Chitosan nanoparticles (CS NPs) exhibit good physicochemical properties as drug delivery systems. The aim of this study is to determine the modulation of preparative parameters on the physical characteristics and colloidal stability of CS NPs. CS NPs were fabricated by ionic interaction with dextran sulphate (DS) prior to determination of their storage stability. The smallest CS NPs of
Endogenous peptides, protein, and oligonucleotides are among the main drugs which attract much attention because of their great potentials in treating chronic diseases [
Several studies had reported the unique features of chitosan nanoparticles (CS NPs) using DS. However, the modulation of preparative parameters on their physical characteristics is still not fully investigated, for example, the influence of DS steric hindrance on the electrostatic attraction between CS and BSA [
Low molecular weight chitosan (70 kDa with the degree of deacetylation 75%–85%), acetic acid glacial, phosphate buffered saline (PBS), bovine serum albumin (BSA, 46 kDa), and Bradford reagent was purchased from Sigma-Aldrich Inc., USA. Double-stranded siRNA (sense: 5′-GAUUAUGUCCGGUUAUGUAUU-3′, antisense: 3′-UACAUAACCGGACAUAAUCUU-5′) was purchased from Thermoscientific Dharmacon, USA. Dextran sulphate (DS) was purchased from Fisher Scientific, UK. Protein ladder (High range), Laemmli sample buffer, 10x tris/glycine/sodium dodecyl sulfate buffer, ammonium persulfate, tetramethylenediamine (TEMED), 2% bis solution, and 40% acrylamide solution were purchased from Bio-Rad, USA. Tris-HCl buffer was obtained from Invitrogen, USA. All other chemicals used were of analytical grade.
CS and DS solution were dissolved in 1% v/v acetic acid and distilled water, respectively. pH of CS solution was adjusted to pH 4 by adding 1 M NaOH or 1 M HCl. DS solution (0.05%, 0.1%, 0.15%, 0.2%, and 0.25% w/v) was added dropwise into CS solution (0.1% w/v) under magnetic stirring (WiseStir Digital Multipoint Magnetic Stirrer MS-MP8, DAIHAN Scientific, Korea) at 250 rpm for 15 min to form nanoparticles. All samples were made in triplicate. The resultant nanoparticles were washed and harvested by ultracentrifugation (Optima L-100 XP Ultracentrifuge with a rotor NV 70.1, Beckman-Coulter, USA) twice at 12 500 rpm for 15 min at 10°C. For BSA association into CS NPs, BSA was dissolved in CS solution (0.1% w/v, pH 4) to produce a final concentration of 1 mg/mL. BSA-loaded CS NPs were then prepared by the above method. For siRNA association into CS NPs, 3
Electrophoretic mobility measurements (
Particle size, surface charge, and polydispersity index (PDI) of freshly prepared CS NPs were measured using a Zetasizer Nano ZS (Malvern Instruments, UK), based on the Photon Correlation Spectroscopy (PCS) techniques. No dilutions were performed during the analysis. Each sample was analyzed in triplicate. The measurements were made at 25°C.
Morphological characterization of unloaded CS NPs, BSA/siRNA loaded CS NPs (DS : CS weight ratio of 0.5 : 1, 1 : 1) was carried out by using transmission electron microscopy (TEM), Tecnai Spirit, FEI, Eindhoven (The Netherlands).
BSA/siRNA loaded CS NPs were separated from the solution by ultracentrifugation (Optima L-100 XP Ultracentrifuge with a rotor NV 70.1, Beckman-Coulter, USA) at 14000 rpm for 30 min. Supernatants recovered from centrifugation were decanted. BSA content in the supernatant was analyzed by a UV-Vis spectrophotometer at 595 nm (U.V-1601; Shimadzu, Japan) using the Bradford protein assay as per manufacturer instruction. siRNA content in the supernatant was analyzed by a UV-Vis spectrophotometer at 260 nm. Samples were prepared and measured in triplicate. The BSA/siRNA entrapment efficiency (EE) was calculated using the following equation:
Freshly prepared CS NPs (made from 0.05% and 0.1% w/v of DS and CS solution, resp.) were centrifuged at 12 500 rpm for 15 min prior to storing. After ultracentrifugation, the obtained pellets were resuspended in either distilled water (measured pH of 6.6) or PBS pH 7.4. The particle size and surface charge were measured at predetermined storage time durations (0, 1, 2, 3, 5, 8, and 14 days), and at either ambient temperature or 4°C.
The release of BSA/siRNA was determined from CS NPs with the highest EE (DS : CS ratio 1 : 1, EE = 98% ± 0.2 and
The integrity of BSA released from CS NPs was determined by SDS-PAGE (12% resolving and 10% stacking gel) using Mini-Protein System (Bio-Rad, USA). BSA samples were mixed with Laemmli sample buffer in 1 : 1 ratio and heated at 95°C for 5 min. Samples (15
All the data were presented as mean ± standard deviation (SD). Statistical analysis (ANOVA test and Tukey’s posthoc analysis) was performed by using the Statistical Package for the Social (SPSS) programme version 15. A
Figure
Effects of DS : CS weight ratios on the physical characteristics of unloaded (top) and BSA loaded (below) CS NPs,
DS (% w/v) | CS (% w/v) | DS : CS weight ratio | pH of nanoparticle dispersion | Particle size, nm ± SD | PDI ± SD | Surface charge, mV ± SD |
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0.05 | 0.10 | 0.5 : 1 | 3.84 |
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0.10 | 0.10 | 1 : 1 | 3.79 |
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0.15 | 0.10 | 1.5 : 1 | 3.80 |
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0.20 | 0.10 | 2 : 1 | 3.81 |
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0.25 | 0.10 | 2.5 : 1 | 3.82 |
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DS (% w/v) | CS (% w/v) | DS : CS weight ratio | Particle size, nm ± SD | PDI ± SD | Surface charge, mV ± SD | EE, % ± SD |
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0.05 | 0.10 | 0.5 : 1 |
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0.10 | 0.10 | 1 : 1 |
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0.15 | 0.10 | 1.5 : 1 |
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0.20 | 0.10 | 2 : 1 |
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Electrophoretic mobility as a function of time (a) and effect of the final concentrations of CS and DS on the particle size of nanoparticles (b),
Table
Table
Effects of DS : CS weight ratios on the physical characteristics of siRNA loaded CS NPs,
DS (% w/v) | CS (% w/v) | DS : CS weight ratio | Particle size, nm ± SD | PDI ± SD | Surface charge, mV ± SD | EE, % ± SD |
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0.05 | 0.10 | 0.5 : 1 |
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0.10 | 0.10 | 1 : 1 |
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0.15 | 0.10 | 1.5 : 1 |
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0.20 | 0.10 | 2 : 1 |
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The images of the CS NPs were obtained by TEM (Figure
TEM images of CS NPs. (a) and (b) Unloaded CS NPs at 0.5 : 1 and 1 : 1, (c) and (d) BSA loaded CS NPs at 0.5 : 1 and 1 : 1, and (e) and (f) siRNA loaded CS NPs at 0.5 : 1 and 1 : 1, respectively. All the images were taken at 60 kX magnification.
Both nanoparticles made from 0.05 and 0.10% w/v DS were increased in size over time as shown in Figure
(a) Particle size and (b) surface charge of CS NPs prepared at 0.05 and 0.01% w/v DS solution and stored at 25°C. Nanoparticles were suspended in distilled water (pH in the range of 6-7),
(a) Particle size and (b) surface charge of CS NPs prepared at 0.05 and 0.10% w/v and stored at 4°C. Nanoparticles were suspended in distilled water (pH in the range of 6-7),
Figure
(a) The release profile of BSA loaded CS NPs at DS : CS ratio of 1 : 1 at pH 7.4,
Figure
The release profile of siRNA loaded CS NPs at DS : CS ratio of 1 : 1 at pH 7.4,
The method used to produce CS NPs in the present study is a mild process, and it enables control of the particle size by varying certain parameters for example, concentration of added salts, viscosity, quantity of nonsolvent, and molecular weight of polymer. This study was started with the investigation to obtain information regarding electrical state of ionizable groups of CS NPs by determining the stabilization time of e.d.l. This step is important to obtain reliable and reproducible
A study was also carried out to determine the influence of polymer concentration on particle formation. The study was aimed at establishing the range of polyelectrolytes concentration to produce nanoparticles with the desired size. To study the effects of the varying concentrations of CS and DS on the formation of nanoparticles, CS and DS solution of 0.1, 0.25, and 0.5% w/v were prepared. Variable volumes of DS solution (1, 2, 3, 4, 5, 5.8, and 10 mL) were mixed with 5 mL of each CS concentration (0.1–0.5% w/v). The final concentration of CS and DS was calculated, and sizes of samples were categorized either as 100–500, 501–1000, or more than 1000 nm. It was found that particle size was affected by the DS concentration. This finding corroborated with the results of CS-TPP NPs [
Furthermore, the results revealed that only DS concentration of 0.05% w/v was able to produce nanoparticles with particle size less than 500 nm as shown in Table
In present study, the incorporation of BSA into CS NPs was achieved by simply mixing the acidic CS solution containing dissolved BSA molecules with the DS solution at room temperature without addition of stabilizer. BSA is frequently used as a model protein because it embraces the general characteristic of other proteins and it is biocompatible to humans. It was found that CS NPs were comparatively larger in size after loading with BSA. Particle size was expected to increase when BSA was successfully being loaded into nanoparticles. This trend may be possibly due to the molecular weight and size of the added BSA molecules. These large particle sizes may limit their use in delivery of protein. Nanoparticles of 150–300 nm are found mainly in the liver and the spleen [
For instance, tumor growth will induce the development of neovasculature characterized by discontinuous endothelium with large fenestrations of 200–780 nm [
Positively charged cationic polymers can effectively bind to and protect nucleic acids such as DNA, oligonucleotides [
Ideally, a successful delivery system should have a high degree of associating drugs. The siRNA loaded CS NPs showed higher entrapment efficiency (<90%) for all DS : CS weight ratios. The entrapment efficiency of nanoparticles at DS : CS weight ratio of 1 : 1, 1.5 : 1, and 2 : 1 was higher than the weight ratio of 0.5 : 1. This phenomenon was most probably due to higher proportion of DS presented in the nanoparticles. As more DS added, it would facilitate more BSA to be entrapped into nanoparticles. This could be explained by the fact that BSA is a zwitterionic molecule. At the pH of formulation medium of 3.5–4.0, the solubility of BSA could be highly increased due to increased positive charges possessed by it [
In the study, the electrostatic interaction was present between BSA and CS, instead of BSA and TPP. It was also suggested that BSA should dissolve in a solution with pH higher than its isoelectric point in order for BSA to possess negative charge and interact with the positively charged CS molecules. This finding therefore demonstrated that electrostatic interaction is the main contributing factor to promote the incorporation of BSA into nanoparticles either via CS-protein interaction or DS-protein interaction.
TEM allows nanoscale visualization of individual nanoparticles and provides information of both size and morphology. The particle morphology is an important factor for the colloidal and chemical stability as well as the bioactivity of nanoparticles. siRNA loaded CS NPs showed irregular morphology; however, BSA loaded CS NPs showed elongated morphology. This could be due to larger size of BSA which may entangle or act like a shield to CS, thus limiting the overall exposure of CS within structure.
Stability profile of CS NPs upon storage is also important. This information could provide a view about the stability of nanoparticles under different media and temperature. The stability of nanoparticles was investigated by assessing their variation in mean particle size and surface charge over time. At first, the nanoparticles were resuspended in distilled water at pH 6.6 which was filtered by 0.2
The
In summary, this study shows that CS and DS concentration as well as pH were the parameters controlling particle size and surface charge of CS NPs. Nanoparticles less than 500 nm could be obtained at DS : CS weight ratio of 0.5 : 1 at pH 4. In the case of BSA entrapment, nanoparticles with higher DS : CS weight ratios have possessed higher entrapment efficiencies of more than 88%. The highest percentage of entrapment efficiency achieved was at 0.10% w/v DS (DS : CS ratio of 1 : 1). However, CS NPs loaded with siRNA showed high entrapment efficiency (>90%) for all DS : CS ratios. Storage temperature and suspending medium were found to be the factors that could influence the stability of CS NPs. CS NPs were labile and tend to destabilize at ambient temperature but withhold this labile behavior when cool environment (2–4°C) was provided. In addition, CS NPs had better stability in distilled water than in PBS which might be due to hydrogen bonds that formed between water molecules and ionizable groups of CS NPs.
The authors declare that there is no personal or financial conflict of interests in the current research.
The authors gratefully acknowledge the “Dana Lonjakan Penerbitan” of Universiti Kebangsaan Malaysia (UKM-DLP-2011-001) for funding and supporting the current research project.