Electrospun Poly(ε-caprolactone) Composite Nanofibers with Controlled Release of Cis-Diamminediiodoplatinum for a Higher Anticancer Activity
© The Author(s). 2017
Received: 12 February 2017
Accepted: 20 April 2017
Published: 28 April 2017
Poly(ε-caprolactone) (PCL) nanofibers were prepared by electrospun, on which the cis-diamminediiodoplatinum (cis-DIDP) was loaded, cis-DIDP@PCL, which effectively overcame cis-DIDP from dissociation or premature interaction with other bimolecular groups. Meanwhile, the toxicity and cross-resistance of cis-DIDP were reduced greatly. In vitro, cis-DIDP released from the PCL nanofibers eradicated the tumor cells around twice times more than free cis-DIDP, even better than cisplatin. Furthermore, cis-DIDP@PCL could controllably release cis-DIDP in different sustained-release solution based on our experiment.
In the 1960s, Rosenberg and colleagues accidentally discovered the cytotoxicity of cisplatin (cis-Pt [NH3]2Cl2, Additional file 1: Figure S1a), which showed high anticancer activity . At the end of 1970s, cisplatin became the first platinum anticancer drugs in clinic . Then, it was widely used for the treatment of many malignancies, including testicular, ovarian, bladder, head and neck, small-cell, and non-small-cell lung cancers . Dozens of cisplatin analogs, such as carboplatin, oxaliplatin, nedaplatin, and lobaplatin, were synthesized and used in some limited range . However, the efficacies of cisplatin and its analogs were primarily restricted by their poor water solubility, toxicity, and cross-resistance . The rapid development of nanotechnology had promoted the in-depth study of platinum anticancer drugs . Cis-diamminediiodoplatinum (cis-DIDP) is now mainly used as the intermediate preparing cisplatin and other analogs . With square planar structure, the cis-DIDP is similar to cisplatin, but chlorine ion (Cl−) is substituted by iodine ion (I−). According to the spectrochemical sequence of crystal field theory, cis-DIDP is more unstable than cisplatin. Therefore, in solution, I− is easier to leave than Cl−, so I− is more reactive than Cl−. In other words, in platinum complexes, I− is more readily being substituted than Cl− by solvent water molecules, which makes it possible for cis-DIDP to act as an anticancer reagent with better activity than cisplatin . It is to be expected that cis-DIDP could directly act as an efficient anticancer reagent rather than as an intermediate. There might be some methods to improve the therapeutic indices of platinum anticancer drugs, i.e., the development of cancer-targeting formulations of platinum-containing drugs, including drug carriers such as polymer, long-circulating liposome, and polymeric micelle [9–11]. The development of controlled release drug carrier makes it possible for cis-DIDP to be applied in clinical.
Electrospinning is a direct and relatively easy method to fabricate ultra-fine fibers with average diameters in the range of sub-micrometer down to nanometer [12, 13]. In this process, continuous polymer liquid strand is drawn through a spinneret needle by a high electrostatic force to deposit randomly on a grounded collector as non-woven fibers. These fibers exhibit interesting characteristics, for example, higher surface area to mass or volume ratio, smaller inter-fibrous pore size with high porosity, and vast possibilities for surface fictionalizations [12, 14]. Due to these advantages, fibers prepared by electrospun have been recently used as new controlled release drug carrier, [15–17] which can lower overall medicinal dosages, improve therapeutic efficacy, reduce toxicity by delivering drugs into the lesion location, and release drug at controlled rate . With good biocompatibility, many polymers were used as medical materials,  even used in anticancer drugs . For good drug permeability, poly(ε-caprolactone) (PCL) fibers are now widely used as drug carriers and surgical sutures [21–23]. PCL fibers were selected as the delivery vehicle for some characteristics such as biocompatible, biodegradable characteristic, and PCL could be eliminated from the body dissolved in body fluid without side effect [24, 25].
The first time, we reported that the cis-DIDP was loaded on PCL fibers by electrospun to overcome its instability, poor-water-solubility, toxicity, and cross-resistance. The drug loading efficiency of cis-DIDP@PCL was assessed; releasing profiles and anticancer activity were tested in vitro. Ultraviolet–visible spectroscopy (UV–Vis) had been handily used to detect the hydrolysis of platinum complexes. Degraded in vitro, the cis-DIDP@PCL might be used as a vehicle for anticancer drug to improve cancer chemotherapy both in safety and efficacy. It was interesting to note that cis-DIDP can act not only as intermediate to prepare other platinum-based drugs but also as anticancer reagent.
Chemicals and Materials
Cisplatin and cis-DIDP were purchased from Kunming Guiyan Pharmaceutical Co. Ltd. (China) and stored away from light at −4 °C. PCL (molecular weight 5 × 104), sodium chlorine (A.R.), glycine (B.R.), and glucose (A.R.) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). RPMI1640 (the culture medium) and newborn calf serum were purchased from Shanghai Shichen Reagent Co. Ltd. (China). Human hepatocellular carcinoma cell line SMMC-7721 was newly purchased from Shanghai Cell Center (Chinese Academy of Sciences).
UV of Cisplatin and Cis-DIDP
Cisplatin and cis-DIDP were dissolved respectively away from light in deionized water (different solutions such as normal saline, 5% glucose, and 0.1 mol L−1 glycine were respectively used as alternative solvent to examine the solvent effect) to form 1 mmol L−1 solution. The solution absorbance was determined from time to time by the Agilent 8453 UV–Vis spectrophotometer (Agilent, USA).
Preparation of Cis-DIDP@PCL
PCL was dissolved in dimethylformamide (DMF) to form a polymer solution (PCL wt% 5–15%), and heated by water bath. Then, a predetermined amount of cis-DIDP (1–15% to PCL) was dispersed in DMF. The cis-DIDP dispersion was added into the PCL polymer solution with continuous stirring to form homogeneous PCL polymer solution containing cis-DIDP. In the electrospinning procedure, the polymer solution was firstly transferred to a syringe. Then, the syringe pump was used to deliver the solution through a hollow needle (8#, outside diameter of the needle is 8 mm), the flow rates were 0.5–3.0 mL h−1. A high voltage DC generator was used to produce 10–25 kV voltage to inject polymer solution through the hollow needle. An aluminum foil was used as a collector to gather the random fibers. The distances from the spinneret to the collector were 10–25 cm. All the experiments were performed at room temperature. The fibers were finally taken out and dried under vacuum for 48 h. The blank PCL fibers were fabricated by the same method but without dispersing the cis-DIDP in the DMF dichloromethane solution.
The fibers with average diameters from 50 to 500 nm could be fine-tuned by adjusting electrospinning parameters, such as concentration of cis-DIDP, solvent, electrospinning voltage, polymer solution flow rates, and the distances between needle and collector. Different operation parameters are listed in Additional file 1: Tables S1–S4. Additional file 1: Figures S5–S8 shows the SEM images of the products fabricated under different conditions. After trial and error, the following electrospinning conditions were used: 10/100 (cis-DIDP/PCL), 20 kV (voltage), 1.0 mL h−1 (flow rate), 15 cm (distance) to prepare products for further studies.
The products generally were characterized by SEM, XRD, and FT-IR . An S-4800 high-resolution field-emission scanning electron microscopy (FE-SEM, Hitachi, Japan) was used to observe the morphology of collected fibers. The samples for SEM observation were sputtered and coated with a thin layer of gold for better imaging. The average fiber diameters and its distribution were calculated from the random fibers of a typical SEM image.
The structure of cis-DIDP powders, PCL, and nanofibers were examined by Advance D8 X-ray diffraction (XRD, Bruker, Germany). The XRD patterns were determined with an X-ray diffractometer with Cu Ka radiation (λ = 1.54056 Å, 40 kV, 40 mA) over the 2θ range of 10°–70° with the scanning rate of 0.2°s−1.
FT-IR (Thermo Fisher, USA) was used to analyze the molecular structure of cis-DIDP, blank PCL nanofibers, and cis-DIDP@PCL nanofibers. Drug cis-DIDP was commonly mixed with potassium bromide (KBr) and compressed to pellet; nanofibers were cut into pieces and mixed with KBr and compressed to pellets, then were scanned at the wave number of 4000–400 cm−1.
Release Profile In Vitro and Loading Efficiency
Here, C 0 is the concentration of cis-DIDP in cis-DIDP@PCL (μg mL−1), V 0 is the volume of cis-DIDP@PCL solution (mL), M 0 is the mass of added cis-DIDP@PCL (mg), and wt% is the mass fraction of cis-DIDP in fiber.
Anticancer Activity In Vitro
In vitro, the anticancer activity of the cis-DIDP and cis-DIDP@PCL fibers were examined by MTT assay; cisplatin was selected as control. Human hepatocellular carcinoma cells (SMMC-7721 line cell) were chosen as the target tumor cells. The tumor cells were cultured in RPMI 1640 containing 10% newborn calf serum, 25 μg mL−1 penicillin and 25 μg mL−1 streptomycin, then adjusted to 5 × 104 cells mL−1; 200 μL aliquots of the cell suspension were added into each well of a 108-well plate and incubated in the humidified atmosphere containing 5% CO2 at 30°C for 24 h. Cisplatin, cis-DIDP, and cis-DIDP@PCL were added to the tumor-cell-cultured well and incubated for another 12, 24, 36, 48, and 72 h, respectively. Cisplatin, cis-DIDP, and cis-DIDP in cis-DIDP@PCL contents were 50 μg mL−1. The 20 μL MTT solution (5 mg mL−1) was added to each well and maintained incubation for 4 h. Finally, the supernatant in the wells was discarded carefully, and 150 μL DMSO was added to each of the wells to dissolve the residue. The optical densities of DMSO solutions were determined by a microplate reader at 490 nm, and the cell inhibition was calculated.
Results and Discussion
Comparison of Cis-DIDP and Cisplatin by UV Irradiation
Morphology and Structure of the Products
Release Profile In Vitro and Drug Loading Efficiency
In these equations, y is the absorption and x is the concentration of cis-DIDP. Based on these equations, the amount of the cis-DIDP was measured over time and the release profiles of cis-DIDP from cis-DIDP@PCL were obtained in different solutions. The cumulative concentration of cis-DIDP released from cis-DIDP@PCL in different solution was calculated by Eqs. (2)–(4).
Figure 6 shows the release profiles of cis-DIDP from cis-DIDP@PCL (cis-DIDP, PCL = 1:10) in (a) deionized water, (b) normal saline, and (c) PBS. The release rate of cis-DIDP was faster in normal saline than that in deionized water, but it was a little slower in PBS. When the drug accumulative release reached the maximum, there was a trend that the curve declines at different degrees. This phenomenon could be further confirmed that the hydrolysis of cis-DIDP occurs in solution as discussed above, but cis-DIDP did not hydrolyze completely. The PCL nanofibers were dispersed in deionized water, and the PCL polymers were uniformly distributed in solution, which inhibited the cis-DIDP hydrolysis. The serious burst release did not appear in the initial release of cis-DIDP from cis-DIDP@PCL, indicating that cis-DIDP was better incorporated into nanofibers. The concentration of cis-DIDP was observed to reach its maximum earlier in normal saline (about 24 h) than that in deionized water (about 48 h), and in PBS (about 72 h). Then, the concentration of cis-DIDP decreased gradually, and the downward trend was most obvious in deionized water, moderate in PBS, and weakest in normal saline. The results indicated that the presence of Cl− promoted the release of cis-DIDP from cis-DIDP@PCL but inhibited its hydrolysis. As shown in Fig. 6c, the controlled release of cis-DIDP from cis-DIDP@PCL might be gained for long term in PBS. Cis-DIDP@PCL (100 mg) was dissolved in 100 mL deionized water. The concentration of free cis-DIDP in the solution was measured by UV–Vis spectroscopy for three times. Because of uniform dispersion of cis-DIDP in electrospinning solution and scattering in products, the encapsulation efficiency of product could be calculated to be 88.87% (EE%, Eq. (1)).
Scheme of Electrospinning and Sustained-Release Process
The model process of cis-DIDP sustained-release from cis-DIDP@PCL in solution was exhibited in Additional file 1: Scheme S2. At the beginning, cis-DIDP soon dropped from the cis-DIDP@PCL surfaces and dispersed into the solution. The initial concentration of cis-DIDP was approximately 10%. As time goes on, cis-DIDP continuously released from cis-DIDP@PCL and the concentration of cis-DIDP increased gradually. Finally, the cis-DIDP released almost completely and uniformly dispersed with extremely slow hydrolysis in solution, while PCL nanofibers formed a layer of film.
Anticancer Activity In Vitro
According to the molecular structure analysis, the anticancer activity of cis-DIDP was better than that of cisplatin. However, there was little research on the clinical application of cis-DIDP for its unstability, which defect is overcome by incorporating cis-DIDP into the carriers. Meanwhile, the common toxicity and cross-resistance of platinum-based anticancer drugs have also been inhibited. In this work, the controlled-release systems of cis-DIDP from the electrospun carriers were tested, in which cis-DIDP was finely incorporated into the PCL nanofibers. It is obviously effective that cis-DIDP sustained-releases from the nanofibers inhibit human lung tumor cells in vitro. The results show that cis-DIDP@PCL are ideal controlled-release drug carrier, and the vehicle may be applied in clinic. It is instructive to improve other inorganic anticancer drug anticancer chemotherapy with the same method. The total evaluation of the system in vivo would be confirmed after more perfect evaluation in vitro, and the results would be further verified by animal experiments. If achieved good results, there would be potential for clinical trials.
The authors are grateful to the financial support of the National Natural Science Foundation of China (No. 21471114), the State Major Research Plan (973) of China (No. 2011CB932404).
QSW carried out the overall design of the project, provided the technical guidance, and revised the manuscript. CJM actualized all the experiments and wrote the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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