Preparation And In Vitro Release Study Of Rhegf/Chitosan Nanoparticle-Loaded Fibrin-Binding Amniotic Membrane

ObjectiveTo synthesize a drug-loaded amniotic membrane, i.e. rhEGF/chitosan nanoparticle-loaded fibrin-binding amniotic membrane, and investigate its drug release kinetics in vitro.Method The method included four steps:1. rhEGF-loaded chitosan nanoparticles were prepared and their particle size, zeta potential, polydispersity index and encapsulation efficiency were examined.2. The stability of the encapsulated rhEGF in the rabbit conjunctival homogenate was evaluated and compared with that of the free rhEGF.3. The rhEGF-loaded chitosan nanoparticles with the maximal encapsulation efficiency were added into the thrombin solution. Then the thrombin solution was mixed and reacted with the fibrinogen solution which was placed on the surface of a piece of amniotic membrane to get a rhEGF/chitosan nanoparticle-loaded fibrin-binding amniotic membrane (rhEGF/CS-FBAM). The in vitro kinetic of rhEGF release from the rhEGF/CS-FBAM were investigated and compared with that of rhEGF/CS nanoparticles and rhEGF-FBAM, respectively.4. The rhEGF released from the rhEGF/CS-FBAM was added to cell culture medium and its bioactivity was assessed in vitro by determining its ability to stimulate the proliferation of BALB/c 3T3 cells. As positive control, soluble rhEGF was added daily to the cell medium in amounts equivalent to those released from the fibrin clots. Cell culture in the basal medium without rhEGF served as a negative control.Result1. The average particle size of the prepared nanoparticles ranged from 264.0±4.2 to 288.6±2.8 nm. The zeta potential ranged from 31.3±0.5～43.7±0.8 mV. The polydispersity index ranged from 0.19±0.02～0.26±0.02. The encapsulation efficiency (%) ranged from 54.87±2.94～67.03±1.22.2. The recovery of encapsulated rhEGF was higher than that of free rhEGF after incubation in the rabbit conjunctival homogenate with in 1, 2, 3, 4, 5, 6 h (P< 0.05).3. The release of rhEGF from the rhEGF/CS nanoparticles sustained for approximately 60 h with a burst release at the initial phase. The release of rhEGF from the rhEGF-FBAM sustained for approximately 96 h. Compared with the two systems above, the rhEGF/CS-FBAM released rhEGF more gradually and more steadily and had a release period of approximate 14 d. Moreover, as the concentration of fibrinogen or thrombin was increased in the rhEGF/CS-FBAM, the release of rhEGF from the rhEGF/CS-FBAM would be slowed.4. Significant increases in optical density were seen in the experimental group and positive control compared with the negative control (P < 0.05). There were no differences between experimental group and positive control (P> 0.05).ConclusionIn this study, we successfully developed a novel kind of composite biomaterial by combining rhEGF/CS nanoparticles, fibrin gel and amniotic membrane. It possesses the properties below: (1) chitosan nanoparticles could protect rhEGF and improve its stability; (2) as a drug depot, fibrin gel could release rhEGF for a long time in a sustained and controllable way; (3) amniotic membrane could act as a basal membrane when used in wound healing. As a kind of biomaterial, rhEGF/CS-FBAM could efficiently release therapeutic growth factor at the same time when used in wound site, thus promote wound healing.