The present work aimed to develop and evaluate a colloidal system composed of poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) associated with chlorambucil (CHB) and its effects on cancer cells. The nanoparticles showed %EE (>92%), a mean particle size in the range of 240 to 334 nm and zeta potential of -16.7 to -26.0 mV. In vitro release profile showed a biphasic pattern, with an initial burst for all formulations. The scanning electron microscopy of CHB-nanoparticles showed regular spherical shapes, smooth surface without aggregations. Differential scanning calorimetry thermograms, UV-vis absorption, fluorescence emission and Fourier transform infrared spectroscopy were performed showing the entrapment of the antitumoral in drug delivery system. CHB encapsulated in PLGA nanoparticles decrease the survival rates of the breast cancer cells: 68.9% reduction of cell viability on MCF-7 cell line and 59.7% on NIH3T3. Our results indicated that polymeric nanoparticles produced by classical methods are efficient drug delivery systems for CHB.
Breast cancer is the leading diagnosed cancer and the most common cause of cancer-related death in women worldwide [
A promising approach to prevent harmful side effects and to increase drug bioavailability and the fraction of the drug accumulated in the required zone, is the entrapment of drug in appropriate drug delivery systems [
Chlorambucil, poly(lactic-co-glycolic) acid (PLGA 50:50, Mw 17 kDa) and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) were obtained from Sigma Chemical (St. Louis, MO, USA); poly(vinyl alcohol) (PVA) (13 - 23 kDa, 87% - 89% hydrolyzed) was supplied by Aldrich (Milwaukee, WI, USA). Dichloromethane and dimethylsulfoxide (DMSO) of analytical grade were supplied by Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), penicillin and streptomycin (Gibco- BRL; Grand Island, NY). All other chemicals were of analytical grade and used without further purification.
Nanoparticles (CHB-NP I) were produced by solvent evaporation method [
The preparation CHB-NP II was achieved by adjusting the double emulsion technique (W1/O/W2, water-in-oil- in-water), previously applied to the preparation of other PLGA nanoparticles [
The Encapsulation Efficiencies (%EEs) of both CHB-NP I and CHB-NP II were analyzed in relation to its %EE, for both formulations the volume equivalent to 5.0 mg of particles were weighed and dissolved in 5.0 mL methanol. For this process, the free drug are measured in organic medium methanol and the absorbance of this solution was taken and compared with a calibration curve and calculated in the same way using the Equation (1).
Scanning Electron Microscopy (SM) was used to evaluate the shape and size of both CHB-NP I and CHB-NP II. Samples containing nanoparticles were placed on aluminum stubs coated with 50 nm gold coating under an argon atmosphere. CHB-NP I and II were examined and imaged by a Jeol 840 A (Tokyo, Japan) Scanning Electron Microscope operating at 20 kV in the traditional mode (SEI detector). Particle size was measured by using the software ImageJ® NIH.
Hydrodynamic diameter was determined by photon correlation spectroscopy (PCS) using the quasi-elastic light scattering technique, in a Zetasizer Nano ZS equipment (Malvern Instruments, Worcestershire, UK) equipped with a 10 mW He-Ne 633 nm laser beam, at 25˚C and at a scattering angle of 173˚. For the particle size analysis, a dilute suspension (1.0 mg/mL) of CHB-NP I and CHB-NP II were prepared in double distilled water and sonicated in an ice bath for 30 s. The zeta potential of the CHB-NP I and CHB-NP II in PBS buffer, (0.1 mmol, pH 7.4) was determined by using ZetaPlusTM in the zeta potential analysis mode.
The UV-vis measurements were performed on Hitachi U-3900H UV-vis Spectrophotometer in the wavelength interval of 200 - 800 nm at 37˚C. Fluorescence spectra were recorded on a spectrofluorimeter (Hitachi F-7000) at 37˚C. Each spectrum was an average of three (from 280 to 550 nm) with a resolution of 1.0 nm. The chosen wavelength for excitation was 270 nm for CHB. Slits of 2.0 nm were used for excitation and emission.
The chemical structure of the nanoparticles (unloaded and loaded with CHB-NP I and CHB-NP II) was analyzed by FTIR (IR Prestige-21 FTIR-8400S, Shimadzu, Japan) in transmission mode. For that, dried nanoparticles (1.0 mg) were mixed with KBr (40.0 mg) and then formed into a disc in a manual press. Transmission spectra were recorded using at least 32 scans with 4.0 cm−1 resolution, in the spectral range 4000 - 400 cm−1.
Thermal characterizations of CHB-NP I and CHB-NP II were performed with a Shimadzu DSC-60A. The equip- ment was calibrated with indium. The sample (approximately 3.0 mg) was heated twice from 35˚C to 600˚C at 5˚C/min in a nitrogen atmosphere (flow rate 20 mL/min). The melting temperature (Tm) was determined from the endothermic peak of the DSC curve recorded in the first heating scan. The glass transition temperature (Tg) was determined from the DSC curve recorded in the second heating scan.
The in vitro release analyses of the CHB-NP I and CHB-NP II were carried out in triplicate. Particles were added to 20 mL PBS buffer (10 mM; pH 7.4) in a conical flask shaken at 37˚C in a water bath at 65 rpm. At different time intervals, a sample volume of 1.5 mL was transferred and centrifuged at 25,000 g for 10 min. A quantity of 1.0 mL of the supernatant was taken for UV-vis analysis. 1.0 mL of fresh PBS buffer was added to the remaining 0.5 mL sample. 1.5 mL of the suspension was agitated vigorously by vortexing and replaced in the flasks. The cumulative percentage release profiles were obtained by taking the ratio of the amount of CHB released to the total drug content in the same volume of sample.
To evaluate cytotoxicity, breast cancer cells (MCF-7) and fibroblast cell line (NIH3T3) were obtained from exponentially growing 90% - 95% confluent cultures and seeded at 5000 cells/well in 96-well plates. The cells were kept in 100 μL fresh DMEM media (supplemented by 10% (v/v) FBS, penicillin (50 IU/mL) and streptomycin (50 mg/mL) for 24 h to allow cell adhesion and environmental adaptation. Subsequently, the cells were treated with additional 10 μL PBS containing different concentrations of CHB-NP I and CHB-NP II and in solution for 72 hours. The cell viability was evaluated using the yellow tetrazolium dye (3-(4,5-dimethylthia-zolyl-2) -2,5-diphenyltetrazolium bromide) (MTT) solution. The plates were read at 595 nm using Spectra Max Plate Reader. The percentage of cell viability was calculated with respect to control cells that were incubated without the drug and the nanoparticles as described in previous studies [
All numerical data are presented as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical analysis was conducted using the Prism 5.03 software (GraphPad Software, USA). Statistical differences in multiple groups were determined by one-way ANOVA followed by Tukey’s test. P < 0.05 was considered statistically significant.
Several parameters were systematically investigated to enhance the encapsulation efficiency of CHB in PLGA nanoparticles. Briefly, speed of stirring were modulated to achieve a suitable size for administration; then other properties, such as the particle size distribution, surface charge, crystallinity, and morphology were characterized for both methods of production of CHB-NP I and CHB-NP II.
It is recognized that the degree of agitation influences the stability of the emulsion. Suitable agitation provides the required energy to the system to break up the droplets of the dispersed phase [
All of these results are similar to those obtained by Mao et al. that prepared FITC-dextran loaded PLGA microspheres [
CHB-NP I and CHB-NP II were evaluated by its capacity of carrying drug. The nanoparticles were assessed using fixed amounts of polymer and surfactant and variable quantities of CHB. According to the results shown in
The nanoparticles produced by double emulsion method (CHB-NP II) and nanoparticles prepared with single emulsion method (CHB-NP I) had similar %EE, CHB-NPI (93.6% ± 2.9%) and CHB-NP II (92.6% ± 2.5%)
Nanoparticles | %EE |
---|---|
CHB-NP I (10 mg) | 93.6 ± 2.9 |
CHB-NP I (20 mg) | 75.6 ± 3.4 |
CHB-NP I (30 mg) | 49.3 ± 4.2 |
CHB-NP II (10 mg) | 92.6 ± 2.5 |
CHB-NP II (20 mg) | 73.8 ± 3.7 |
CHB-NP II (30 mg) | 68.3 ± 3.6 |
(
Polyvinyl alcohol (PVA) concentrations in the external water phase are known to be a key factor influencing the particle size of nanoparticles. The sizes and PdI of nanoparticles produced at 0.1%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 4.0% and 5.0% PVA are showed in
Increasing PVA concentration from 0.1 to 5.0% caused approximately 42% particle size decrease. The smaller size particles were obtained when increase PVA concentration from 0.1% to 2.0%, causing reduction in particle size approximately 62%, producing particles having a size of 317 nm. It is well known that the presence of PVA in the external phase stabilizes emulsion droplets against coalescence. The stabilization effect is dominant athigher PVA concentrations and leads to the decrease in the size of nanoparticles [
Scanning electron microscopy (SM) was used to investigate the morphology of NPs. The SM images of CHB- NP I and CHB-NP II showed NPs with regular spherical shapes (
unimodal with sizes in the range of 240 - 320 nm as confirmed by the morphometric analysis using ImageJ® software (
Particle sizes and zeta potentials of CHB-NP I and CHB-NP II and empty-NP were determined by employing DLS (n = 3). Nanoparticles size was 238 - 334 nm in diameter with PdI of 0.12 - 0.26 indicating a narrow size distribution (
The results of the determination of zeta potential for PLGA nanoparticles without (empty-NP) and with CHB (CHB-NP I and CHB-NP II) ranged between −16.7 and −26.0 mV and are shown in
Zeta potential is an important physicochemical parameter that influences the stability of the suspension. Extremely positive or negative zeta potential values cause larger repulsive forces, whereas repulsion between particles with similar electric charge prevents aggregation of the particles and thus ensures easy redispersion [
The presence of CHB in PLGA nanoparticles decreased the negative zeta potential value; probably, there was a masking effect of the superficial carboxylic groups by the drug adsorbed on nanoparticles surface. The results shown in
FTIR was used to evaluate the chemical structure of CHB-NP I and CHB-NP IIentrapped in PLGA nanoparticles (
DSC is a thermal analytical technique, which provides information regarding the physical properties like crystalline or amorphous nature of the samples. This aspect could influence the in vitro and in vivo release of the drug from the systems.
As shown in
Nanoparticles | Size (nm) | PdI | Zeta potential (mV) |
---|---|---|---|
Empty-NP I | 288.90 ± 1.80 | 0.131 ± 0.028 | −26.00 ± 0.45 |
Empty-NP II | 238.40 ± 2.40 | 0.118 ± 0.025 | −23.50 ± 0.35 |
CHB-NP I | 250.30 ± 1.40 | 0.155 ± 0.031 | −17.30 ± 0.2 |
CHB-NP II | 334.40 ± 9.80 | 0.263 ± 0.026 | −16.70 ± 1.07 |
tallization process during the preparation.
The CHB release from PLGA nanoparticles was studied in 10 mM PBS (pH 7.4) at 37˚C, which provides a basic idea of the drug release in physiological systems.
The in vitro release of CHB-NP I and CHB-NP II were presented as the cumulative percentage releases in
The results indicate the decrease the efficiency of the drug delivery system if applied intravenously (the drug could be released before the nanoparticles reach the target site). These great characteristics turn CHB-NP system
more suitable for local application (such as intratumoral injection) [
Also the spectrum profile of the entrapment of CHB in PLGA nanoparticles was done by using UV-vis absorption and fluorescence emission spectrum. The characteristic bands of CHB were observed at 206, 254 and 306 nm, respectively displayed in
We report the effects of CHB and CHB-NP I and CHB-NP II on the growth of the fibroblast cell line (NIH3T3) and human breast cancer cell MCF-7. It has been well established that the MCF-7 cell line is a powerful tool for the study of breast cancer resistance to chemotherapy, because it appears to mirror the heterogeneity of tumor cells in vivo [
Cor L105 lung epithelial-like cells. The results show that the viability of cells treated with free and loaded nanoparticles remains unchanged. Ribeiro-Costa [
In
Our analyses characterizes the formulation of CHB-PLGA nanoparticles applying double emulsion method and solvent evaporation process to improve the physic chemical characteristics of the drug delivery system. The size and entrapment efficiency of the nanoparticles can be adjusted by modifying various parameters, such as, drug/ polymer ratio, surfactant concentration and speed of homogenization. The smaller size and high erentrapment efficiency was obtained directing the lower amount of drug, intermediate concentration surfactant and higher agitation speed. The UV-vis spectroscopic as say indicated the potential use of PLGA nanoparticles for sustained release of lipophilic drugs such as CHB as demonstrated in the release profile. A cytotoxicity study revealed that empty NP had no cytotoxicity, while both CHB-NP I and CHB-NP II showed similar antitumor activities as free chlorambucil after 72 h. In conclusion, the developed PLGA-CHB nanoparticles could be considered as an effective strategy for application of this drug in cancer therapy using nanotechnology.
The authors report no conflict of interest. The authors alone are responsible for the content and writing of this article.
The authors are grateful to CNPq, CAPES, FAPDF, FINATEC and DPP for financial support of this research.