Real time in vitro studies of doxorubicin release from PHEMA nanoparticles
© Chouhan and Bajpai; licensee BioMed Central Ltd. 2009
Received: 10 July 2009
Accepted: 20 October 2009
Published: 20 October 2009
Many anticancer agents have poor water solubility and therefore the development of novel delivery systems for such molecules has received significant attention. Nanocarriers show great potential in delivering therapeutic agents into the targeted organs or cells and have recently emerged as a promising approach to cancer treatments. The aim of this study was to prepare and use poly-2-hydroxyethyl methacrylate (PHEMA) nanoparticles for the controlled release of the anticancer drug doxorubicin.
PHEMA nanoparticles have been synthesized and characterized using FTIR and scanning electron microscopy (SEM), particle size analysis and surface charge measurements. We also studied the effects of various parameters such as percent loading of drugs, chemical architecture of the nanocarriers, pH, temperature and nature of the release media on the release profiles of the drug. The chemical stability of doxorubicin in PBS was assessed at a range of pH.
Suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) results in the formation of swellable nanoparticles of defined composition. PHEMA nanoparticles can potentially be used for the controlled release of the anticancer drug doxorubicin.
The number of reported cases of cancer is steadily increasing in both industrialised and developing countries. The latest world cancer statistics indicate that the number of new cancer cases will increase to more than 15 million in 2020 whereas another report issued by the World Health organization says that there are over 10 million new cases of cancer each year and over 6 million deaths annually are caused by the disease . In spite of the fact that significant progress has been achieved in tumor biology, molecular genetics and in the prevention, detection and treatment of cancer over the last few years, adequate therapy remains elusive due to late diagnosis, inadequate strategies for addressing aggressive metastasis, and the lack of clinical procedures overcoming multidrug resistant (MDR) cancer . The integration of nanotechnology and medicine has the potential to uncover the structure and function of biosystems at the nanoscale level. Nanobiotechnology may provides a reliable and effective tool to treat diseases at a molecular scale. Nanobiotechnology offers an unprecedented opportunity to rationalize delivery of drugs and genes to solid tumours following systemic administration . Examples of nanotechnologies applied in pharmaceutical product development include polymer-based nanoparticles, lipid-based nanoparticles (liposomes, nanoemulsions, and solid-lipid nanoparticles), self-assembling nanostructures such as micelles and dendrimers-based nanostructures among others. In recent years, much research has gone into the characterisation of nanoparticles and their biological effects and potential applications. These include bottom-up and molecular self-assembly, biological effects of naked nanoparticles and nano-safety, drug encapsulation and nanotherapeutics, and novel nanoparticles for use in microscopy, imaging and diagnostics .
To be successful a cancer treatment approach needs to overcome physiological barriers such as vascular endothelial pores, heterogeneous blood supply, heterogeneous architecture to name just a few , and and it strongly depends on the method of delivery. In the past, many anticancer drugs had only limited success and had major adverse side effects [6, 7]. Nanoparticles have attracted considerable attention worldwide because of their unique functional characters such as small particle size, high stability, lower toxicity, tuneable hydrophilic-hydrophobic balance and the ability to bear surface features for target specific localization, etc. Thus, polymeric nanoparticles constitute a versatile drug delivery system , which can potentially overcome physiological barriers, and carry the drug to specific cells or intracellular compartments by passive or ligand-mediated targeting approaches . The use of some polymers also allows, at least in principle, to achieve controlled release and the sustained drug levels for longer periods of time. Numerous biodegradable polymeric nanoparticles made of natural polymers such as proteins or polysaccharides have been tried for drug delivery and controlled drug release. More recently the focus of such studies moved onto synthetic polymers, and much progress have been achieved in this area. Recent examples include, for example polycationic nanoparticles for encapsulation and controlled release of amphotericin B by Vieria and Carmona-Ribeiro ; or encapsulation of curcumin for human cancer therapy by Maitra et al .
Results and Discussion
Preparation and characterization of Nanoparticles
Surface potential of nanoparticles
Modeling of the release mechanism
Release exponent and diffusion coefficient of nanoparticles
0.46 ± 0.014
1.81 ± 0.054
0.61 ± 0.018
1.98 ± 0.059
0.50 ± 0.015
1.81 ± 0.054
0.55 ± 0.016
1.69 ± 0.051
0.40 ± 0.012
2.16 ± 0.065
0.44 ± 0.013
2.04 ± 0.061
0.45 ± 0.013
2.04 ± 0.061
0.58 ± 0.017
1.81 ± 0.054
0.47 ± 0.014
2.48 ± 0.074
0.58 ± 0.017
1.98 ± 0.059
0.86 ± 0.026
2.61 ± 0.078
0.60 ± 0.018
1.81 ± 0.054
Effect of % loading on drug release
Effect of monomer on drug release
Effect of Cross-Linker on Drug (Doxorubicin) Release
Effect of initiator on drug release
Effect of pH on drug release
Since the pH change occurs at many specific or physiological sites in the body, it is one of the important parameters in the design of drug delivery systems. Several methods have been proposed for targeting the specific regions. Among these, utilization of pH changes within the GI tract and exploitation of bacterial enzymes localized within the colon are of especial interest for the controlled drug delivery . Differences in pH in the target site may allow a specific drug to be delivered to that target site only. The underlying principle for targeted drug delivery is the pH controlled swelling of hydrogel which normally results from the change in relaxation rate of network chains with changing pH of the medium. The pH profile of normal tissue is different from that of pathological tissues such as cancerous and infected tissues. Amiji et al  reported that pH of normal tissue is higher than the pH of infected and tumourous tissues. The physical properties of stimuli-responsive carriers such as swelling/deswelling, particles disruption and aggregation vary according to changing environmental conditions. These change the nanocarriers-cells interactions, and therefore the release of the drug at tumour site may be achieved. Drug loaded nanoparticles undergo rapid dissolution and release the drug content in the acidic microenvironments of a tumour .
Effect of temperature on drug release
Effect of physiological fluids on drug release
Chemical stability of the entrapped doxorubicin
The PHEMA nanoparticles can be prepared by suspension polymerization method and characterized by techniques such as FTIR, SEM and particle size analysis. The addition of model drug, doxorubicin, to polymeric nanoparticles results in 28% drug entrapment. Release profiles of doxorubicin can be greatly modified by varying the experimental parameters such as percent loading of doxorubicin and concentrations of HEMA, cross-linker and initiator. Swelling of nanoparticles and the release of doxorubicin increases with the increase in percentage loading of drug. The amount of released drug decreases with increasing HEMA and EGDMA content of the nanoparticles. Increase in the concentration of the initiator, benzoyl peroxide, from 0.082 mM to 0.248 mM results in the increase of drug release, but this effect is reduced at higher concetration of benzoyl peroxide. The best combination of individual components for making PHEMA nanoparticles for doxorubicin delivery was 12.37 mM HEMA, 1.06 mM EGDMA and 0.248 mM Bz2O2. Fast drug release was observed at acidic pH1.2 at 37°C whilst physiological and alkaline pH and lower temperature slow down the release of doxorubicin. alts and additives affecting osmotic pressure also suppress the extent of drug release. Absorption spectra of doxorubicin do not change following its capture and release form the nanoparticles, indicating that chemical structure of the drug is likely to be unaffected by the procedure.
2-Hydroxyethyl methacrylate (HEMA) and ethyleneglycol dimethacrylate (EGDMA) were purchased from Sigma Aldrich Co. USA. Benzoyl peroxide (BPO) (MERCK) and polyvinyl alcohol (PVA) (Mol. Wt. 14000) (MERCK) were used as the initiator and the stabilizer, respectively. Toluene (MERCK) was used as the diluent. All chemicals were of analytical grade and doubly distilled water was used throughout the experiments.
HEMA monomer was purified by using a previously reported method . The purity of distilled HEMA was determined by high-pressure liquid chromatography (HPLC), [Backmen System (Gold 127)] equipped with a ultraviolet detector and a 25 cm × 46 mm id separation columns ODS (C18) of 5 μm particle size. The UV detector was set at 217 nm. The mobile phase was methanol-water (60:40 v/v) and the flow-rate was kept at 1 mL/min. All samples were diluted with pure methanol to 1/1600. 10 μL samples were injected for each analysis. Samples of known concentrations of MAA and EGDMA were injected into the HPLC and the resultant chromatogram was used to construct a standard curve of known concentrations vs. area under the curve. The chromatogram showed two distinct peaks. The first peak, at 3.614 min was identified as MAA. The next peak at 5.503 min was the major peak due to HEMA monomer. The amounts of impurities of MAA and EGDMA in the monomer samples were found to be less than 0.01 mol% MAA and 0.001 mol% EGDMA.
Preparative methods for making nanoparticles for pharmaceutical use are broadly divided into two categories, those based on physiochemical properties such as phase separation and solvent evaporation , and chemical reactions such as polymerization , and polycondensation. In the present study cross-linked PHEMA nanoparticles of defined composition were prepared by using a modified suspension polymerization technique, as previously reported by Kaparissides et. al. . In particular, the polymerization was carried out in an aqueous phase containing PVA, which was used as the stabilizing agent. The mixture containing the 12.37 mM HEMA (the monomer), 1.06 mM EGDMA (the cross-linker) and 0.248 mM Bz2O2 (the initiator) dispersed in toluene was added into 500 mL conical flask containing the suspension medium (200 mL aqueous PVA solution (0.5% W/V)). The reaction mixture was flushed by bubbling nitrogen and then sealed. The reaction mixture was then placed on magnetic stirrer and heated by vigorous stirring (600-700 rpm) at 80°C for 2 h and then at 90°C for 1 h. The cross-linking reaction was completed within three hours. After cooling, the polymeric particles were separated from the polymerization medium by washing thrice with toluene and twice with acetone. The collected nanoparticles were dried at room temperature to obtain the fine white powder and thereafter stored in airtight polyethylene bags.
The IR spectra of cross-linked PHEMA nanoparticles were recorded on a FTIR spectrophotometer (Perkin-Elmer, 1000 Paragon) (Shimadzu). While recording FTIR spectra KBr disc method was used for preparation of samples.
Morphological studies of cross-linked PHEMA nanoparticles were performed on scanning electron micrographs (SEM). SEM observations were carried out with a Philips, 515, fine coater. Drops of the polymeric nanoparticles suspension were placed on a graphite surface and freeze dried. The sample was then coated with gold by ion sputter. The coating was performed at 20 mA for 4 min, and observation was made at 10 KV. Nanoparticles were further characterized by particle size analysis for size and size distribution. The particle size analysis of prepared nanoparticles was performed on a particle size analyzer (Malvern Mastersizer 2000).
Zeta potential studies were performed with a digital potentiometer (Model No. 118, EI Product, Mumbai, India). In a typical experiment 0.1 g nanoparticles were dispersed in 20 mL of respective pH solution and emf was recorded using a compound electrode system. A similar experiment was also repeated for drug-loaded nanoparticles.
where, Wt is the weight of swollen nanoparticles at time t, and W0 is the initial weight of dry nanoparticles (at time 0).
where, Wd and W0 are the weights of loaded and unloaded nanoparticles, respectively.
In-vitro release of the loaded doxorubicin was carried out by placing the dried and loaded nanoparticles (0.1 g) in a test tube containing a definite volume (10 mL) of phosphate buffer saline (PBS) as the release medium (pH = 7.4) (1.2 mM KH2PO4, 1.15 mM Na2HPO4, 2.7 mM KCl, 1.38 mM NaCl). The amount of doxorubicin released from the polymeric nanoparticles was measured spectrophotometrically at 496 nm (Shimandzu 1700 Phama Spec.) and the released amount of drug was determined from the calibration plot.
To study the kinetics of the release process, drug-loaded nanoparticles were added to the release medium and the suspension was shaken for 3.5 h. For monitoring the progress of the release process, aliquots were withdrawn at desired time intervals and the amount of drug released was estimated spectrophotometrically.
where, D is the diffusion constant of the drug and L being the diameter of the dry nanoparticles.
In order to check the chemical stability of entrapped drug in different release media, the UV spectral studies (Shimandzu 1700 Pharma Spec) were performed as described in .
All experiments were done at least thrice and a fair reproducibility was observed. The data summarized in Tables have been expressed as mean ± SD of at least three independent determinations. The plots were drawn taking the mean values and each curve has been shown to include error bars.
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