Enhanced localization of anticancer drug in tumor tissue using polyethylenimine-conjugated cationic liposomes
© Han et al.; licensee Springer. 2014
Received: 4 December 2013
Accepted: 24 April 2014
Published: 5 May 2014
Liposome-based drug delivery systems hold great potential for cancer therapy. However, to enhance the localization of payloads, an efficient method of systemic delivery of liposomes to tumor tissues is required. In this study, we developed cationic liposomes composed of polyethylenimine (PEI)-conjugated distearoylglycerophosphoethanolamine (DSPE) as an enhanced local drug delivery system. The particle size of DSPE-PEI liposomes was 130 ± 10 nm and the zeta potential of liposomes was increased from -25 to 30 mV by the incorporation of cationic PEI onto the liposomal membrane. Intracellular uptake of DSPE-PEI liposomes by tumor cells was 14-fold higher than that of DSPE liposomes. After intratumoral injection of liposomes into tumor-bearing mice, DSPE-PEI liposomes showed higher and sustained localization in tumor tissue compared to DSPE liposomes. Taken together, our findings suggest that DSPE-PEI liposomes have the potential to be used as effective drug carriers for enhanced intracellular uptake and localization of anticancer drugs in tumor tissue through intratumoral injection.
Liposome-based approaches, which show great potential for cancer therapy, allow for the development of a broad armamentarium of targeted drugs [1–3]. However, one of the key challenges in the application of liposomal drug delivery for chemotherapy is the requirement of efficient drug localization in tumor tissue. These liposomal systems are normally injected intravenously for systemic application. The effectiveness of intravenously delivered liposomes, however, is plagued by problems such as rapid opsonization and uptake by the reticuloendothelial system (RES), resulting in inefficient delivery [4–6]. Therefore, novel delivery systems to overcome such limitations are thus in urgent need.
Under localized conditions, drug delivery systems formulated to deliver high concentration of drugs over an extended period could be an ideal strategy to maximize the therapeutic benefit and avoid possible side effects . However, because low molecular weight drugs can rapidly pass into the bloodstream after intratumoral injection and because the retention time of such drugs in tumors is considerably short, new strategies to enhance the drug delivery and therapeutic effects in tumor tissues are needed.
In this study, we present a novel method for drug delivery using polyethylenimine (PEI)-incorporated cationic liposomes, which can be injected directly into the tumor site. PEI is a synthetic cationic polymer that has been extensively used to deliver oligonucleotides, siRNA, and plasmid DNA in vitro and in vivo[8–10]. Moreover, the cationic charge of the carrier surface can be enhanced through the intracellular uptake of vehicles to negatively charged tumor cells or tissues [11–13]. After penetration of cationic PEI liposomes into the cells, PEI has a protonatable nitrogen atom, which enables the ‘proton sponge’ effect over a wide range of pHs in the endosome. Consequently, PEI buffers acidification within the endosome after endocytosis, resulting in osmotic swelling and cell rupture allowing for endosomal escape of the PEI/siRNA polyplexes . Although cationic PEI has promising potential as a vehicle, it also presents some of the toxicity problems associated with other non-viral vectors [15, 16]. PEI can, however, be modified for reduced toxicity, and its free amine groups can be used to conjugate cell binding or targeting ligands [17–19]. Therefore, we selected PEI to increase localization of liposomes in tumor micro-environment in this study.
Cationic liposomes can also be simply injected at the target site without the need for surgical procedures. The PEI-incorporated cationic liposomes system, thus, has the potential to enhance the concentrations of therapeutic payloads at the tumor site, minimize possible side effects, and ultimately increase the therapeutic index of therapies. Although many cancers metastasize, several types of external cancers such as skin, breast, or neck cancer may be amenable to treatment using DSPE-PEI liposomes. Here, we demonstrate that the anticancer drug delivery system based on cationic liposomes is potentially a novel and powerful local drug delivery system for therapeutic agents.
Polyethylenimine (PEI, MW, 600 g/mol), glutaric anhydride (GA), 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxy-succinimide (NHS) were purchased from Sigma Aldrich Co. (Milwaukee, WI, USA). Chemicals 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l-α-phosphatidylcholine (soy-hydrogenated) (HSPC), and cholesterol (CHOL) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). The anticancer drug doxorubicin (DOX) was obtained from Boryung Pharm. Co. (Ansan, Korea) and calcein was purchased from Sigma Co. (St. Louis, MO, USA). All other materials and solvents were of analytical grade and used without further purification.
Synthesis of DSPE-PEI
Preparation of liposomes
DOX-loaded cationic liposomes were prepared using the remote loading method by employing ammonium sulfate gradient [21, 22]. Lipid compositions of the prepared control (DSPE) and DSPE-PEI liposomes were HSPC/CHOL (4 mg of lipid) and HSPC/CHOL/DSPE-PEI (0.1 mg, 0.4 mg, 0.7 mg, and 1 mg of DSPE-PEI based on HSPC/CHOL formulation), respectively. Lipids were dissolved in chloroform, dried on a thin film on a rotary evaporator (Buchi Rotavapor R-200, Switzerland), and finally suspended in a 250 mM of ammonium sulfate solution. The liposomal solution was extruded by passing it through a polycarbonate filter (pore size, 100 nm, Whatman, Piscataway, NJ, USA) using an extruder (Northern Lipids Inc., Burnaby, Canada). Free ammonium sulfate was removed by dialysis for 48 h at 4°C using cellulose dialysis tubing (MWCO 3500, Viskase Co., Darien, USA). The liposomal solution was mixed with a 2 mg/ml DOX solution and incubated for 2 h at 60°C after which the mixture was dialyzed to facilitate the removal of free DOX. DOX-loaded liposomes were stored at 4°C until use. In addition, to DOX-loaded liposomes, calcein-loaded liposomes were prepared for assessment of the localization in tumor-bearing mice. Calcein-loaded liposomes with the above-mentioned compositions were prepared by loading calcein serving as a model drug in liposomes using the pH gradient method .
The particle size and zeta potential of liposomes were measured by laser light scattering using a particle size analyzer (ELS-8000, Outskate, Seongnam, South Korea). The loading efficiency of DOX into liposomes was measured by fluorescence spectrophotometry (Barnstead, Apogent Tech., Dubuque, IA, USA) at excitation and emission wavelengths of 490 and 590 nm, respectively.
Cell line and mice
The human lung carcinoma cell line A549 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin-streptomycin, 2 mM l-glutamine, 1 mM sodium pyruvate, 2 mM non-essential amino acids, and 0.4 mg/ml G418. The cell culture was sustained at 37°C in a 5% CO2 incubator, and the cells were maintained in the exponential growth phase. Male BALB/c nu/nu nude mice (5 weeks old, 20 to 22 g) were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan). All procedures involving animals were performed according to approved protocols and in accordance with the recommendations specified in the NIH guidelines for proper use and care of laboratory animals.
Flow cytometry analysis
A549 cells were plated in a 6-well plate at a density of 2 × 105 cells per well and cultured in medium supplemented with 10% FBS and 1% penicillin (Life Technologies, Carlsbad, CA, USA) at 37°C. Culture medium was replaced with 2 ml per well of culture medium containing liposomal solutions (30 μg DOX/ml). The cells were incubated with liposomes for 2 h at 37°C in a 5% CO2 incubator. After incubation, the cells were washed three times with phosphate-buffered saline (PBS). The intracellular uptake efficiency of liposomes by A549 cells was monitored by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA) using CELLQuest software (Becton Dickinson Immunocytometry System, Mountain View, CA, USA), and the morphology of tumor cells containing DOX-loaded liposomes was observed by fluorescence microscopy (Olympus CKX 41, Shinjuku-ku, Tokyo, Japan).
The cytotoxicity of liposomes in A549 cells was determined by MTT assay. A549 cells were seeded into 96-well plates at a density of 1 × 103 cells per well and cultured in liposomal solution containing culture medium 37°C for a predetermined time. The absorbance was measured at 590 nm using a microplate reader (EL808, Bio-Tek, Instruments, Winooski, VT, USA).
Localization of DSPE-PEI liposomes in tumor tissue
A549 (1 × 106) cells were subcutaneously injected into BALB/c nu/nu nude mice. Four weeks after injection, free calcein was used as a model drug or liposomal calcein was injected intratumorally into the mice, after which the tumor tissue was monitored continuously for 4 h. The localization efficiency of liposomes in tumor tissues of the live tumor-bearing mice was directly observed under a fluorescence microscope (Macro-Imaging System Plus LT-9macimstsplus, Lightools Research, Encinitas, CA, USA) equipped with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).
Results and discussion
The synthesis of DSPE-PEI conjugate was confirmed by proton NMR analysis. Figure 1 shows the chemical structures and 1H-NMR spectra of the synthesized DSPE-PEI conjugate. As shown in Figure 1B, peaks corresponding to the CH3 (1) and CH2 (2,3, and 4) protons were observed at 0.8 to 1.0 ppm (1), 1.1 to 1.4 ppm (2), 2.1 to 2.3 ppm (3), and 3.7 to 3.8 ppm (4), respectively. In addition, the PEI peaks were observed at 2.5 to 3.5 ppm. The synthesis yield was approximately 93%.
Characteristics of liposomes
Intracellular delivery of DSPE-PEI liposomes
Tumor tissue localization of liposomes
Intratumoral injection is an effective method for liposome-mediated drug delivery into tumor tissues. The use of DOX-loaded DSPE-PEI cationic liposomes was found to result in significantly increased in vitro intracellular uptake compared with control liposomes. Notably, the conjugation of PEI to the liposomal membrane effectively improved the localization of drug-loaded liposomes at the tumor site through electrostatic interaction, which occurred in the tumor tissue of tumor-bearing mice treated with intratumorally injected liposomes. Our results demonstrate a promising approach to improve the intracellular uptake and localization effect of cationic liposomes. Although DSPE-PEI liposomes exhibit enhanced intracellular uptake, additional studies on the localization, injection route, and stability of these carriers is required for validation of their potential clinical application. The cationic liposome delivery strategy presented here has considerable potential as a drug delivery platform for the treatment of a broad range of human diseases and can be adapted for other injection applications in various therapeutic fields.
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2009–0078434) (BCS) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2059167) (HDH). This work was supported by Basic Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. 2013R1A4A1069575) (HDH).
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