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Cellulose-based drug carriers for cancer therapy: Cytotoxic evaluation in cancer and healthy cells
Materials Letters 132 (2014) 432–435
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Cellulose-based drug carriers for cancer therapy: Cytotoxic evaluation in cancer and healthy cells Aikaterini-Foteini Metaxa, Eleni K. Efthimiadou n, George Kordas n Sol–Gel Laboratory, Institute of Nanoscience & Nanotechnology, 15310 Aghia Paraskevi, Attikis, Greece
art ic l e i nf o
a b s t r a c t
Article history: Received 19 February 2014 Accepted 21 June 2014 Available online 30 June 2014
Conventional chemotherapy drugs show lack of specificity, inducing reduced activity on the cancer treatment. They exhibit high toxicity and after a prolonged period of administration the cancer cells develop resistance (multiple-drug resistance, MDR). This situation leads to increasing the side effects of drugs and affect the quality of the patients' life. In order to overcome the above mentioned problems, the recent research community is focused primarily on developing smart drug delivery systems which respond in different stimulus, in the range of nanometer [1–3]. The objective of this work is the biological evaluation of synthesized double- layer microspheres based on cellulose coating, for delivery of chemotherapeutic drugs with low water-solubility. In a previous published work, we have designed double-layer microspheres, based on the co-polymerization of widelyused sensitive polymers (Methyl acrylic acid and N-isopropylacrylamide) and modified cellose's coating. Briefly, we have synthesized and fully characterized Poly(methyl acrylic acid-co-N-isopropylacrylamideco-ethyleneglycol dimethacrylate)@cellulose succinate (P(MAA-co-NIPAAM-co-EGDMA)@CS) microspheres by a combination of sol–gel method, distillation precipitation co-polymerization and chemical deposition. The resulting spheres were characterized structural through spectroscopy as well as morphological. The anticancer agent daunorubicin was encapsulated in the spheres, and drug's release behavior was evaluated at acidic and slightly basic pH conditions. The highlight of the previous evaluation is the microspheres' response at acidic pH environment for targeted release of the drug at the tumor affected area in contrary to neutral pH. This fact was a sign that our system may be a potential drug delivery vehicle for cancer treatment, as it is well known that tumor's environment is acidic. For that purpose we evaluated the cytotoxic effects of spheres, spheres loaded with daunorubicin and free daunorubicin via MTT assay in MCF-7 (breast cancer) and HeLa cells (cervical cancer) as a function of concentration and in different time intervals. Finally, HEK 293 (Human Embryonic Kidney) cells were incubated with unloaded microspheres in order to evaluate the cytotoxic activity in healthy cells. & 2014 Elsevier B.V. All rights reserved.
Keywords: Polymeric microspheres Drug delivery Cytotoxicity Cellulose HeLa cells MCF-7 MTT assay
1. Introduction Traditional chemotherapeutic agents present poor specificity against cancer cells and high toxicity in healthy cells.[1–3] Aiming at decreasing the toxicity and increasing the therapeutic efficacy of the drug agents, scientific research focus on nanoscale drug delivery systems. These drug-vehicles allow the uptake of drugs into the body, improving in that way the tumor efficacy by controlling the way and the rhythm of the releasing process at the pathogenic area.[4]
n
Corresponding author. Tel.: þ 30 210 6503301. E-mail addresses: [email protected] (E.K. Efthimiadou), [email protected] (G. Kordas). http://dx.doi.org/10.1016/j.matlet.2014.06.134 0167-577X/& 2014 Elsevier B.V. All rights reserved.
According to literature drug nano-vehicles are mainly fabricated by water-soluble polymers (such as poly(ethyleneglycol) (PEG), poly(ethylene oxide) (PEO), dextran, poly(acrylic acid) (PAA), biodegradable aliphatic polyesters or polyacrylamides. [5] Although, polysaccharides such as cellulose, are promising raw materials for the synthesis of drug-carriers, nevertheless the potential of these drug carriers is still under-represented and deserves more attention.[6,7] Scheme 1 Polysaccharides are natural biopolymers with a plenty of advantages, based on that they are broadly used as basic components in drug delivery systems. They are non-toxic, biocompatible and have a low cost of production.[8] Nanoparticles made of polysaccharides could prolong the resistance time inside the body circulation and increase the absorption of encapsulated drugs. Cellulose as a raw material for drug delivery-vehicle has
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
433
Scheme 1. Graphical abstract.
significant advantages as biocompatibility, antimicrobial activity, can be easily chemically modified to increase the solubility and others. Although that the last years there is an increase of the publications about delivery systems based on polysaccharidesespecially chitosan [9,10] there are less more on cellulose-based drug delivery systems and the most studies have not focused on specific drug formulations for specific diseases and in-vitro/in-vivo applications.[11] In previous paper we have synthesized double-layered P(MAAco-NIPAAM-co-EGDMA)@CS microspheres. Using as sacrificed templates silica nanospheres via Stober method and modified with 3methacryloxypropyltrimethoxysilane (MPS), in order to initiate a distillation precipitation copolymerization of methyl acrylic acid (MAA), N-isopropylacrylamide (NIPAAM) and ethyleneglycol dimethacrylate (EGDMA) as a cross-linker, to create the first shell. The second shell was created via chemical deposition of cellulose succinate and cellulose powder onto microspheres' surface. Cellulose succinate was synthesized with a neat reaction of cellulose powder and succinic anhydride Finally, the two shells were cross linked by esteric bonds with carbodiimide chemistry coupling and the core was removed by buffer solution NH4Cl/NH3. The anticancer agent daunorubicin was loaded in the spheres, and its release behavior was evaluated at acidic and slightly basic pH conditions, in order to evaluate the behavior at the healthy (slightly basic environment) and pathogenic tissues (acidic environment). In this work we evaluate the cytotoxic activity of the fabricated spheres with/without the loaded drug via MTT assay, an established colorimetric method, in MCF-7 cells (breast cancer) and HeLa cells (cervical cancer). We also investigated the toxicity of unloaded spheres in healthy cells HEK 293 (Human Embryonic Kidney).
HCl (DNR) was provided by Pharmacia & Upjohn and used as received. Fetal Bovine Serum (FBS) was purchased from Antisel. High glucose Dulbecco's modified Eagle Medium (DMEM) and MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma. Trypsin-EDTA, L-glutamine, penicillin–streptomycin solution and heat inactivated fetal bovine serum (FBS) were obtained from Biochrom KG, Berlin, Germany.
3. Characterization Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were obtained on an FEI Inspect microscope with W (Tungsten) filament operating at 25 kV and a FEI CM20 microscope operating at 200 kV, respectively. the spectra were scanned over the range 4000–500 cm 1. NMR spectra were recorded with a Bruker Avance 500 MHz instrument and were processed by X-WIN NMR 2.6 (Bruker Analytik GmbH). In the data presented in this study, each measurement represents the average value of 10 measurements, with 20 s integration time for each measurement. UV–vis absorption spectra in the wavelength range of 200–800 nm were obtained on a Jusco V-650 spectrometer, UV–vis at 480 nm spectrometer. An ultrasonic bath was used for sonication (Elma Sonic, S. 30H). 3.1. Morphological characterization The morphological characterization of the synthesized P(MAAco-NIPAAM-co-EGDMA)@CS microspheres has been carried out by SEM and TEM microscopy (Fig. 1A and B). According to SEM results it is observed the characteristic raspberry-like surface and through TEM microscopy it is confirmed the shell and cavity formation.
2. Materials and methods
4. Loading and release study of daunorubicin
SiO2@P(MAA-co-NIPAAM-co-EGDMA)@CS spheres were synthesized using published methods.[12] Cellulose powder, MW ¼78.000–98.000 g/mol, was purchased from Riedel-de Haen Ag Seelze-Hannover. Succinic anhydride was purchased from Sigma-Aldrich and was purified with recrystalization 2,2-azobis (2-methylpropionitrile) (AIBN), N-hydroxysuccinimide (NHS), 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 3-methacryloxypropyltrimethoxysilane (MPS) were purchased from Acros Organics and used as received. Ethyleneglycol dimethacrylate (EGDMA) was purchased from Sigma Aldrich. Tetraethyl orthosilicate (TEOS) was purchased from Fluka. Methacrylic acid (MAA) was purified by distillation before using and acetonitrile was used as received from Aldrich. Daunorubicin
8 mg of daunorubicin hydrochloride (DNR) were dissolved in 4 ml of PBS (phosphate-buffered saline, pH ¼ 7.4) and then 4.0 mg of hollow microspheres were added. The mixture was gently stirred for 72 h at room temperature. The resulting suspension was centrifuged (12.000 rpm for 5 min) and the unloaded DNR was removed. The loading amount of DNR was determined by using ultra violet spectroscopy (UV) according to a standard curve. The calculation of DNR was carried out by the difference of DNR concentration between the starting amount of DNR and the supernatant solution after loading. Standard curve was prepared by different concentrations of DNR in a PBS solution and in acidic solution. The drug release has taken place in acidic (pH ¼ 4.0, 100 mM, Acetic Acid glacial, Sodium Acetate Trihydrate) and in
434
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
Fig. 1. (A) SEM and (B) TEM images of hollow P(MAA-co-NIPAAM-co-EGDMA)@CS drug carriers.[12].
Fig. 2. Cell viability of HeLa and MCF-7 cells after incubation with P(MAA-co-NIPAAM-co-EGDMA)@CS carriers (blue), P(MAA-co-NIPAAM-co-EGDMA)@CS carriers loaded with daunorubicin (purple) and pure daunorubicin (pink).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
slightly basic (pH ¼ 7.4, 100 mM, KH2PO4, Na2HPO4 dihydrate, NaCl, KCl). The concentration of DNR, which was released from hollow microspheres in different buffer solutions, was quantified by using UV–vis spectroscopy via the standard curve method[12].
5. in vitro cytotoxicity – MTT assay In order to evaluate the therapeutic ability of P(MAA-co-NIPAAMco-EGDMA)@CS spheres as potential drug vehicles, we investigated the in-vitro cytotoxicity against MCF-7 (breast cancer) and HeLa (cervical cancer ) cells. Furthermore, we assessed the toxicity of carriers in healthy cells (HEK 293, Human Embryonic Kidney healthy cells) aiming at determining the induced toxicity in normal cells.. The cells were cultivated in DMEM (Dulbeccos's Modified Eagle Medium) with extra L-glutamine (2 mM), penicillin/streptomycin (1000/ 0.1 mg/ml), and 10% v/v fetal bovine serum at 37 1C, in a 5% CO2 atmosphere as exponentially growing monolayers. For the determination of cell viability we used MTT assay– an established colorimetric method. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) is a water-soluble yellow tetrazole that can be reduced to water-insoluble purple formazan crystals in living cells by cellular reductase. The amount of formazan is directly proportional to the number of living cells. The formazan crystals can be diluted in DMSO to perform a colored solution and the absorbance of this solution can be
Table 1 IC50 values.
DNR Spheres P(MAA-co-NIPAAM-co-EGDMA)@CS with DNR
MCF-7 IC50 (μM)
HeLa IC50 (μM)
0.87 1.22
0.31 0.81
measured by a spectrometer at 540/620 nm. For experiments, cells were plated in 96-well plates and in each well, they were added 10.000 cells diluted in 100 μl DMEM. For the determination of cells' number we used a hemocytometer. After an incubation period of 24 h, we prepared solutions of material, material with DNR and pure DNR in various concentrations (10, 5, 1, 0.5, 0.1, 0.05 and 0.01 μM of DNR) and they were added in wells (100 μl per well) in triplicates. After 24, 48 and 72 h of incubation at 37 1C, 5% CO2/95% air humidified atmosphere, the medium was removed and cells were purified with PBS and 100 μl of MTT solution in PBS (1 mg/ml) were added. After 4 h of incubation at 37 1C, the MTT solution was removed and 100 μl DMSO were added to dissolve formazan's crystals. Finally, drug effect was quantified as the percentage of control absorbance of reduced dye at λmax ¼490 nm. The absorbance was measured by a micro plate reader Sirio S. Seac Radim.
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
435
Fig. 3. Cell viability of HeLa and MCF-7 cells after incubation with P(MAA-co-NIPAAM-co-EGDMA)@CS carriers loaded with daunorubicin at different time intervals of incubation ([24 h (pink), 48 h (purple), 72 h (blue)])(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
6. Conclusions
Fig. 4. Toxicity in healthy embryonic cells.
Polymeric microspheres coated with polysaccharides hold a predominant location in drug delivery systems. Based on that we fabricate smart pH and Thermo sensitive polymeric microspheres P(MAA-co-NIPAAM-co-EGDMA) coated with modified cellulose. Due to their biocompatible character we evaluated their cytotoxicity behavior. As it is concluded by the in vitro results the loaded microspheres present the desired behavior as acidic and thermo -sensitive release system with equal cytotoxicity comparing to free DNR. Evaluating the emerged data,the most significant result which is concerned is the induced cytotoxicity in two cancer cell lines, MCF-7 and HeLa in contrary to its in healthy cells after treatment with unloaded microspheres. Therefore, it is of interest to explore the potential use of cellulose-based microspheres as non-toxic carriers of antitumor drugs. Further investigation into the mechanisms of cytotoxic and antitumor activity of cellulose – based drug devices, in vitro and in vivo would be useful in the treatment of various classes of cancer in future.
The percentage of livings cells was calculated using the formula below: Cell viability ð%Þ ¼ As=Acx100%; As ¼ Absorbancevalueofsample; Ac ¼ Absorbancevalueofcontrol The above experiment was repeated three times and the final results are resumed in Fig. 2A and B. From the sigmoidal graphs it is calculated the half maximal inhibitory concentration (IC50) for spheres loaded with DNR and free DNR. The results are presented in Table 1. As shown in Fig. 2 the empty carriers P(MAA-co-NIPAAM-co-EGDMA)@CS do not show cytotoxicity on the MCF-7 and HeLa cells even at the higher concentrations tested. On the other hand the carriers loaded with drug, shows comparable growth inhibition with pure daunorubicin. Specifically, the DNR-loaded cellulose containers' IC50 values were determined to be 0.87 μM on MCF-7 and 0.31 μM on HeLa cells when DNRs' IC50 values were 1.22 and 0.81 respectively. The sustained release behavior of the drug carriers is supported by Fig. 3, by which the cytotoxic activity is depended on incubation time period. Finally, the MTT assay also used in Embryonic Kidney cells as previously described. After 24 h incubation by different concentrations of polymeric materials (32, 17, 3, 1.7, 0.03 and 0.001 μg/ml) the results are depicted in Fig. 4. As it is observed in above figure, spheres are non-toxic even in high concentration (32 μg/ml).
Acknowledgments We thank the European Research Council (ERC) for financial support of this work under the “IDEAS” project called “A Novel Micro-container drug carrier for targeted treatment of prostate cancer” with the acronym NANOTHERAPY and the reference number 232959.
References [1] Arruebo M, et al. Nano Today 2007;2(3):22. [2] Cho K, et al. Clin Cancer Res: Off Journal Am Assoc Cancer Res 2008;14 (5):1310. [3] Sunderland CJ, et al. Drug Dev Res 2006;67(1):70. [4] Drug Delivery Systems, Methods in Molecular Biology, 437, Human press, Springer; 2008. [5] Cheng R, et al. Biomaterials 2013;34(14):3647. [6] Alvarez-Lorenzo C, et al. Adv Drug Deliv Rev 2013;65(9):1148. [7] Zhang Y, et al. Adv Drug Deliv Rev 2013;65(1):104. [8] Doshi N, Mitragotri S. Adv Funct Mater 2009;19(24):3843. [9] Uchegbu IF, et al. Polym Int 2014 (n/a). [10] Wang JJ, et al. Int J Nanomed 2011;6:765. [11] Plackett. Nordic Pulp Pap Res J 2014;29(01):105. [12] Metaxa AF, et al. J Colloid Interface Sci 2012;384(1):198.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Cellulose-based drug carriers for cancer therapy: Cytotoxic evaluation in cancer and healthy cells Aikaterini-Foteini Metaxa, Eleni K. Efthimiadou n, George Kordas n Sol–Gel Laboratory, Institute of Nanoscience & Nanotechnology, 15310 Aghia Paraskevi, Attikis, Greece
art ic l e i nf o
a b s t r a c t
Article history: Received 19 February 2014 Accepted 21 June 2014 Available online 30 June 2014
Conventional chemotherapy drugs show lack of specificity, inducing reduced activity on the cancer treatment. They exhibit high toxicity and after a prolonged period of administration the cancer cells develop resistance (multiple-drug resistance, MDR). This situation leads to increasing the side effects of drugs and affect the quality of the patients' life. In order to overcome the above mentioned problems, the recent research community is focused primarily on developing smart drug delivery systems which respond in different stimulus, in the range of nanometer [1–3]. The objective of this work is the biological evaluation of synthesized double- layer microspheres based on cellulose coating, for delivery of chemotherapeutic drugs with low water-solubility. In a previous published work, we have designed double-layer microspheres, based on the co-polymerization of widelyused sensitive polymers (Methyl acrylic acid and N-isopropylacrylamide) and modified cellose's coating. Briefly, we have synthesized and fully characterized Poly(methyl acrylic acid-co-N-isopropylacrylamideco-ethyleneglycol dimethacrylate)@cellulose succinate (P(MAA-co-NIPAAM-co-EGDMA)@CS) microspheres by a combination of sol–gel method, distillation precipitation co-polymerization and chemical deposition. The resulting spheres were characterized structural through spectroscopy as well as morphological. The anticancer agent daunorubicin was encapsulated in the spheres, and drug's release behavior was evaluated at acidic and slightly basic pH conditions. The highlight of the previous evaluation is the microspheres' response at acidic pH environment for targeted release of the drug at the tumor affected area in contrary to neutral pH. This fact was a sign that our system may be a potential drug delivery vehicle for cancer treatment, as it is well known that tumor's environment is acidic. For that purpose we evaluated the cytotoxic effects of spheres, spheres loaded with daunorubicin and free daunorubicin via MTT assay in MCF-7 (breast cancer) and HeLa cells (cervical cancer) as a function of concentration and in different time intervals. Finally, HEK 293 (Human Embryonic Kidney) cells were incubated with unloaded microspheres in order to evaluate the cytotoxic activity in healthy cells. & 2014 Elsevier B.V. All rights reserved.
Keywords: Polymeric microspheres Drug delivery Cytotoxicity Cellulose HeLa cells MCF-7 MTT assay
1. Introduction Traditional chemotherapeutic agents present poor specificity against cancer cells and high toxicity in healthy cells.[1–3] Aiming at decreasing the toxicity and increasing the therapeutic efficacy of the drug agents, scientific research focus on nanoscale drug delivery systems. These drug-vehicles allow the uptake of drugs into the body, improving in that way the tumor efficacy by controlling the way and the rhythm of the releasing process at the pathogenic area.[4]
n
Corresponding author. Tel.: þ 30 210 6503301. E-mail addresses: [email protected] (E.K. Efthimiadou), [email protected] (G. Kordas). http://dx.doi.org/10.1016/j.matlet.2014.06.134 0167-577X/& 2014 Elsevier B.V. All rights reserved.
According to literature drug nano-vehicles are mainly fabricated by water-soluble polymers (such as poly(ethyleneglycol) (PEG), poly(ethylene oxide) (PEO), dextran, poly(acrylic acid) (PAA), biodegradable aliphatic polyesters or polyacrylamides. [5] Although, polysaccharides such as cellulose, are promising raw materials for the synthesis of drug-carriers, nevertheless the potential of these drug carriers is still under-represented and deserves more attention.[6,7] Scheme 1 Polysaccharides are natural biopolymers with a plenty of advantages, based on that they are broadly used as basic components in drug delivery systems. They are non-toxic, biocompatible and have a low cost of production.[8] Nanoparticles made of polysaccharides could prolong the resistance time inside the body circulation and increase the absorption of encapsulated drugs. Cellulose as a raw material for drug delivery-vehicle has
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
433
Scheme 1. Graphical abstract.
significant advantages as biocompatibility, antimicrobial activity, can be easily chemically modified to increase the solubility and others. Although that the last years there is an increase of the publications about delivery systems based on polysaccharidesespecially chitosan [9,10] there are less more on cellulose-based drug delivery systems and the most studies have not focused on specific drug formulations for specific diseases and in-vitro/in-vivo applications.[11] In previous paper we have synthesized double-layered P(MAAco-NIPAAM-co-EGDMA)@CS microspheres. Using as sacrificed templates silica nanospheres via Stober method and modified with 3methacryloxypropyltrimethoxysilane (MPS), in order to initiate a distillation precipitation copolymerization of methyl acrylic acid (MAA), N-isopropylacrylamide (NIPAAM) and ethyleneglycol dimethacrylate (EGDMA) as a cross-linker, to create the first shell. The second shell was created via chemical deposition of cellulose succinate and cellulose powder onto microspheres' surface. Cellulose succinate was synthesized with a neat reaction of cellulose powder and succinic anhydride Finally, the two shells were cross linked by esteric bonds with carbodiimide chemistry coupling and the core was removed by buffer solution NH4Cl/NH3. The anticancer agent daunorubicin was loaded in the spheres, and its release behavior was evaluated at acidic and slightly basic pH conditions, in order to evaluate the behavior at the healthy (slightly basic environment) and pathogenic tissues (acidic environment). In this work we evaluate the cytotoxic activity of the fabricated spheres with/without the loaded drug via MTT assay, an established colorimetric method, in MCF-7 cells (breast cancer) and HeLa cells (cervical cancer). We also investigated the toxicity of unloaded spheres in healthy cells HEK 293 (Human Embryonic Kidney).
HCl (DNR) was provided by Pharmacia & Upjohn and used as received. Fetal Bovine Serum (FBS) was purchased from Antisel. High glucose Dulbecco's modified Eagle Medium (DMEM) and MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma. Trypsin-EDTA, L-glutamine, penicillin–streptomycin solution and heat inactivated fetal bovine serum (FBS) were obtained from Biochrom KG, Berlin, Germany.
3. Characterization Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images were obtained on an FEI Inspect microscope with W (Tungsten) filament operating at 25 kV and a FEI CM20 microscope operating at 200 kV, respectively. the spectra were scanned over the range 4000–500 cm 1. NMR spectra were recorded with a Bruker Avance 500 MHz instrument and were processed by X-WIN NMR 2.6 (Bruker Analytik GmbH). In the data presented in this study, each measurement represents the average value of 10 measurements, with 20 s integration time for each measurement. UV–vis absorption spectra in the wavelength range of 200–800 nm were obtained on a Jusco V-650 spectrometer, UV–vis at 480 nm spectrometer. An ultrasonic bath was used for sonication (Elma Sonic, S. 30H). 3.1. Morphological characterization The morphological characterization of the synthesized P(MAAco-NIPAAM-co-EGDMA)@CS microspheres has been carried out by SEM and TEM microscopy (Fig. 1A and B). According to SEM results it is observed the characteristic raspberry-like surface and through TEM microscopy it is confirmed the shell and cavity formation.
2. Materials and methods
4. Loading and release study of daunorubicin
SiO2@P(MAA-co-NIPAAM-co-EGDMA)@CS spheres were synthesized using published methods.[12] Cellulose powder, MW ¼78.000–98.000 g/mol, was purchased from Riedel-de Haen Ag Seelze-Hannover. Succinic anhydride was purchased from Sigma-Aldrich and was purified with recrystalization 2,2-azobis (2-methylpropionitrile) (AIBN), N-hydroxysuccinimide (NHS), 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 3-methacryloxypropyltrimethoxysilane (MPS) were purchased from Acros Organics and used as received. Ethyleneglycol dimethacrylate (EGDMA) was purchased from Sigma Aldrich. Tetraethyl orthosilicate (TEOS) was purchased from Fluka. Methacrylic acid (MAA) was purified by distillation before using and acetonitrile was used as received from Aldrich. Daunorubicin
8 mg of daunorubicin hydrochloride (DNR) were dissolved in 4 ml of PBS (phosphate-buffered saline, pH ¼ 7.4) and then 4.0 mg of hollow microspheres were added. The mixture was gently stirred for 72 h at room temperature. The resulting suspension was centrifuged (12.000 rpm for 5 min) and the unloaded DNR was removed. The loading amount of DNR was determined by using ultra violet spectroscopy (UV) according to a standard curve. The calculation of DNR was carried out by the difference of DNR concentration between the starting amount of DNR and the supernatant solution after loading. Standard curve was prepared by different concentrations of DNR in a PBS solution and in acidic solution. The drug release has taken place in acidic (pH ¼ 4.0, 100 mM, Acetic Acid glacial, Sodium Acetate Trihydrate) and in
434
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
Fig. 1. (A) SEM and (B) TEM images of hollow P(MAA-co-NIPAAM-co-EGDMA)@CS drug carriers.[12].
Fig. 2. Cell viability of HeLa and MCF-7 cells after incubation with P(MAA-co-NIPAAM-co-EGDMA)@CS carriers (blue), P(MAA-co-NIPAAM-co-EGDMA)@CS carriers loaded with daunorubicin (purple) and pure daunorubicin (pink).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
slightly basic (pH ¼ 7.4, 100 mM, KH2PO4, Na2HPO4 dihydrate, NaCl, KCl). The concentration of DNR, which was released from hollow microspheres in different buffer solutions, was quantified by using UV–vis spectroscopy via the standard curve method[12].
5. in vitro cytotoxicity – MTT assay In order to evaluate the therapeutic ability of P(MAA-co-NIPAAMco-EGDMA)@CS spheres as potential drug vehicles, we investigated the in-vitro cytotoxicity against MCF-7 (breast cancer) and HeLa (cervical cancer ) cells. Furthermore, we assessed the toxicity of carriers in healthy cells (HEK 293, Human Embryonic Kidney healthy cells) aiming at determining the induced toxicity in normal cells.. The cells were cultivated in DMEM (Dulbeccos's Modified Eagle Medium) with extra L-glutamine (2 mM), penicillin/streptomycin (1000/ 0.1 mg/ml), and 10% v/v fetal bovine serum at 37 1C, in a 5% CO2 atmosphere as exponentially growing monolayers. For the determination of cell viability we used MTT assay– an established colorimetric method. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) is a water-soluble yellow tetrazole that can be reduced to water-insoluble purple formazan crystals in living cells by cellular reductase. The amount of formazan is directly proportional to the number of living cells. The formazan crystals can be diluted in DMSO to perform a colored solution and the absorbance of this solution can be
Table 1 IC50 values.
DNR Spheres P(MAA-co-NIPAAM-co-EGDMA)@CS with DNR
MCF-7 IC50 (μM)
HeLa IC50 (μM)
0.87 1.22
0.31 0.81
measured by a spectrometer at 540/620 nm. For experiments, cells were plated in 96-well plates and in each well, they were added 10.000 cells diluted in 100 μl DMEM. For the determination of cells' number we used a hemocytometer. After an incubation period of 24 h, we prepared solutions of material, material with DNR and pure DNR in various concentrations (10, 5, 1, 0.5, 0.1, 0.05 and 0.01 μM of DNR) and they were added in wells (100 μl per well) in triplicates. After 24, 48 and 72 h of incubation at 37 1C, 5% CO2/95% air humidified atmosphere, the medium was removed and cells were purified with PBS and 100 μl of MTT solution in PBS (1 mg/ml) were added. After 4 h of incubation at 37 1C, the MTT solution was removed and 100 μl DMSO were added to dissolve formazan's crystals. Finally, drug effect was quantified as the percentage of control absorbance of reduced dye at λmax ¼490 nm. The absorbance was measured by a micro plate reader Sirio S. Seac Radim.
A.-F. Metaxa et al. / Materials Letters 132 (2014) 432–435
435
Fig. 3. Cell viability of HeLa and MCF-7 cells after incubation with P(MAA-co-NIPAAM-co-EGDMA)@CS carriers loaded with daunorubicin at different time intervals of incubation ([24 h (pink), 48 h (purple), 72 h (blue)])(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
6. Conclusions
Fig. 4. Toxicity in healthy embryonic cells.
Polymeric microspheres coated with polysaccharides hold a predominant location in drug delivery systems. Based on that we fabricate smart pH and Thermo sensitive polymeric microspheres P(MAA-co-NIPAAM-co-EGDMA) coated with modified cellulose. Due to their biocompatible character we evaluated their cytotoxicity behavior. As it is concluded by the in vitro results the loaded microspheres present the desired behavior as acidic and thermo -sensitive release system with equal cytotoxicity comparing to free DNR. Evaluating the emerged data,the most significant result which is concerned is the induced cytotoxicity in two cancer cell lines, MCF-7 and HeLa in contrary to its in healthy cells after treatment with unloaded microspheres. Therefore, it is of interest to explore the potential use of cellulose-based microspheres as non-toxic carriers of antitumor drugs. Further investigation into the mechanisms of cytotoxic and antitumor activity of cellulose – based drug devices, in vitro and in vivo would be useful in the treatment of various classes of cancer in future.
The percentage of livings cells was calculated using the formula below: Cell viability ð%Þ ¼ As=Acx100%; As ¼ Absorbancevalueofsample; Ac ¼ Absorbancevalueofcontrol The above experiment was repeated three times and the final results are resumed in Fig. 2A and B. From the sigmoidal graphs it is calculated the half maximal inhibitory concentration (IC50) for spheres loaded with DNR and free DNR. The results are presented in Table 1. As shown in Fig. 2 the empty carriers P(MAA-co-NIPAAM-co-EGDMA)@CS do not show cytotoxicity on the MCF-7 and HeLa cells even at the higher concentrations tested. On the other hand the carriers loaded with drug, shows comparable growth inhibition with pure daunorubicin. Specifically, the DNR-loaded cellulose containers' IC50 values were determined to be 0.87 μM on MCF-7 and 0.31 μM on HeLa cells when DNRs' IC50 values were 1.22 and 0.81 respectively. The sustained release behavior of the drug carriers is supported by Fig. 3, by which the cytotoxic activity is depended on incubation time period. Finally, the MTT assay also used in Embryonic Kidney cells as previously described. After 24 h incubation by different concentrations of polymeric materials (32, 17, 3, 1.7, 0.03 and 0.001 μg/ml) the results are depicted in Fig. 4. As it is observed in above figure, spheres are non-toxic even in high concentration (32 μg/ml).
Acknowledgments We thank the European Research Council (ERC) for financial support of this work under the “IDEAS” project called “A Novel Micro-container drug carrier for targeted treatment of prostate cancer” with the acronym NANOTHERAPY and the reference number 232959.
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