Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for cancer therapy
Graphical abstract
Introduction
Chemical drugs are generally considered to be one of the most efficient forms of cancer therapy. To achieve maximum therapeutic efficacy with minimal side effects, specific delivery of anticancer drugs to the target tumor site is highly desirable in cancer treatment [1]. For this purpose, many research groups have developed various nano-size drug carriers, such as liposomes, polymer conjugates, inorganic particles, and polymeric nanoparticles [2], [3], [4], [5]. Among these, polymeric nanoparticles have received much attention from biomedical researchers because they possess many useful properties and can be easily modified for clinical purposes [6], [7]. Moreover, nanoparticles provide prolonged circulation in the blood and accumulation to high levels in tumor tissue by avoiding rapid renal clearance when injected intravenously [8].
A large number of chemical drugs for cancer therapy are hydrophobic, and two methods are used to introduce hydrophobic drugs into polymeric nanoparticles: 1) physically loading onto polymeric nanoparticles and 2) chemical conjugation to polymeric nanoparticles [9]. The physical loading technique has been widely used with amphiphilic nanoparticles, but problems, such as the instability of nanoparticles during blood circulation causing a burst of release and loss of the loaded drugs, have been encountered [10]. The chemical conjugation technique allows the conjugation of hydrophobic drugs to hydrophilic polymers, which can then self-assemble to form spherical nanoparticles in aqueous conditions [11]. Importantly, chemically conjugated drugs in nanoparticles demonstrate increased stability, and unintended release is less frequent than with loaded drugs. Even with similar polymers and drugs, the in vitro and in vivo characteristics of nanoparticles can be largely changed by the method employed to introduce the drugs into the nanoparticles. However, to the best of our knowledge, few studies have been performed for analyzing these changes. Therefore, a comparative study of nanoparticles physically loaded with or chemically conjugated to similar polymers and drugs is expected to be helpful to researchers who develop or use nanoparticles for drug delivery in cancer treatment.
Photodynamic therapy (PDT) with photosensitizers has emerged as an effective therapeutic option for various tumors and other diseases [12], [13]. When the proper wavelength of light irradiates photosensitizers, highly reactive singlet oxygen is generated, causing damage to tumor tissues [14]. Along with singlet oxygen, photosensitizers can emit fluorescence, which enables easy detection and tracking of the photosensitizers during both in vitro and in vivo studies [15]. Especially under in vivo conditions, this fluorescence is beneficial for the imaging and quantification of photosensitizers in target tumor tissues or other organs [16]. Moreover, even if they are conjugated to other molecules like polymers, photosensitizers remain therapeutic unlike most other drugs [17]. Because of these characteristics, photosensitizers are suitable as model drugs for a comparative study of loaded and conjugated polymeric nanoparticles under both in vitro and in vivo conditions.
Chitosan is a natural polymer and a form of deacetylated chitin. Chitosan is both biodegradable and biocompatible. Consequently, chitosan has been widely used in various biomedical and pharmaceutical formulations [18]. Glycol chitosan (GC) is a chitosan derivative with ethylene glycol groups on its backbone, and its water solubility is highly enhanced by these glycol groups. In previous papers, we developed tumor-homing glycol chitosan nanoparticles and applied them as drug carriers for cancer therapy [5]. When hydrophobic molecules, such as 5β-cholanic acid or protophorphyrin IX, were conjugated to a GC polymer, the resulting amphiphilic conjugates formed self-assembled hydrophobic GC nanoparticles (HGCs) with hydrophilic GC shells and hydrophobic cores under aqueous conditions [19], [20]. These HGCs harbored various anticancer drugs in their hydrophobic inner cores, and showed prolonged circulation in the blood and specific delivery of drugs to tumors for cancer therapy [21], [22].
Herein, we synthesized two kinds of tumor targeting nanoparticles containing photosensitizers for PDT. We selected chlorin e6 (Ce6) as the photosensitizer because of its hydrophobicity, its activation by near infrared wavelengths, allowing it to act in deep tissue layers, and high singlet oxygen generation efficiency [23]. We obtained Ce6-loaded glycol chitosan nanoparticles (HGC-Ce6) and Ce6-conjugated chitosan nanoparticles (GC-Ce6), and compared the in vitro and in vivo characteristics of these two nanoparticles for PDT in cancer therapy. We developed new photosensitizer-containing glycol chitosan nanoparticles and demonstrated their potential for efficient PDT of tumors. Furthermore, this comparative study provides valuable information about the in vivo behaviors of nanoparticles for drug delivery.
Section snippets
Materials
Glycol chitosan (average molecular weight = 250 kDa; degree of deacetylation = 82.7%), chlorin e6(Ce6), N-hydroxysuccinimide (NHS), 5β-colanic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), fluorescein isothiocyanate (FITC), and p-nitroso-N,N′-dimethylaniline (RNO) were purchased from Sigma (St. Louis, MO). Anhydrous methanol and dimethyl sulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). All other chemicals and solvents were analytical grade and were used
Physicochemical properties of HGC-Ce6 and GC-Ce6
Hydrophobically modified glycol chitosan nanoparticles (HGC) were prepared as nano-size drug carriers for cancer treatment (Fig. 1A) [5]. HGC containing no drugs formed stable self-assembled nanoparticle with an average diameter of 250 nm in aqueous condition. Many hydrophobic anticancer drugs can be successfully loaded into the hydrophobic inner-cores of HGC by a simple dialysis method [15]. To make photosensitizer-loaded nanoparticles, both HGC and Ce6 were dissolved in DMSO and then dialyzed
Conclusions
In this study, we have shown that drug-loaded and conjugated nanoparticles perform differently during both in vitro and in vivo experiments. We selected Ce6 and GC as a photosensitizer and a biocompatible polymer, respectively, and developed drug-loaded and drug-conjugated nanoparticles as model drug delivery systems in cancer treatment. Both nanoparticles were well dispersed in the aqueous system and formed stable nano-structures. HGC-Ce6 showed time dependant release of Ce6 and more efficient
Acknowledgements
This work was financially supported by the Real-Time Molecular Imaging Project, the Global Research Laboratory Project, Fusion Technology Project (2009-0081876) of MEST, National R&D Program for Cancer Control of Ministry for Health and Welfare from Republic of Korea (1020260), and the Intramural Research Program of KIST.
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These authors contributed equally to this paper.