Prototype of biliary drug-eluting stent with photodynamic and chemotherapy using electrospinning
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 15 April 2014
Accepted: 14 August 2014
Published: 19 August 2014
The combination of biliary stent with photodynamic and chemotherapy seemed to be a beneficial palliative treatment of unresectable cholangiocarcinoma. However, by intravenous delivery to the target tumor the distribution of the drug had its limitations and caused serious side effect on non-target organs. Therefore, in this study, we are going to develop a localized eluting stent, named PDT-chemo stent, covered with gemcitabine (GEM) and hematoporphyrin (HP).
The prototype of PDT-chemo stent was made through electrospinning and electrospraying dual-processes with an electrical charge to cover the stent with a drug-storing membrane from polymer liquid. The design of prototype used PU as the material of the backing layer, and PCL/PEG blends in different molar ratio of 9:1 and of 1:4 were used in two drug-storing layers with GEM and HP loaded respectively.
The optical microscopy revealed that the backing layer was formed in fine fibers from electrospinning, while drug-storing layers, attributed to the droplets from electrospraying process. The covered membrane, the morphology of which was observed by scanning electron microscopy (SEM), covered the stent surface homogeneously without crack appearances. The GEM had almost 100% of electrosprayed efficiency than 70% HP loaded on the covered membrane due to the different solubility of drug in PEG/PCL blends. Drug release study confirmed the two-phased drug release pattern by regulating in different molar ratio of PEG/PCL blends polymer.
The result proves that the PDT-chemo stent is composed of a first burst-releasing phase from HP and a later slow-releasing phase from GEM eluting. This two-phase of drug eluting stent may provide a new prospect of localized and controlled release treatment for cholangiocarcinoma disease.
Cholangiocarcinoma is the second most common hepatobiliary tumor, which is generally a locally invasive tumor that occludes the biliary tree and leads to cholangitis and liver failure. Until now, tumor resection has been the only potential cure for cholangiocarcinoma [1, 2]. Unfortunately, even with resection, the survival rate with five years can decrease to 11% at most and more than 50% of patients still remained at unresectable stage [3, 4]. Inoperable patients with advanced cholangiocarcinoma typically have obstructive cholestasis. So far, the primary standard method of treatment has been biliary stenting . However, this treatment can prolong survival time slightly by providing temporary biliary drainage. Therefore, the secondary method of treatment is required to prolong the survival time by reducing tumor burden. Chemotherapy and radiotherapy are classical treatments but their results are also disappointing [3, 5].
Photodynamic therapy (PDT) is a new and promising treatment option, which contains a photosensitizer, light source, and oxygen . The concept of PDT is based on a photosensitizer exposed to the specific wavelength of light, which can generate cytotoxic reactive oxygen species (ROS) to kill tumor cells . Additionally, previous studies have shown that PDT could also inhibit the P-glycoprotein efflux of drug. A combination of PDT and chemotherapy can improve the accumulation of chemo-drug in tumor cells, and reduce the chemo-drug resistance from the P-glycoprotein efflux . Another advantage of combination therapy with PDT and chemo-drug is the capability to induce antitumor immunity . However, by intravenous delivery to target tumor, the distribution of the drug had its limitations and caused serious side effect on non-target organs. After receiving PDT treatment, patients have to stay indoors, away from bright light for 3 to 4 days to avoid the skin photosensitivity from the side effect .
In order to decrease the side effect during the treatment, the aim of this study is to develop a localized drug eluting stent, named PDT-chemo stent, by incorporating gemcitabine (GEM) with hematoporphyrin (HP) to cover the stent surface. Drug-eluting stent has been considered a method to maximize the drug concentration immediately on the localized tumor environment, while minimizing the non-target organs exposure [11, 12]. In clinical practice, this PDT-chemo stent could be inserted to the tumor area via endoscopic retrograde cholangiography , followed by the simultaneous specific light source from endoscopy to activate the photosensitizer for PDT. Meanwhile, the chemo-drug of GEM will be released continuously as the second step for chemotherapy. The multimodal function of PDT-chemo stent will not only aim to increase the accumulation of drug within the neoplastic tissue, but also decrease the side effect on non-target tissues.
Polyurethane (PU), Polycaprolactone (PCL, Mw = 80,000), Polyethylene glycol (PEG, Mw = 20,000), Tetrahydrofuran (THF), 1,1,1,3,3,3-Hexafluro-2-propanel (HFIP) and Hematoporphyrin (HP) were purchased from Sigma-Aldrich (St Louis, MO). Gemcitabine (GEM) of clinical grade was supplied by National Taiwan University Hospital. All other chemicals were of analytical grade and used as received.
Preparation of PDT-chemo stent
The PDT-chemo stent, consisting of tri-layers of membranes, was made by electrospinning and electrospraying dual-processes. The metallic stent was provided from the laboratory of Dr. Fuh-Yu Chang in National Taiwan University of Science and Technology, and the femtosecond laser was used to carve the 316 L stainless metallic tube. The unit of electrospinning contained a high-voltage power supply, a motor to rotate the stent, a syringe pump, and a 19G-needle that was connected by a tube to a syringe. The metallic stent rotated by a motor was horizontally placed 15 cm away from the needle. The solution of PU in HFIP (10 m/v%) was used for electrospinning process, while PCL/PEG blends were mixed in HFIP/THF (1:1) solution for electrospraying process. Both PU and PCL/PEG blends solution were extruded from the syringe at a rate of 5 μL/min. The backing layer of PU was first electrospun with voltage 14 kV, and was followed by the drug-storing layer of PCL/PEG blends elecrosprayed with higher voltage 22 kV. Herein, the drug-storing layer of PCL/PEG blends in molar ratio of 9:1 was loaded with GEM covering the backing layer, followed by the HP coating on the top in PCL/PEG molar ratio of 1:4 (Figure 1). The extruded polymer from the syringe of electrspinning/electrospraying was collected for only a short period time on cover glass for optical microscopy (OM, Leica, Germany). Samples collected covered stent were prepared by coating with thin gold film by sputtering PVD and visualized by scanning electron microscopy (SEM, JSM-7000 F, Japan) operated at 15 KV.
Drug electrosprayed efficiency
To further confirm the electrsprayed efficiency of loading drug in state of covered membrane, the membrane was collected from the stent and absolutely dissolved in dimethyl sulfoxide (DMSO) solution. After that, high-performance liquid chromatography (HPLC, Waters e2695, USA) and ultraviolet–visible spectroscopy (UV/vis, JASCO V-550, USA) were used to examine the extruded drugs of GEM and HP from the covered membrane respectively.
The covered membrane was incubated in a sealed glass bottle with 0.5 ml phosphate-buffered saline (PBS) as the releasing medium. The bottle was placed in a shaking incubator at 37°C at a shaking speed of 50 rpm. At the predetermined time, 0.5 ml sample was withdrawn and replaced with the same volume of fresh medium. Residual concentration of drug in the membrane was counted by dissolving the membrane in DMSO solution as the eluting medium. Samples were collected and analyzed under the UV–vis spectrometer and HPLC. The morphology of membrane after 72 h of release was assessed by SEM imaging. The values were presented as mean ± standard error (STD) in triplicate. Statistical analysis was performed using the analysis student’s t-test. Values of p < 0.05 was considered being statistically significant.
Results and discussion
Prototype of PDT-chemo stent
To make the stent with covered membrane more uniformly adhered to the surface and easily regulate the thickness according to the clinical needs, electrospinning/electrospraying has been regarded as the appropriate means for demonstrating the PDT-chemo stent [18, 19]. The covered stent can be manufactured by several electrospinning methods: post-spinning modification, drug/polymer blends, emulsion electrospinning and core-shell electrospinning . Drug/polymer blends technique could easily mix the drug with polymer directly and form a layer of membrane to achieve sustained drug release. Therefore, in this study, the prototype of PDT-chemo stent was constructed by drug/polymer blends technique via electrospinning and electrospraying dual-processes. The backing layer was electrospun first from PU polymer solution, followed by the electrospraying process from PCL/PEG blends solution with drug loaded. Electrospinning is the process with voltage to extrude polymer solution into fine fibers for the production of fine-fibers-covered stents. In our case, fine fibers could support the superior mechanical properties of the membrane and be introduced as the backing layer for effectively controlling majority of the inner drugs released to the surrounding tissue. During electrospinning, the organic solvent, which could be toxic to cells, will be completely evaporated due to its high volatility . Electrospraying has similar preparation process to electrospinning but is usually used with higher voltage and lower polymer density, which makes the polymer solution more easily broken up into droplets [23, 24]. The concept of electrospraying process was used to increase layer-to-layer adhesion, which could avoid drug-storing layer cracking and separating from backing layer during the stent expending.
Effect of drug release
Herein, GEM is one of the first line chemo-drugs in the treatment of advanced cholangiocarcinoma, which is a prodrug belonging to an analog of deoxycytidine. Once GEM is transported into the cell, it will be phosphorylated to an active form to inhibit DNA synthesis . Therefore, low initial burst of GEM may help to prevent undesired toxicity associated with high concentration of GEM, and the burst release of HP can provide a simultaneous treatment for PDT, triggered by the light source from endoscopy when the stent is localized in bile duct. Overall, the prototype of PDT-chemo stent has demonstrated the proof of concept of localized combination therapy for cholangiocarcinoma. Based on the theory, the drug-releasing rate could be further regulated by changing the initial electrospraying blend polymer solution, concentration, structure and type of fibers and the amount of additives for the clinical needs [34, 35].
In preliminary study, we have successfully developed a prototype of tri-layered covered stent with PDT and chemotherapy. This PDT-chemo stent was prepared by electrospinning and electrospraying dual-processes. The membrane is composed of PU backing layer as the base and PCL/PEG drug-storing layers with GEM and HP on the top. The mixing of drugs with different PCL and PEG composition demonstrated an effective strategy for regulating the drugs release from the membrane. The release study has confirmed a two-phased drug release pattern, which provides a proof of concept for the hypothesis that the PDT-chemo stent is composed of a first burst-releasing phase from HP and a later slow-releasing phase from GEM eluting. This two-phase of drug eluting stent may provide a new prospective of localized controlled release treatment for cholangiocarcinoma disease.
The study was financially supported by National Research Program for Biopharmaceuticals NRPB [100INP015-2]. The authors would like to thank the laboratory members from Dr. Ming-Jium Shieh for the HPLC technical support and thank Mr. Ko-Chung Yen for the assistance of eletrospinning technique. The authors would also like to thank Ms. Sami Corber and Ms. Yu-Wen Fang for grammatical corrections.
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