Preparation Of Monodisperse Chitosan Nanoparticles And Their Applications As Carrier System

Chitosan is a natural cationic biopolymer, which has important value in the application of drug carrier, owing to its biocompatibility, low toxicity, biodegradability, strong bioadhesive, and so on. The design and applications of chitosan based micro/nanoparticles drug delivery system have become one of the research hotspots in the interdisciplinary science between materials and biology. The important technological advantages of nanoparticles used as drug carriers are high carrier capacity, controlled release, high biological activity and well-designable. But as a kind of synthetic carrier, when nanoparticles used in human circulatory system, the body's immune system will quickly remove the nanoparticles from the circulatory system, which is not conducive to maintenance long-term blood-drug concentration and targete drug to the diseased tissues or organs.Erythrocytes carrier, as a biocompatible drug carrier system, has a significant longer circulation lifetime than other drug carriers. The main problems exist in erythrocytes carrier is that the lack of controllability of drug release. By using crosslinking agent to modify erythrocytes membrane can slow down the release of drug, however, this will usually destroy the normal physical structure of the erythrocytes. In other case, some drugs may cause damage to the structure of erythrocytes, all these limit the application of erythrocytes as long circulation lifetime carrier.In this paper, we creatively combine chitosan nanoparticles and erythrocytes carrier. By loading chitosan nanoparticles into erythrocytes or making nanoparticles adhesive onto erythrocytes membrane surface, we may take full advantage of both chitosan nanoparticles and erythrocytes and avoid their weaknesses, intend to achieve the purpose of long-term and controlled release of drug. Explore the feasibility and potential applications of this novel erythrocytes-chitosan nanoparticles drug carrier system.This paper first reviewed the preparations and applications of chitosan particles, erythrocytes and erythrocytes-particles drug carrier systems. In the experiment part, we successfully prepared the monodisperse low molecular weight chitosan nanoparticles by using a modified method of ionic crosslinking. Through the use of dynamic light scattering particle size analyzer, we system investigated the affects of a variety of experimental variables on particle size and distribution of chitosan particles, and we also analyzed the reasons for these effects from the points of molecular mechanism. The investigation of experimental variables included concentration of chitosan solution, concentration of sodium tripolyphosphate (TPP) solution, mass ratio of chitosan/TPP, pH value of chitosan solution, stirring speed, temperature of chitosan solution, concentration of acetic acid and environmental temperature during the crosslinking reaction. By the further use of transmission electron microscopy (TEM), we found that chitosan nanoparticles prepared at the optimal conditions had an average hydrodynamic diameter of 138nm, zeta potential of 35mv and the particles had good monodispersity, of which the polydispersity index (PDI) could be controlled below 0.05, significantly lower than that reported in current literatures. We first found that at least for the low molecular weight chitosan, by reducing the concentration of acetic acid solution, we could significantly improve the monodispersity of chitosan/TPP nanoparticles. Moreover, we first found that environmental temperature has a significant effect on the monodispersity of chitosan nanoparticles, that is low environmental temperature is helpful to improve the monodispersity of chitosan nanoparticles.In order to investigate the feasibility of applying the chitosan/TPP nanoparticles in erythrocytes carrier, we researched the hemolysis rate and the erythrocytes aggregation behavior caused by chitosan nanoparticles. The results showed that in the test nanoparticles concentration range, the hemolysis rate was far less than 5%, which meets the requirements of medical application materials. On the other hand, it was found that the erythrocytes aggregation behavior relyed on the ratio betwwen erythrocytes and nanoparticles, that is excess of chitosan nanoparticles would lead to erythrocytes aggregation, while an appropriate amount of chitosan nanoparticles would not cause the aggregation of erythrocytes.Studies have shown that the positive charged protonated amino groups in chitosan could form electrostatic interactions with negatively charged glycoprotein (sialic acid) in erythrocytes membrane surface. However, sialic acid play an important role in avoiding erythrocytes aggregation by keeping erythrocytes surface negatively charged. Therefore, in order to avoid erythrocytes aggregation, we reduced the positive potential of chitosan nanoparticles and the ratio of nanoparticles/erythrocytes. In further experiments, fluorescein isothiocyanate (FITC) was labeled at the amino groups of chitosan and FITC-chitosan nanoparticles were prepared. By the use of Laser Scanning Confocal Microscope (LSCM), we investigated the feasibility of loading chitosan nanoparticles into erythrocytes or making nanoparticles adhesive onto erythrocytes membrane surface.