Preparation andcharacterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
(PHBHHX) based nanoparticles for targeted cancer therapy
Ebru Kılıçay
a, Murat Demirbilek
b, Mustafa Türk
c, Eylem Güven
b, Baki Hazer
a, Emir Baki Denkbas
b,d,⇑a
Karaelmas University, Chemistry Department, Physical Chemistry Division, Zonguldak, Turkey
b
Hacettepe University, Nanotechnology and Nanomedicine Division, Beytepe, Ankara, Turkey
c
Kırıkkale University, Biology Department, Yahsßihan, Kırıkkale, Turkey
d
Hacettepe University, Chemistry Department, Biochemistry Division, Beytepe, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 8 March 2011
Received in revised form 8 August 2011 Accepted 15 August 2011
Available online 23 August 2011 Keywords:
Targeted cancer therapy PHBHHX nanoparticles Etoposide
Folic acid
Nanoparticle–cell interactions
a b s t r a c t
Targeted drug delivery systems are one of the most promising alternatives for the cancer therapy. Rapid developments on nanomedicine facilitated the creation of novel nanotherapeutics by using different nanomaterials. Especially polymer based nanoparticles are convenient for this purpose. In this study; a natural polymer (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBHHX) was used as a base matrix for the production of a novel nanotherapeutic including antineoplastic agent, Etoposide and attached folic acid as a ligand on the nanoparticles. Modified solvent evaporation technique was used for the produc-tion of PHBHHX nanoparticles and the average size of the obtained PHBHHX nanoparticles were observed in the range of 180 nm and 1.5lm by the change in experimental conditions (i.e., homogenization rate, surfactant concentration and polymer/solvent ratio). By the increase in homogenization rate and surfac-tant concentration, size of the nanoparticles was decreased, while the size was increased by the increase in polymer/solvent ratio. Drug loading ratio was also found to be highly affected by polymer/drug ratio. Surface charge of the prepared nanoparticles was also investigated by zeta potential measurements. In the cytotoxicity tests; Etoposide loaded and folic acid attached PHBHHX nanoparticles were observed as more effective on HeLa cells than Etoposide loaded PHBHHX nanoparticles without attached folic acid. The cytotoxicity of folic acid conjugated PHBHHX nanoparticles to cancer cells was found to be much higher than that of normal fibroblast cells, demonstrating that the folate conjugated nanoparticles has the ability to selectively target to cancer cells. In addition, apoptotic/necrotic activities were evaluated for all formulations of the PHBHHX nanoparticles and parallel results with cytotoxicity tests were obtained. These studies demonstrate that the folic acid attached and Etoposide loaded PHBHHX nanopar-ticles seem as promising for the targeted cancer therapy.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
Cancer is still one of the most destructive disease group for human body due to the complexity and progressive nature of the cancer diseases (Stewart and Kleihues, 2003). In the last decade, there are 10% of decrease in the death caused by cancer diseases by using conventional techniques (Eschenbach von, 2004). On the other hand conventional cancer treatment strategies such as surgery, chemotherapy, radiotherapy or immunotherapy which are widely used in all over the world do not seem satisfactory and do not supply selective therapy. Meaning that almost all of the mentioned strategies have very destructive side effects for the heathy cells and tissues which surround the cancerous tissues
(Catane et al., 2006; Llombart et al., 2006; Gabriel, 2007). It is also desirable to maintain a steady infusion of the drug into the tumor interstitium to accomplish continuous extermination of the divid-ing cells that eventually results in tumor regression. Therefore can-cer diseases still need novel and both effective diagnosis and treatment strategies. Development of suitable active agent delivery systems that carry the therapeutically active agent molecules only to the tumor site without affecting healthy organs and tissues has to be developed. At this point, nanotechnology plays an important role in therapies of the future as ‘‘nanomedicines’’ by enabling this situation to happen, thus lowering doses required for efficacy as well as increasing the therapeutic indices and safety profiles of new therapeutics (Koo et al., 2005).
Many different types of nanomaterials including liposomes, mi-celles, nanoemulsions, nanoparticulate systems (polymer, lipid, ceramic and albumin based nanoparticles and nanogels), dendri-mers, carbon nanotubes and peptide–protein nanotubes are still under investigation for convenient drug delivery (Brigger et al.,
0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.08.013
⇑Corresponding author at: Hacettepe University, Chemistry Department, Bio-chemistry Division, 06532 Beytepe, Ankara, Turkey. Tel.: +90 312 297 6195; fax: +90 312 299 2163.
E-mail address:denkbas@hacettepe.edu.tr(E.B. Denkbas).
Contents lists available atSciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
2002; Hughes, 2005). Polymeric nanoparticles are one of the most popular group throughly the mentioned nanomaterials due to their easy production and process diversity into the required character-istics for the design of suitable drug delivery systems. Especially biocompatible and biodegradable polymeric structures such as natural polymers are preferred for this purpose.
Targeted delivery is a viable route to enhance intracellular up-take of drug containing nano-carriers within cancerous cells at the tumor site thereby increasing the effective use of the drug and minimizing undesirable side effects and toxicity. Various tar-geting moieties or ligands against tumor-cell specific receptors have been immobilized on the surface of polymeric nanoparticles to achieve active targeting. Among them, vitamin folic acid (folate) is a stable, inexpensive and nonimmunogenic chemical with a high affinity for the folate receptor which overexpresses on many hu-man epithelial cancer cell surfaces such as uterus, colon, lung and ovary cancer (Mathew et al., 2010). Therefore conjugation of macromolecules or drugs can enhance drug uptake and targeting (Yang et al., 2010a,b).
Polyhydroxyalkanoates are bacteria-based natural polymers and they can be used in many different application fields due to their renewable nature and favorable biological characteristics (i.e., biocompatibility, biodegradability, easy processability into desired shape or geometry, cost effectiveness, etc.) which are required in these mentioned applications (Anderson and Dawes, 1990; Byrom, 1992; Zinn et al., 2001; Lee, 1996). In the last decade these polymers have been used in the production of certain drug
delivery systems (Bayram et al., 2008). Among them
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is a promis-ing drug carrier, especially for hydrophobic drugs due to its ester backbone and alkane side chain. In addition to its total biodegrad-ability and nontoxic-nonimmunogenic degradation products (i.e., 3-hydroxybutyrate), this polymer has better elastic properties (Lu et al., 2010a,b).
Etoposide (is also called vepesid, code designation VP-16-213, abbreviated VP16), a topoisomerase II inhibitor, is an important chemotherapeutic agent currently in clinical use and has a signifi-cant activity on different tumors such as lung, stomach, ovarian, testicular cancers and lymphomas (Hande, 1998). Its activity is mediated by its interaction with topoisomerase II, a nuclear en-zyme which passes an intact helix through a transient double-stranded break in DNA to modulate DNA topology by using ATP. Following the strand passage, the DNA backbone is religated and the structure of DNA restored. Etoposide damage DNA by interac-tion with topoisomerase II to form cleavable complexes that pre-vent religation of DNA leading to double-strand DNA breaks in the genome of treated cells (Hande, 2006). Etoposide is poorly sol-uble in water and has a short biological half-life (3.6 h). As the effective chemotherapy depends on prolonged exposure of cancer-ous cells to anticancer agents, drug delivery systems has been used to deliver Etoposide with higher efficiency and also with fewer adverse side effects (Zhang et al., 2011; Dhanarajua et al., 2010; Reddy and Murthy, 2005).
In this study; PHBHHX was used for the production of antican-cer agent loaded nanoparticles for targeted canantican-cer therapy and the modified solvent evaporation technique was used for this purpose. In the active agent loading and release experiments Etoposide was used as a model anticancer agent and in vitro release studies were performed spectrophotometrically. Additionally folic acid was at-tached onto the PHBHHX nanoparticles as a ligand for cancer cell targeting. The physicochemical properties of nanoparticles were studied by FTIR, zetasizer, zeta potential, scanning electron micros-copy (SEM) and atomic force microsmicros-copy (AFM). In the PHBHHX nanoparticle–cell interaction studies both cell cytotoxicity over the model healthy cells, mouse fibroblast-like cell line (i.e., L-929 cell line) and model cancer cells, human epithelial carcinoma cell
line (i.e., HeLa cells) were investigated. Apoptosis and necrosis in-dexes were also evaluated.
2. Materials
PHBHHx [weight-average molecular weight (Mw) = 454,000]
containing 12 mol% of 3-hydroxyhexanoate (3-HHx) units was supplied by Procter & Gamble Company (USA). Dichloromethane and Tween-80 were purchased from Merck (Germany). Tripsin-Ethylenediamine tetra acetic acid (Tripsin-EDTA), Dulbecco’s Mod-ified Eagle’s Medium (DMEM F-12), Fetal Calf Serum (FCS) and DMSO (Dimethylsulfoxide) were purchased from Biological Indus-tries (Israel). 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva (USA). Folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) were purchased from Sigma (USA). Hoechst dye 33342 and propidium iodide were supplied from Roeche (Germany). All reagents were of analytical grade and used without further purification. 3. Methods
3.1. Preparation of PHBHHX nanoparticles
PHBHHX nanoparticles were prepared by the modified solvent evaporation technique and all the system parameters were chan-ged to optimize the size and the size distribution of the nanoparti-cles. Briefly, 10 mg of PHBHHX was dissolved in 5 ml of dichloromethane to obtain 0.2% (w/v) PHBHHX solution as organic phase. Aqueous phase as a dispersion medium for the nanoparticle production were prepared by using 3 ml of Tween-80 and 50 ml of distilled water. Organic phase was added dropwise to the aqueous phase and homogenized using IKA T 125 Digital Ultra turrax homogenizer for 2 h. During the homogenization, mechanical stir-rer was also used to get circular motion for the solution and to pro-vide spherical shaped nanoparticles. The formed PHBHHX nanoparticles were recovered by centrifugation at 12,000 rpm for 30 min followed by washing with distilled water several times and lyophilized. For the cell culture studies, nanoparticles were sterilized under UV light for 30 min and later filtered with micro-filter including 0.45
lm pore size.
To prepare nanoparticles loaded with drug, PHBHHX and Etopo-side were completely dissolved in dichloromethane at different polymer/drug weight ratios (i.e., 1/0.5, 1/0.25 and 1/0.125) in the initial step of nanoparticle preparation procedure, followed by the same sequence as above. For the cell culture studies, nanopar-ticles were sterilized under UV light for 30 min and later filtered with microfilter including 0.45
lm pore size.
3.2. Immobilization of folic acid on PHBHHX nanoparticles
In this study; folic acid was covalently conjugated with PHBHHX nanoparticles in the presence of 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDAC) by the well-established carbo-diimide method. EDAC was first reacted with carboxylic acid groups of PHBHHX which exist in the two ends of each polymer chain to form o-acrylisourea derivative. Then it was reacted with amine group of folic acid to yield amide bond between PHBHHX and folic acid, and leave the isourea derivative as a byproduct (Lin et al., 2004; Kavaz et al., 2010). Typically, folic acid (i.e., 250,
500, 750 and 1000
lg/mL) was dissolved in 1 ml of water by
adjusting pH to weak alkaline. PHBHHX nanoparticle-folate conju-gates were obtained by adding folate solution (2.5%, 5%, 7.5%, 10% w/w) into the activated 10.0 ml of PHBHHX nanoparticle suspen-sion (1.0 mg/ml) corresponding to a PHBHHX:EDAC weight ratio of 1:1 and 2 h of activation time. The mixture was stirred at room
temperature for 24 h. Next, the resulting folic acid conjugated-PHBHHX nanoparticles were centrifuged and the supernatants were stored for further analysis. Nanoparticles were then washed with distilled water and lyophilized. The unreacted folic acid remaining in the supernatant was quantified using a UV-spectro-photometer (Mini UV 1240, Schimadzu, Japan). The absorbance of the supernatant was converted into a folic acid concentration by using a calibration curve constructed with standard folic acid
solutions (wavelength = 280 nm, R2 of the calibration curve:
0.9994 and 0.0125–0.2 mg/ml of concentration range) and folic acid conjugation on the surface of the nanoparticles was calculated.
3.3. Characterization of nanoparticles
The morphology of the PHBHHX nanoparticles was evaluated by using a scanning electron microscope (SEM, Jeol, Japan) and an atomic force microscope (AFM) (Nanomagnetics, Turkey). The Fou-rier transform infrared analysis was conducted for the structural characterization of the prepared nanoparticles (FTIR, Schimadzu, DR8101, Japan). The mean size-size distribution and surface charge of the prepared nanoparticles were determined by using Zeta Sizer (Malvern Instruments, Model 3000 HSA, England). For nanoparticle size analysis freeze-dried PHBHHX nanoparticles were first sus-pended in filtered water and subjected to sonication before analy-sis. Zeta potential of nanoparticles was determined in 0.001 M HEPES buffer, pH 7.4 (0.5 mg/ml).
3.4. Determination of drug incorporation efficiency
Nanoparticles were dissolved in dichloromethane and the amount of drug in the solution was measured using ultraviolet spectroscopy (Thermo Scientific, Nanodrop 1000) at a wavelength of 284 nm. Drug content and entrapment efficiency were calcu-lated according to Eqs. (1) and (2);
Drug content ð%; w=wÞ
¼ ðMass of drug in nanoparticles
=Mass of nanoparticles recoveredÞ 100 ð1Þ Entrapment efficiency ð%Þ
¼ ðMass of drug in nanoparticles
=Mass of drug used in the formulationÞ 100 ð2Þ
3.5. In vitro Etoposide release studies
Five milliliters of phosphate buffer solution (pH: 7.4) was put into two cells separated by a dialysis membrane (with 12–14 kDa pore size) and 50 mg of PHBHHX nanoparticles including specific amounts of Etoposide were added into one of the cells. Nanoparti-cle samples were incubated at 37 °C under mild agitation in a
water bath. At pre-determined time intervals, 100
lL-samples
were withdrawn from the cell including no nanoparticles and ana-lysed for Etoposide with UV spectrophotometer (Mini UV 1240, Schimadzu, Japan) at 284 nm. The absorbance was converted into an Etoposide concentration by using a calibration curve con-structed with standard Etoposide solutions (R2 of the calibration
curve: 0.9999 and 0.008–0.429 mg/ml of concentration range) and Etoposide release was calculated.
3.6. In vitro cytotoxicity tests for PHBHHX nanoparticles
Cytotoxicity studies were performed using 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Hela cells (12 103cells/ml) and L929 cells (7 103cells/ml) were seeded in
96-well microassay plates and incubated overnight. The nanoparti-cles were diluted (5, 10, 25 and 50
lg/ml) with the cell culture
medium, inoculated to the cells and incubated for 24 h. Followingthe incubation, mediums were removed and 100
ll of fresh
med-ium and 13
ll of MTT solution (5
lg/ml, diluted with RPMI 1640
without phenol red) were added to the each well. Incubation was allowed for another 4 h in dark at 37 °C. Living cells metabolize MTT in their mitochondria and form blue formazan crystals. There-fore 100ll/well isopropanol–HCl (absolute isopropanol containing
0.04 M HCl) solution was added to dissolve the formed formazan crystals. The wells were read at 570 nm on an ELISA plate reader and percentage of cell viability was calculated. For each MTT assay, the control HeLa cell viability was defined as 100% (Kavaz et al., 2010).The cell viability (%) was calculated according to Eq.(3);
Cell viability ð%Þ ¼ ðOptical Density; OD570ðsampleÞ
=Optical Density; OD570ðcontrolÞÞ 100 ð3Þ
The OD570(sample) represents the measurement from the wells
treated with nanoparticles and the OD570(control) represents the
measurements from the wells that were not treated with nanoparticles.
3.7. Analysis of apoptosis and necrosis 3.7.1. Double staining
Double staining were performed to quantify the number of apoptotic cells in the culture on the basis of scoring apoptotic cell nuclei. HeLa cells (20 103cells per well) were grown in
DMEM-F12 withoutL-glutamine supplemented with 10% fetal bovine
ser-um and 1% penicillin-streptomycin at 37 °C in a 5% CO2humidified
atmosphere by using 24-well plates. HeLa cells were treated with different concentrations of etoposide (5, 10, 20, 40, 80 and
100
lM), PHBHHX nanoparticles (drug loading ratio; 1/0.5, 1/
0.25, 1/0.125) including the same amount of etoposide and, folic acid attached PHBHHX nanoparticles (drug loading ratio; 1/0.5, 1/0.25, 1/0.125) including the same amount of etoposide for 24 h period. On the other hand, cancer cells were treated with drug free PHBHHX nanoparticles having no folic acid on the surface and only cell medium as a control. Both attached and detached cells were collected, then washed with PBS and stained with Hoechst dye 33342 (2
lg mL
1), propidium iodide (PI) (1lg mL
1) and DNAsefree-RNAse (100
lg mL
1) for 15 min at room temperature. Next,10–50
lL of cell suspension was smeared on slide and cover slip
for examination by fluorescence microscopy. The nuclei of normal cells were stained as light blue but apoptotic cells were stained as dark blue by the Hoechst dye. The apoptotic cells were identified by their nuclear morphology as a nuclear fragmentation or chro-matin condensation. Necrotic cells were stained red by PI. Necrotic cells lacking plasma membrane integrity allow PI dye to cross cell membrane, providing that PI dye does not cross non-necrotic cell membrane. The number of apoptotic and necrotic cells in 10 ran-domly chosen microscopic fields were counted and the results were expressed as a ratio of apoptotic and necrotic cells to normal cells. The number of apoptotic and necrotic cells were determined by Fluorescence Inverted Microscope (Leica, Germany) with DAPI filter, and FITC filter, respectively (Türk et al., 2010).3.7.2. Immunocytochemical staining
Approximately, 2 ml of HeLa cells (20 103cells per well) were
treated with different concentrations of etoposide (5, 10, 20, 40, 80 and 100
lM), PHBHHX nanoparticles (drug loading ratio; 1/0.5, 1/
0.25, 1/0.125) including the same amount of etoposide and, folic acid attached PHBHHX nanoparticles (drug loading ratio; 1/0.5, 1/0.25, 1/0.125) including the same amount of etoposide for 24 hperiod. On the other hand, cancer cells were treated with drug free PHBHHX nanoparticles having no folic acid on the surface and only cell medium as a control. For an indirect immunocytochemical pro-cedure, cytology specimens were treated with 3% H2O2for 10 min,
diluted with water, and then rinsed in PBS (pH 7.4) for 5 min. The primary antibody, caspase-3 (Lab Vision) was used at 1:300 dilu-tion, and then incubated for 1 h at room temperature. Specimens were washed with PBS buffer (pH 7.4) and incubated in biotinyla-ted secondary antibody solution for 10 min. For the negative con-trol the primary antibody was omitted in one of the slides (Dinçer et al., 2010).
The immune reactivity of the caspase-3 antibody is confined to the cytoplasm of apoptotic cells. The numbers of the caspase-3-positive cytoplasm stained cells in all fields were found at 400 final magnification. For each image (at least 100 cells/field), three randomly selected microscopic fields were evaluated.
3.8. Statistical analysis
Cytotoxicity, apoptosis and necrosis studies were performed in triplicate. The data are represented as mean ± standard deviation (SD). Data were analyzed using the independent samples t-test (for two groups). Statistical significance was set at P < 0.05. 4. Results and discussions
In this presented study; Etoposide loaded and folic acid conjugated PHBHHX nanoparticles were prepared by the modified solvent evaporation technique. The nanoparticle size–size distribu-tion has been optimized by changing the homogenizadistribu-tion rate of dispersion medium, surfactant concentration and polymer/solvent ratio. The obtained optimum formulation was used for drug load-ing/release, ligand binding and in vitro studies.
4.1. Characterization of nanoparticles
Morphological investigations were performed using SEM and AFM. Well shaped Etoposide loaded and folic acid attached-PHBHHX nanoparticles were obtained with nearly narrow size dis-tribution as seen in bothFigs. 1 and 2.
Folic acid modification of PHBHHX nanoparticles were exam-ined by Fourier Transform Infrared (FTIR) spectroscopy. The FTIR spectra of PHBHHX nanoparticles, pure folic acid and folic acid con-jugated PHBHHX nanoparticles were shown inFig. 3A–C, respec-tively. Characteristic FTIR absorption peaks of folic acid at 1450
and 1605 cm1 were observed in the spectrum of folic acid
attached PHBHHX nanoparticles. In the pure folic acid and folic acid–PHBHHX conjugates, the absorption bands at 1450 and 1605 cm1were assigned to the stretching vibrations of C@C in
the backbone of the aromatic ring. Also the new peak appeared
at around 1550 cm1 in the spectrum of folic acid attached
PHBHHX nanoparticles corresponds to the amide linkage between folate and PHBHHX confirming the conjugation of folic acid on PHBHHX nanoparticles. Similar results were obtained in the
re-lated literatures (Chatterjee and Zhang, 2007; Yang et al.,
2010a,b). Bands were observed at reduced intensity due to the small concentration on the surface of the nanoparticles.
Due to the energy requirement for the homogenization of the dispersion medium to obtain smaller droplets of polymeric parti-cles such as nanopartiparti-cles, homogenization rate is one of the important parameters effective on size and size distribution (Mohanraj and Chen, 2006). Therefore homogenization rate (or acceleration rate) was evaluated as an effective parameter and acceleration rate of the ultrasound probe was changed as 60%, 70% and 90% for this purpose during the nanoparticle preparation. In all cases all the other parameters or conditions were kept con-stant. Obtained results were shown inTable 1and the average size of the PHBHHX nanoparticles were decreased significantly by the increase in homogenization rate. Increasing the acceleration rate caused the increase in the energy level that transferred into the dispersion medium leading to the decrease in polymeric droplet size down to nanoscale. Briefly it can be considered that higher amount of acceleration rates produce nanoparticles with smaller in size. Additionally size distribution was also decreased by increasing the acceleration rate, it means that it was caused to nar-rower size distribution of nanoparticles.
Surfactant causes a decrease in the interfacial tension between the organic droplets (or polymeric droplets) and the aqueous dis-persion medium (Kwon et al., 2001). Therefore increasing the amount of surfactant cause a decrease in the nanoparticle size and hence in this part of the study the amount of Tween-80 was varied while the other parameters were kept constant to investi-gate the surfactant concentration on nanoparticle size. The ob-tained results were given inTable 1. As a result the average size of the PHBHHX nanoparticles were decreased by increasing the amount of Tween-80.
Another effective parameter was selected as polymer/solvent ratio due to the increase in the difficulties for more viscous
Fig. 1. Scanning electron microscopy photograph of Etoposide loaded and folic acid attached-PHBHHX nanoparticles.
Fig. 2. Atomic force microscopy image of Etoposide loaded and folic acid attached-PHBHHX nanoparticles.
polymer solution to be dispersed in dispersion medium with the same acceleration rates leading larger nanoparticles. Obtained
re-sults were also given in Table 1 and the average size of the
PHBHHX nanoparticles were increased by increasing the poly-mer/solvent ratio as expected. Similar tendencies were obtained in our earlier studies, in which polylactide and poly(ethylene
C A -0 10 20 30 40 50 60 70 80 90 100 110 120 %T 600 800 1000 1200 1400 1600 1800 2000 Wavenumbers (cm-1) B
Fig. 3. FTIR spectra of (A) PHBHHX nanoparticle, (B) folic acid (pure), (C) folic acid attached PHBHHX nanoparticle.
Table 1
Effect of the concentration of surfactant, polymer/solvent ratio and homogenization rate on the size of PHBHHX nanoparticles. Sample No. PHBHHX concentration
(mg/mL dichloromethane) Tween-80 concentration (ml/ml suspension medium) Homogenization rate (% amplitude) Size (nm) Polydispersity Effect of polymer concentration
1 10 0.06 90 290 ± 10 0.167 ± 0.012
2 5 0.06 90 226 ± 8 0.145 ± 0.042
3 2 0.06 90 199 ± 5 0.137 ± 0.035
Effect of surfactant concentration
3 2 0.06 90 199 ± 5 0.137 ± 0.035
4 2 0.04 90 392 ± 10 0.189 ± 0.046
6 2 0.02 90 550 ± 20 0.161 ± 0.034
Effect of homogenization rate
3 2 0.06 90 199 ± 5 0.137 ± 0.035
6 2 0.06 70 626 ± 15 0.148 ± 0.018
7 3 0.06 60 1695 ± 30 0.129 ± 0.020
Table 2
Z-average diameter and zeta-potential of the PHBHHX nanoparticles prepared under different conditions.
Sample Size (nm) Polydispersity Zeta-potential (mV) PHBHHX NP 199 ± 5 0.137 ± 0.035 28.8 ± 2.56 Eto-PHBHHX NPa 201 ± 5 0.145 ± 0.072 24.6 ± 4.72 Eto-PHBHHX NPb 204 ± 2 0.162 ± 0.052 24.7 ± 3.62 Eto-PHBHHX NPc 203 ± 3 0.182 ± 0.046 25.9 ± 5.73 f-PHBHHX NP 210 ± 17 0.163 ± 0.048 22.1 ± 2.29 f-Eto-PHBHHX NPa 210 ± 15 0.153 ± 0.064 20.1 ± 3.45 f-Eto-PHBHHX NPb 212 ± 17 0.126 ± 0.043 20.3 ± 2.44 f-Eto-PHBHHX NPc 219 ± 10 0.164 ± 0.068 20.2 ± 1.21 a 1/0.5 PHBHHX/Etoposide ratio. b 1/0.25 PHBHHX/Etoposide ratio. c 1/0.125 PHBHHX/Etoposide ratio. Table 3
Percentage of drug content and entrapment efficiencies of prepared nanoparticles. Drug Polymer/drug Drug content (%) Entrapment efficiency (%)
Etoposide 1/0.5 8.77 35.52
1/0.25 5.45 45.78
1/0.125 2.92 53.78 Fig. 4. In vitro release behavior of Etoposide from PHBHHX nanoparticles with different initial feeding ratios (PHBHHX/Etoposide).
glycol)-polylactide microspheres (Denkbasß et al., 1997, Çelikkaya et al., 1996) and polyhydroxybutyrate microspheres (Kassab et al., 1997) were prepared, and in those studies performed by other researchers (Rak et al., 1985).
In clinical use, the smaller particles lead to an easier intrave-nous injection, tend to accumulate in the tumor site and avoid spleen filtration (Zhang et al., 2011). As the smallest nanoparticles (199 nm) was obtained using 2 mg of PHBHHX/ml dichlorometh-ane, 0.06 ml of tween 80/ml suspension medium and 90% ampli-tude of homogenization rate, this formulation (sample no. 3) was used for all of the subsequent experiments.
Table 2shows the z-average diameter and zeta-potential, of the PHBHHX nanoparticles prepared under different conditions which were used for in vitro studies. The z-average diameter of the
prepared nanoparticles did not vary significantly with folic acid modification or Etoposide loading. The zeta-potential analysis re-vealed that all the formulations had a net negative charge in the range of (20(29 mV)). However, when PHBHHX nanoparticles were modified with folic acid (f-PHBHHX NP) and loaded with Eto-poside (f-Eto-PHBHHX NP), the zeta-potential increased slightly. In addition, the loading of different amounts of Etoposide resulted in no observable change in size and surface integrity.
4.2. Determination of drug incorporation efficiency
Drug content and entrapment efficiency were observed to be greatly affected by the initial polymer/drug ratio as seen inTable 3. Drug content of PHBHHX nanoparticles increased from 2.92% to
Fig. 5. The diagrams of cell viability (A) for various concentrations of the free drug (B) for PHBHHX nanoparticles at various polymer/drug ratios (C) for folic acid conjugated-PHBHHX nanoparticles at various polymer/drug ratios under 24 h of treatment. Data are expressed as means of a representative of three similar experiments.
8.77% by the increase in the drug loading from 1/0.125 to 1/0.5. On the other hand, entrapment efficiency decreased from 53.78% to 35.52% with the increase in drug loading. This decrease in entrap-ment efficiency can be explained by the constant polymer amount used in all of the formulations and increased drug loss from the polymer matrix to the outer aqueous phase due to the enlarged concentration gradient resulted with the increased drug amounts in nanoparticles in the case of higher initial feeding amount of drug in the fabrication process (Bayram et al., 2008; Lu et al., 2010a,b). Loading efficiencies was found identical for folic acid conju-gated PHBHHX nanoparticles (f-Eto-PHBHHX NPa,b,cinTable 2).
4.3. In vitro Etoposide release study
In the in vitro release studies; all different formulations pre-pared with different amounts of Etoposide (i.e., pi) were investi-gated for release studies. Released Etoposide were measured spectrophotometrically at certain time intervals. Obtained release rates were given inFig. 4as percentage cumulative release rates for different Etoposide loading ratios. An initial burst release fol-lowed by a slower release rate was observed. Maximum release rate was obtained in the case of 1/0.125 PHBHHX/Etoposide initial ratio while the lowest value was obtained with 1/0.5 ratio. This re-sult can be due to the lower drug content in the case of 1/0.125 ini-tial ratio as explained in previous section. These results are similar with the related literature (Lu et al., 2010a,b). Increased initial
burst was caused by the drug closer to the surface of the nanopar-ticles while the drug in the core of the nanoparnanopar-ticles is responsible for the prolonged drug release from the nanoparticles (Avinash et al., 2007).
4.4. Folic acid conjugation efficiency
2.5%, 5.0%, 7.5%, 10.0% (w/w) of folic acid solutions yielded dif-ferent ligand binding efficiencies on PHBHHX nanoparticles as 71%, 81%, 86% and 93%, respectively. As the highest folic acid binding efficiency was obtained with 10.0% (w/w) folic acid solution, PHBHHX nanoparticles to be used in in vitro studies were prepared at this concentration of folic acid and at different polymer/drug ratios (1/0.5, 1/0.25 and 1/0.125 PHBHHX/Etoposide ratios) as summarized inTable 2. Additionally we observed that concentra-tion of drug did not alter the binding efficiency significantly. 93.1%, 95.1% and 96.4% binding efficiencies were observed for 1/0.5, 1/0.25 and 1/0.125 PHBHHX/Etoposide ratios, respectively. 4.5. In vitro cytotoxicity
Cytotoxicities of various PHBHHX nanoparticle formulations (i.e., folic acid attached and non-modified PHBHHX nanoparticles) prepared at different initial polymer/drug ratios (i.e., 1/0.5, 1/0.25 and 1/0.125) and also pure Etoposide were quantified using con-ventional MTT assay. For HeLa cells, cytotoxicity was observed to
Fig. 6. The diagram of cell viability for folic acid conjugated-PHBHHX nanoparticles at various polymer/drug ratios on L929 cells under 24 h of treatment. Data are expressed as means of a representative of three similar experiments.
Table 4
Apoptotic percentage indices obtained as a result of the interaction of Etoposide (E), nanoparticles loaded (E/P) with different ratios of Etoposide, and folic acid-targeted nanoparticles loaded (E/PF) with different ratios of Etoposide in HeLa cell cultures. Data represent the mean ± SD.
Drug amount (lM) Apoptotic percentage indices (%)
E P E/P (1:0.125) E/P (1:0.25) E/P (1:0.5) E/PF (1:0.125) E/PF (1:0.25) E/PF (1:05)
0 1 ± 1 1 ± 1 2 ± 1 1 ± 1 2 ± 1 1 ± 1 2 ± 1 3 ± 1 5 4 ± 1 1 ± 1 5 ± 1a 4 ± 1a 6 ± 1a 7 ± 1b 11 ± 1b 14 ± 2b 10 7 ± 1 2 ± 1 9 ± 1a 8 ± 1a 12 ± 1a 16 ± 2b 18 ± 1b 20 ± 2b 20 11 ± 1 4 ± 1 13 ± 1a 15 ± 1a 16 ± 2a 18 ± 2b 23 ± 1b 26 ± 2b 40 16 ± 1 6 ± 1 21 ± 1a 21 ± 1a 22 ± 1a 26 ± 2b 29 ± 2b 35 ± 2b 80 22 ± 2 9 ± 1 27 ± 1a 29 ± 2a 30 ± 1a 34 ± 2b 36 ± 2b 39 ± 2b 100 27 ± 2 11 ± 1 32 ± 2a 31 ± 2a 34 ± 2a 39 ± 2b 47 ± 2b 54 ± 3b a
Statistical difference (P > 0.05) was not found for apoptotic activity between free Etoposide (E) and Etoposide loaded nanoparticle (E/P) in different ratios.
b
Statistical difference (P < 0.05) was found for apoptotic activity between free Etoposide (E) and Etoposide loaded folic acid attached nanoparticles (E/PF) in different ratios and P values were 0.029, 0.004, and 0.001 at 1:0.125, 1:0.25, and 1:0.5 Etoposide loading ratios, respectively.
be highly affected by the initial polymer/drug ratio and also folic acid conjugation to PHBHHX nanoparticles. A non-significant de-crease in mortality was observed when the cells were incubated with free etoposide at different concentrations (i.e., 5, 10, 25 and 50
lg/ml). Higher drug concentration caused lower cell viability
or equivalently higher mortality of the cells. For example for50
lg/ml drug concentration cell mortality was observed as
13.2 ± 7.79% and 5
lg/ml drug concentration resulted with
3.9 ± 2.48% cell mortality (Fig. 5A). For the cytotoxicity tests of PHBHHX nanoparticle formulations (i.e., folic acid attached and non-modified PHBHHX nanoparticles), the same concentration of the drug (i.e., 5, 10, 25 and 50
lg/ml) which was encapsulated in
the nanoparticles at different initial polymer/drug ratios (i.e., 1/ 0.5, 1/0.25 and 1/0.125) were applied (Fig. 5B and C). They exhib-ited more potent cytotoxic effect on hela cells than free Etoposide. Folic acid attached nanoparticles caused more cell mortality than non-modified nanoparticles and also in the case of higheretopo-side loading ratio, more cytotoxicity was observed as very well cor-related with drug incorporation efficiency studies. For example, 50
lg/ml drug concentration and 1/0.5 polymer/drug ratio resulted
with 44.2 ± 4.68% cell mortality for folate attached PHBHHX nano-particles, while 30.1 ± 3.950% cell mortality was observed for non-modified PHBHHX nanoparticles.In the evaluation of the cytotoxicity of folic acid attached PHBHHX nanoparticles at different initial polymer/drug ratios (i.e., 1/0.5, 1/0.25 and 1/0.125) on L929 cells, folic acid attached and Etoposide loaded nanoparticles were observed to be less cyto-toxic on healthy cells (L929 cells) than cancer cells (HeLa cell line) (Fig. 6). For example, cytotoxicity of PHBHHX nanoparticles having
50
lg/ml drug concentration and 1/0.5 polymer/drug ratio was
found as 21.3 ± 7.54%. This formulation was also observed to be the most cytotoxic nanoparticle formulation on L929 cell and when compared to the cytotoxicity on Hela cells, this nanoparticle for-mulation was more cytotoxic on HeLa cells (44.2 ± 4.68%) than
Fig. 7. Photographs of immunocytochemical staining performed by Caspase-3 antibody (A) HeLa cells stained by Caspase-3 (control group); since cells were not interacted with nanoparticles, their cytoplasms look blue, i.e., no apoptosis occurred. (B) HeLa cells stained by Caspase-3; some of the cells were exposed to apoptosis as a result of their interaction with NP loaded with Etoposide in the ratio of 1/0.5 (40lM), their cytoplasms were stained in brown, non-apoptotic cells look blue in color. (C) HeLa cells interacted only with Etoposide (40lM). (D) Image of HeLa cells interacted with folic acid attached NP (100lM) loaded with Etoposide in the ratio of 1/0.125. Cytoplasms of cells look dark brown. (E) Image of HeLa cells interacted only with Etoposide (100lM). (F) Image of HeLa cells interacted with NP (100lM) loaded with Etoposide in the ratio of 1/0.125. The arrows show some of the apoptotic cells. Photographs were taken by Zeiss light microscope at 400 magnification. The scale shows a distance of 20lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Apoptotic cell photographs obtained from the double staining performed by using Hoechst 33342 fluorescent stain (A) HeLa cells stained by Hoechst 33342 and not treated with Etoposide and nanoparticles (control group); since cells were not treated with nanoparticles, they were not exposed to apoptosis and cell nuclei appear pale blue in color, nucleus borders are normal, nucleus did not decompose. (B) HeLa cells interacted with NP (40lM) loaded with Etoposide in the ratio of 1/ 0.25. (C) HeLa cells interacted only with Etoposide (100lM). (D) Image of HeLa cells interacted with folic acid attached-NP (100lM) loaded with Etoposide in the ratio of 1/0.25. (E) Image of HeLa cells interacted with NP (100lM) loaded with Etoposide in the ratio of 1/0.125. (F) Image of HeLa cells interacted with folic acid attached NP (100lM) loaded with Etoposide in the ratio of 1/0.125. The arrows show some of the apoptotic cells. Cell nuclei exposed to apoptosis look bright and decomposed and non-apoptotic ones look pale blue. Photographs were taken by Leica inverted fluorescent microscope at 400 magnification. The scale shows a distance of 20lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
L929 cells (21.3 ± 7.54%). These results demonstrate that the folate conjugated and Etoposide loaded PHBHHX nanoparticles can selec-tively target to cancer cells with overexpressed folic acid receptors. 4.6. Apoptosis and necrosis studies
4.6.1. Caspase-3 apoptotic index results obtained by immunocytochemical staining
Apoptotic index was obtained in two different ways: by caspase staining and by double staining method.Table 4 introduces the average of these two methods. Light microscope photographs ob-tained from the study were given inFig. 7. According to apoptotic results obtained from the study, apoptotic effect was observed to increase with higher drug concentrations. The same result was also achieved in drug-loaded PHBHHX nanoparticles as well as in folic acid attached drug loaded-nanoparticles (Table 4). It was observed that apoptotic index was increased by 5% when etoposide was loaded into PHBHHX nanoparticles and interacted with cancer cells. When folic acid was attached on drug-loaded particles,
apoptotic effect was observed to be increased by 10–25% with re-spect to the drug and by 10–20% in drug-loaded nanoparticles hav-ing no folic acid on the surface. This effect was achieved at the highest ratio especially in nanoparticles at 1/0.125 polymer/drug ratio and targeted with folic acid. While no significant difference was found between the apoptotic effects of nanoparticles loaded with different ratios of drug, the difference in apoptotic effect was remarkable when targeted with folic acid. These results reveal that the application of folic acid-attached, drug loaded-nanoparti-cles results in a higher reception of the drug by cancer cells. In addition, as particles release the drug in a controlled manner, the effect lasted longer than free drug, resulting in an increase in apop-totic effect. This effect can be seen more clearly in the photographs
Fig. 9. Photographs showing apoptotic cell DNA obtained from the double staining performed by using Hoechst 33342 fluorescent stain (A) Photographs of folic acid-targeted NPs loaded with Etoposide (containing 80lM drug) in a ratio of 1/0.25 in HeLa cell culture. DNA is decomposed but within nucleus borders. (B) Photographs of folic acid-targeted NPs loaded with Etoposide (containing 40lM drug) in a ratio of 1/0.125 in HeLa cell culture. The arrows show some of the cells with decomposed DNA. DNA was completely decomposed and spread inside cell cytoplasm in the shape of beads. Photographs were taken by DAPI filter 400 magnification. The bar shows a distance of 20lm.
Fig. 10. Necrotic cell photographs obtained from double staining performed by using propidium iodide (PI) fluorescent stain. (A) HeLa cells stained by double staining method and not treated with drug and nanoparticles (control group); since cells were not interacted with nanoparticles, cells did not necrose and cell nuclei look green in color (stained by Hoechst 33342). (B) HeLa cells interacted with NP (100lM) loaded with Etoposide in the ratio of 1/0.125. (C) HeLa cells interacted only with Etoposide (100lM). (D) Image of HeLa cells interacted with folic acid attached-NP (100lM) loaded with Etoposide in the ratio of 1/0.125; necrosed cell nuclei look red and non-necrosed cell nuclei look green. (E) Image of HeLa cells interacted with NP (100lM) loaded with Etoposide in the ratio of 1/0.25. (F) Image of HeLa cells interacted with folic acid-attached NP (100lM) loaded with Etoposide in the ratio of 1/0.25. Fig. 12E and F, the photographs taken by using DAPI filter, show that bright blue nuclei indicate apoptotic cells and purple nuclei indicate necrotic cells. The arrows show some of the necrotic cells. Photographs were taken by Leica inverted fluorescent microscope at 400 magnification. The scale shows a distance of 20lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
obtained by caspase staining. EspeciallyFig. 7A reveals no brown color caused by caspase-3 staining in cytoplasm, since there was no apoptosis in the control group; whileFig. 7B–F show a brown color in cell cytoplasms due to the reaction formed by caspase-3 antibodies. Moreover, Fig. 7D demonstrates the effect of folic acid-attached nanoparticles at 1/0.125 polymer/drug ratio and cell cytoplasms are stained in dark brown.
4.6.2. Apoptosis and necrosis results obtained by double staining Double staining is another method used in the determination of apoptosis. Hoechst 33342 fluorescent stain used in this method penetrates through the membranes of living cells and stains the nucleus, and gives the nuclei a blue color when observed with DAPI filter under fluorescent microscope. It also enables the evaluation of nuclear morphology. Apoptotic index obtained by the double staining were given inTable 4 and the photographs taken under fluorescent inverted microscope were given inFig. 8. Apoptotic ef-fect was observed to be parallel with the results of caspase-3 immunocytochemical staining. Apoptotic effect can be seen very clearly especially in wells treated with folic acid-attached, drug-loaded nanoparticles.Fig. 8A shows the photograph of the control group, and no morphological difference was observed in cell nuclei. However, it was observed that apoptotic cell nuclei was decom-posed and appeared in brighter blue color compared to non-apop-totic cells in the case of treatment with etoposide alone and drug-loaded, ligand-bound nanoparticles (Fig. 8C and D). When drug-loaded nanoparticles were targeted with folic acid, apoptotic effect increased as shown inFig. 8B, E and F. The effect of folic acid-attached nanoparticles containing drug in the ratio of 1/0.125 and 1/0.25 on DNA were given inFig. 9. As seen inFig. 9, DNA in the nucleus decomposed completely and spread into cell cytoplasm like beads. These images were obtained from the cells that adhered to the well bottom. Cells that were detached from the well bottom did not reveal such detailed images. The nanoparticles differing in drug loading (i.e., 1/0.5, 1/0.25 and 1/0.125) and targeted with folic acid or non targetted did not differ significantly (P > 0.05) with re-gard to the apoptotic effect.
PI (propidium iodide) fluorescent stain is another stain used in double staining method, and activity was detected in cancer cells interacted with drug and drug-loaded particles. This stain pene-trates through dead cells and cells with damaged plasma mem-brane, and causes nuclei to look red under fluorescent light. Nuclei of healthy and apoptotic cells look blue in color and they look green when scanned by FITC fluorescent filter. In our study, necrotic index deviation was examined under fluorescent light (by FITC filter) at a wavelength of 480–520 nm, and at this wave-length necrotic cell nuclei were observed to be red and healthy or apoptotic cell nuclei were observed to be green at simultaneous times. Photographs of necrotic cells were given in Fig. 10 and necrotic percentage index is given inTable 5. As seen inTable 5,
necrotic index is not very high at low concentrations. However, higher concentrations resulted in an increase in toxicity, and therefore, an increase in necrosis. It was determined that especially folic acid attached and drug-loaded nanoparticles had high necro-tic effect. As shown inFig. 10, necrotic effect of nanoparticles that were not conjugated with folic acid was lower (Fig. 10B and E) compared to folic acid attached-nanoparticles. Necrotic effect of folic acid attached, drug loaded-nanoparticles was found to be 20% higher compared to samples that were treated with drug alone, and 10% higher in non-targeted drug-loaded nanoparticles. Although the highest necrotic index of approximately 40% was ob-tained in nanoparticles targeted with folic acid in the ratio of 1/ 0.125 and loaded with 100
lM drug (
Fig. 10D), no statistical differ-ence (P > 0.05) was found regarding to necrotic effect between non-targeted drug loaded NP and folic acid targeted drug loaded NP at each concentration and different loading ratios. Statistical difference (P < 0.05) was found for necrotic activity between free Etoposide (E) and Etoposide loaded folic acid targeted nanoparti-cles (E/PF) in different ratios.5. Conclusion
Etoposide-loaded and folic acid conjugated PHBHHX nanoparti-cles were prepared by the modified solvent evaporation technique for folate-receptor-targeted cancer therapy. Nanoparticles were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), FTIR spectroscopy, size and zeta potential mea-surements. SEM and AFM showed that the nanoparticles were in a well-defined spherical shape. The size of the prepared nanoparti-cles were affected significantly by the preparation condition (i.e., homogenization rate, surfactant concentration and polymer/sol-vent ratio). A net negative charge was observed by the zeta poten-tial analysis on the surface of the nanoparticles. Drug loading and release studies were performed to investigate drug incorporation efficiency and in vitro Etoposide release profile. The drug-release behavior of the nanoparticles was characterized by two stages involving an initial rapid release, followed by a controlled release. Ligand binding was confirmed by FTIR and ligand binding effi-ciency was investigated. Cytotoxicity of the prepared nanoparticles was determined by MTT assay. The cytotoxicity of the folic acid conjugated and Etoposide loaded PHBHHX nanoparticles to cancer cells was much higher than free etoposide or Etoposide loaded PHBHHX nanoparticles without folic acid. Also the cytotoxicity of folic acid conjugated and Etoposide loaded PHBHHX nanoparticles to cancer cells was found to be higher than that of normal fibro-blast cells, demonstrating that the folic acid conjugated PHBHHX nanoparticles has the ability to selectively target to cancer cells. In addition, apoptotic and necrotic activities were evaluated which confirmed the cytotoxicity studies. These studies revealed that the folate conjugated and Etoposide loaded PHBHHX nanoparticles can
Table 5
Necrotic percentage indices obtained as a result of the interactions of Etoposide (E), nanoparticles loaded (E/P) with different ratios of Etoposide, and folic acid-targeted nanoparticles loaded (E/PF) with different ratios of Etoposide in HeLa cell cultures. Data represent the mean ± SD.
Drug amount (lM) Necrotic percentage indices (%)
E P E/P (1:0.5) E/P (1:0.25) E/P (1:0.125 E/PF (1:0.5) E/PF (1:0.25) E/PF (1:0.125)
0 2 ± 1 1 ± 1 1 ± 1 1 ± 1 1 ± 1 2 ± 1 2 ± 1 3 ± 1 5 1 ± 1 2 ± 1 2 ± 1a 1 ± 1a 3 ± 1a 2 ± 1b 3 ± 1b 3 ± 1b 10 3 ± 1 1 ± 1 4 ± 1a 6 ± 1a 6 ± 1a 7 ± 1b 8 ± 1b 8 ± 1b 20 8 ± 1 2 ± 1 10 ± 1a 11 ± 1a 10 ± 1a 13 ± 1b 13 ± 1b 14 ± 1b 40 13 ± 1 6 ± 1 20 ± 2a 17 ± 1a 19 ± 2a 23 ± 2b 21 ± 2b 24 ± 2b 80 18 ± 2 10 ± 1 24 ± 2a 23 ± 2a 24 ± 2a 28 ± 2b 30 ± 2b 35 ± 2b 100 21 ± 2 15 ± 1 31 ± 2a 32 ± 2a 34 ± 2a 37 ± 2b 38 ± 2b 40 ± 2b
a Statistical difference (P > 0.05) was not found for necrotic activity between free Etoposide (E) and Etoposide loaded nanoparticle (E/P) in different ratios.
b Statistical difference (P < 0.05) was found for necrotic activity between free etoposide (E) and etoposide loaded folic acid targeted nanoparticle (E/PF) in different ratios
effectively target the site of tumor via the folate receptor mediated recognition. Therefore, the prepared folic acid conjugated PHBHHX nanoparticles loaded with Etoposide may be used as a potential drug delivery system for the targeted delivery to cancer cells. Fur-ther in vivo studies are planned to demonstrate the efficacy of these PHBHHX nanoparticles.
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