Yu-Hsiang Lee1,2,3* and Yun-Han Lai1
1Graduate Institute of Biomedical Engineering, National Central University, Taoyuan City, Taiwan
2Department of Biomedical Sciences and Engineering, National Central University, Taoyuan City, Taiwan
3Department of Chemical and Materials Engineering, National Central University, Taoyuan City, Taiwan
Received: 25 August, 2015; Accepted: 25 September, 2015; Published: 28 September, 2015
Yu-Hsiang Lee, Department of Biomedical Sciences and Engineering, National Central University. No. 300, Jhongda Rd., Taoyuan City, 32001, Taiwan R.O.C. Tel: (+886)-3-422-7151; Ext #: 37352; Fax: (+886)-3-280-4627; E-mail:
Lee YH, Lai YH (2015) Fabrication and Characterization of HER2 Cell Receptor-Targeted Indocyanine Green-Encapsulated Poly (Lactic-co-Glycolic Acid) Nanoparticles. Peertechz J Biomed Eng 1(1): 015-020.
© 2015 Lee YH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Indocyanine green; PLGA nanoparticle; PEG; anti-HER2 antibody; Near Infrared; Phototherapy
Introduction: The aim of this study is to fabricate and characterize human epidermal growth factor receptor 2 (HER2)-targeted indocyanine green (ICG)-loaded poly (lactic-co-glycolic acid (PLGA) nanoparticles (HIPNPs).
Methods: The HIPNPs were fabricated by a modified emulsification in association with solvent evaporation approach. The size and surface charge of the manufactured nanoparticles were determined by dynamic light scattering technique. The morphology of the HIPNPs was detected by SEM. The activity of surface anchored anti-HER2 antibodies was detected by spectro fluorometry and fluorescent microscopy. The encapsulation rate of ICG, percentage of ICG content, and the degradation efficiencies of entrapped ICG under different temperatures were measured through UV-Vis spectrometry.
Results: All HIPNPs exhibited particulate morphology with size of 302 ± 1.8 nm and surface charge of -15 ± 0.15 mV where the polydispersity index was in the range of 0.02 - 0.07. The encapsulation rate of ICG and percentage of ICG content in the HIPNPs were 70% and 23%, respectively. The stability of ICG can be improved after encapsulated into the PLGA nanoparticles, by which the degradation rates of entrapped ICG in 4 oC and 37 oC aqueous medium significantly reduced about 6- and 3-fold, respectively, as compared to the freely dissolved ICG within 48 h. All HIPNPs exhibited particulate morphology with size of 302 ± 1.8 nm and surface charge of -15 ± 0.15 mV where the polydispersity index was in the range of 0.02 - 0.07. The encapsulation rate of ICG and percentage of ICG content in the HIPNPs were 70% and 23%, respectively. The stability of ICG can be improved after encapsulated into the PLGA nanoparticles, by which the degradation rates of entrapped ICG in 4 oC and 37 oC aqueous medium significantly reduced about 6- and 3-fold, respectively, as compared to the freely dissolved ICG within 48 h.
Conclusions: We have successfully fabricated and characterized the HIPNPs in this study. Based on the enhanced stability, bioavailability, biocompatibility, and target-ability of the HIPNPs, the developed ICG Nano-carriers exhibited a high potential for use in near infrared-based diagnostics and therapeutics in vitro and/or in vivo for HER2-expressing cells.
Indocyanine green (ICG), also known as cardio green, is an U.S. Food and Drug Administration-approved water-soluble tricarbocyanine dye that enables to absorb and fluoresce in the near-infrared (NIR) wavelength region (650 - 850 nm) . As water and most intrinsic biomolecules in tissue do not absorb strongly within the NIR range , interest in using ICG as optical probe for deeper tissue structure is increasing. So far ICG has been extensively used and/or investigated in a variety of diagnostic applications including evaluation of cardiac output , NIR-Fluorescence image-guide surgery , lymph node detection in multiple types of cancers [5-7], and assessment of liver function . In addition, since it can generate heat and singlet oxygen upon NIR irradiation, ICG has further motivated investigations into its utility on therapeutic purpose such as applications in photodynamic therapy, photo thermal therapy, and tissue welding [9-11].
However, currently the clinical efficacy of ICG remains limited by various factors including molecular instability, rapid circulation kinetics, and lack of target specificity. It has been reported that the degradation of ICG in aqueous medium follows first-order kinetics , and can be induced/accelerated by light exposure (photo-degradation) and/or heating (thermal-degradation) , leading to functionless of the ICG product since the degraded ICG molecules fail to fluoresce in NIR wavelength region . Moreover, as being a fluorescence probe and/or photosensitizing agent for use in vivo, reagents are usually required to be equipped with properties of long circulation half-life, target-ability, and capability of accumulation at the disease site and/or prolong stay at the site of action. However, ICG administered intravenously can provide only about 2 - 4 min of plasmatic half-life and exhibit extensive protein binding in the duration of circulation [13,14]. Such circumstances seriously hampered the applicability of ICG and thus a strategy that enables to overcome the aforementioned drawbacks is certainly needed for development of ICG-based application.
Among various polymeric drug carriers, poly(lactic-co-glycolic acid) (PLGA) has been recognized as one of the most commonly used biomaterials with U.S. FDA approval for the encapsulation of therapeutics (e.g., ICG) due to its biocompatibility and biodegradability . Furthermore, the degradation efficiency of PLGA can be modulated by adjusting the molecular weight and/or ratio of lactide to glycolide in the PLGA molecules that renders a feature of controlled drug release to the drug carrier. Regarding the issue of drug carrier size, it has been known that the nanometer-sized particles enable to provide (1) improved bioavailability by enhancing aqueous solubility; (2) enhanced permeability on the target cells and (3) increasing retention time in the vasculature and/or tissue as compared to micro particles (d ≥ 1 μm). These features may result in decreased amount of payload required and therefore reduce dosage toxicity, offering an efficient delivery of therapeutics (e.g., ICG) with low side effects for non-targeted tissues and/or cells .
In this paper, we aimed to develop ICG-loaded PLGA nanoparticles with ligands-mediated target-ability for ICG delivery. The human epidermal growth factor receptor 2-targeted ICG-loaded PLGA nanoparticles (HIPNPs) were prepared by a modified emulsification in association with solvent evaporation approach, and the physicochemical properties of the resulting products including particle size, surface charge, morphology, activity of anchored ligands, encapsulation rate, percentage of ICG content, and the efficiency of ICG degradation in vitro were comprehensively investigated in this work.
Materials and Methods
Poly(lactic-co-glycolic acid) (PLGA; 50:50, MW = 7000 - 17000 kDa), polyvinyl alcohol (PVA), dichloromethane (DCM), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), hetero-bifunctional polyethylene glycol (COOH-PEG-NH2), and indocyanine green (ICG, absorption wavelength = 780 nm) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-human epidermal growth factor receptor 2 monoclonal antibody (Anti-HER2-mAb) and Anti-mouse IgG secondary antibody were purchased from Cell Signaling (Danvers, MA). All chemicals were used as received.
Fabrication of HIPNPs
The HIPNPs were fabricated by a modified emulsification in association with solvent evaporation approach. Briefly, 30 mg of PLGA was first mixed with 1 mg of ICG in 1 mL DCM-methanol solution (v/v = 7:3). The mixture was then added to 15 mL of PVA solution (0.2 wt%) and emulsified by sonication at 100 W for 90 sec in an ice bath. The emulsified medium was then stirred for another 4 h at 700 rpm, followed by centrifugation at 20000 ×g for 20 min. The nanoparticles formed were then washed twice with PBS solution and re-suspended in 1-mL PBS. Afterward the collected nanoparticles were stepwise assembled with hetero-bifunctional PEG molecules and anti-HER2-mAb on the particle surface using EDC and NHS cross linkers to conjugate carboxyl and amide groups as reported previously . The duplicate PBS washes were performed after completion of each time of crosslinking on the particle surface. Ultimately the HIPNPs were lyophilized for 48 h and stored at-20 oC until further use. The overall procedures of HIPNP manufacture was illustrated in Figure 1.