Preparation And Characterization Of Protein-Polysaccharide Self-assembled Nanogels-based Drug Delivery Systems

Natural polymer materials based on proteins and polysaccharides exhibiting appealing properties such as safety, low or non-toxicity, biodegradability are extensively investigated as a drug delivery system in the pharmaceutical research. Nanoparticle drug delivery systems made from natural proteins and polysaccharides have outstanding advantages in the protection of premature degradation and against rapid metabolic activation, enhancement of absorption into a target tissue, improvement of intracellular penetration; reversion of multidrug resistance in tumour cells, restoration the tumoral cells'sensitivity to anticancer drugs and provision an affinity to tumor cell membrane. On top of that, nanocarriers based on proteins and polysaccharides have great practical application prospects in drug loading and delivery.Compared to the well-researched drug delivery systems, the nutraceutical delivery systems were studied less. In this research, four kinds of edible materials were selected, including low-density lipoprotein (LDL), bovine serum albumin (BSA), chitosan (CS) and water-soluble chitin (WSC), to fabricate LDL-CS and BSA-WSC nanocarriers through a simple self-assembly method. The optimum production conditions were established. After that, the formation mechanism of the nanogels was discussed. Nanoparticles were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Considering of difficulty of nutrient effect evaluation, doxorubicin hydrochloride was used as a drug model to study the loading behavior and anticancer activity in two kinds of nanocarriers. The objective of this research is to improve the effect of gastric cancer chemotherapy, and lay the foundation for the design, build and evaluation of nutraceutical delivery system.The main results were as follows:1. Nanoparticles were fabricated by self-assembly of LDL and CS. The optimal technology parameters and conditions were as follows:0.5 mg/mL lipoprotein aqueous solution was prepared before reacted with chitosan with the ratio 6:1 in a 120 r/min oscillating water bath for 2 h. After that, the pH of the system was adjusted to 2.5 with 1M HC1 before heated at ? for 45 min. The results of the analyses by DLS, TEM and AFM showed that the average particle size and ?-potential of LDL-CS nanoparticles was 160nm and+52.8 mV, respectively. The nanoparticles were very stable against long-term storage as the size distribution of the nanogels did not change within 90 days of storage at 4?. It was indicated that the protonated chitosan chains in acidic condition promoted the formation of strong electrostatic repulsion which could prevent the particles from agglomerating.2. Bovine serum albumin and water-soluble chitin were used as raw materials to prepare bovine serum albumin-chitosan nanoparticles (BSA-WSC) by self-assembly. The spherical nanogels with a diameter of 290 nm, PDI of 0.13 and ?-potential of+18.5 mV were obtained when mixing the albumin (0.5 mg/mL) and chitin solution (5 mg/mL) with a mass ratio of 4:1 before stirred for 60 min and heated at the 90? for another 60 min. After that, the system was stirred for another 30 min after the pH was adjusted to 5.95. According to the results of SEM?TEM?XPS and DLS, it could be proposed that nano-network structure was formed by the agglomeration between part of the chitin chain segments and BSA, leaving the unconjugated chitin chain being exposed on the surface of the nanogel.3. Both LDL-CS and BSA-WSC were used to entrap DOX with the entrapment rate being 51.7% and 46.3%, respectively. The particle diameter of the nanocarriers after encapsulation of DOX did not change obviously, maintaining a small narrow particle size distribution. Cytotoxicity experiments showed that the drug-loaded nanocarrier systems had a similar inhibitory effect with the bare drug on cancer toxicity. However, nanocarriers loaded with DOX showed less cytotoxicity than DOX alone. The IC50 values of free DOX, DOX-LDL-CS and DOX-BSA-WSC obtained by MTT assay in SGC7901 cells were 0.05-0.058,0.15,0.22 ?g/mL, respectively. The cytotoxicity of DOX decreased significantly within 24 h after it was encapsulated by the nanocarriers, indicating that the loaded drug was not 100% released within 24 h. The cellular uptake experiments indicated that the nanoparticles could be endocytosed by tumor cells and slowly release the loaded DOX to improve drug utilization. In addition, it was proven by flow cytometry and TUNEL assay that the DOX loaded in nanogels could induce the apoptosis of SGC7901 cells. The findings are expected to be used for the treatment of gastric cancer.