The objective of this study was to investigate the potential of water soluble chitosan as a carrier in the preparation of protein-loaded nanoparticles. Nanoparticles were prepared by ionotropic gelation of water-soluble chitosan (WSC) with sodium tripolyphosphate (TPP). Bovine serum albumin (BSA) was applied as a model drug. The size and morphology of the nanoparticles were investigated as a function of the preparation conditions. The particles were spherical in shape and had a smooth surface. The size range of the nanoparticles was between 100 and 400 nm. Result of the in vitro studies showed that the WSC nanoparticles enhance and prolong the intestinal absorption of BSA. These results also indicated that WSC nanoparticles were a potential protein delivery system.
Peptides and proteins have become the drugs of choice for the treatment of numerous diseases as a result of their incredible selectivity and their ability to provide effective and potent action [
Nanoparticles have received much attention for the delivery of macromolecular drugs, such as peptides, proteins, and genes, due to their ability to protect protein and peptides from degradation in the gastrointestinal track by proteolytic enzymes [
Chitosan received attention as a material for nanoparticles for the decade [
Despite of its superiority as a biomaterial, chitosan is not fully soluble in water and then soluble in acidic solution. Aqueous solubility of chitosan only in acidic solution because of its rigid crystalline structure and the deacetylation which limits its application to bioactive agents such as gene delivery carriers, peptide carriers, and drug carriers. Water-soluble chitosan is easily soluble in neutral aqueous solution. Its advantage is ease of modification, useful as gene or peptide drug carriers. Therefore, water-soluble chitosan and functional property have been developing for pharmaceutical and new drug candidate.
Therefore, the major goal of the present study is to create a kind of new biodegradable nanoparticle as protein delivery system. The nanoparticles have been characterized in terms of size, polydispersity index, and association efficiency. Also in vitro release was investigated to determine the efficacy of this system and the effective factors on absorption via this system.
WSC (degree of deacetylation 87%, molecular weight 21 kDa) was made from a shrimp shell and obtained from Shandong Aokang Biotech Ltd. (Shandong, China). BSA with Mw 68 kDa and Coomassie brilliant blue G250 were purchased from Sigma Chemical Co. (USA). All other reagents and solvents were of analytical grade.
WSC–BSA nanoparticles were prepared by methods adapted from that reported by Calvo et al. [
The particle size and size distributions of the nanoparticles were performed by particle sizer (Zetasizer 3000 HAS, Malvern Instruments Ltd., Worcs, UK). Chitosan nanoparticles separated from suspension were dried by a freeze dryer; their FTIRs (Shimadzu, FT-IR 8700, Japan) were taken with KBr pellets on Perkin-Elmer Spectrum one FTIR.
To determine the encapsulation efficiency and loading capacity, nanoparticles with the different formation were centrifuged at
BSA release from chitosan nanoparticles was determined as follows. A known amount of freeze-dried nanoparticles was transferred to a 25 mL tube and 10 mL of the respective dissolution buffer was added to the tube. The temperature and rotation were adjusted to 37°C and 90 rpm, respectively. At predetermined time of 0.5, 2, 4, 6, 8, 10, 12, and 24 hour 5 mL of sample was removed and ultracentrifuged at
The structure of BSA-loaded lyophilized nanoparticles was examined by TEM (Tokyo, Japan). They showed that the particles have a uniform spherical shape and smooth surface (Figure
(a) Particle size distribution of WSC nanoparticles. (b) TEM image of WSC nanoparticles.
FTIR of BSA nanoparticles, WSC nanoparticles, WSC and BSA.
In most nanoparticle delivery systems, the drug carrying capacity is defined as an encapsulation efficiency. In the present study, BSA was carried on the nanoparticles via the ionic interaction. In water, the long hydrophilic chains extended to the water, and some BSA might be encapsulated among the positive hydrophilic chains, which indicated that the BSA was not only on the surface of the nanoparticles but also was distributed in the outer hydrophilic area. So, the BSA carrying capacity of the nanoparticles could be termed as encapsulation efficiency. In this study, BSA encapsulation efficiency from 40% to 95% was significantly affected by the initial BSA concentration in Figure
The influence of BSA initial concentration on encapsulation efficiency (WSC 1 mg/mL, TPP 1 mg/mL,
The influence of BSA initial concentration on loading capacity (WSC 1 mg/mL, TPP 1 mg/mL,
When TPP concentration was 1 mg/mL, decreasing chitosan concentration down to 0.5 mg/mL, aggregates with large diameter were formed; increasing chitosan concentration up to 4 mg/mL made encapsulation extremely difficult. Formation of nanoparticles is only possible for some specific concentration of chitosan and TPP. As for gelation between TPP solution of 1 mg/mL and chitosan solution of 1–3 mg/mL, we usually observed that some opalescent suspension was formed, which was examined as nanoparticles. Adding TPP solution of 1 mg/mL to chitosan solution of 4 mg/mL, firstly we observed some opalescent suspension and then disappeared immediately, and the nanoparticles formation was extremely difficult. Figure
The influence of chitosan concentration on BSA encapsulation efficiency (BSA 0.5 mg/mL, TPP 1 mg/mL,
Many studies have reported that the quantity of TPP in a given formulation has a significant effect on the protein encapsulation and characteristic of NP [
The influence of TPP concentration on BSA encapsulation efficiency (WSC 1 mg/mL, BSA 0.5 mg/mL,
On the basis of above result, TPP did not affect the entrapment efficiency of BSA-chitosan nanoparticles when nanoparticles were formed at optimal chitosan/BSA mass of
The preliminary protein release test from BSA nanoparticles in vitro proves that they have a sustained release form as shown in Figure a first initial burst release of 30%, due to the drug desorbed from the particles surface, a plateau for the following 8h, resulting from the only diffusion of the drug dispersed in the polymermatrix, a constant sustained release of the drug, resulting from the diffusion of the protein through the polymer wall as well as its erosion.
BSA release (WSC 1 mg/mL, BSA 0.5 mg/mL, TPP 1 mg/mL).
In summary, novel ionic crosslinking nanoparticles composed of WSC and TPP loaded with BSA were successfully prepared. The size distribution was in the range of 200–400 nm. The nanoparticles were spherical shape and smooth surface. WSC nanoparticles showed higher protein encapsulation efficiency. In addition, the BSA release from the nanoparticles was sustained release. Therefore, WSCs have a potential carrier in a controlled drug delivery system for protein drugs.