j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Laser-induced drug release for local tumor controlda proof of concept Andreas Lambertz, MD,a,* Christian D. Klink, MD,a Anjali Ro¨th, MD,a Dominik Schmitz,b Andrij Pich, PhD,b Katalin Feher,c Elke Bremus-Ko¨bberling,d Ulf P. Neumann, MD, PhD,a and Karsten Junge, MDa a
Department of General, Visceral and Transplantation Surgery, RWTH Aachen University Hospital, Aachen, Germany Leibniz Institute of Interactive Materials and RWTH Aachen University, Aachen, Germany c Institute of Textile Technology, (ITA), RWTH Aachen, Aachen, Germany d Fraunhofer Institute for Laser Technology, (ILT), RWTH Aachen, Aachen, Germany b
article info
abstract
Article history:
Background: The systemic palliative chemotherapy of locally extended gastrointestinal and
Received 9 April 2014
hepatobiliary tumors is associated with a considerable burden for the patient. The aim of this
Received in revised form
project was to develop a new drug release system to improve the local stent therapy in these
7 May 2014
patients as a proof of concept study. For this purpose, polymer filaments were modified with
Accepted 16 July 2014
drug-loaded polymer microgels that allow selective release of the active substance by
Available online 22 July 2014
photochemical triggering using laser radiation. Integrated into a stent system, the better local tumor control could thus contribute to a significant increase in the quality of life of patients.
Keywords:
Methods: A standard mammalian cell line and two carcinoma cell lines were established. By
Laser-induced drug release
Fluorescence activated cell sorting (FACS), the cytotoxicity of the different materials was
Tumor reduction
determined in vitro before and after drug loading with the chemotherapeutic agent 5-
5-FU dimer
Fluorouracil (5-FU). For this purpose, the locally applied 5-FU concentration was previously
Cytotoxicity
determined by Bromdesoxyuridin assay. 5-FU dimer was synthesized by photo-induced
Carcinoma cell lines
dimerization of 5-FU in the presence of benzophenone in methanol. The chemical structure of 5-FU dimer was confirmed with Hydrogen-1 nuclear magnetic resonance and Fluorine-19 nuclear magnetic resonance. 5-FU dimer is nonsoluble in water and can be easily incorporated in polymer microgels modified with hydrophobic binding domains (cyclodextrin). After laser irradiation, 5-FU dimer decomposes and 5-FU can be released from microgels. Finally, the measurements were repeated after this laser-induced drug release. Results: In FACS analysis, neither the microgels nor the microgel cumarin complexes showed a significant difference in comparison with the negative control with H2O and therefore no toxic effect on the cell lines. After loading with the 5-FU dimer, there was no significant cell death (contrary to the pure 5-FU monomer, which dose had been previously tested as highly toxic). After laser-induced dissociation back to monomer and the associated drug release, FACS analysis showed cytotoxicity. Conclusions: It was possible to develop 5-FU dimerloaded microgels, which show no cytotoxic effect on cell lines before laser irradiation. After dissociation back to 5-FU monomer
* Corresponding author. Department of General, Visceral and Transplantation Surgery, RWTH Aachen University Hospital, Pauwelsstr. 30, 52074 Aachen, Germany. Tel.: þ49 241 80 89500; fax: þ49 241 80 82417. E-mail address: [email protected] (A. Lambertz). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.07.036
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
313
by selective photochemical triggering using laser irradiation, the active substance was released. Thus, a new drug release system has been created and tested in vitro. For further development, integration into a stent system and for in vivo follow-up evaluation more studies need to be conducted. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Systemic palliative chemotherapy is often the only therapeutic option for patients with locally extended gastrointestinal and hepatobiliary tumors [1,2]. Especially in pancreatic cancer, the majority of patients are diagnosed at advanced stages with irresectable local tumors [3e5]. In these cases, the systemic therapy is associated with a considerable burden for the patients and many of them cannot tolerate the side effects [6]. Because this concerns a relevantly high number of patients, pancreatic cancer is the fourth leading cause of cancer death [3], therapeutic alternatives are urgently needed. Thus, the aim of this study was the interdisciplinary development of a new drug delivery system by photochemical triggering using laser irradiation as a proof of concept study. By integrating this concept into a stent system, the tumor would be treated locally, the systemic side effects would decrease and a significant increase in the patients’ quality of life would be reached. Whereas drug delivery systems have been an object of research for recent years, most authors did not use chemotherapeutic agents [7] or they did not try to avoid cytotoxicity before irradiation [8]. In contrast, we used 5-Fluorouracil (5-FU) as active substance and synthesized a 5-FU dimer without cytotoxic properties. Polymer filaments were prepared with drug-loaded microgels. The hydrophobic 5-FU dimer was loaded into the microgels. After laser irradiation, a drug release should be reached by dissociation of the dimer back to water-soluble 5-FU monomer (Fig. 1).
In clinical practice, such a drug-eluted stent system could be implanted endoscopically in cases of irresectable esophageal and pancreatic cancer. Apart from the mechanical aspects of stent therapy, the active substance would be released by percutaneous or endoscopic laser irradiation, and the tumor would be treated locally.
2.
Materials and methods
To evaluate the created single elements in vitro, different cell lines were established. These were L929 mouse fibroblasts as a standard mammalian cell line and two carcinoma cell lines, fast growth cells, which are epidermal growth factor receptor positive adherent human pancreas adenocarcinoma cells and COLO-680N cells as human esophageal squamous carcinoma cell line (Fig. 2). This way, a clinical situation with healthy human cells beside potentially stenosing carcinoma cells was simulated. By Fluorescence activated cell sorting (FACS), the cytotoxicity of the single elements was tested. First, the microgels modified with beta-cyclodextrins and afterward the microgels loaded with 5-FU dimer were investigated. Therefore, the materials were incubated with 105 cells of each line on a 24well plate for 24 h. Materials were diluted with medium and measured in different concentrations as well as stained and unstained pure medium was tested. As negative control we used H2O, whereas the positive control substance was ethanol (C2H5OH) (Figs. 3 and 5).
Fig. 1 e Diagram of laser-induced drug release from microgel-modified polymer filaments. (Color version of this figure is available online.)
314
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
Fig. 2 e L929- and fast growthecell lines. (Color version of this figure is available online.)
Next, the locally applied 5-FU concentration was determined by Bromdesoxyuridin (BrdU) assay. A total of 5000 cells per well were incubated with increasing 5-FU concentrations (microgram per milliliter) until cell death began (Fig. 4). To load the active substance into the microgel, a 5-FU dimer was synthesized. After loading of the 5-FU dimer into the microgels, the cytotoxicity measurements were repeated. Two complexes with different 5-FU dimer concentrations were created (Gel A and Gel B). In these concentrations, the 5-FU monomer had shown a highly cytotoxic effect before (Fig. 5). Finally, the drug-eluted complexes were irradiated with a 266-nm laser for 30 min. Afterward, they were incubated with 105 cells of each line on a 24-well plate in different concentrations for 24 h and cytotoxicity was measured by FACS analysis again (Fig. 6).
3.
Results
To be able to load the active substance into the microgels, beta-cyclodextrinemodified microgels were synthesized. In
Fig. 3 e FACS analysis of beta-cyclodextrin (beta CD) microgels with different cell lines. (Color version of this figure is available online.)
FACS analysis, the microgels did not show a significant difference in comparison with the negative control with H2O and therefore no toxic effect on the cell lines. After incubation with ethanol as positive control, nearly all cells died (Fig. 3). In BrdU assay, the locally applied 5-FU concentration was determined. It was shown that cell proliferation decreased with increasing concentrations of 5-FU. Between 6 and 12 mg/ mL 5-FU, the minimum of cell proliferation was reached (Fig. 4). To create an active substance, which can be activated by laser irradiation, a 5-FU dimer was synthesized and loaded into microgels. According to the BrdU assay, the microgels were loaded with 6 mg/mL (Gel A) and 12 mg/mL (Gel B) of 5-FU dimer. After incubation of Gel A and Gel B with the different cell lines for 24 h, about 90% of the cells were still alive in FACS analysis. There was no significant difference compared with the negative control with H2O and therefore, the two drugloaded microgel samples did not show any cytotoxic effect. Between the mammalian cell line and the carcinoma cell lines, no significant difference was seen (Fig. 5). These drug-loaded microgels were irradiated with a 266-nm laser for 30 min. FACS analysis showed after laser
Fig. 4 e BrdU assay of 5-FU and L929 cells. (Color version of this figure is available online.)
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
Fig. 5 e FACS analysis of Gel A and B with different cell lines. (Color version of this figure is available online.)
irradiation and dissociation back to 5-FU monomer a comparable cell death like the previously tested 5-FU itself had shown (Fig. 6). Laser irradiation of the pure microgels did not show any cytotoxic effect.
4.
Discussion
In palliative situations due to irresectable pancreatic or esophageal cancer, the therapeutic concept involves two major parts: (1) on the one hand, the systemic chemotherapy [9,10] to improve the patients’ survival and (2) on the other hand, local stent implantations to treat complications like jaundice or swallowing disorders [11]. Both therapy regimes are associated with specific complications. Although there are several approaches to improve the systemic treatment strategies [12], they are still connected with a considerable burden for the seriously sick patients due to their side effects [6]. Furthermore, locally implanted stents may also cause problems like coughing or hemorrhage [13], and they mostly need to be changed after a period of time. Thus, the patients are affected by both the systemic side effects and the potential local complications caused by the implanted stents.
Therefore, the approach of this proof of concept study was to create a new drug release system, which might be integrated into a stent system to minimize the systemic side effects by reaching a better local tumor control and to increase the quality of life of patients this way. The development of drug delivery systems has been an object of research for recent years. Kenawy et al. [14] first described the release of tetracycline from polymer filaments by ultraviolet light for the treatment of periodontal disease. There are several publications focusing on binding and releasing drugs like paracetamol or paclitaxel [15e18]. In contrast to other authors, we focused on creating a chemotherapeutic agent without cytotoxic properties before laser irradiation. This 5-FU dimer would be applicable without harming human cells. To simulate this situation, L929 mouse fibroblasts were used as a standard mammalian cell line and in vitro evaluation. Neither the single elements of the microgels nor the drug-eluted variants showed any significant cytotoxic effects (Figs. 3 and 5). Thus, the 5-FU dimer showed biocompatibility before laser irradiation. After laser irradiation, we found a comparable cell death like the previously tested 5-FU itself had shown. This irradiation did not destroy the active substance, which is a frequently discussed problem in similar experiments [19e21]. Instead, the used 266-nm light caused a dissociation of the 5-FU dimer back to the 5-FU monomer, and afterward, the cytotoxic effect was seen. Thereby, a new drug release system was created. To mention some major limitations of this study, it was not possible to control the described 5-FU release temporally. The induced effect was a single boost of active substance release, which was not stoppable or repeatable. Furthermore, there was no difference seen between the L929 mouse fibroblasts and the carcinoma cell lines. It would have been expected that carcinoma cells react earlier because of their higher cell proliferation rates, which has been described by other authors [22]. In clinical application, the local side effects on healthy cells could be reduced by choosing a 5-FU concentration, which influences the carcinoma cells selectively. As mentioned before, it was not possible to study the drug releasing process in detail in our experiments. Thus, a delayed cell death of the mouse fibroblasts may not have been observed. Furthermore, results generated by cell experiments cannot be transferred directly to the situation in humans. Finally, the microgel-modified polymer filaments have to be formed into a stent structure for future clinical application. In these areas, our developed system needs further studies until in vivo evaluation. Other parts, the duration of laser irradiation for 30 min would be already feasible for clinical practice.
5.
Fig. 6 e FACS analysis of Gel A and B and FG cells after laser irradiation and drug release. (Color version of this figure is available online.)
315
Conclusions
It was possible to develop 5-FU dimereloaded microgels, which show no cytotoxic effect on cell lines before laser irradiation. After dissociation back to 5-FU monomer by selective photochemical triggering using laser irradiation, the active substance was released. Thus, a new drug release system has been created and tested in vitro. For further
316
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
development, integration into a stent system and for in vivo follow-up evaluation more studies need to be conducted.
Acknowledgment The authors thank Mrs Ellen Krott and Mr Jochen Nolting for their most excellent and careful assistance during this investigation. This study was supported by a boost fund grant (OPBo24) of the RWTH Aachen, Germany. Authors’ contributions: A.L., D.S., A.P., K.F, E.B.-K., and K.J. conceived and designed the study and collected, analyzed and interpreted the data. A.L., C.D.K., A.R., U.P.N., and K.J wrote the manuscript and provided critical revisions that were important for the intellectual content. All authors approved the final version of the manuscript.
Disclosure The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
references
[1] Ahmed WB, Ng J, Wazer DE, Saif MW. New tools and novel approaches in treating locally advanced pancreatic adenocarcinoma. JOP 2012;13:354. [2] Nieman DR, Peters JH. Treatment strategies for esophageal cancer. Gastroenterol Clin North Am 2013;42:187. [3] Li J, Ng J, Allendorf J, Saif MW. Locally advanced pancreatic adenocarcinoma: are we making progress? Highlights from the “2011 ASCO Annual Meeting”. Chicago, IL, USA; June 3-7, 2011. JOP 2011;12:347. [4] Merl MY, Abdelghany O, Li J, Saif MW. First-line treatment of metastatic pancreatic adenocarcinoma: can we do better? Highlights from the “2010 ASCO Annual Meeting”. Chicago, IL, USA. June 4-8, 2010. JOP 2010;11:317. [5] Sarris EG, Syrigos KN, Saif MW. Novel agents and future prospects in the treatment of pancreatic adenocarcinoma. JOP 2013;14:395. [6] Shahrokni A, Saif MW. Metastatic pancreatic cancer: the dilemma of quality vs. quantity of life. JOP 2013;14:391. [7] Chen J, Liu H, Zhao C, et al. One-step reduction and PEGylation of graphene oxide for photothermally controlled drug delivery. Biomaterials 2014;35:4986.
[8] Zheng M, Yue C, Ma Y, et al. Single-step assembly of DOX/ICG loaded lipidepolymer nanoparticles for highly effective chemo-photothermal combination therapy. ACS Nano 2013; 7:2056. [9] Rosemurgy AS, Serafini FM. New directions in systemic therapy of pancreatic cancer. Cancer Control 2000;7:437. [10] Douglass HO. Current approaches to multimodality management of advanced pancreatic cancer. Hepatogastroenterology 1993;40:433. [11] Sreedharan A, Harris K, Crellin A, Forman D, Everett SM. Interventions for dysphagia in oesophageal cancer. Cochrane Database Syst Rev 2009;7:CD005048. [12] Kulke MH. Recent developments in the pharmacological treatment of advanced pancreatic cancer. Expert Opin Investig Drugs 2003;12:983. [13] Peng Z, Xu S, Li H, Sun C, Fu M. Metallic expandable stents in the management of malignant tracheal stenosis due to esophageal cancer with lymph node metastasis. Oncol Lett 2013;6:1461. [14] Kenawy el R, Bowlin GL, Mansfield K, et al. Release of tetracycline hydrochloride from electrospun poly(ethyleneco-vinylacetate), poly(lactic acid), and a blend. J Control Release 2002;81:57. [15] Xie J, Wang CH. Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. Pharm Res 2006;23:1817. [16] Peng H, Zhou S, Guo T, et al. In vitro degradation and release profiles for electrospun polymeric fibers containing paracetanol. Colloids Surf B Biointerfaces 2008;66:206. [17] Cui W, Li X, Zhu X, Yu G, Zhou S, Weng J. Investigation of drug release and matrix degradation of electrospun poly(DLlactide) fibers with paracetanol inoculation. Biomacromolecules 2006;7:1623. [18] Huang HH, He CL, Wang HS, Mo XM. Preparation of coreshell biodegradable microfibers for long-term drug delivery. J Biomed Mater Res A 2009;90:1243. [19] Kim HC, Hartner S, Behe M, Behr TM, Hampp NA. Twophoton absorption-controlled multidose drug release: a novel approach for secondary cataract treatment. J Biomed Opt 2006;11:34024. [20] Sinkel C, Schwarzer MC, Frenking G, Greiner A, Agarwal S. Polymer-bound 4-methylcoumarin/1-heptanoyl-5fluorouracil photodimers: NMR elucidation of dimer structure. Magn Reson Chem 2011;49:70. [21] Levrand B, Herrmann A. Light induced controlled release of fragrances by Norrish type II photofragmentation of alkyl phenyl ketones. Photochem Photobiol Sci 2002; 1:907. [22] Strianese M, Basile A, Mazzone A, Morello S, Turco MC, Pellecchia C. Therapeutic potential of a pyridoxal-based vanadium(IV) complex showing selective cytotoxicity for cancer versus healthy cells. J Cell Physiol 2013;228:2202.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Laser-induced drug release for local tumor controlda proof of concept Andreas Lambertz, MD,a,* Christian D. Klink, MD,a Anjali Ro¨th, MD,a Dominik Schmitz,b Andrij Pich, PhD,b Katalin Feher,c Elke Bremus-Ko¨bberling,d Ulf P. Neumann, MD, PhD,a and Karsten Junge, MDa a
Department of General, Visceral and Transplantation Surgery, RWTH Aachen University Hospital, Aachen, Germany Leibniz Institute of Interactive Materials and RWTH Aachen University, Aachen, Germany c Institute of Textile Technology, (ITA), RWTH Aachen, Aachen, Germany d Fraunhofer Institute for Laser Technology, (ILT), RWTH Aachen, Aachen, Germany b
article info
abstract
Article history:
Background: The systemic palliative chemotherapy of locally extended gastrointestinal and
Received 9 April 2014
hepatobiliary tumors is associated with a considerable burden for the patient. The aim of this
Received in revised form
project was to develop a new drug release system to improve the local stent therapy in these
7 May 2014
patients as a proof of concept study. For this purpose, polymer filaments were modified with
Accepted 16 July 2014
drug-loaded polymer microgels that allow selective release of the active substance by
Available online 22 July 2014
photochemical triggering using laser radiation. Integrated into a stent system, the better local tumor control could thus contribute to a significant increase in the quality of life of patients.
Keywords:
Methods: A standard mammalian cell line and two carcinoma cell lines were established. By
Laser-induced drug release
Fluorescence activated cell sorting (FACS), the cytotoxicity of the different materials was
Tumor reduction
determined in vitro before and after drug loading with the chemotherapeutic agent 5-
5-FU dimer
Fluorouracil (5-FU). For this purpose, the locally applied 5-FU concentration was previously
Cytotoxicity
determined by Bromdesoxyuridin assay. 5-FU dimer was synthesized by photo-induced
Carcinoma cell lines
dimerization of 5-FU in the presence of benzophenone in methanol. The chemical structure of 5-FU dimer was confirmed with Hydrogen-1 nuclear magnetic resonance and Fluorine-19 nuclear magnetic resonance. 5-FU dimer is nonsoluble in water and can be easily incorporated in polymer microgels modified with hydrophobic binding domains (cyclodextrin). After laser irradiation, 5-FU dimer decomposes and 5-FU can be released from microgels. Finally, the measurements were repeated after this laser-induced drug release. Results: In FACS analysis, neither the microgels nor the microgel cumarin complexes showed a significant difference in comparison with the negative control with H2O and therefore no toxic effect on the cell lines. After loading with the 5-FU dimer, there was no significant cell death (contrary to the pure 5-FU monomer, which dose had been previously tested as highly toxic). After laser-induced dissociation back to monomer and the associated drug release, FACS analysis showed cytotoxicity. Conclusions: It was possible to develop 5-FU dimerloaded microgels, which show no cytotoxic effect on cell lines before laser irradiation. After dissociation back to 5-FU monomer
* Corresponding author. Department of General, Visceral and Transplantation Surgery, RWTH Aachen University Hospital, Pauwelsstr. 30, 52074 Aachen, Germany. Tel.: þ49 241 80 89500; fax: þ49 241 80 82417. E-mail address: [email protected] (A. Lambertz). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.07.036
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
313
by selective photochemical triggering using laser irradiation, the active substance was released. Thus, a new drug release system has been created and tested in vitro. For further development, integration into a stent system and for in vivo follow-up evaluation more studies need to be conducted. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Systemic palliative chemotherapy is often the only therapeutic option for patients with locally extended gastrointestinal and hepatobiliary tumors [1,2]. Especially in pancreatic cancer, the majority of patients are diagnosed at advanced stages with irresectable local tumors [3e5]. In these cases, the systemic therapy is associated with a considerable burden for the patients and many of them cannot tolerate the side effects [6]. Because this concerns a relevantly high number of patients, pancreatic cancer is the fourth leading cause of cancer death [3], therapeutic alternatives are urgently needed. Thus, the aim of this study was the interdisciplinary development of a new drug delivery system by photochemical triggering using laser irradiation as a proof of concept study. By integrating this concept into a stent system, the tumor would be treated locally, the systemic side effects would decrease and a significant increase in the patients’ quality of life would be reached. Whereas drug delivery systems have been an object of research for recent years, most authors did not use chemotherapeutic agents [7] or they did not try to avoid cytotoxicity before irradiation [8]. In contrast, we used 5-Fluorouracil (5-FU) as active substance and synthesized a 5-FU dimer without cytotoxic properties. Polymer filaments were prepared with drug-loaded microgels. The hydrophobic 5-FU dimer was loaded into the microgels. After laser irradiation, a drug release should be reached by dissociation of the dimer back to water-soluble 5-FU monomer (Fig. 1).
In clinical practice, such a drug-eluted stent system could be implanted endoscopically in cases of irresectable esophageal and pancreatic cancer. Apart from the mechanical aspects of stent therapy, the active substance would be released by percutaneous or endoscopic laser irradiation, and the tumor would be treated locally.
2.
Materials and methods
To evaluate the created single elements in vitro, different cell lines were established. These were L929 mouse fibroblasts as a standard mammalian cell line and two carcinoma cell lines, fast growth cells, which are epidermal growth factor receptor positive adherent human pancreas adenocarcinoma cells and COLO-680N cells as human esophageal squamous carcinoma cell line (Fig. 2). This way, a clinical situation with healthy human cells beside potentially stenosing carcinoma cells was simulated. By Fluorescence activated cell sorting (FACS), the cytotoxicity of the single elements was tested. First, the microgels modified with beta-cyclodextrins and afterward the microgels loaded with 5-FU dimer were investigated. Therefore, the materials were incubated with 105 cells of each line on a 24well plate for 24 h. Materials were diluted with medium and measured in different concentrations as well as stained and unstained pure medium was tested. As negative control we used H2O, whereas the positive control substance was ethanol (C2H5OH) (Figs. 3 and 5).
Fig. 1 e Diagram of laser-induced drug release from microgel-modified polymer filaments. (Color version of this figure is available online.)
314
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
Fig. 2 e L929- and fast growthecell lines. (Color version of this figure is available online.)
Next, the locally applied 5-FU concentration was determined by Bromdesoxyuridin (BrdU) assay. A total of 5000 cells per well were incubated with increasing 5-FU concentrations (microgram per milliliter) until cell death began (Fig. 4). To load the active substance into the microgel, a 5-FU dimer was synthesized. After loading of the 5-FU dimer into the microgels, the cytotoxicity measurements were repeated. Two complexes with different 5-FU dimer concentrations were created (Gel A and Gel B). In these concentrations, the 5-FU monomer had shown a highly cytotoxic effect before (Fig. 5). Finally, the drug-eluted complexes were irradiated with a 266-nm laser for 30 min. Afterward, they were incubated with 105 cells of each line on a 24-well plate in different concentrations for 24 h and cytotoxicity was measured by FACS analysis again (Fig. 6).
3.
Results
To be able to load the active substance into the microgels, beta-cyclodextrinemodified microgels were synthesized. In
Fig. 3 e FACS analysis of beta-cyclodextrin (beta CD) microgels with different cell lines. (Color version of this figure is available online.)
FACS analysis, the microgels did not show a significant difference in comparison with the negative control with H2O and therefore no toxic effect on the cell lines. After incubation with ethanol as positive control, nearly all cells died (Fig. 3). In BrdU assay, the locally applied 5-FU concentration was determined. It was shown that cell proliferation decreased with increasing concentrations of 5-FU. Between 6 and 12 mg/ mL 5-FU, the minimum of cell proliferation was reached (Fig. 4). To create an active substance, which can be activated by laser irradiation, a 5-FU dimer was synthesized and loaded into microgels. According to the BrdU assay, the microgels were loaded with 6 mg/mL (Gel A) and 12 mg/mL (Gel B) of 5-FU dimer. After incubation of Gel A and Gel B with the different cell lines for 24 h, about 90% of the cells were still alive in FACS analysis. There was no significant difference compared with the negative control with H2O and therefore, the two drugloaded microgel samples did not show any cytotoxic effect. Between the mammalian cell line and the carcinoma cell lines, no significant difference was seen (Fig. 5). These drug-loaded microgels were irradiated with a 266-nm laser for 30 min. FACS analysis showed after laser
Fig. 4 e BrdU assay of 5-FU and L929 cells. (Color version of this figure is available online.)
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
Fig. 5 e FACS analysis of Gel A and B with different cell lines. (Color version of this figure is available online.)
irradiation and dissociation back to 5-FU monomer a comparable cell death like the previously tested 5-FU itself had shown (Fig. 6). Laser irradiation of the pure microgels did not show any cytotoxic effect.
4.
Discussion
In palliative situations due to irresectable pancreatic or esophageal cancer, the therapeutic concept involves two major parts: (1) on the one hand, the systemic chemotherapy [9,10] to improve the patients’ survival and (2) on the other hand, local stent implantations to treat complications like jaundice or swallowing disorders [11]. Both therapy regimes are associated with specific complications. Although there are several approaches to improve the systemic treatment strategies [12], they are still connected with a considerable burden for the seriously sick patients due to their side effects [6]. Furthermore, locally implanted stents may also cause problems like coughing or hemorrhage [13], and they mostly need to be changed after a period of time. Thus, the patients are affected by both the systemic side effects and the potential local complications caused by the implanted stents.
Therefore, the approach of this proof of concept study was to create a new drug release system, which might be integrated into a stent system to minimize the systemic side effects by reaching a better local tumor control and to increase the quality of life of patients this way. The development of drug delivery systems has been an object of research for recent years. Kenawy et al. [14] first described the release of tetracycline from polymer filaments by ultraviolet light for the treatment of periodontal disease. There are several publications focusing on binding and releasing drugs like paracetamol or paclitaxel [15e18]. In contrast to other authors, we focused on creating a chemotherapeutic agent without cytotoxic properties before laser irradiation. This 5-FU dimer would be applicable without harming human cells. To simulate this situation, L929 mouse fibroblasts were used as a standard mammalian cell line and in vitro evaluation. Neither the single elements of the microgels nor the drug-eluted variants showed any significant cytotoxic effects (Figs. 3 and 5). Thus, the 5-FU dimer showed biocompatibility before laser irradiation. After laser irradiation, we found a comparable cell death like the previously tested 5-FU itself had shown. This irradiation did not destroy the active substance, which is a frequently discussed problem in similar experiments [19e21]. Instead, the used 266-nm light caused a dissociation of the 5-FU dimer back to the 5-FU monomer, and afterward, the cytotoxic effect was seen. Thereby, a new drug release system was created. To mention some major limitations of this study, it was not possible to control the described 5-FU release temporally. The induced effect was a single boost of active substance release, which was not stoppable or repeatable. Furthermore, there was no difference seen between the L929 mouse fibroblasts and the carcinoma cell lines. It would have been expected that carcinoma cells react earlier because of their higher cell proliferation rates, which has been described by other authors [22]. In clinical application, the local side effects on healthy cells could be reduced by choosing a 5-FU concentration, which influences the carcinoma cells selectively. As mentioned before, it was not possible to study the drug releasing process in detail in our experiments. Thus, a delayed cell death of the mouse fibroblasts may not have been observed. Furthermore, results generated by cell experiments cannot be transferred directly to the situation in humans. Finally, the microgel-modified polymer filaments have to be formed into a stent structure for future clinical application. In these areas, our developed system needs further studies until in vivo evaluation. Other parts, the duration of laser irradiation for 30 min would be already feasible for clinical practice.
5.
Fig. 6 e FACS analysis of Gel A and B and FG cells after laser irradiation and drug release. (Color version of this figure is available online.)
315
Conclusions
It was possible to develop 5-FU dimereloaded microgels, which show no cytotoxic effect on cell lines before laser irradiation. After dissociation back to 5-FU monomer by selective photochemical triggering using laser irradiation, the active substance was released. Thus, a new drug release system has been created and tested in vitro. For further
316
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 2 ( 2 0 1 4 ) 3 1 2 e3 1 6
development, integration into a stent system and for in vivo follow-up evaluation more studies need to be conducted.
Acknowledgment The authors thank Mrs Ellen Krott and Mr Jochen Nolting for their most excellent and careful assistance during this investigation. This study was supported by a boost fund grant (OPBo24) of the RWTH Aachen, Germany. Authors’ contributions: A.L., D.S., A.P., K.F, E.B.-K., and K.J. conceived and designed the study and collected, analyzed and interpreted the data. A.L., C.D.K., A.R., U.P.N., and K.J wrote the manuscript and provided critical revisions that were important for the intellectual content. All authors approved the final version of the manuscript.
Disclosure The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
references
[1] Ahmed WB, Ng J, Wazer DE, Saif MW. New tools and novel approaches in treating locally advanced pancreatic adenocarcinoma. JOP 2012;13:354. [2] Nieman DR, Peters JH. Treatment strategies for esophageal cancer. Gastroenterol Clin North Am 2013;42:187. [3] Li J, Ng J, Allendorf J, Saif MW. Locally advanced pancreatic adenocarcinoma: are we making progress? Highlights from the “2011 ASCO Annual Meeting”. Chicago, IL, USA; June 3-7, 2011. JOP 2011;12:347. [4] Merl MY, Abdelghany O, Li J, Saif MW. First-line treatment of metastatic pancreatic adenocarcinoma: can we do better? Highlights from the “2010 ASCO Annual Meeting”. Chicago, IL, USA. June 4-8, 2010. JOP 2010;11:317. [5] Sarris EG, Syrigos KN, Saif MW. Novel agents and future prospects in the treatment of pancreatic adenocarcinoma. JOP 2013;14:395. [6] Shahrokni A, Saif MW. Metastatic pancreatic cancer: the dilemma of quality vs. quantity of life. JOP 2013;14:391. [7] Chen J, Liu H, Zhao C, et al. One-step reduction and PEGylation of graphene oxide for photothermally controlled drug delivery. Biomaterials 2014;35:4986.
[8] Zheng M, Yue C, Ma Y, et al. Single-step assembly of DOX/ICG loaded lipidepolymer nanoparticles for highly effective chemo-photothermal combination therapy. ACS Nano 2013; 7:2056. [9] Rosemurgy AS, Serafini FM. New directions in systemic therapy of pancreatic cancer. Cancer Control 2000;7:437. [10] Douglass HO. Current approaches to multimodality management of advanced pancreatic cancer. Hepatogastroenterology 1993;40:433. [11] Sreedharan A, Harris K, Crellin A, Forman D, Everett SM. Interventions for dysphagia in oesophageal cancer. Cochrane Database Syst Rev 2009;7:CD005048. [12] Kulke MH. Recent developments in the pharmacological treatment of advanced pancreatic cancer. Expert Opin Investig Drugs 2003;12:983. [13] Peng Z, Xu S, Li H, Sun C, Fu M. Metallic expandable stents in the management of malignant tracheal stenosis due to esophageal cancer with lymph node metastasis. Oncol Lett 2013;6:1461. [14] Kenawy el R, Bowlin GL, Mansfield K, et al. Release of tetracycline hydrochloride from electrospun poly(ethyleneco-vinylacetate), poly(lactic acid), and a blend. J Control Release 2002;81:57. [15] Xie J, Wang CH. Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. Pharm Res 2006;23:1817. [16] Peng H, Zhou S, Guo T, et al. In vitro degradation and release profiles for electrospun polymeric fibers containing paracetanol. Colloids Surf B Biointerfaces 2008;66:206. [17] Cui W, Li X, Zhu X, Yu G, Zhou S, Weng J. Investigation of drug release and matrix degradation of electrospun poly(DLlactide) fibers with paracetanol inoculation. Biomacromolecules 2006;7:1623. [18] Huang HH, He CL, Wang HS, Mo XM. Preparation of coreshell biodegradable microfibers for long-term drug delivery. J Biomed Mater Res A 2009;90:1243. [19] Kim HC, Hartner S, Behe M, Behr TM, Hampp NA. Twophoton absorption-controlled multidose drug release: a novel approach for secondary cataract treatment. J Biomed Opt 2006;11:34024. [20] Sinkel C, Schwarzer MC, Frenking G, Greiner A, Agarwal S. Polymer-bound 4-methylcoumarin/1-heptanoyl-5fluorouracil photodimers: NMR elucidation of dimer structure. Magn Reson Chem 2011;49:70. [21] Levrand B, Herrmann A. Light induced controlled release of fragrances by Norrish type II photofragmentation of alkyl phenyl ketones. Photochem Photobiol Sci 2002; 1:907. [22] Strianese M, Basile A, Mazzone A, Morello S, Turco MC, Pellecchia C. Therapeutic potential of a pyridoxal-based vanadium(IV) complex showing selective cytotoxicity for cancer versus healthy cells. J Cell Physiol 2013;228:2202.
