Study On Rapamycin And CsA Drug Delivery System For Prevention Of Rabbit High-risk Corneal Allograft Rejection

ObjectiveEstablish rapamycin(RAPA) and cyclosporine A(CsA) Drug deliver system and the model of rabbit high-risk corneal allograft rejection;study the drug concentration in the aqueous humor,biocompatibility and how long been on the eye ball surface of rapamycine drug deliber system; study the immunosuppressive effect and mechanism of different therapeutic modalities and immunosuppressants drug deliber system in the corneal allograft of rabbit model.Methods1.Rapamycin-loaded PLA/Cs-Chol particles and rapamycine or cyclosporine A-loaded PLA/PEG wafers were prepared respectively.The comparion of encapsulation ratio, encapsulation yield and in vitro release of drug deliver system were measured by UV method. How long RAPA nano-particles on eye ball surface with SPECT.2.Thirty six New Zealand rabbits(36eyes) were randomly divided into4groups to receive the4different therapeutic modalities and observed4weeks. study RAPA releasing and biocompatibility.3.A was Syngeneic control whose both donor and recipient are health newzeland rabbit. Gray donor corneas were implanted into the105recipients of New Zealand albino rabbits with corneal neovascularization who were randomly divided into B,C,D,E,F,G,H7groups to receive the diferrent types of therapy:B was no therapy control;C was eye drop of nanometer vector but no RAPA twice a day,28days;D1%RAPA eye drop three times a day for28days; E was0.5%RAPA eye drop of nanometer vector twice a day,28days;F was PLA wafers of RAPA in the anterior chamber of rabbit eyes;G was PLA wafers of CsA in the anterior chamber of rabbit eyes; H was PLA wafers of CsA in the anterior chamber of rabbit eyes and 0.5%RAPA eye drop of nanometer vector eye drop twice a day for28days together.Postoperative evaluation included slit-lamp biomicroscopy,histopathology and immunohistology,Cytokines related with neovascularization and immunosuppression in the corneal tissue and the peripheral blood by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA),respectively.Results1.Rapamycin-loaded PLA/Cs-Chol particles that the particles were of regular round shape with smooth surface and narrow size distribution.Using chitosan-g-cholestorol amphiphilic polymer as surfactant with different condition to produce different particles.With the Cs-Chol concentration increased drug particles encapsulation ratio and encapsulation yield increased but narrow size;With the RAPA concentration increased drug particles encapsulation yield increased but encapsulation ratio not changed;When use different ratio Ch2Cl2and acetone as oil solvent with Ch2Cl2ratio increased;With molecular weight of PLA increasing drug particle yield gently but drug particle diameter increased.Drug particle releases,dash fast with RAPA content increased of drug particle;Drug particle releases fast with drug particle diameter decreased;Nano-particle can prolong drug on the surface of eye ball.2.Releasing speed of drug wafers with PEG is fastter than the one without PEG;Drug releasing speed of wafers increased with PEG increasing in PLA wafer.3.RAPA drug-deliver system have a good biocompatibility with rabbit eyes. Compared experiment with control group we found slit-lamp biomicroscopy,tonometry,coneal endothelium count, Histopathology,coneal endothelium scan electric-biomicroscopy,retina transmission electric-biomicroscopy, len anterior capsule scan electric-biomicroscopy have no difference.4.Both0.5%RAPA nano-particle eye drop and RAPA wafer supply the RAPA concentration in the aqueous humour;0.5%RAPA nano-particle eye drop can supply a stable one.5.The mean survival time of A group was the longgest(100±0day).the mean survival time of group E (44.8±8.0) day、F (43.3±6.7) day、G (44.1±10.2) day and H (76.9±14.8) day was a statistically significant prolongation compared with group B(10.9±1.7)day,C (11.7±2.8) day and D(17.78±8.41) day(P＜0.05). However,Group H was significantly superior than the group E,F,G (P＜0.01)and group E,F,G was superior than the group B,C,D(P＜0.05).There were no difference in group E,F,G and so were group B,C,D (P＞0.05).Conclusions1.To study the all kind of drug concentration,paticle diameter,producing condition we found that40.2%and300nm RAPA nano-particle matched our request.2.Cyclosporine-loaded or Rapamycin-loaded PLA/PEG wafers were study showed suitable PLA to PEG was50:1(W/W).3.RAPA and drug-deliver system have a good biocompatibility with rabbit eyes.0.5%RAPA nano-particle eye drop can supply a stable RAPA concentration in the aqueous humour.4. RAPA wafer,CsA wafer,0.5%RAPA nano-particle eye drop could prevent acute allograft rejection of rabbit corneal transplantation.5.Combination therapy was significantly superior than single therapy but inferior to the syngeneic group.