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Advanced Fiber Materials

, Volume 1, Issue 2, pp 152–162 | Cite as

Preparation and Characterization of Paclitaxel/Chitosan Nanosuspensions for Drug Delivery System and Cytotoxicity Evaluation In Vitro

  • Yongjia Liu
  • Fengren Wu
  • Yongle Ding
  • Bangshang ZhuEmail author
  • Yue Su
  • Xinyuan Zhu
Research Article
  • 224 Downloads

Abstract

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.

Graphic Abstract

Keywords

Paclitaxel Chitosan Nanosuspensions Drug release Cytotoxicity in vitro 

Introduction

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) [20], Panzem® (2-methoxy estradiol) [21], and Abraxane® (Paclitaxel) [22] 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 [23]. 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 [30]. Under an ultrasound field, the crystal nucleus would become smaller and more uniform, compared with those counterparts which are prepared by other methods [31].

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 [34]. 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 [37]. 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 [38], 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 [39]. Some groups are focused on preparation of paclitaxel/chitosan drug delivery systems. Wang et al. [40] 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. [41] 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. [42] 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.

Experimental Section

Materials

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.

Water Removal

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.

Physicochemical Characterization

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.

To evaluate the PTX drug release behavior under different pH condition (pH 5.0 and 7.4), 1 mg of freeze-dried PTX/CS NSs was added into 1 mL of PBS solution (0.2 M, pH 7.4 or 5.0). Subsequently, the mixture was transferred into dialysis bags with a molecular weight cut-off of 3.5 kDa, immersed into containers with 29 mL of PBS, and shaken at 120 rev/min at 37 °C. The released PTX was extracted from the medium and removed by dichloromethane (DCM) at specific time intervals. Then, the separated PTX was re-dissolved in 4 mL of EtOH/HAc (1:1) solvent and the concentration of the released PTX was measured by using a UV–Vis spectrophotometer at the wavelength of 227 nm. The drug loading efficiency (DLE) was calculated according to the following formulas (Eq. 1):
$${\text{DLE }}\left( {{\text{wt.}}\% } \right)\, = \,{\text{weight of loaded drug}}/{\text{weight of drug in feed}}\, \times \, 100\% .$$
(1)

Cell Culture

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 Assays

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.

ROS Detection

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

Nanosuspension of insoluble drugs is a facile strategy to overcome the poor solubility of anti-cancer drugs. Drug NSs with nearly 100% drug-loading capacity and alternative routes (e.g., oral, parenteral) of administration are potentially demanded in both commercial and clinical aspects. In this study, we use the bottom-up method using probe-sonication disruption technique to obtain PTX/CS NSs. Size (average length and diameter), morphology and dispersion status of the NSs were investigated by TEM (Fig. 1). The average diameter of the PTX/CS NSs was approximately 170–210 nm and their average length was determined to be about 1–10 μm. It is also observed that diameter of PTX/CS NSs decrease with the increase of PTX content. The process of ultrasonication was applied on equal concentrations (2 mg/mL) for both PTX NS and PTX/CS samples. However, yielding of suspension was relatively higher for PTX/CS NSs. This reveals that CS plays a dominant role in formation of NS. Meanwhile, further PTX/CS NSs were formed with increased content of CS. In this basis, we insinuate that CS acts as a hydrophilic stabilizer, adhering on the surfaces of the PTX NSs through noncovalent interactions. In term of morphology and function, coverage by CS can enhance the state of suspension and increase the biocompatible trait of system [43, 44].
Fig. 1

TEM and size distribution of PTX/CS NSs a, a′ PTX/CS (1.5:2), b, b′ PTX/CS (2:2), c, c′ PTX/CS (2.5:2). TEM images (scale bar 5 μm) and the NSs distribution was determined via Nano measure software (by Fudan University)

Characterization of PTX/CS Nanosuspensions

Zeta potential of PTX/CS NSs is tabulated in Table 1. It indicated that the PTX/CS NSs are positively charged. Crystalline structure of PTX/CS NSs was examined with differential scanning calorimetry (DSC) and X-ray diffraction (XRD). As shown in Fig. 2a, there is a clear endothermic peak in the DSC spectrum of PTX/CS NSs. The onset melting temperature (To) and the melting temperature (Tm) of the PTX drug are, respectively, 211.4 and 220.6 °C. Meanwhile, the To and Tm of the PTX NSs are shown to be 207.9 and 218 °C, respectively. The To and Tm of the simply blended PTX/CS are almost equal to those of bare PTX; as expected, since To and Tm cannot be changed by a simple physical mixing. However, the To and Tm of PTX/CS NSs with different PTX/CS ratios (1.5:2, 2:2, 2.5:2) were higher than those of bare PTX and PTX NSs. This generally indicate that the crystalline state of  PTX/CS NSs is favored by CS, as a better crystalline structure can be obtained by increase of the content of CS. Crystalline structure of NSs was further characterized by XRD. As shown in Fig. 2b, four typical peaks of PTX are appeared at 2θ of 5.1°, 10.8°, 12.5°, and 13.5° in the both spectrum of PTX NSs and PTX/CS NSs. High intensity of characteristic peaks indicate that PTX is formed in high-crystalline mode in both PTX NSs and PTX/CS NSs samples.
Table 1

Zeta potential of the PTX/CS NSs, PTX drug, and PTX/CS blends

Sample

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

PTX drug

0.7 ± 1.2

PTX/CS blends

63.5 ± 3.3

Fig. 2

a DSC thermograms of PTX drug, PTX NSs, PTX/CS blend, PTX/CS NSs (1.5:2), (2:2) and (2.5:2). b XRD patterns of PTX drug, PTX NSs, PTX/CS NSs (1.5:2), (2:2) and (2.5:2)

Improvement of the PTX Saturation Solubility and Drug Release In Vitro

Solubility probation in aqueous probe station manifests a saturation solubility of PTX in water is approximately 5 µg/mL, while in PTX/CS NSs composition a 150 µg/mL saturation solubility achieved. This significant revolution in solubility behavior is unarguably assigned to reduction of size and association of CS background. DLE of PTX/CS NSs was determined to be 80.7 wt.%, demonstrating the high drug loading capability of CS. In vitro release of PTX from PTX/CS NSs was evaluated in PBS buffer medium (pH 5.0 and 7.4) for over 8 days (Fig. 3). In a pH 5.0 PBS buffer, in first day, drug showed a release trend against the content of CS, i.e. 50.1 ± 5.4 wt.% (PTX/CS 1.5:2), 33.8 ± 2.8 wt.% (PTX/CS 2:2), and 23.6 ± 4.4 wt.% (PTX/CS 2.5:2). Changing the medium to pH 7.4 PBS buffer, fastened drug release was fasten to 60.9 ± 1.8 wt.% (PTX/CS 1.5:2), 48.0 ± 1.4 wt.% (PTX/CS 2:2), and 36.1 ± 1.1 wt.% (PTX/CS 2.5:2) at the first day. After 1 day, in vitro drug release reached a steady trend in all PTX and PTX/CS NSs samples. Then in the second week of observation, trend undertook a drop to 20–25 wt.% and 15–30 wt.% in pH 5.0 and 7.4 PBS systems, respectively. The release rate of NSs in a pH 5.0 PBS buffer was totally less than that in a 7.4 PBS buffer. This might be attributed to the fact that the preparation of the PTX and PTX/CS NSs occurred in the presence of HAc solution, leading to stable products. Also looking at the formulation solely PTX/CS NSs showed a slower release rate compared with the bare counterpart, i.e. PTX NSs, and among the NSs, PTX/CS NSs (2.5:2) demonstrated slowest release rate. This is consistent with the results of the XRD analysis. The CS participation can promote the crystallinity of the PTX/CS NSs; thus, the dissolution rate of PTX in PTX/CS NSs would be decreased leading to a prolonged release.
Fig. 3

The PTX loading efficiencies of PTX NSs and PTX/CS NSs (1.5:2), (2:2) and (2.5:2). a In vitro release profile of PTX NSs and PTX/CS NSs (1.5:2) (2:2) and (2.5:2) in PBS solution (pH 5.0) at 37 °C. b In vitro release profile of PTX NSs and PTX/CS NSs (1.5:2) (2:2) and (2.5:2) in PBS solution (pH 7.4) at 37 °C

Cell Cytotoxicity and LDH Assay

The PTX/CS NSs were evaluated by the MTT assay against NIH/3T3 and HeLa cells to determine their cytotoxicity in vitro (Fig. 4a, b). The dose response profile presents the MTT assay of PTX/CS NSs for 24 h. More than 70% of the NIH/3T3 cells still had cell viability when the amount of PTX/CS NSs reached around 20 μg/mL. Moreover, IC50 value of the PTX/CS NS (1.5:2 and 2.5:2) applied on HeLa cells was approximately 2 μg/mL. The results indicate that PTX/CS NSs has a trivial toxicity for normal cells, while efficiently kills the human tumor cells.
Fig. 4

Cytotoxicity evaluation in vitro of PTX NSs and PTX/CS NSs (1.5:2), (2:2) and (2.5:2). a NIH/3T3 cells. b HeLa cells

The lactate dehydrogenase (LDH) leakage assay is performed based on monitoring of the loss of intracellular lactate dehydrogenase into the culture medium due to cell membrane damage. As shown in Fig. 5, experimental results demonstrate that PTX NSs and PTX/CS NSs significantly increase the LDH release in HeLa cells. Moreover, LDH release in NIH/3T3 cells show a minor increase through the treatment by PTX and PTX/CS NSs. The PTX and PTX/CS NSs could enter into tumors via their leaky vasculatures. Basically the cancerous cells possess a thicker and denser membrane compared with the normal cells. Therefore the dosage drugs, either PTX or PTX/CS NSs would have challenges of penetration and diffusion through the cell membrane. Our data strongly support this hypothesis that the PTX and PTX/CS NSs might generate a temporary rupture in the cell membranes and exhibit a greater cellular uptake in tumor cells.
Fig. 5

Content of LDH release in cell culture medium treated by PTX NSs and PTX/CS NSs (2:2). a NIH/3T3. b HeLa

ROS Generation

Production of ROS and oxidative stress are the main mechanisms for nanomaterials toxicity. With respect to PTX/CS NSs treatments, a significant increase of ROS was found in HeLa cells, while little increase of ROS was found in NIH/3T3 cells (Fig. 6). The ROS fluorescence intensity slightly increased at 2, 4, and 6 h, and then displayed a downward trend after exposure time of 12 and 24 h. However, a high-level ROS fluorescence intensity for PTX/CS NSs treated HeLa cells could be observed with long exposure time. In the CLSM images, the HeLa incubated with PTX/CS NSs (2:2) for 24 h showed a strong shiny green fluorescence, which reflects overproduction of ROS. Overall results indicate that the PTX/CS NSs could cause ROS overproduction and oxidative stress, which eventually led to apoptosis.
Fig. 6

The ROS generation in cells after exposure to 10 mg/mL of PTX NSs and PTX/CS NSs (2:2). Data was shown as fold of ROS fluorescence intensity to control. a NIH/3T3, b HeLa, c the confocal images of HeLa incubated with PTX/CS NSs (2:2) stained by Hochst (blue) and ROS probe (green)

Morphological Changes of PTX/CS Nanosuspensions Treated HeLa Cells

To explore the morphological changes of PTX/CS NSs, the HeLa cells treated with PTX/CS NSs were investigated by TEM (Fig. 7). The red arrows are used to denote the PTX/CS NSs and the green arrows are used to denote the membranaceous structures of the cell and organelle. In addition, it is important to note that all micrographs shown in Fig. 7 were acquired from cells sliced in the plane of the nucleolus. The PTX/CS NS treated cells show fiber-like structures in the cell membrane (Fig. 7c, d) in comparison with the control group (Fig. 7a, b), in which the nuclear membrane and organelle membrane of the control cell can be observed clearly. In Fig. 7c, d, the nuclear membrane and organelle membrane of the PTX/CS NS treated cells become indistinct. These phenomena might result from both physical and chemical effects. PTX/CS NSs are probably able to enter the cell through the endocytosis and macropinocytosis effects on the cell membrane, which definitely cause physical damage on the cell membrane. Moreover, PTX/CS NSs could induce significant increase of ROS in the cancer cells, by which the free-radicals mainly destroy the membranaceous structures of cell and organelles.
Fig. 7

TEM images of morphological changes in HeLa cells. a, b Control cells; c, d PTX/CS NSs (2:2) treated HeLa cells

Conclusions

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.

Notes

Acknowledgements

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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Copyright information

© Donghua University, Shanghai, China 2019

Authors and Affiliations

  1. 1.Instrumental Analysis Center, National Infrastructures for Translation Medicine (Shanghai), State Key Laboratory of Metal Matrix Composites, School of Chemistry and Chemical EngineeringShanghai Jiao Tong UniversityShanghaiChina
  2. 2.State Key Laboratory for Modification of Chemical Fibers and Polymer MaterialsDonghua UniversityShanghaiChina

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