Agarose Grafting Hyaluronan Copolymer As Peptide Carrier

Agarose(abbr Ag) is a naturally polysaccharide derived from red-purple seaweed.To find its new features and broaden its application,the key work is to tailor agarose to the need in biomedical fields.The major drawbacks of agarose are that it is slowly degraded and has no specific binding motif for protein or cell.So it is necessary to lower molecular weight of agarose for easier solution at lower temperature or quicker degradation,and composite with some quickly degradable polymer for promoting its degradation rate,or with some bioactive agents(such as hyaluronan,abbr HA) to provide advantages in specific interaction with protein, peptide or cell.Hyaluronan not only can bind to cell surface CD44 receptor,but also can be used as a component to induce the ionic interaction with peptide for efficient loading and sustained release.In addition,peptides can keep their activation when complexing with hydrophilic polymer by a physical loading process.Agarose grafting hyaluronan copolymer might be a promising carrier for protein,peptides or a regenerative tissue scaffold..Agarose was degraded by hydrogen peroxide to lower molecular weight,then activated with epichlorohydrin,and had epoxy group content of over 1.42 mmol/g, then grafted with 5.7 kDa hyaluronan to obtain agarose grafting hyaluronan copolymer(Ag-g-HA).Results of IR and EA experiments proved that HA was succeedly grafted into agarose chain and the grafting ratio was changed with the adding amount of HA.The result of the implanted experiments Showed that Ag-g-HA copolymer with HA content 37.7%totally degraded in 28d,but the agarose degraded with H₂O₂ for 8h only lost 23.65±3.33%under mice subdermis.Results of preliminary biocompatibility evoluations disclosed that the copolymer had no pyrogen reaction and no irritant toxicity reaction,haemolysis rate being less than 5%.It is concluded that Ag-g-HA copolymer is a degradable biomaterial with good biocompatibility.As an acidic peptide model,insulin was capsuled into Ag-g-HA copolymer microparticles by an ionic interaction process,and its release behavior in the copolymer was tested in different pH environments.Using Caco-2 cell monolayer model,we also evaluated if the copolymer enhanced insulin transport effects or not in vitro.The copolymer could load 40%weight percentage of insulin and capsule 80% of insulin added into solution.It might disclose that the gel effect of agarose chain in the copolymer does faver to increasing capsulation rate of the copolymer to insulin. Results suggested that the stability of the insulin loaded Ag-g-HA complexes in distinct pH environments significantly affected their insulin release profiles and these microparticles had sustained-release effect in the environment of pH 6.8 with a complete release within 3 h.The result of Caco-2 cell model experiment showed that Ag-g-HA could specificly enhance insulin transportation.It means that HA maintains its biological effect in the copolymer.As a basic peptide model,basic fibroblast growth factor(bFGF) was loaded into Ag-g-HA sponge for accelerating wound skin healing with an ionic interaction process.Water uptake ratio of the sponge was 20 to 22 times.Results of in vitro measurements showed that the release of bFGF in the sponge was sustained over 14 days and the cumulative release percent was 41.42±5.07%.Also bFGF could be effectively detected in mice plasma which proved again that the bFGF loading sponge could sustainedly release bFGF.MTT test result revealed that the sponge had good biocompatibility and bFGF in it remained active to accelerate cell proliferation. Animal experiments disclosed that the Ag-g-HA sponge loading 5μg/mL bFGF was excellent one for accelerating wound skin healing,and most f tissues in skin are regenerated.In conculsion,the degradable Ag-g-HA copolymer is a safety biomaterial,and is convienent to load peptides.It has the potential to be a novel peptide carder and be widely applied in biomedical fields.