Preparation and Characterization of Paclitaxel/Chitosan Nanosuspensions for Drug Delivery System and Cytotoxicity Evaluation In Vitro
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In this study, we prepared paclitaxel/chitosan (PTX/CS) nanosuspensions (NSs) with different mass ratios of PTX and CS (1.5:2, 2:2, and 2.5:2), for controlled drug delivery purposes. For attachment and dispersion in water medium, a simple ultrasonic disruption technique was employed. The water-dispersed PTX/CS NSs exhibited a rod-shape morphology with an average diameter of 170–210 nm and average length of about 1–10 µm. Transmission electron microscopy, differential scanning calorimetry and X-ray diffraction indicated that the obtained PTX/CS NSs contain a nanocrystalline PTX phase. It was also inferred that presence of CS can promotes the crystalline nature of PTX up to 80%. In addition, efficiency of PTX loading reached over 85% in freeze-dried PTX/CS NSs, showing a slow rate of drug release in vitro for 8 days. The MTT and LDH assessments revealed that PTX/CS NSs significantly inhibit the growth of tumor cells (HeLa), while it is slightly toxic for the normal cells (NIH/3T3). Therefore, PTX/CS NSs is suggested as a potential nanodrug delivery system for cancer therapy.
KeywordsPaclitaxel Chitosan Nanosuspensions Drug release Cytotoxicity in vitro
Numerous novel chemical compounds discovered in pharmaceutical programs are poorly soluble in water [1, 2, 3]. In order to obtain appropriate dissolution rates, a decrease in particle size and increase in surface area are usually implemented in various drug delivery systems [4, 5]. The use of nanotechnology to achieve a stable nano-sized drug has become a novel strategy and attracted significant attention in recent years [6, 7, 8].
The drug nanocrystal systems, which are simply prepared by facile and scalable methods has been considered as a promising approach [9, 10]. Via this principle, poorly soluble drugs can be converted to nanocrystals solid state particles [so-called nanosuspensions (NSs)] in a liquid system [11, 12, 13]; the use of surface-active agents for a better dispersion is also frequently suggested [14, 15, 16]. PTX/CS NSs is typically available with an average size of 100–1000 nm. With decrease of size and just evenly dispersion in digestion medium (water as model system) the area of contact will be enhanced, favoring saturation solubility and absorption into the target organ consequently [15, 17]. On the other hand such a manipulation allows a continuous trend of release, accelerates the effect and corresponding responses, elevates the level of bioavailability and decreases the possible side effects [18, 19]. These advantages facilitate the commercial and clinical application of NSn drugs. So far multitude of NSs drug systems such as Theralux® (Thymectacin) , Panzem® (2-methoxy estradiol) , and Abraxane® (Paclitaxel)  have been approved and admitted by the Food and Drug Administration (FDA).
In a technical and processing point of view, drug NSs are prepared following two approaches: top-down and bottom-up processing . Top-down method starts from large crystals, which are broken down into smaller-sized NSs using pearl milling or high-pressure homogenization [24, 25]. The bottom-up method begins from molecules, which are transformed into NSs using solvent exchange, precipitation, supercritical methods, or non-covalent interactions. The bottom-up approach is an energy-economy modelling compared with the top-down methods. The ultrasonic method is one of the bottom-up preparation methods, which requires simple instruments and cheap processes [26, 27]. In the pharmaceutical industry, ultrasonic force is often used to prepare nanosized oral formulations or hypodermic injections of suspensions for small and even nanodrug crystals [28, 29]. The cavitation effect of ultrasound is capable of promoting the crystallization nucleation process at low concentration . Under an ultrasound field, the crystal nucleus would become smaller and more uniform, compared with those counterparts which are prepared by other methods .
Paclitaxel (PTX) is a popular tumor therapy clinical drug for ovarian and breast cancers in clinic [32, 33]. PTX is also a kind of water-insoluble drug, and the use of Cremophor EL® (polyethoxylated castor oil) can improve its water solubility, but it brings the risk of allergic effects . To overcome this issue, docetaxel, paclitaxel liposome, and albumin paclitaxel (Abraxane®) were developed, which caused remarkable clinical and commercial enhancements [35, 36]. However, the modified PTX dosage forms are only limited to intravenous application, and rarely applied for oral administration. On the other hand taking PTX by injection may lead to serious allergic responses on injection site, which usually require pretreatment with corticosteroids or antihistamines . Oral administration can partly reduce the toxicity and side effects of PTX. In this context, investigation on oral administration of PTX would be benefit to solve the problems above. Chitosan (CS) is a type of biocompatible natural polymer material , which is nontoxic for oral administration. Thus far CS has been successfully used for drug delivery purposes, as literature reported the CS can prevent the colon implanted drugs to be dissolved by gastric acid . Some groups are focused on preparation of paclitaxel/chitosan drug delivery systems. Wang et al.  developed a chitosan microparticle for loading paclitaxel as an oral delivery system by synthesizing folate acid–chitosan (FA-CS) and prepared FA-CS-PTX microparticles (MPs). The FA-CS-PTX/MPs showed low cytotoxicity on L929 cells and could significantly decrease the viability of HepG2 cells. Lee et al.  conjugated low molecular weight chitosan (average MW: 6 kDa) onto paclitaxel for oral administration, which this system could be easily absorbed in the small intestine and effectively inhibited tumor growth. Li et al.  used a double emulsion crosslinking method to prepare paclitaxel-loaded chitosan nanoparticles with an average particle size of 116 ± 15 nm. The paclitaxel-loaded chitosan nanoparticles were shown to induce A2780 cancer cell apoptosis.
In this study, we prepared PTX/CS NSs through a bottom-up approach and the ultrasonic disruption method. Structural and in vitro analysis unveil that PTX/CS NSs is a high-potential system for an efficient and low-risk oral administration.
Paclitaxel (PTX) was purchased from Boshi Biological Technology Co., Ltd. Chitosan (CS) (average Mw, 144 kDa, deacetylation degree 79%), dehydrated alcohol (EtOH), acetic acid (HAc), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich and directly used without post-treatment. The reagents and solvents were bought from domestic suppliers. The ultrapure water generated by the Milli-Q ultrapure water system was used in all the experiments.
Preparation of PTX/CS Nanosuspensions
A series of paclitaxel ethanol solutions (1.5, 2.0, and 2.5 mg/mL) were prepared by dissolving PTX in EtOH. 2.0 mg CS was dissolved in 1 mL HAc/water solution (1% V/V) to obtain the 2.0 wt.% CS solution. The paclitaxel ethanol solution was added into the same volume of CS solution and treated by a probe-sonication system for 20 min. The PTX NSs were prepared by mixing 2 mg/mL of the paclitaxel ethanol solution with HAc/water solution (1% V/V) at the same volume under the same conditions mentioned above.
After ultrasonication, the prepared PTX/CS mixed solutions and PTX solution were concentrated using an ultrafiltration device (Amicon Ultra-15 Centrifugal Filter Unit, 10 kDa molecular weight cutoff). Subsequently, the concentrated solution was dialyzed in ultrapure water for 120 h to diminish EtOH and HAc. Finally, the dry PTX/CS samples were obtained by freeze-drying, and easily re-dispersed in water or PBS, forming PTX NSs with different concentrations.
Particle Size and Morphology
Morphology of the PTX/CS NSs was observed using transmission electron microscopy (TEM, JEM-1200EX, Japan). A single drop of prepared NSs was placed onto carbon-coated copper grids, dried at room temperature, to be observed by TEM. The TEM images were analyzed using the Nano Measurer software (Fudan University, China). The diameter and length of the NSs were measured, and for each sample 100 trails of measurements were performed and subjected to statistical calculation.
The zeta potential was measured for PTX/CS NSs, PTX/CS blend, and bare PTX in ultrapure water, using zeta potential analyzer (ZS90, Malvern Instruments Ltd., UK). Differential scanning calorimetry (DSC) analysis was performed using a DSC 204F1 apparatus (Netzsch, Germany). In DSC experiment, the samples were heated from 0 to 250 °C at a heating rate of 10 °C/min under a nitrogen flow. X-ray diffraction spectrometer (XRD, D8 Advance da Vinci, Bruker, Germany) was used to investigate the physical state (crystallography) of PTX in the NSs.
Evaluation of PTX Solubility and Drug Release In Vitro
The solubility of PTX in water was measured by adding an excessive amount of drug into the solvent. After freeze-drying PTX/CS NSs was magnetically stirred at room temperature for 48 h. The dissolved PTX was filtered to be separated from the NSs and removed by the dichloromethane (DCM). Finally, separated PTX was redissolved in 4 mL of EtOH/HAc (1:1) and concentration of the released PTX was measured using UV–Vis spectrophotometry (EV300, ThermoFisher, USA), at the wavelength of 227 nm. Each sample was analyzed in triplicate.
NIH/3T3 cells (a mouse embryonic fibroblast cell line) and HeLa cells (human cervical carcinoma cells) were incubated at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% FBS and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin). The cultivation environment was humid and contained a 5% CO2. The cultures were passed with trypsin/EDTA (0.05%/0.2% in PBS).
NIH/3T3 cells and HeLa cells were treated with PTX NSs and PTX/CS NSs (1.5:2, 2:2 and 2.5:2) to evaluate the cytotoxicity in vitro.
Cytotoxicity of PTX/CS NSs (1.5:2, 2:2 and 2.5:2) in NIH/3T3 cells and HeLa cells were examined using the methyl tetrazolium (MTT) assay and the lactate dehydrogenase leakage (LDH) assay. The NIH/3T3 cells and HeLa cells were seeded in 96-well plates with 1.0 × 104 cells/well in 200 μL DMEM. After 24 h incubation, the culture medium was removed and replaced with 200 μL fresh DMEM. The lyophilized samples were dispersed in DMEM and then added to the well (PTX concentration as a benchmark). Then, 20 μL of 5 mg/mL MTT assays stock solution was added to each well. After 4 h incubation, the MTT solution was removed and the cells were treated with 200 μL dimethylsulfoxide (DMSO). Plates were slightly shaken until the crystals were completely dissolved at room temperature. The OD values of the each well were measured by the multi-scan plate reader (BioTek, Synergy H4, USA) at the wavelength of 490 nm.
According to LDH assay, the cell culture followed procedure was the same as MTT assay described above, and the cell-free culture supernatants were collected by centrifugal separation. Following the manufacturer’s protocol (CytoTox 96, Promega, USA), the culture medium without cells was used as the control sample, the lysis solution treated cells was used as positive control sample, and the results were calculated as fold of LDH release to control. Each MTT and LDH test was repeated for 6 times.
The intracellular ROS levels in NIH/3T3 cells and HeLa cells were measured using ROS Detection Kit (Life Technologies, Burlington, ON, Canada). The NIH/3T3 cells and HeLa cells were seeded in 12-wells plate at a density of 1.0 × 105 cells/well, and incubated separately with 2 mg/mL of PTX NSs and PTX/CS (2:2) NSs for 2, 4, 6, 12, 24 and 48 h at 37 °C. The cell-permeable CellROX® Green reagent is a fluorogenic probe for measuring oxidative stress in live cells. This green probe is non-fluorescent in a reduced state and exhibits bright green photostable fluorescence upon oxidation by reactive oxygen species (ROS) and subsequent binding to DNA, with absorption/emission maxima ~ 485/520 nm. The intracellular ROS was analyzed by a flow cytometry (BD Accuri C6, USA). The ROS levels were expressed as fold of collected fluorescence intensity to control. The ROS detection was 3 replicates.
The HeLa cells were incubated with 2 mg/mL PTX/CS (2:2) NSs for 24 h and fixed in 4% paraformaldehyde. Then, the fixed cells were counterstained with CellROX® green reagent and Hoechst 33342 in sequence. Finally, the cell was mounted on a glass slide, covered with a glass coverslip, and the edges sealed with transparent fingernail polish. The prepared samples were examined with confocal laser scanning microscopy (CLSM, TCS SP8 STED 3X, Leica, Germany) at 488 nm excitation fluorescence to detect the presence of the green fluorescence, which is indicative of ROS stress.
Ultrastructure Observation of PTX/CS Nanosuspensions Treated HeLa Cells
The TEM observation was used to investigate the morphology changes induced by PTX/CS NSs. The HeLa cells were cultured with 2 μg/mL of PTX/CS NSs in 6-well plates for 4 h. Then, the cells were collected and fixed with 2% glutaraldehyde in 0.1 M pH 7.4 PBS buffer for 30–60 min, followed by incubation with 1% OsO4 pH 7.4 PBS buffer for 2 h, dehydrated with analytical gradient ethanol (50%, 75%, 95%, and 100%), and embedded in epoxy resin at 60 °C for 48 h. The ultrathin cross sections (60–70 nm) of the cell layers were cut by using a Leica EM UC6 ultra-microtomy, collected on the copper grids, and stained with uranyl acetate dihydrate and lead citrate.
Results and Discussion
Morphology and Size Distribution of PTX/CS Nanosuspensions
Characterization of PTX/CS Nanosuspensions
Zeta potential of the PTX/CS NSs, PTX drug, and PTX/CS blends
Zeta potential (mV)
PTX/CS NSs (1.5:2)
61.8 ± 3.2
PTX/CS NSs (2:2)
60.9 ± 3.0
PTX/CS NSs (2.5:2)
59.7 ± 4.8
0.7 ± 1.2
63.5 ± 3.3
Improvement of the PTX Saturation Solubility and Drug Release In Vitro
Cell Cytotoxicity and LDH Assay
Morphological Changes of PTX/CS Nanosuspensions Treated HeLa Cells
In this study a simple procedure based on ultrasonic disruption was successfully employed to prepare PTX/CS NSs (paclitaxel/chitosan Nanosuspension). Produced PTX/CS NSs exhibited rod-shape morphology with average diameter of 170–210 nm and length of 1–10 μm. DSC and XRD characterization indicated that PTX in NSs forms a crystalline state, and presence of CS would promote the crystallinity of PTX. In vitro drug release showed that the PTX/CS NSs shows a slower release trend compared with PTX NSs and PTX drug. In addition, in vitro cytotoxicity demonstrated that PTX/CS NSs are highly cytotoxic for tumor cells but slightly cytotoxic to the normal cells. PTX/CS NSs could cause physical damage to tumor cell membranes by endocytosis and micropinocytosis. ROS overproduction and subsequent cellular oxidative stress would destroy the membranaceous structures of cell and organelle. Thus, the PTX/CS NSs are promising for oral administration, and nanodrug delivery system for cancer therapy.
This work is financially supported by National Natural Science Foundation of China (Grant No: 51373099) State Key Laboratory of open funds of China from Donghua University (LK1411).
Compliance with Ethical Standards
Conflict of interest
The authors declared that they have no conflicts of interest to this work.
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