar ratio of NIPAAm/PEGMA. The release of ZnPc4 loaded in Aurod@pNIPAAm-PEGMA nanogels increased with the extension of irradiated time and the increase of the power of the NIR laser. The loaded ZnPc4 in Aurod@pNIPAAm-PEGMA nanogels could generate singlet oxygen efficiently. The in vitro study showed obvious PDT effect on Hela cells. On these bases, the Aurod@pNIPAAm-PEGMA nanogels might serve as a promising drug carrier in PDT.
RL, TXH, and LDH are Ph.Ds. and professors. ST, WCD, KXB, YAQ, and CM are M.D. students in the Department of Biomaterials, College of Materials, Xiamen University.
This work was financially supported by the National Basic Research Program of China (2010CB732402, 2013CB933703) and the National Natural Science Foundation of China (30970733, 81171448).
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