- Nano Express
- Open Access
Antitumor Activity of Doxorubicin-Loaded Carbon Nanotubes Incorporated Poly(Lactic-Co-Glycolic Acid) Electrospun Composite Nanofibers
© Yu et al. 2015
- Received: 8 June 2015
- Accepted: 10 August 2015
- Published: 26 August 2015
The drug-loaded composite electrospun nanofiber has attracted more attention in biomedical field, especially in cancer therapy. In this study, a composite nanofiber was fabricated by electrospinning for cancer treatment. Firstly, the carbon nanotubes (CNTs) were selected as carriers to load the anticancer drug—doxorubicin (DOX) hydrochloride. Secondly, the DOX-loaded CNTs (DOX@CNTs) were incorporated into the poly(lactic-co-glycolic acid) (PLGA) nanofibers via electrospinning. Finally, a new drug-loaded nanofibrous scaffold (PLGA/DOX@CNTs) was formed. The properties of the prepared composite nanofibrous mats were characterized by various techniques. The release profiles of the different DOX-loaded nanofibers were measured, and the in vitro antitumor efficacy against HeLa cells was also evaluated. The results showed that DOX-loaded CNTs can be readily incorporated into the nanofibers with relatively uniform distribution within the nanofibers. More importantly, the drug from the composite nanofibers can be released in a sustained and prolonged manner, and thereby, a significant antitumor efficacy in vitro is obtained. Thus, the prepared composite nanofibrous mats are a promising alternative for cancer treatment.
- Electrospun nanofiber
- Poly(lactic-co-glycolic acid)
- Carbon nanotubes
- Drug release
- Antitumor efficacy
Up to date, cancer is still one of the deadliest killers to human lives, because both the incidence and mortality rates of cancers (including lung, stomach, liver, prostate, colorectum, breast cancers, and so on) are continuously rising [1, 2]. Unfortunately, it can be said that, as of yet, despite tremendous efforts have been made in the cancer therapy, there is still no efficient methods for prevention of cancer relapse, as well as the prevention of their spread and metastasis . A wholesale and thorough cure for cancers remains elusive for a number of reasons. Some of these reasons including late stage diagnosis, inadequate resection during surgery, and cancer cell migration often lead to cancer recurrence [4, 5]. Local cancer recurrence after “curative” treatment remains a major clinical problem for most cancers. As we all know, current therapeutic approaches for malignant tumors include surgery, radiotherapy, chemotherapy, hyperthermia, immunotherapy, hormone therapy, stem cell therapy, and combinations of these modalities . Chemotherapy, as a general therapeutic approach, has been widely investigated to treat a variety of malignant cancer cells [7, 8]. Doxorubicin (DOX), a class I anthracycline antibiotic, is an excellent broad-spectrum anticancer drug for treating many types of cancers . It can effectively kill cancerous cells by damaging DNA and its replication via the mechanisms of intercalation between nucleotides, inhibition of topoisomerase II, and generating oxygen free radicals . However, the clinical application of free DOX is known to have short life and low therapeutic index, and a large dosage of administration dosages are required to achieve the desirable therapeutic effect, which also causes severe toxicity to normal tissues, for example, cardiotoxicity and myelosuppression [10, 11]. Therefore, the development of a suitable carrier system for resisting local cancer recurrence is necessary.
Nanotechnology as an emerging technology may be a reasonable choice and an effective strategy for solving the above problem. According to the report from the National Cancer Institute (NCI) in USA, nanotechnology seeks to exploit distinct technological advances towards cancer prevention and treatment . Electrospinning is a versatile technique to fabricate two-dimensional nanofibers. Electrospun nanofibers have recently received significant attentions in biomedical applications because they possess large surface area-to-volume ratio, high interfiber porosity with tunable pore size, low hindrance for mass transfer, flexible handling, adjustable morphology, and well mechanical strength, which would make nanofibers use as therapeutic patch for drug delivery [13, 14]. The controlled drug release from drug-loaded nanofibrous mat at a rate according to the need could be realized by properly designed architecture, porosity, fiber diameter, drug incorporation manner, and composition of nanofibers . In addition, the medicated nanofibrous products can be easily set to the targeted site by adjusting their morphology . Thus, the drug-loaded nanofibers can be targeted delivery to the desired target tissues with controlled release where drug is released in a certain way for preventing local tumor recurrence after surgery. Aliphatic biodegradable polyester, such as poly(lactic acid-co-glycolic acid) (PLGA), is widely used to form electrospun nanofibers in various clinic and xsimplebiomedical research applications because it has excellent biocompatibility and biodegradability [10, 16]. However, the simple introduction of drug into polymer matrix always leads to inevitable burst drug release, since the drug molecules might migrate on or near the fiber surfaces because of the high ionic strength in drug/polymer solution and the rapid evaporation of the solvent during electrospinning. To address this limitation, it is highly desirable to develop efficient nanocarrier-mediated nanofibrous delivery systems which may serve as barrier for improving the safety of anticancer drugs and avoid drug premature burst release under physiological conditions [7, 17–21]. In fact, several nanoscale carriers such as mesoporous silica nanoparticles , hydroxyapatite , and liposome  have recently been incorporated into electrospun nanofibers for potential anticancer therapy, from which the prolonged drug release with tunable drug release kinetics could be achieved. Carbon nanotubes (CNTs), which are rolled-up seamless cylinder of graphene sheets, have gained extensive attention in the past decade . CNTs are classified as single-walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs), which depend on the number of graphene layer which a single nanotubes is composed . In recent years, efforts have also been devoted to explore the potential biomedical application of CNTs. Benefitting from their superior heir distinct properties, especially, the biocompatibility in the physiological condition, CNTs have been one of the most promising inorganic nano-sized vectors for anticancer drugs, proteins, genetic therapeutics, and biological imaging agent delivery [8, 24, 25]. Up to now, the development of CNTs-doped PLGA nanofibers for cancer therapy has not been reported to the best of our knowledge. The objective of this work, therefore, was to examine the hypothesis of preparing a sustained anticancer drug release system by doping drug-loaded CNTs into PLGA nanofibers.
In this present work, we have successfully fabricated DOX-loaded PLGA/CNTs composite nanofibrous mats by using electrospinning technique and then have assessed its antitumor efficacy against HeLa cells. The morphology and structure of electrospun nanofibers were characterized. After the evaluation of the drug loading capacity and efficiency, the in vitro release characteristics of DOX from the composite nanofibers were also measured. And further cytotoxicity experiments demonstrate that the drug-loaded nanofibers exhibit obviously therapeutic effect for cancer cell, implying that it may become a novel therapy strategy for preventing local cancer recurrence in future cancer therapy.
PLGA with lactic acid/glycolic acid ratio of 75:25 (Mw = 110 kDa) was purchased from Daigang Biomaterials Inc. (Jinan, China). Carbon nanotube (CNTs) was purchased from Chengdu Organic Chemicals Co., Ltd. Doxorubicin (DOX) hydrochloride was purchased from Beijing Huafeng United Technology Co., Ltd. HeLa cells were supplied by Institute of Biochemistry and Cell Biology (the Chinese Academy of Sciences, Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), trypsin, penicillin (100 U/mL), and streptomycin (100 mg/mL) were all purchased from Shanghai Yuanxiang Medical Equipment Co., Ltd. All other chemicals were obtained from Sino-pharm Chemical Reagents Co., Ltd. (Shanghai, China).
Fabrication of DOX-loaded CNTs
The DOX-loaded CNTs (DOX@CNTs) were fabricated by the previously described method with some modifications . Briefly, DOX aqueous solution (1 mg/mL) was prepared, and 20 mg CNTs was dispersed into DOX solution. The mixture was stirred under dark conditions for 12 h and vacuumed slowly at room temperature for 3 h. The DOX@CNTs were collected by centrifugation (10,000 rpm, 10 min) and washed with phosphate-buffered saline (PBS) (pH = 7.4) solution to remove the dissociative DOX. The obtained DOX@CNTs was vacuum-dried at room temperature and stored by sealing for future use. To evaluate the loading efficiency of DOX, the supernatant was collected, and the DOX concentration in the supernatant was analyzed by using UV-vis spectrophotometer at 488 nm. The loading percentage of DOX in CNTs was calculated as follows:
Loading percentage (%) = (initial weight of DOX−residual weight of DOX)/weight of DOX-loaded CNTs × 100 %.
Preparation of PLGA/DOX@CNTs Nanofibers
The PLGA/DOX@CNTs composite nanofibers were fabricated by using a blend electrospinning. Firstly, the prepared DOX@CNTs was completely dispersed in hexauoroisopropanol (HFIP) and the PLGA was added into the above solution at 20 w/v %, and then stirred thoroughly to form a homogenous spinning solution. The solution was placed into a 5-mL plastic syringe with an 18-gauge blunt-ended needle. The electrospinning parameters were set as follows: the applied voltage of 20 kV, the collection distance of 15 cm, and the solution flow rate of 1.0 mL/h controlled by a syringe pump (789100C, Cole-Parmer Instruments, USA). The collected nanofibers were vacuum-dried at least 72 h to remove the residual solvent before further use.
The CNTs content in the nanofibers were 0.5, 1, and 2 wt. % (CNTs relative to PLGA), and the corresponding DOX contents in nanofibers can be calculated from the loading efficiency of CNTs. For comparison, the PLGA/DOX composite nanofibers were also prepared by electrospinning with the same DOX contents.
The morphology of the composite nanofibers was observed by a scanning electron microscope (SEM, Hitachi TM-1000, Japan), and the distribution of DOX@CNTs in the nanofibers was characterized by a transmission electron microscope (TEM, JEM-2100, Japan) at an operating voltage of 200 kV. At least 40 nanofibers were selected from different SEM image, and the nanofiber diameter was measured using Image J 1.40 G software (NIH, USA). The fluorescent images of the different composite nanofibers were observed by a fluorescence microscope (Nikon TS100, Japan). The thermogravimetric analysis (TGA) was conducted on a thermal analyzer (TG 209 F1, Germany) from the room temperature to 600 °C at a heating rate of 10 °C/min. The tensile testing of the composite nanofibers with a planar area of 50 × 10 mm was measured using a universal material tester (H5K-S, Hounsfield, UK) with a cross-head speed of 10 mm/min.
In Vitro Drug Release
The DOX release behaviors from the DOX-loaded samples including PLGA/1.5 % DOX and PLGA/1.5 % DOX@2 % CNTs electrospun nanofibers were carried out in PBS at pH 7.4. Briefly, a certain amount of the different DOX-loaded samples with the same content of DOX were put in a dialysis bag (cutoff molecular weight 7000 Da), and the bag was immersed in 15 mL of PBS (pH 7.4) at 37 °C in a thermostatted shaker with shaking at a rate of 100 rpm. At selected time intervals, the release medium (5 mL) was taken out and supplied with the same volume of fresh PBS. The concentration of DOX in the release media was determined by recording the absorbance of DOX at 480 nm using UV-vis spectrophotometer.
Cytotoxicity Evaluation of Different DOX-Loaded Samples
HeLa cells were cultured in the DMEM medium supplemented with 10 % FBS 100 U/mL penicillin and 100 μg/mL streptomycin. The cells were cultured at 37 °C in a humidified incubator containing 5 % CO2. MTT assay was employed to evaluate the viability of the HeLa cells cultured with different samples. For all experiments, cells were harvested by using trypsin solution and resuspended in the fresh DMEM medium. Before cell seeding, the samples were sterilized under UV light for 3 h and washed with PBS for three times.
For MTT assay, HeLa cells at a density of 1 × 104 cells/well were seeded in 24-well plates and cultured for 12 h to allow cells to attach. Then, the medium was replaced with a fresh medium (negative control) and the medium containing the PLGA/2 % CNTs, free DOX (positive control), PLGA/1.5 % DOX, and PLGA/1.5 % DOX@2 % CNTs composite nanofibers, and the total DOX concentrations was designed 10 mg/mL, 25 mg/mL, and 50 mg/mL. After the cells were cultured for 24 h, the cells were washed twice with PBS and the 360-μL fresh culture medium and 40-μL MTT solution (5 mg/mL in PBS) were then added to each well. The cells were incubated for another 4 h. Thereafter, the 400 μL suspension was discarded, and 400 μL of DMSO was added to each well to completely dissolve the precipitate by stirring for 15 min. Then, 100 μL of the supernatant was transferred to 96-well microplates, and the OD value was measured with a microplate reader (MK3, Thermo, USA) at 492 nm. The relative cell viability was calculated by dividing the mean OD value of the control group, and the average value was obtained from five parallel samples.
For confocal microscopy observation, HeLa cells were seeded into 24-well plates at a density of 1 × 104 cells/well and incubated for 24 h. Then, the culture medium was removed, and the cells were incubated with PLGA/2 % CNTs, free DOX, PLGA/1.5 % DOX, and PLGA/1.5 % DOX@2 % CNT composite nanofibers (DOX concentrations at 25 μg/mL) for another 24 h. After washing with PBS twice, the cells were fixed with 4 % paraformaldehyde for 10 min. The cells were then permeabilized in 0.1 % Triton X-100 in PBS for 5 min, followed by blocking with 1 % BSA for 20 min. The F-actin of cells was stained by using Alexa Fluor 488® phalloidin solution for 10 min. Finally, all samples were washed several times with PBS and observed by confocal laser scanning microscope (CLSM).
Characterization of Composite Nanofibers
Tensile mechanical properties of PLGA nanofibers and PLGA/CNTs nanofibers
CNTs content (%)
Tensile strength (MPa)
Elongation at break (%)
Young’s modulus (MPa)
6.78 ± 1.00
177.38 ± 24.82
170.71 ± 46.22
2.87 ± 0.30
65.43 ± 18.32
76.50 ± 2.30
4.08 ± 0.11
78.13 ± 12.94
145.53 ± 8.67
4.45 ± 0.40
97.33 ± 2.10
142.25 ± 3.30
DOX Loading and Release
In Vitro Antitumor Effect
In summary, we have reported a very effective and reproducible route to prepare a drug delivery system based on PLGA/CNTs composite nanofibers. The highly uniform and smooth nanofiber is successfully designed and developed. By combining the CNTs, as a proof-of-concept, we demonstrated that the PLGA/CNTs composite nanofibers could be used for the sustained release of the anticancer drug molecule DOX, which is important for biomedical applications requiring the drug molecule to maintain long-term anticancer efficacy. And the release profiles clearly indicated that the DOX can be loaded in the inner cavities or on the outside surface of the CNTs weakening the initial burst release of DOX. In addition, our results indicated that the prepared PLGA/DOX@CNTs nanofibers platform could effectively inhibit the cell viability of HeLa cells in vitro. Our results shed light on a promising use of the PLGA/DOX@CNTs composite nanofibers as a long-term drug release nanoplatform for chemotherapy in clinical cancer treatment.
This research was financially supported by the National Natural Science Foundation of China (51401031) and Natural Science Foundation of Shandong Province (ZR2013HL004).
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