Article pubs.acs.org/Biomac
Biocompatible Drug Delivery System for Photo-Triggered Controlled Release of 5-Fluorouracil Qiao Jin, Fabian Mitschang, and Seema Agarwal* Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein Strasse, D-35032, Marburg, Germany ABSTRACT: The synthesis of a photo-triggered biocompatible drug delivery system on the basis of coumarinfunctionalized block copolymers is reported. The coumarinfunctionalized block copolymers poly(ethylene oxide)-b-poly(n-butyl methacrylate-co-4-methyl-[7-(methacryloyl)oxyethyloxy]coumarin)) (PEO-b-P(BMA- co-CMA)) were synthesized via atom transfer radical polymerization (ATRP). The micelle−drug conjugates were made by covalent bonding of anticancer drug 5-fluorouracil (5-FU) to the coumarin under UV irradiation at wavelength >310 nm. These micelle−drug conjugates possessed spherical morphology with diameters of 70 nm from TEM images. In vitro drug release experiments showed the controlled release of anticancer drug 5-FU from the micelle−drug conjugates under UV irradiation (254 nm). These micelle−drug conjugates also showed excellent biocompatibility by the in vitro cytotoxicity experiments. The results suggest that these micelle−drug conjugates could be a promising candidate for the delivery of anticancer agents with low side effects on normal cells and excellent therapeutic efficacy to cancer cells.
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administration, which might due to its small size and poor stability in the core.21 Unlike polymeric micelles, polymer− drug conjugates (polymeric prodrugs) are usually water-soluble polymers that have drugs covalently conjugated via labile linkers which can be cleaved in certain conditions.8−10,22 The drugs cannot be released before the degradable linking was cleaved. Therefore, polymer−drug conjugates exhibit excellent storage stability, low systemic toxicity in circulation, and localized drug releases. To better control the biocompatibility, undesirable diffusion during transportation and selective targeting with precisely structured drug carriers, a new class of polymeric micelles has been designed to have the anticancer drugs covalently conjugated to the micelles via labile linkers.23−28 These micelle−drug conjugates combine the advantages of conventional drug-encapsulated micelles and polymer−drug conjugates. The drugs can only be released after the degradable bonds were cleaved under stimulus conditions. Acid-labile linkers, such as hydrazone23,29 and carbamate,30 were widely used to design micelle−drug conjugates. Kataoka and coworkers synthesized acid-labile amphiphilic block copolymer, poly(ethylene glycol)−poly(aspartate-hydrazone-adriamycin) (PEG-p(Asp-Hyd-ADR)), which can be self-assembled into micelles. The micelles were stable in physiological condition (pH 7.4). On the other hand, anticancer drug adriamycin can be gradually released in acid condition (pH 5).23
INTRODUCTION It is well-known that cancer is a multifaceted disease and one of the leading causes of mortality in the world. A lot of anticancer drugs have been developed.1,2 However, the high efficacy of anticancer drugs is always associated with severe side effects to normal cells. In particular, the dose-dependent cardiotoxicity induced by anticancer drugs is cumulative and life-threatening. 3 Various polymeric drug delivery systems, such as polymeric micelles, 4−7 polymer−drug conjugates (polymeric prodrugs), 8−10 polymeric nanoparticles, 11,12 and polymeric gels,13,14 have been developed to address these problems. Since polymeric micelles were first introduced as drug delivery vehicles in the early 1980s by Helmut Ringsdorf, 15 there has been considerable interest in polymeric micelles formed from amphiphilic block copolymers as drug-delivery devices.4−7,16−18 Polymeric micelles have several advantages, such as a simple preparation, efficient drug loading without chemical modification of the parent drug, and controlled drug release. Compared to free drugs, drugs incorporated into polymeric micelles exhibit favorable therapeutic advantages, such as prolonged circulation time by inhibiting phagocytic and renal clearance,19 passive targeting to the tumor tissues by the enhanced permeability and retention (EPR) effect,20 decreased side effects and selective tumor accumulation, enhanced drug solubility in water, and improved drug bioavailability. However, since the amphiphilic polymeric micelles have hydrophobic inner cores that load and stabilize anticancer drugs via hydrophobic association, only hydrophobic drugs can be physically encapsulated into the micelles. When physically encapsulated, the anticancer drugs tend to rapidly diffuse and release out of the polymeric micelles upon intravenous © 2011 American Chemical Society
Received: July 3, 2011 Revised: August 22, 2011 Published: August 24, 2011 3684
dx.doi.org/10.1021/bm2009125 | Biomacromolecules 2011, 12, 3684−3691
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Tetrahydrofuran (600 mL) and water (300 mL) were added. Argon was bubbled through the colorless solution for half an hour to remove oxygen. Subsequently, the reaction mixture was distributed to six sealable reaction tubes equipped with a magnetic stir bar. These tubes were sealed under argon, placed in a rotating sample holder to ensure uniform irradiation, and irradiated for 20 h in a Rayonet type photoreactor with broadband UV light. The mixture was concentrated under reduced pressure. The polymer was precipitated from ethanol. For further purification, the polymer was dissolved in chloroform and extracted with water twice. After the solvent was removed under reduced pressure, the polymer was precipitated again from ethanol. The polymer was then dried in high vacuum. Preparation of Micelle−Drug Conjugates. PEO−P(BMA-coCMA)−FU was first dissolved in THF (10 mg/mL). Then 1 mL of the THF solution was injected into 6 mL of water. After stirring for 2 h, the solution was dialyzed against water to remove the cosolvent THF. The final polymer concentration was adjusted to 1 mg/mL. In Vitro Drug Release Experiments. In vitro release of anticancer drug 5-FU from the micelle−drug conjugates was determined as follows. 5 mL of PBS solution with micelle−drug conjugates (pH 7.4, 1 mg/mL) was introduced into a dialysis tube (MWCO 1000) which was placed in 15 mL of PBS release media. The drug release experiments started with or without UV irradiation (254 nm) at 37 °C. At predetermined time intervals, 2 mL aliquots were collected from the PBS release media, and another 2 mL of fresh PBS was added into the release media. Then the concentration of 5-FU released was monitored using a UV spectrophotometer at 266 nm by the calibration curve of 5-FU in PBS. In Vitro Cytotoxicity Experiments. MTT-tests with a L292 (mouse fibroblast) cell line (DSMZ) were utilized to investigate cytotoxicity because L929 cells are considered to be a standard cell line for cytotoxicity test according to ISO 10993-5 and the US Pharmacopeia.27 Living cells are able to reduce methylthiazolyldiphenyltetrazolium bromide (MTT, water-soluble dye) to a water-insoluble formazan derivative with a different absorption by oxidoreductase. This enzyme is not active in dead cells, so this dye can be used for the determination of viable cells. The L292 cells were cultured in low glucose DMEM (Dulbecco’s modified Eagle medium) with 10% fetal calf serum (FCS, Gibco) as well as 2 mM L-glutamine at 37 °C in humidified air with 8.5% CO2 and seeded in 96-well plates (NunclonTM, Nunc, Germany) at a density of 8000 cells per well. After incubation for 24 h at 37 °C, 10% humidity, and 8.5% CO2, the cell culture medium was removed, and the cells were exposed to a geometric dilution series of polymer in tissue culture medium containing 2 mM L-glutamine, sterilized by filtration (0.2 μm), as well as a blank control with culture medium; each 200 μL of solution (100 μL of fresh medium plus 100 μL of polymer solution) was added per well. After incubation for 24 h (37 °C, 10% humidity, 8.5% CO2) 20 μL of MTT was added to the wells from a sterile filtered stock solution in DMEM without serum to achieve the final concentration of 0.5 mg/mL. Subsequent to incubation for 4 h in the dark, unreacted dye was removed, and DMSO was added to the aspirated wells in order to dissolve the formed MTT-formazan. The absorption of the DMSO solutions was determined at 570 and 690 nm utilizing a Titertek Plus MC 212 ELISA reader by ICN. By comparison with the blank control of cells cultured in culture medium, the relative cell viability was calculated; these data were fitted to a logistic dose− response function to determine the IC50 values. Characterization. The molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC) at 25 °C using chloroform (CHCl3) as the eluent at a flow rate of 0.5 mL/min. PMMA standards were used for conventional calibration. The OmniSEC 4.2 software (Viscotek) was used for data recording and interpretation. The 1H NMR spectra in CDCl3 were recorded on Bruker Avance 300 A/Avance 300 B. UV−vis spectra were carried out with a Perkin-Elmer Lambda 9 UV−vis/nearIR (NIR) spectrophotometer. Dynamic laser scattering (DLS) was performed on the Beckman Coulter Delsa Nano C particle analyzer at the 90° scattering angle. Intensity-average hydrodynamic diameter was adopted in this research. The samples were cleaned using a 0.45 μm
5-Fluorouracil (5-FU) is the first class of compounds that has been subjected to intensive research as a chemotherapeutic agent.31−33 It is widely used to treat solid cancers, such as colon, breast, rectal, and pancreatic cancers. However, because 5-FU is particularly toxic to dividing tissues, its clinical application is limited by its extremely severe side effects on normal cells. Because 5-FU is hydrophilic, it cannot be encapsulated into micelles by hydrophobic interaction. We and some other groups have introduced “prodrug” strategy to improve the clinical utility of 5-FU as an important cancer chemotherapeutic agent.34−37 Recently, photoreversible immobilization of 5-FU prodrug on coumarin-functionalized polymers was introduced by us and others.34,37−40 The 5fluorouracil prodrug can be attached directly to coumarinfunctionalized polymers via a [2 + 2] cycloaddition reaction under UV irradiation at wavelength >310 nm. The release of 5FU can be triggered with UV irradiation at wavelength 310 nm.42,43 To prevent the polymer chains from undergoing the favored chain self-dimerization instead of drug loading, the amount of the photoresponsive coumarin groups was kept relatively low (three coumarin groups in every polymer chain in average). At the same time, a 100-fold excess of the anticancer 3686
dx.doi.org/10.1021/bm2009125 | Biomacromolecules 2011, 12, 3684−3691
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Figure 2. 1H NMR spectra of block copolymer PEO−P(BMA-co-CMA) and its polymer−drug conjugate PEO−P(BMA-co-CMA)−FU in CDCl3.
drug 5-fluorouracil (5-FU) was employed. The block copolymer PEO−P(BMA-co-CMA) cannot be molecularly dissolved in water. 5-FU can be dissolved in water but shows very low solubility in organic solvents. As a result, a mixed solvent of THF and water (THF/water 2:1) was employed in the drug loading experiments. After the polymer PEO− P(BMA-co-CMA) and drug 5-FU were dissolved, no micellar aggregations were observed by DLS (data not shown), which showed that PEO−P(BMA-co-CMA) and 5-FU were molecularly dissolved in this mixed solvent. After the solution was irradiated under UV light (350 nm) for 20 h, the characteristic absorption peak of coumarin groups at 310 nm (π−π* transition in the coumarin chromophore) disappeared, which confirmed the successful [2 + 2]-cycloaddition reaction (Figure 3). The successful synthesis of drug-attached polymer PEO− P(BMA-co -CMA)−FU was further analyzed by 1H NMR and GPC. From the GPC results as shown in Figure 1, a slight increase of the number-average molecular weights was observed after photoinduced drug loading. At the same time, we can just find a single peak from the molecular-weight-distribution profile of PEO−P(BMA-co -CMA)−FU, which showed no indications of intermolecular cross-linking. Most of the coumarin groups formed coumarin−fluorouracil heterodimerization. The distinct differences of the 1H NMR spectra between PEO−P(BMA-co-CMA) and PEO−P(BMA-co-CMA)−FU are shown in Figure 2. After UV irradiation (350 nm), the characteristic resonance signals of coumarin groups (2.35, 6.09 ppm) disappeared from the 1H NMR spectra of PEO−P(BMAco -CMA)−FU, indicating the conversion of coumarin moieties into coumarin−fluorouracil conjugates. Furthermore, we can find characteristic cyclobutane proton signals (5.03−5.17 ppm) from the 1H NMR spectra of PEO−P(BMA-co -CMA)−FU,
Figure 3. UV/vis spectra of the drug loading reaction before and after UV irradiation (350 nm).
which was absent in the 1H NMR spectra of PEO−P(BMA-coCMA). Furthermore, the magnified inset in Figure 2 showed two resonances that were assigned to the cyclobutane proton at 5.03−5.17 ppm. After the fluorouracil addition, the double bond proton of coumarin moved to 5.13 ppm, which was designated i′. The double bond of 5-FU moved to 5.07, which was designated k′. It was the further proof for the formation of cyclobutane-type linkages to 5-FU. We can also calculate that there was 36 μg of anticancer drug 5-FU in 1 mg of the 3687
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polymer−drug conjugate PEO−P(BMA-co -CMA)−FU from the 1H NMR spectra. Formation of the Micelle−Drug Conjugates. The amphiphilic block copolymer PEO−P(BMA-co-CMA)−FU can self-assemble to form micelles in an aqueous solution. The micelle−drug conjugates were prepared by the cosolvent strategy as described in the Experimental Section. After dialysis, the micellar aggregates were observed by dynamic lightscattering measurements (DLS). The intensity-average hydrodynamic diameter (Dh) of PEO−P(BMA-co-CMA)−FU micelle−drug conjugates was about 87 nm with a polydispersity index of 0.255 (Figure 4). TEM images of the micelle−drug
photoresponsive drug depot. In contrast to long-wavelength UV irradiation (>310 nm), which promotes [2 + 2] cycloaddition reaction (drug loading), the resulting dimers can also be photocleaved by exposure to UV light at wavelength
