Preparation And Evaluation Of Plasmin-sensitive Hydrogel Microspheres

Objective This experiment tends to synthesize plasmin-sensitive polymer and prepare plasmin-sensitive hydrogel microspheres as well as sustained-release hydrogel microspheres, with myoglobin as model drug. The quality of microspheres were evaluated in vitro. The purpose of this experiment is to provide evidence for preparing plasmin-sensitive self-regulated drug delivery system.Methods1. Preparation and characterization of myoglobin PEGDA hydrogel microspheresPEGDA monomer was synthesized by chloride esterification and identified by ’H-NMR. PEGDA hydrogel were prepared by free radical solution polymerization using APS and TEMED oxidation-reduction initiator system to initiate the polymerization of PEGDA. The swelling ratio of the hydrogel was measured and calculated as volume fraction and mass fraction. PEGDA hydrogel microspheres were prepared through aqueous two-phase emulsion polymerization, with PEGDA solution being the dispersed phase,30% w/w of DextranT500 solution being the continuous phase, respectively. Microscope and SEM were employed to observe the morphology, particle size and particle distribution of the microspheres. With myoglobin as model drug, the drug loading and encapsulation efficiency of myoglobin sustained-release hydrogel microspheres were determined by UV-spectrophotometry. The in vitro release of myoglobin from microspheres were calculated by ELISA and the release profile was studied.2. Preparation and characterization of myoglobin plasmin-sensitive hydrogel microspheresH2N-GGVRNGGK-COOH was selected as the plasmin-sensitive substrate pep tide sequence. The peptide was synthesized by Fmoc solid-phase method. The pu rity and molecular weight of the peptide were identified by high performance liq uide chromatography (HPLC) and mass spectrometry (MS), respectively. The plas min-sensitive polymer ACRL-PEG-peptide-PEG-ACRL was synthesized by couplin g ACRL-PEG-NHS with peptide. MALDI-TOF was used to detect the molecular weight in order to identify the reaction product. Plasmin-sensitive microspheres were prepared through aqueous two-phase emulsion polymerization (ACRL-PEG-p eptide-PEG-ACRL/Dextran T500). Myoglobin as model drug, the drug loading an d encapsulation efficiency of microspheres can be determined by UV-spectrophoto metry. The in vitro release behavior was calculated by ELISA.Results 1. The result of 1H-NMR spectrum proved that the product was PEGDA and the average yield and esterification rate were 65.12% and 80.00%, respectively. Through free radical solution polymerization we have obtained colorless and transparent hydrogel. The equilibrium mass swelling ratio and volume swelling ratio of the hydrogel were 296.76% and 582.74%, respectively. Prepared by aqueous two-phase emulsion method, the microspheres obtained were round and smooth and in uniform particle size, with the average particle size being 46.73 μm. The Drug loading and encapsulation efficiency of microspheres were (4.41± 1.05)% and (77.21±2.66)%, respectively. In vitro release results showed that myoglobin released from PEGDA hydrogel microspheres slowly.75.98% was released in 5 days, with a burst of 54.52% release in 10hr. The release kinetics can be described with Peppas equation 1gQ=0.12171gt+0.4724, R2=0.9898.2. plasmin-sensitive substrate peptide was successfully synthesized. The amino acid sequence was H2N-GGVRNGGK-COOH. The purity and the molecular weight were 99.06% and 743.50, respectively. The result of MALDI-TOF proved the molecular weight conform the ACRL-PEG-peptide-PEG-ACRL. The microspheres were round and smooth and in uniform particle size with the average particle size was 50.56μm. The Drug loading and encapsulation efficiency of microspheres were (3.96±0.62)% and (73.83±0.67)%, respectively. In vitro release results showed that plasmin-sensitive hydrogel microspheres have sensitivity to plasmin.93.58% was released in 5 days, with a burst of 72.11% release in 10hr. The release kinetics meet Peppas equation lgQ=0.09581gt+0.4584, R2=0.9897。Conclusions 1. PEGDA with a relatively high esterification rate was prepared by chloride esterification method. PEGDA hydrogel and PEGDA hydrogel microspheres were obtained by free radical solution polymerization and aqueous two-phase emulsion polymerization. The preparation process of both the polymer derivative and the hydrogel microspheres is stable.2. Plasmin-sensitive hydrogel microspheres entrapped with the model drug myoglobin was obtained by free radical solution polymerization and aqueous two-phase emulsion. Experimental results showed that plasmin-sensitive microspheres release more drug under the present of plasmin, which provided experimental evidence for preparing plasmin-sensitive drug delivery system.