J Nanopart Res (2008) 10:1045–1052 DOI 10.1007/s11051-007-9336-1
RESEARCH PAPER
Cellular uptake and radiosensitization of SR-2508 loaded PLGA nanoparticles Cheng Jin Æ Ling Bai Æ Hong Wu Æ Zenghui Teng Æ Guozhen Guo Æ Jingyuan Chen
Received: 10 August 2007 / Accepted: 17 November 2007 / Published online: 8 December 2007 Ó Springer Science+Business Media B.V. 2007
Abstract SR-2508 (etanidazole), a hypoxic radiosensitizer, has potential applications in radiotherapy. The poly(D,L-lactide-co-glycolide)(PLGA) nanoparticles containing SR-2508 were prepared by w/o/w emulsification-solvent evaporation method. The physicochemical characteristics of the nanoparticles (i.e. encapsulation efficiency, particle size distribution, morphology, in vitro release) were studied. The cellular uptake of the nanoparticles for the two human tumor cell lines: human breast carcinoma cells (MCF-7) and human carcinoma cervices cells Cheng Jin and Ling Bai contributed equally to this work. C. Jin G. Guo (&) Department of Radiation Medicine, Fourth Military Medical University, Xi’an 710032, China e-mail:
[email protected] L. Bai Department of Clinical Laboratories, Xi’an Gaoxin Hospital, Xi’an 710075, China H. Wu Department of Pharmacy, Fourth Military Medical University, Xi’an 710032, China Z. Teng Department of Pharmacology, Fourth Military Medical University, Xi’an 710032, China J. Chen Department of Occupational and Environmental Health, Fourth Military Medical University, Xi’an 710032, China e-mail:
[email protected]
(HeLa), was evaluated by fluorescence microscopy and transmission electronic microscopy. Cell viability was measured by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical in shape with size between 90 nm and 190 nm. The encapsulation efficiency was 20.06%. The drug release pattern exhibited an initial burst followed by a plateau for over 24 h. The cellular uptake of nanoparticles was observed. Co-culture of MCF-7 and HeLa cells with SR-2508 loaded nanoparticles showed that released SR-2508 retained its bioactivity and effectively sensitized two hypoxic tumor cell lines to radiation. The radiosensitization of SR-2508 loaded nanoparticles was more significant than that of free drug. Keywords SR-2508 Drug delivery Nanoparticle Radiation Hypoxia Human tumor cells Radiotherapy Nanomedicine
Introduction Poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles have been prepared as a biodegradable delivery system by many researchers (Panyam and Labhasetwar 2003; Bala et al. 2004; Avgoustakis 2004; Panyam et al. 2004; Astete and Sabliov 2006; Costantino et al. 2006; Yin et al. 2006). The advantages of such a formulation include the sustained drug
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action on the lesion, reduced systemic side effects, facilitated extravasation into the tumor, high capability to cross various physiological barriers as well as controlled and targeted delivery of the drug (Brigger et al. 2002; Yuan et al. 1994; Desai et al. 1997). The inability of radiotherapy to eradicate completely certain human tumors may be due to the presence of resistant hypoxic cells (Gray et al. 1953; Howes 1969; Bush et al. 1978; Brown 1979). Oxygen-mimetic or electro-affinic compounds such as misonidazole and other nitroimidazoles were developed specifically to sensitize hypoxic cells and thus improve the efficacy of radiation therapy in controlling human tumors (Adams 1981; Brown 1982a, b; Dische 1985; Adams and Stratford 1986), but doselimiting neurotoxicity renders misonidazole ineffective for clinical use (Dische 1985). The secondgeneration sensitizer SR-2508 (etanidazole) has the advantage of the lower toxicity, gram-level doses to be administered to patients to attain tumor concentrations reasonable for radiosensitization (Coleman et al. 1986). The efficacy of the SR-2508 is currently being determined under Phase II and Phase III clinical trials in a few countries (Lee et al. 1995; Lawton et al. 1996; Eschwege et al. 1997; Riese et al. 1997; Urtasun et al. 1998). However, SR-2508 has a very short half-life, e.g. 2 ± 3 h in a dog’s body, and can be eliminated rapidly by kidney without significant changes in the urine (O’Dwyer and LaCreta 1991). Therefore, a much higher dosage is usually required to achieve the desired administration efficiency, resulting in peripheral neuropathy (Coleman et al. 1986). This is one possible reason why some clinical trials for SR-2508 did not exhibit benefits for the overall improvement of survival rate for cancer patients (Lee et al. 1995; Eschwege et al. 1997). The systemic administration used in these clinical trials may also be responsible for the failure to achieve the desired treatment efficacy. This may be due to the restriction of dose used and the fact that drug cannot reach the target tumor in sufficient quantity. Although SR-2508 has been reported to be successfully encapsulated in the PLGA, PDLA and PLLA microspheres as a radiosensitizer by the spraydrying technique and the solvent extraction/evaporation method (Wang and Wang 2002a, b, 2003; Lee et al. 2002), the formulation proposed in this paper may have advantages over the previous work in
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resulting in smaller size, better morphology, desired in vitro drug release kinetics. This study was carried out to evaluate the physicochemical characteristics and cellular uptake of SR2508 loaded nanoparticles and determine the ability of the released SR-2508 to radiosensitize two different tumor cell lines: human breast carcinoma cells (MCF-7) and human carcinoma cervicis cells (HeLa).
Materials and methods Materials SR-2508 (etanidazole), 1,6-Diphenyl-1,3,5-hexatriene (DPH), Propidium iodide (PI) and Polyvinyl acohol (PVA, Mw = 30,000–70,000 Da) were purchased from Sigma-Aldrich (St. Louis, USA). Poly (D,L-lactide-co-glycolide) (PLGA, L/G = 50/50; Mw = 25,000 Da) was from Chengdu Institute of Organic Chemistry, Chinese Academy of Science (China). Dichloromethane (DCM) was purchased from Tianjin Chemical Factory (China). DMEM and fetal bovine serum (FBS) were from Gibco, Life Technologies (USA). The other chemicals were of analytical grade. MCF-7 and HeLa cells were cultured as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C, and subcultured twice weekly. For experiments, cells were grown in glass culture flasks and used when in exponential growth phase. To obtain hypoxia, the incubator was flushed with 95%N2 and 5%CO2 for 24 h.
Methods Preparation of SR-2508 loaded nanoparticles The preparation of SR-2508 loaded nanoparticles was based on w/o/w emulsification-solvent evaporation method. 0.2 mL of SR-2508 aqueous solution (25 mg/mL) was first emulsified by magnetic and vigorous stirring into a solution of PLGA (50 mg) in DCM (2 mL) with emolsifiers 10 lL of span and 10 lL of Tween 80. This first emulsion (w/o) was poured into 10 mL of pH-3 purified water (1% w/v
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PVA). The pH-3 purified water was made by adding hydrochloric acid and measured by pH paper. A w/o/ w emulsion was formed by extensive stirring and sonicated for 3 min. The resulting emulsion was then placed on the magnetic stirrer plate and continuously stirred at room temperature to evaporate DCM for 6 h. The nanoparticles were collected by centrifugation and washed thrice with purified water. The nanoparticles were then lyophilized and stored at 4 °C before further analysis. Preparation of fluorescent nanoparticles Fluorescent nanoparticles were prepared adding 10 mg of DPH (0.25% (w/v) calculated on the whole microemulsion) as fluorescent marker to the internal phase of DCM containing 2.5% (w/v) of PLGA and maintaining the other components of the formulations fixed. The fluorescent nanoparticles were obtained as described above for drug-loaded nanoparticles.
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same extraction procedure as described above was done. The resulting factor was 100%, which means that about 100% of the original amount of the SR2508 could be detected. The percentage yield was calculated based on the amount of lyophilized nanoparticles of each formulation obtained to the amount of solid material used in the dispersed phase. Loading efficiency and encapsulation efficiency were calculated as following: Loading efficiencyð%Þ ¼ ðamount of drug in nanoparticles =amount of drug loaded nanoparticlesÞ 100 Encapsulation efficiencyð%Þ ¼ ðamount of drug in nanoparticles =initial amount of drugÞ 100
Morphology and particle size distribution of the nanoparticles
Determination of drug content in the nanoparticles About 5 mg of SR-2508 loaded nanoparticles were dissolved in 1 mL of DCM and 10 mL of phosphate buffer saline (PBS) medium (pH 7.4) was then added and extracted. A nitrogen stream was introduced to evaporate the DCM and clarified by centrifugation at 4,000 rpm for 15 min. PBS solution containing the extracted drug was determined using high performance liquid chromatography (HPLC) system. The experiment was repeated thrice. The HPLC assay (Agilent 1100 series) for SR-2508 was performed on a reverse phase Zorbax1 C18 column. The mobile phase was a mixture of acetonitrile: water (5:95, (v/v)) delivered at a flow rate of 1.0 mL/min. SR-2508 was detected at 324 nm with a variable wavelength detector (VWD). The calibration curve for the quantification for SR-2508 was linear over the range of standard concentration between 50 and 100,000 ng/ mL with a correlation coefficient of R2 = 1.0. The recovery efficiency factor of the extraction procedure on encapsulation efficiency was determined according to the following method. A certain weight of pure SR2508 which was similar to the amount loaded in a certain amount of nanoparticles and 3.0–5.0 mg of placebo nanoparticles or polymer were dissolved in 1 mL of DCM. About 5 mL of PBS was added. The
Nanoparticle morphology was examined by scanning electron microscope (SEM) (JSM-6700F, JEOL, Japan). Nanoparticles were analysed for their size distribution using laser diffraction in a particle size analyzer (Zetasizer Nano S, Malvern Instruments, UK). In vitro release The release rate of SR-2508 from nanoparticles was measured in PBS by an HPLC assay in triplicate. SR2508 loaded nanoparticles were suspended in 10 mL of PBS in screw capped tubes and placed in an orbital shaker maintained at 37 °C and shaken at 120 rpm. At predetermined time intervals, the tubes were taken out of the shaker and centrifuged at 3,000 rpm for 5 min. The supernatant was taken for analysis. The precipitated nanoparticles were resuspended in 10 mL of fresh buffer and placed back in the shaker. The drug content in PBS was analyzed via HPLC as previously described. Transmission electronic microscopy MCF-7 and HeLa cells were plated at 1 9 105 cells per well in six-well tissue culture plates for 24 h. The
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cells were incubated with SR-2508 loaded nanoparticles for 24 h. The cells were washed twice with PBS and harvested by trypsinization. The cells were centrifuged at 1,000 rpm for 10 min. At this point, special care was taken when removing the sediment cells. The pellets of cells were fixed by 3% glutaraldehyde solution. Fixed cells were washed with PBS and dehydrated thrice sequentially in a graded series of ethanol solutions (50, 70, 90, 95 and 100%). The cells were then soaked overnight in a 1:1 ratio of 100% alcohol and embedding resin. The resinembedded cells were placed in capsules and the capsules were placed in a Pelco UV-2 Cryo Chamber at 4 °C for 48 h for polymerization of the resin by UV radiation. The polymerized blocks were sectioned and the ultrathin sections were prepared. The cellular uptake of nanoparticles was evaluated by transmission electronic microscope (TEM) (JEM2000EX, JEOL, Japan).
Fluorescence microscopy The cellular uptake of nanoparticles was further studied using fluorescence microscopy. MCF-7 and HeLa cells were grown on coverslips for 24 h in a six-well tissue culture plate at 37 °C. The cells were then incubated for 24 h with the fluorescent nanoparticles at a concentration of 2 mg/mL employed in the experiment. After rinsing with PBS, the cells were fixed by 95% ethanol solution for 30 min. The nuclei of the cells were then stained using 5 lg/mL of PI for 8 min at 37 °C. The stained coverslips were mounted on a glass slide and photographed using fluorescence microscope (TE2000-S, Nikon, Japan). DPH and PI show blue and orange, respectively.
Cell survival assay Cell viability was measured by the ability of single cell to form colonies in vitro. The cells were plated at 500 cells per well in six-well tissue culture plates and allowed to adhere for 24 h. Then cells were incubated in hypoxic condition with non-drug nanoparticles (control), SR-2508 loaded nanoparticles (20.86 lg/ mL SR-2508 released at 24 h) for 24 h and 21.4 lg/ mL free SR-2508 for 80 min. Whereafter, radiation was delivered at room temperature utilizing 60Co
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source. The radiation was delivered in a single fraction at 0, 225.77, 452.30, 668.78, 893.97, 1126.86 cGy/min, respectively, for duration of 53 s to produce doses of 0, 2, 4, 6, 8, 10 Gy. Following radiation, cells were allowed to grow in a 37 °C incubator under standard culture conditions for 10– 14 days. After this time interval, macroscopic colonies were stained with Giemsa and were counted manually. The experiment was repeated thrice. All the data are presented as means plus or minus SD. Significance of differences between the groups were determined with the one-way ANOVA (SPSS10.0 statistical software), with the level of significance set at p \ 0.05.
Results and discussion Characterization of nanoparticle delivery system These studies examined encapsulation efficiency (EE), morphology, particle size distribution and release kinetics of SR-2508 loaded PLGA nanoparticles. Drug EE is a factor to be considered, especially for such a drug as SR-2508. In the present work, HPLC analysis indicated that the loading efficiency of 1.86% and the encapsulation efficiency of 20.06% were lower, because SR-2508 was a highly hydrophilic drug with a relatively low molecular weight (214.2 Da) and easy to migrate to aqueous phase in preparation. Particle size plays an important role in determining the drug release behavior of the SR-2508 loaded nanoparticles as well as their fate after administration. It was reported that smaller particles tended to accumulate in the tumor sites due to the facilitated extravasation (Yuan et al. 1994) and a greater internalization was also observed (Desai et al. 1997). Less than 200 nm particles can prevent spleen filtering (Moghimi et al. 1991). In addition, smaller particles make intravenous injection easier and their sterilization may be simply done by filtration (Konan et al. 2002; Kwon and Kataoka 1995). During the study, morphology of the nanoparticles was studied through scanning electron microscopy. As seen from the electron micrographs in Fig. 1, the nanoparticles were smooth and spherical in shape. The size distribution illustrated in Fig. 2 was narrow with a diameter between 90 nm and 190 nm, about 120 nm on average.
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Fig. 1 Scanning electron microscopic photographs of SR-2508 loaded nanoparticles
Fig. 2 Particle nanoparticles
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In the release study, SR-2508 release from the nanoparticles was better controlled. The result was shown in Fig. 3. The kinetic data showed that majority of the drug release from the biodegradable polymeric delivery system occurred in the first day (approximately 90%) and the release amount almost remained unchanged after 24 h. A release phenomenon termed the burst effect, where drug is released in the very early stages of the study, was found to be very large (about 50% in 3 h) in this SR-2508 loaded nanoparticle delivery system. This was probably due to the hydrophilic nature of the drug. When nanoparticles are prepared by the w/o/w method, watersoluble drugs show significant tendency to migrate to
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Fig. 3 The release profile of SR-2508 loaded nanoparticles in vitro
the aqueous dissolution medium, thereby concentrating at the surface of the nanoparticles and enhancing the burst effect. The release data showed that SR2508 was released proportionately from the nanoparticles during initial 12 h. The kinetic data showed that the degree and the rate of SR-2508 release could be controlled by time. This ability is vital in the design of a degradable nanoparticle delivery system. The advantage of the nanoparticle system is that certain parameters can be altered to control the release of drug.
Evaluation of SR-2508 bioactivity and radiation response The cellular uptake of SR-2508 loaded nanoparticles was evidenced by TEM. Figure 4 showed the internalization of the nanoparticles in MCF-7 and HeLa cells following 24 h treatment. Further, Fig. 5 demonstrated that the tumor cells swallowed the drugloaded nanoparticles. In order to view and locate the cells, the nuclei of cells were stained with PI for fluorescence microscopy. SR-2508 was gradually released from the nanoparticles to produce directly a therapeutical effect in the cells. The result of the bioactivity/radiation study is shown in Figs. 6, 7. Plating efficiency was 80 and 51% for MCF-7 and HeLa cells, respectively. In the radiation study, to compare the radiosensitizing effects, hypoxic MCF-7 and HeLa cells cultured with non-drug nanoparticles, SR-2508 loaded nanoparticles and free SR-2508 were exposed to varying doses of radiation. Clearly then, treatment with SR2508 loaded nanoparticles and free SR-2508 resulted in enhancement in the fraction of cell of clonogenic incompetent for two hypoxic cell lines (p \ 0.05).
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Fig. 4 SR-2508 loaded nanoparticles swallowed in MCF-7 and HeLa cells (a and b), respectively
Fig. 5 Cellular uptake 24 h after incubation with fluorescent nanoparticles. (a) MCF-7 cells, (b) HeLa cells
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Fig. 6 Cell surviving fraction versus absorbed dose of 60Co gamma rays for hypoxic MCF-7 cells with non-drug nanoparticles (control), SR-2508 loaded nanoparticles and free SR-2508
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Fig. 7 Cell surviving fraction versus absorbed dose of 60Co gamma rays for hypoxic HeLa cells with non-drug nanoparticles (control), SR-2508 loaded nanoparticles and free SR-2508
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This data indicated that SR-2508 was successfully released from the nanoparticle system as a radiosensitizer. Due to the cytotoxicity of SR-2508, the colony counts revealed that the colony numbers droped with the addition of SR-2508 loaded nanoparticles and free SR-2508 with no radiation. The radiosensitization of SR-2508 loaded nanoparticles was more significant than that of free SR-2508 (p \ 0.05), because SR-2508 was gradually released from nanoparticles and had persistent therapeutic effect on the tumor cells. On the another hand, the higher sensitivity of the cells to the drug-loaded nanoparticles than to the drugs in solution may be related to the marked uptake and accumulation of nanoparticles in the cells, where the drug-loaded nanoparticles should release the drugs, so enhancing their action. It is clear that the therapeutic effects of the drug-loaded nanoparticles would depend on internalization and sustained retention of nanoparticles by the diseased cells. This plays an important role in the cancer therapy. However, there is a possibility that most drug-loaded nanoparticles may be trapped by the reticuloendothelial system in body. In order to overcome the problem, the further study on the drug-loaded nanoparticles specially targeting the tumor cells is necessary. The advantage of a nanoparticle-based carrier system is that localized doses of drug will be delivered to tumor sites to radiosensitize hypoxic tumor cells. This kind of a localized delivery system may eliminate the toxic side effects that are accompanied with systemic administration of SR-2508. Further, this method may also prolong the radiosensitizing effect of SR-2508 and avoid the disadvantage of its short half-life. Based on these findings, studies in vivo with SR-2508 loaded nanoparticles, including tumor radiosensitization evaluation, should be encouraged.
Conclusions The results have demonstrated the release of bioactive SR-2508 from a degradable PLGA nanoparticle delivery system. SR-2508 was released in a controlled manner as demonstrated by HPLC data. The cellular uptake of SR-2508 loaded nanoparticles was observed. The presence of SR-2508 and radiation was found to significantly lower colony counts of hypoxic
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tumor cells compared to radiation alone. The radiosensitization of SR-2508 loaded nanoparticles was more significant than that of free SR-2508. Based on these results, this nanoparticle system shows promise as a carrier for SR-2508 and shows potential for the future in vivo development of a radiosensitizer delivery system. Acknowledgement The authors would like to thank Dr. JY Liu (Department of Radiation Medicine, Fourth Military Medical University) for technical assistance.
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