Prolonging The Survival Period Of Vascularized Composite Allotransplantation Using Sustained-release Nanoparticles

Objective:In clinical practice,many patients suffer from severe composite tissue injuries due to various causes,such as trauma,diseases,and surgery,which impact their quality of life.Traditional treatment methods involve using autologous tissue repair and replacement,which can not only cause donor site morbidity but also yield unsatisfactory results in terms of morphology and function due to inadequate matching.An ideal alternative is to apply vascularized composite allotransplantation(VCA)with anatomically and functionally consistent allografts.However,it is well known that composite tissues possess stronger antigenicity compared to solid organs(e.g.,heart,liver,kidney),and necessitating lifelong administration of high-dose immunosuppressive drugs.Prolonged use of these drugs brings inconvenience to patients’ lives and leads to a range of drug-related toxic side effects,potentially shortening their expected lifespan.This limits the treatment and development of VCA.To address these limitations,this study aims to develop a nanoparticle-based system with immunosuppressive,precise recognition,and sequential treatment capabilities,ultimately prolonging graft survival period.Methods:We performed vascularized composite allotransplantation surgeries on animals using microsurgical techniques.Postoperatively,we observed the changes in the grafts,documented them with photographs,and collected skin and muscle tissue samples at different intervals for hematoxylin and eosin(H&E)staining.When the animal models experienced acute immune rejection,we obtained skin and muscle tissue samples for mi RNA transcriptome sequencing.Bioinformatics analysis was conducted based on the sequencing results,and significantly differentially expressed mi RNAs were screened for further investigation of their functional mechanisms.Finally,the differentially expressed mi RNAs were validated using reverse transcription-quantitative polymerase chain reaction(RT-q PCR).We prepared MECA79-TAC/MMF nanoparticles and Anti CD25-MECA79-TAC/MMF nanoparticles using an emulsification method.The morphology of the nanoparticles was observed under a scanning electron microscope,and the particle size distribution was analyzed.Fouriertransform infrared(FTIR)spectroscopy was used to characterize the various components within the nanoparticles.Live-dead cell staining and the CCK-8 assay were employed to assess cell proliferation.FITC,phalloidin,and DAPI fluorescent dye were used to stain the nanoparticles,cytoskeleton,and nuclei,respectively,to confirm that cells can engulf the nanoparticles.We administered MECA79-TAC/MMF nanoparticles and Anti CD25-MECA79-TAC/MMF nanoparticles to animal models and recorded the survival periods of grafts in different groups.We documented the macroscopic appearance of the grafts with photographs and performed survival analysis.After treatment,tissue samples(skin and muscle)were collected at the terminal time point and subjected to H&E,Masson,and CD31 staining.Fluorescently labeled nanoparticles were injected into the animals,and an in vivo small animal fluorescence imaging system was utilized to observe the fluorescence intensity,verifying the targeting efficacy of the nanoparticles.Results:Following the completion of the surgical procedures,we successfully established animal models of vascularized composite allotransplantation.Tissue samples were collected at different intervals,and H&E staining of the skin and muscle tissues indicated the occurrence of acute immune rejection in the grafts,confirming the successful establishment of the animal models.Based on the mi RNA sequencing results of skin tissues,we identified9 upregulated and 13 downregulated mi RNAs.Bioinformatics analysis suggested that the upregulated mi RNAs were mainly concentrated in the cell periphery and plasma membrane,while the downregulated mi RNAs were primarily located in the cell periphery,playing roles in cellular response to stimuli,multicellular organismal processes,and signaling.The sequencing results of muscle tissues revealed 15 upregulated and 12 downregulated mi RNAs.The upregulated mi RNAs were mainly enriched in the intracellular,and were involved in protein binding and developmental processes,primarily associated with the PI3K-Akt/MAPK signaling pathway.The downregulated mi RNAs were mainly enriched in the intracellular,and involved in protein binding and regulation of cellular,highly related to the Hippo pathway.RT-q PCR validation revealed that rnomir-340-5p and rno-mir-21-5p were significantly upregulated,while rnomir-145-5p and rny-mir-195-5p were significantly downregulated in skin tissues.In muscle tissues,RT-q PCR validation showed that rno-mi R-21-5p and rno-mi R-340-5p were significantly upregulated,whereas rno-mi R-1-3p and rno-mi R-195-5p were significantly downregulated.Under the scanning electron microscope,both MECA79-TAC/MMF and Anti CD25-MECA79-TAC/MMF nanoparticles appeared spherical,uniformly dispersed,and at the nanometer scale.The diameter of the MECA79-TAC/MMF nanoparticles was 0.85 ± 0.04 um,while that of the Anti CD25-MECA79-TAC/MMF nanoparticles was 0.88 ± 0.04 um.Fourier-transform infrared spectroscopy revealed characteristic absorption peaks for Tacrolimus(TAC)、Mycophenolate mofetil(MMF)、Maleimide(MAL)、and Polylactic Acid(PLA)in the infrared spectrum of MECA79-TAC/MMF nanoparticles,confirming the presence of these four components.In the Anti CD25-MECA79-TAC/MMF nanoparticles,the infrared spectrum displayed characteristic absorption peaks for Poly(ethylene glycol)(PEG),TAC,MMF,MAL,and PLA,confirming the presence of these five components.After treatment with the nanoparticles,the average survival time of the grafts in the MECA79-TAC/MMF-NPs treatment group was extended to 108.00 ± 3.06 days,while that of the Anti CD25-MECA79-TAC/MMFNPs treatment group was extended to 131.30 ± 4.10 days.Histological staining revealed that,compared to other groups,both the MECA79-TAC/MMF-NPs and Anti CD25-MECA79-TAC/MMF-NPs treatment groups showed reduced inflammatory cell infiltration,increased collagen fiber deposition,higher microvessel density,and thicker vessel walls in the skin and muscle tissues.In the in vivo fluorescence experiment,both the MECA79-TAC/MMF-NPs and Anti CD25-MECA79-TAC/MMF-NPs treatment groups exhibited stronger fluorescence signals compared to the control group,with no significant difference between the two groups,indicating that both nanoparticles have a certain targeting effect.Conclusions:(1)The rat vascularized composite allotransplantation animal model was successfully constructed.(2)At the molecular level,rno-mir-340-5p and rno-mir-21-5p were significantly upregulated,while rno-mir-145-5p and rno-mir-195-5p were significantly downregulated in the skin tissues.In the muscle tissues,rnomir-340-5p and rno-mir-21-5p were significantly upregulated,and rnomir-1-3p and rno-mir-195-5p were significantly downregulated.(3)MECA79-TAC/MMF-NPs targeted drug-loaded nanoparticles exhibit good biocompatibility.Anti CD25-MECA79-TAC/MMF-NPs sequential drug-loaded nanoparticles also demonstrate good biocompatibility.(4)MECA79-TAC/MMF-NPs targeted drug-loaded nanoparticles can inhibit acute immune rejection and extend the survival time of rat hindlimb allografts,with an average survival time of approximately 108.00 ± 3.06 days.(5)Anti CD25-MECA79-TAC/MMF-NPs sequential drug-loaded nanoparticles can more effectively inhibit acute immune rejection in rat hindlimb allografts,resulting in a longer graft survival time,with an average survival time of 131.30 ± 4.10 days.