Validation of a Janus role of methotrexate-based PEGylated chitosan nanoparticles in vitro
- Fanghong Luo†1, 2, 4,
- Yang Li†1, 2,
- Mengmeng Jia2,
- Fei Cui2,
- Hongjie Wu3,
- Fei Yu1,
- Jinyan Lin1, 2,
- Xiangrui Yang2,
- Zhenqing Hou2Email author and
- Qiqing Zhang5Email author
© Luo et al.; licensee Springer. 2014
Received: 17 June 2014
Accepted: 18 July 2014
Published: 23 July 2014
Recently, methotrexate (MTX) has been used to target to folate (FA) receptor-overexpressing cancer cells for targeted drug delivery. However, the systematic evaluation of MTX as a Janus-like agent has not been reported before. Here, we explored the validity of using MTX playing an early-phase cancer-specific targeting ligand cooperated with a late-phase therapeutic anticancer agent based on the PEGylated chitosan (CS) nanoparticles (NPs) as drug carriers. Some advantages of these nanoscaled drug delivery systems are as follows: (1) the NPs can ensure minimal premature release of MTX at off-target site to reduce the side effects to normal tissue; (2) MTX can function as a targeting ligand at target site prior to cellular uptake; and (3) once internalized by the target cell, the NPs can function as a prodrug formulation, releasing biologically active MTX inside the cells. The (MTX + PEG)-CS-NPs presented a sustained/proteases-mediated drug release. More importantly, compared with the PEG-CS-NPs and (FA + PEG)-CS-NPs, the (MTX + PEG)-CS-NPs showed a greater cellular uptake. Furthermore, the (MTX + PEG)-CS-NPs demonstrated a superior cytotoxicity compare to the free MTX. Our findings therefore validated that the MTX-loaded PEGylated CS-NPs can simultaneously target and treat FA receptor-overexpressing cancer cells.
Nanotechnology is a rapidly advancing and key field of drug delivery. A great variety of nanoparticle (NP)-based therapeutic products have entered clinical development or been approved for clinical use . As an excellent biocompatible and biodegradable nanomaterial with low toxicity and immunogenicity, chitosan (CS)-based nanocarriers presented great advantages for drug, protein, and gene delivery in therapeutics [2–5]. However, most CS-based nanocarriers were easily sequestered by macrophages in the mononuclear phagocyte system (MPS) after intravenous administration. To avoid the rapid clearance of the CS-NPs during circulation, PEGylation can be used to improve the physiological stability, reduce the opsonization, and increase the possibility reaching the tumor by the enhanced permeation and retention (EPR) effect (40 to 400 nm) [6–8].
Despite these advantages of the passive targeting, the main obstacle encountered with the clinical use of the PEGylated CS-NPs is how to facilitate their internalization in the target cells while reducing the unintended side effects. One strategy is the further functionalization of the PEGylated CS-NPs with active targeting agents. For instance, some ligands or antibodies could specifically recognize the receptors or antigens on the surface of various cancer cells . Notably, the exploitation of folate (FA) receptor for targeted drug delivery has long been persued. FA receptors were overexpressed in a wide variety of cancer cells, including ovarian, lung, breast, kidney, and brain cancer cells, but its level is very low in normal cells [10, 11]. Previously, we synthesized the CS-NPs by the combination of ionic gelation and chemical cross-linking method and prepared the (FA + PEG)-CS-NPs by dual-conjugation with mPEG-SPA and FA ; the enhanced cellular uptake and tumor accumulation also inspired our motivation of adopting the CS-NPs as drug carriers to continue our studies for an extensively used anticancer drug methotrexate (MTX).
All chemical reagents were of analytical grade and used without further purification unless otherwise stated. Chitosan (CS, Mw = 70,000 Da, 95% degree of deacetylation) was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. (Zhengjiang, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and crude proteases from bovine pancreas were purchased from Sigma Chemical Corp (St. Louis, MO, USA). Folate (FA) and methotrexate (MTX) were purchased from Bio Basic Inc. (Markham, Ontario, Canada). N-Succinimidyl ester of methoxypolyethylene glycol propionic acid (mPEG-SPA, Mw = 2,000 Da) was purchased from Jiaxing Biomatrix Inc. (Zhengjiang, China). A dialysis bag (Mw = 8,000 to 14,000 Da) was ordered from Greenbird Inc. (Shanghai, China). A Spectra/Por dialysis membrane (Mw = 6,000 to 8,000 Da) was purchased from Spectrum Laboratories (Rancho Domingues, CA, USA). Deionized (DI) water was used throughout. Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (AG, Zug, Switzerland). Trypsin-EDTA (0.25%) and penicillin-streptomycin solution was from Invitrogen. All solvents used in this study were high-performance liquid chromatography (HPLC) grade. HeLa cells and MC 3 T3-E1 cells were provided by American Type Culture Collection (ATCC, Manassas, VA, USA).
Preparation of the (MTX + PEG)-CS-NPs
Firstly, the CS-NPs were prepared by the ionic gelation combined with chemical cross-linking method according to our previous work . Secondly, mPEG-SPA (50 mg) was added into the CS-NPs suspensions (5 mL, 10 mg/mL) accompanied by vigorous stirring for 4 h. The prepared PEG-CS-NPs were dialyzed against DI water to remove excess of mPEG-SPA using a dialysis bag (Mw = 8,000 to 14,000 Da) and lyophilized for 24 h. Lastly, MTX (5 mg), EDC (8 mg), and NHS (5 mg) were dissolved in 5 mL of PBS (pH = 7.4). The pH was adjusted to 6.0 by the addition of 0.2 M HCl. The mixture was allowed to react for 30 min and added dropwise to the PEG-CS-NPs suspension (5 mL, 10 mg/mL). The pH was adjusted to 8.0 with 0.2 M NaOH. The reaction was allowed to occur at room temperature for 48 h. Following MTX conjugation, the (MTX + PEG)-CS-NPs NPs were centrifuged at 20,000 rpm for 30 min at 4°C, washed with PBS/DI water, and lyophilized for 24 h. All of the supernatants were collected for further indirect calculation of the drug-loading content. The (FA + PEG)-CS-NPs were prepared by the same method.
Physicochemical characterization of (MTX + PEG)-CS-NPs
Fourier transform infrared spectroscopy (FTIR) spectrum analysis of (MTX + PEG)-CS-NPs was performed using a NicoletAVTAR36 FTIR Spectrometer (Thermo Scientific, Salt Lake City, UT, USA). For comparison, The CS-NPs, PEG, PEG-CS-NPs, and MTX were used as controls.
Average particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Zeta potential was evaluated by electrophoretic light scattering (ELS) with Zetaplus (Brookhaven Instruments Corporation, Holtsville, NY, USA). Particle size was evaluated by intensity distribution. Atomic force microscopy (AFM) study was performed on a Nanoscope Multimode atomic force microscope (Veeco Instruments Inc., New York, USA). Transmission electron microscopy (TEM) image was obtained on a JEM 2100 transmission electron microscope (JEOL, Tokyo, Japan).
The amount of drug in the supernatant was assayed using a high-performance liquid chromatography (Waters Associates, Milford, MA, USA) system with the following conditions: stationary phase, Hypersill ODS column (250 mm × 4.6 mm, 5 μm); mobile phase, potassium dihydrogen phosphate buffer (pH 4.5)-acetonitrile (88:12); elution flow rate, 1 mL/min; and detection wavelength, 303 nm. The drug-loading content was calculated according to the previous report .
In vitro stability tests
PBS stability test against ionic strength and plasma stability test against protein adsorption were evaluated immediately after preparation and subsequently at regular intervals. Briefly, 5 mg of the lyophilized (MTX + PEG)-CS-NPs were suspended in PBS (pH 7.4) or 10% (v/v) plasma/heparin in PBS and stored at 37°C for 120 h. The particle size was determined at 0, 24, 48, 72, 96, and 120 h, respectively.
In vitro drug release
In vitro release of MTX from the (MTX + PEG)-CS-NPs was evaluated by a dialysis method. The lyophilized (MTX + PEG)-CS-NPs suspended in 10% plasma (with or without the presence of crude proteases) were added into a dialysis bag (Mw = 6,000 to 8,000 Da) and immersed into the release medium at 37°C with agitation. At the predesigned time points, 2 mL of the release medium was completely withdrawn and subsequently replaced with the same volume of fresh PBS. For comparison, in vitro release of the free MTX was evaluated as a control.
HeLa cells were cultured in FA-deficient Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. MC 3 T3-E1 cells were cultured in Minimum Essential Medium, Alpha Modified (α-MEM), under similar conditions. The two cell lines have different levels of FA receptor expression. In particular, HeLa cells (cancer cells) are FA receptor positive, and MC 3 T3-E1 cells (normal cells) are FA receptor negative. All of the cells were cultivated in a 5% CO2-humidified atmosphere at 37°C.
In vitro cellular uptake
To qualitatively investigate the cellular uptake of the PEG-CS-NPs, (FA + PEG)-CS-NPs or (MTX + PEG)-CS-NPs, fluorescein isothiocyanate (FITC) was conjugated to different formulations to prepare the FITC-PEG-CS-NPs, FITC-(FA + PEG)-CS-NPs or FITC-(MTX + PEG)-CS-NPs. HeLa cells were seeded at a density of 8 × 104 cells per well into 6-well plates with their specific cell culture medium. The cells were incubated at 37°C and 5% CO2 for 24 h. One hundred microliters of the FITC-PEG-CS-NPs, FITC-(FA + PEG)-CS-NPs, or FITC-(MTX + PEG)-CS-NPs was added to HeLa cells at the equivalent concentration of FITC and incubated further for 6 h. HeLa cells were washed with PBS and stained with Hoechst 33258. Then, HeLa cells were washed with PBS and fixed with 4% formaldehyde. The cells were observed using a Leica TCS SP5 laser confocal scanning microscopy (Leica Microsystems, Mannheim, Germany).
To quantitatively investigate the internalization of the FITC-labeled (MTX + PEG)-CS-NPs, (FA + PEG)-CS-NPs or PEG-CS-NPs, HeLa cells were incubated in 6-well plates at a density of 2 × 105 cells/mL and allowed to grow for 24 h. The FITC-(MTX + PEG)-CS-NPs, FITC-(FA + PEG)-CS-NPs, or FITC-PEG-CS-NPs at the equivalent concentration of FITC were then added to each well. After incubation for 4 h, the cells were washed with cold PBS twice, harvested by 0.25% (w/v) trypsin/0.03% (w/v) EDTA, centrifuged at 1,000 rpm for 5 min at 4°C and resuspended in PBS for the analysis by a Coulter EPICS XL Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA).
In vitro cell viability studies
Cytotoxicity of the PEG-CS-NPs, (FA + PEG)-CS-NPs, (MTX + PEG)-CS-NPs, and free MTX were evaluated by MTT assay. HeLa cells (cancer cells) or MC 3 T3-E1 cells (normal cells) were seeded at a density of 3 × 103 cells per well into 96-well plates with their specific cell culture medium. The cells were incubated at 37°C in humidified atmosphere containing 5% CO2 for 24 h. The medium was then replaced with fresh medium, and different formulations were added to incubate with the cells. After 24 h of incubation, the medium was removed; each well was rinsed with PBS; and 20 μL of MTT solution was added followed by incubation for 4 h. Then, the metabolized product MTT formazan was dissolved by adding 200 μL of DMSO to each well. Finally, the plate was shaken for 20 min, and the absorbance of the formazan product was measured at 570 nm in a microplate reader (Bio-Rad, Model 680, Bio-Rad Laboratories, Richmond, CA, USA).
To further understand the mechanisms of in vitro cell viability studies, we investigated the subcellular localization using a laser confocal scanning microscopy. After the predesigned incubation times with the FITC-labeled (MTX + PEG)-CS-NPs, HeLa cells were washed with PBS and stained with LysoTracker Red following the manufacturer’s instructions. The cells were then washed with PBS, fixed with 4% formaldehyde for 15 min and observed by a laser confocal scanning microscopy.
Results and discussion
Preparation of the (MTX + PEG)-CS-NPs
Physicochemical characterization of the (MTX + PEG)-CS-NPs
Drug-loading content. CS-NPs possessing peripheral amino groups provided us great opportunities to easy surface biofunctionalization. In our study, the γ-carboxyl groups of MTX were conjugated to the residual amino groups of the PEGylated CS-NPs. The drug-loading content of the (MTX + PEG)-CS-NPs was calculated as 7.23 ± 0.11%. The simple conjugation chemistry and appropriate drug-loading content could favor the dual-acting role of Janus-like MTX.
In vitro stability tests
No significant variation of the particle size was observed in the (MTX + PEG)-CS-NPs even after incubation with PBS for a long period of time (Figure 4F). Notably, the CS-NPs (without PEGylation) could precipitate after 48 h in the presence of salts. It was implied that PEG could protect the (MTX + PEG)-CS-NP against ionic strength. No significant change of the particle size was also shown in the (MTX + PEG)-CS-NPs after incubation with 10% plasma for 120 h (Figure 4G). It should be inferred that PEG could reduce the plasma proteins adsorption, and more importantly, preserve the targeting potential of MTX. All of the results suggested that the (MTX + PEG)-CS-NPs were sufficiently stable to sustain physiological conditions for extended blood circulation.
In vitro drug release profiles
It was well established that the amide bonds could be selectively cleaved at acidic pH by proteases (also called proteolytic enzymes) overexpressed in the tumor cells [33–36]. To simulate the physiological environment of the acidic compartments of the tumor cells, the (MTX + PEG)-CS-NPs were incubated with 10% plasma containing crude proteases at pH 5.0. MTX was released at a constant rate up to 10 h, reaching the accumulated release amounts more than 30%, we believed that proteases exerted a significant promotion effect to control drug release. As is reported, several kinds of particle-bound MTX attached by an amide linkage have been shown to be sensitive to the protease-mediated cleavage in the acidic environments, and hence, the lysosomal proteases could be responsible for the release of MTX from the particles [19, 20, 37, 38]. Once the NPs were internalized by the target cells, the drug release could be significantly speeded up because of the long-lasting activity of proteases inside the cells, which can help to provide a sufficient intracellular level of MTX, and hence efficiently enhance the drug efficacy.
All of the results suggested that the covalent chemistry, preferring over physical adsorption, could be advantageous to preserve the targeting role of MTX. This could be of utmost importance, especially in vivo, where the avoidance of premature drug release and the untimely role change (from targeting to anticancer) of Janus-like MTX are pivotal.
In vitro cellular uptake
These quantitative results were consistent with those qualitative results, giving a further proof of high targeting efficacy of the (MTX + PEG)-CS-NPs to HeLa cells. The possible reason is that the integral binding avidity of the (MTX + PEG)-CS-NPs towards FA receptor presents a great advantage of targeting efficacy outperformed that of the (FA + PEG)-CS-NPs towards FA receptor. As mentioned above, MTX has a suboptimal affinity to FA receptor compared with FA and may be less efficient to target to FA receptor than FA. Nevertheless, it was reported that multivalent binding avidity can be kinetically limited if the binding affinity of an individual receptor-ligand pair is too tight [44, 45]. Well consistent with the above theoretical analysis, our result further suggested that the targeting specificity of the nanoscaled drug delivery systems for a particular cell type can be enhanced by the weaker binding affinity of each individual receptor-ligand pair. Indeed, the integral binding avidity plays a predominant role in the targeting efficacy; the higher integral binding avidity increases the targeting efficacy. Detailed in vivo targeting studies are necessary to further assess this possibility.
In vitro cell viability studies
The cytotoxicity of the (MTX + PEG)-CS-NPs (10 μg/mL) towards HeLa cells and MC 3 T3-E1 cells after 24 h of incubation was shown in Figure 8B. FA receptors were expressed at a high level on the surface of HeLa cells (cancer cells) but at a much lower level on MC 3 T3-E1 cells (normal cells). On the one hand, the cytotoxicity of the (MTX + PEG)-CS-NPs towards cancer cells was significantly higher compared to that of the free MTX. However, in the case of normal cells, the situation was opposite. On the other hand, the (MTX + PEG)-CS-NPs induced a marked cytotoxicity towards targeted cancer cells, but a slight cytotoxicity was observed for nontargeted normal cells, whereas the free drug affected both cell lines equally. The result indicated that the MTX modification played an important role in selectively enhanced cytotoxicity of the nanoscaled drug delivery systems . All of these results also suggested that MTX was not prematurely released from the (MTX + PEG)-CS-NPs outside of HeLa cell, but was preferentially released inside HeLa cell after the cellular uptake of the (MTX + PEG)-CS-NPs.
To investigate the intrinsical mechanisms of the cytotoxicity of the (MTX + PEG)-CS-NPs, we investigated the subcellular localization of the FITC-labeled (MTX + PEG)-CS-NPs in HeLa cells by laser scanning confocal microscopy. As shown in Figure 8C, the internalized (MTX + PEG)-CS-NPs were found initially to be localized in the lysosomes, as evidenced by the yellow spots in the merged image obtained from the images of the (MTX + PEG)-CS-NPs (green) and late endosomes/lysosomes (red). The result indicated that the (MTX + PEG)-CS-NPs were internalized via the endocytosis pathway into the late endosomes/lysosome . Indeed, after incubation for 4 h, some green fluorescent FITC-labeled (MTX + PEG)-CS-NPs were no longer located in the red fluorescent late endosomes/lysosomes, indicating the successful endo/lysosomal escape. In agreement with other reports [37, 48], these results combined with the results of in vitro drug release and cell viability studies further proved that MTX was released from the (MTX + PEG)-CS-NPs inside the cells by the intracellular protease-mediated selective cleavage of peptide bond. These findings were also in agreement with other reports in the literature  that CS possessed the activity to some extent to escape the endo/lysosome.
We presented the versatile, robust, and easy MTX-based PEGylated CS-NPs while validating MTX as a successful targeting ligand coordinated with a simple anticancer drug, and established the (MTX + PEG)-CS-NPs as a cocktail platform of specific targeting cooperated with enhanced anticancer activity. MTX was not prematurely released at off-target site but was intensively released at target site due to its sustained/protease-mediated drug release characteristic. To the best of our knowledge, the work for the first time explored the validation of Janus role of MTX based on the nanoscaled drug delivery system in vitro. Additionally, as MTX (a targeting ligand/a first drug) was introduced into one kind of drug carriers, one further advantage was that the drug delivery systems allowed the further introduction of a second ligand or a second drug for synergistic co-targeted delivery or synergistic co-delivery of drugs. Nevertheless, more details about in vivo targeting and anticancer investigations are indispensable to obtain a better understanding of the therapeutic effect of the (MTX + PEG)-CS-NPs, and relevant studies are in process.
Both authors FL and YL contributed equally and should be considered as co-first authors.
Fanghong Luo acknowledges the financial support by the Natural Science Foundation of Fujian Province of China (Grant No. 2013 J01384) and Science and Technology Foundation of Xiamen of China (Grant No. 3502Z20113012). Dr. Yuan Jiang is acknowledged for useful discussions and editing the manuscript.
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