| Hyaluronan (HA) is a natural moisturizing factor that widely exists in livingorganisms in nature.1g of Hyaluronan can absorb water600g at least. HA iscomposed of disaccharide repeats of D-glucuronic acid (GlcUA) andN-acetylglucosamine (GlcNAc) joined alternately by b-1,3and b-1,4glycosidic bonds.It has unique physicochemical properties and biological functions. It finds variousapplications in the cosmetic, biomedical, and food industries. However, theenzyme-resistant properties of HA in vivo is really poor. Thus, its scope of applicationis lessened heavily. This paper is dedicated to obtain particles of cross-linkedhyaluronan gels which has wonderful ability of enzyme resistant and is easily to use.Firstly, the cross-linked hyaluronan hydrogels were prepared with differentcross-linking agents, such as polyethylene glycol (PEG), poly diglycidyl ether (PDE),1,4-butanediol diglycidyl ether (BDDE). Precipitate the cross-linked hyaluronate gelwith7-10times of95%alcohol in volume for two times, dry and crush the precipitateto grain or powder, re-dissolve it in121℃for15-20min partly. Then, this researchmeasures the range of particle diameter and study for its free polyethylene glycol andenzyme-resistant properties in vitro.Secondly, the particle gels were precipitated by anhydrous ethanol. Finally, thesupernatant which can be used for the determination of PEG residues was collectedthrough centrifugation. The standard solutions of PEG20000of concentrations at0,0.5,10.5,20.5,30.5,40.5μg/ml were prepared with the centrifugal supernatant fromthe precipitate of non-cross-linked sodium hyaluronate gel by anhydrous alcohol. Thecalibration curve was ploted and regression equation was established on the basis ofthe optical density of the complexes solution from the reaction of PEG with bariumion and iodide ion (1:1) with the content of PEG having linear relationship in therange of certain concentration at535nm. The precision of the method (n=9), therecovery rate with marker, and the reproducibility were tested and verified by theactual detection of PEG residues of SH-PEG gel. The reliability of the method wasfurther verified by the appearance percentage of higher R2values of the repeated plots of the working curve for35times.Thirdly, this paper studies on the effects of molecular of HA, particle diameter,crosslinking agent, the addition amount and time of lidocaine hydrochloride toenzyme-resistant properties in vitro of particles of cross-linked hyaluronan gels.The last but not least, this study obtain that the best volume range of95%ethanol is6.5-9times of the volume of cross-linked hyaluronate gel. The best dryingtemperature is45℃~60℃. The enzyme resistant of cross-linked particle gels isvery excellent. The particles were observed a uniform under the microscope. Thediameter of the maximum particle is400μm; the detection method of trace PEGcontained in cross-linked sodium hyaluronate gel has been established whichpossesses the properties of simple operation, quickness, excellent stability. Thedetection method has a excellent linear relationship in the range of0-40.5μg/ml. Theregression equation is Y=0.0108X+0.2047(R2=0.9994). The standard deviation of thedetection method is1.10%(n=9). The recovery rate with marker is97.8%and therepeatability is perfect; the result of enzyme-resistant properties in vitro of particlegels is BDDE-HA＞PDE-HA＞PEG-HA. The particle size has a smaller effect onenzyme-resistant properties in vitro. The antienzyme properties increase with theaddition amount of lidocaine hydrochloride. The time of lidocaine hydrochlorideaddition is also very important to the antienzyme properties in vitro.In conclusion, the preparation process of the particle gel is wonderful. It getswell antienzyme particles and easy to injection. This method can be not only appliedfor the trace detection of PEG residues contained in sodium hyaluronate gelcross-linked by PEG and the quality control of gel products, but also for the other gelscross-linked by PEG and liquid samples containing PEG. The method set thefoundation for the trace detection and product quality control with other cross-linkersin the process of gel production. |
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