Biotin-Avidin System-based Sustained Delivery System Enhances The Therapeutic Performance Of MSC-Derived Exosomes

MSC-EXs is a promising approach to promote wound healing and tissue regeneration,with multiple functions including anti-inflammation,pro-migratory,pro-proliferation,pro-osteogenesis,and pro-angiogenesis.However,when used alone in vivo,the short half-life and rapid clearance of MSC-EXs often limits its therapeutic use.Inspired by the interaction of biotin-avidin system,we presented a facile and versatile method to immobilize MSC-EXs in the hydrogel via biotin-avidin system,thereby achieving sustained MSC-EXs release.First,Bio-Gel MA was fabricated by grafting NHS-PEG₁₂-biotin onto the amino groups of Gel MA.Then,biotinylated MSC-EXs（Bio-EXs）was synthesized using an in situ self-assembling biotinylation strategy,which provided enough binding sites for MSC-EXs delivery with little effect on their cargo composition.Thereafter,MSC-EXs was immobilized in Bio-Gel MA via streptavidin to generate Bio-Gel MA@Bio-EXs hydrogels.In vitro analysis demonstrated that Bio-EXs could be taken up by cells and showed immunomodulatory effects similar to MSC-EXs,and Bio-Gel MA@Bio-EXs hydrogels provided sustained release of MSC-EXs for 7 days.After subcutaneous transplantation,the retention of MSC-EXs in Bio-Gel MA@Bio-EXs hydrogels was observed for up to 28 days in vivo.When placed in an artificial periodontal multi-tissue defect,the functionalized hydrogels exhibited optimized therapeutic performance to regrow complex periodontal tissues,including alveolar bone,periodontal ligament（PDL）and acellular cementum.In this context,Bio-Gel MA@Bio-EXs hydrogels exhibited a robust immunomodulatory effect that promoted macrophage polarization toward an M2 phenotype.In this study,the biotin-avidin system was first applied to sustained delivery of MSC-EXs,enhanced the therapeutic effect of MSC-EXs,and promoted the clinical translation of MSC-EXs.Our findings suggest a novel approach to developing MSC-EXs-functionalized biomaterials for advanced therapies.PartⅠFabrication and Characterization of Bio-Gel MA[Purpose]Condition optimization for the synthesis of Bio-Gel MA with highest degree of biotinylation.[Methods]1.Bio-Gel MA was prepared through the grafting of NHS-PEG₁₂-biotin on the amino groups of Gel MA and purified by dialysis for 24 hours.2.By varying the reaction time and reaction ratio,a highest biotinylaion degree of Bio-Gel MA was achieved.3.The proton nuclear magnetic resonance（H¹NMR）spectroscopy and the fourier transform infrared（FTIR）were used to analyze the chemical structures of NHS-PEG₁₂-biotin,Gel MA and Bio-Gel MA,thereby confirming that biotin was successfully grafted onto Gel MA.4.The effect of biotinylation on the gelation behavior of Gel MA was determined by tube inversion.The effect of biotinylation on the structural morphology of Gel MA was characterized by scanning electron microscopy（SEM）.The mechanical property of Gel MA was characterized by uniaxial compression experiments.[Results]1.NHS-PEG₁₂-biotin had bound stably to Gel MA.After 24 hours of dialysis,the excess free biotin could be completely removed.2.The highest biotinylation degree of Bio-Gel MA was obtained when the NHS-PEG₁₂-biotin and Gel MA with a molar ratio of 20:1 were reacted for 8 hours.3.FTIR and H¹NMR demonstrated that NHS-PEG₁₂-biotin reacted effectively with Gel MA to form a stable amide bond and Bio-Gel MA was successfully synthesized.4.Tube inversion,SEM,and uniaxial compression experiments showed that biotinylation did not influence the UV-induced gelation properties,structural morphology,and mechanical properties of Gel MA.[Conclusion]Bio-Gel MA was successfully synthesized and retained its original properties.PartⅡSynthesis,Identification and Functional Verification of Bio-EXs[Purpose]Screening and optimization of the conditions for Bio-EXs preparation.Assessing the biological functions of Bio-EXs.[Methods]1.Isolation of primary rat bone marrow mesenchymal stem cells by plastic-adherent method.Flow cytometry and trilineage differentiation were used to identify MSCs.2.MSC-EXs was isolated using ultracentrifugation and characterized by nanoparticle tracking analysis（NTA）,transmission electron microscopy,Western blot,and nanoflow cytometry.3.MSC-EXs was incubated with gradient concentrations of DSPE–PEG–FITC or DSPE–PEG–biotin（10-100μmol/L）to obtain FITC/biotin modified MSC-EXs.The proportion of MSC-EXs positively labeled by DSPE–PEG–FITC/biotin was analyzed by nano flow cytometry and confocal microscopy.4.The effect of biotinylation on cellular uptake of MSC-EXs was examined by co-incubation of PKH26-labeled MSC-EXs or Bio-EXs with cells.5.The LPS-induced RAW264.7 macrophage line inflammatory model was used to examine the immunomodulatory functions of Bio-EXs by quantitative real-time polymerase chain reaction（q RT?PCR）,enzyme-linked immunosorbent assay（ELISA）and immunofluorescent staining.[Results]1.The primary MSCs was positive for CD29,CD44,and CD90 and are capable of adipogenic,chondrogenic,and osteogenic differentiation.MSC-EXs exhibited a cup-shaped morphology with a mean diameter of 40-220 nm,and were positive for CD63,HSP70,and TSG101.Nano-flow cytometry analysis showed that the percentages of MSC-EXs that positively expressed CD63 and CD81 were 56.16%and 65.36%,respectively.2.DSPE–PEG–FITC could self-assemble into the membrane of MSC-EXs,yielding54.93%、70.19%、80.61%and 84.19%FITC positive MSC-EXs following incubation with10、20、50、and 100μmol/L DSPE–PEG–FITC.DSPE–PEG–biotin could self-assemble into the membrane of MSC-EX via in situ self-assembling biotinylation strategy,yielding61.03%、80.20%、82.68%and 84.19%biotin positive MSC-EXs following incubation with10,20,50,and 100μmol/L DSPE–PEG–FITC.3.Both PKH26-labeled MSC-EXs and Bio-EXs could be taken up by macrophages.There were no significant differences in the uptake ratios between MSC-EXs and Bio-EXs.4.q RT?PCR analysis showed that incubation with MSC-EXs or Bio-EXs significantly decreased the expression levels of M1 polarization-related genes（IL-1α,IL-1β,and IL-6）while increasing the expression levels of M2 polarization-related genes（Arg-1 and IL-10）.Similarly,the ELISA data indicated that incubation with EXs or Bio-EXs significantly suppressed the production of M1-related cytokines（TNF-αand IL-1β）while promoting the production of M2-related cytokines（IL-10）.Immunofluorescence staining and quantitative analysis showed that incubation with MSC-EXs and Bio-EXs significantly downregulated the presence of M1-polarization-related proteins（i NOS and CCR7）while upregulating M2-polarization-related proteins（CD206）.[Conclusion]The primary MSCs was isolated and cultured successfully,and MSC-EXs was extracted successfully.Bio-EXs was successfully prepared with a biotinylation rate of 80%,and the biotinylation did not affect the uptake of MSC-EXs.MSC-EXs and Bio-EXs can both inhibit the polarization of proinflammatory M1 macrophage and promote the polarization of anti-inflammatory M2 macrophage.Biotinylation did not diminish the immunomodulatory functions of EXs.PartⅢConstruction of Sustained MSC-EXs Delivery System Based on Biotin-Avidin system[Purpose]Bio-EXs was immobilized in Bio-Gel MA using avidin to form Bio-Gel MA@Bio-EXs hydrogel which could sustainably deliver MSC-EXs.[Methods]1.Bio-Gel MA hydrogels carrying streptavidin plus MSC-EXs without biotin grafting（Bio-Gel MA@EXs hydrogels）were used as controls.The Bio-EXs and streptavidin were reacted at various molar ratios（1:1,2:1 and 3:1）to explore the influences of the biotin–avidin system on MSC-EXs retention in Bio-Gel MA@Bio-EXs hydrogels.2.MSC-EXs without biotin grafting or avidin-conjugated Bio-EX were labeled with PKH26.The hydrogels were incubated in PBS and the fluorescence intensity at specific times（1,4,7 days）is divided by initial fluorescence intensity to calculate retention rate.MSC-EXs release profiles from Bio-Gel MA@Bio-EXs hydrogels in vitro was determined by exosomes ELISA kit.3.MSC-EXs without biotin grafting or avidin-conjugated Bio-EXs were labeled with Di R.Retention of Di R-labeled MSC-EXs in vivo was analyzed by in vivo imaging at 0,1,4,7 and 28 days post-surgery.4.For visualization of the uptake of MSC-EXs in vivo,Bio-EXs and MSC-EXs were first labeled with PKH26 and then the prepared hydrogels were subcutaneously implanted as previously described.At 7 days post-surgery,the in vivo distribution of MSC-EXs or Bio-EXs released from the hydrogels was detected.5.The biocompatibility of Bio-Gel MA@Bio-EXs hydrogels was detected by HE staining of the full-thickness skins covering the transplanted hydrogels and major organs after 28 days of subcutaneous implantation.[Results]1.The fluorescence intensities in the Bio-Gel MA@EXs hydrogels group were lower than Bio-Gel MA@Bio-EXs hydrogels in vitro on days 1 and 4.On day 7,the fluorescence intensities of Bio-Gel MA@Bio-EXs（2:1）and Bio-Gel MA@Bio-EXs（3:1）hydrogels were still much higher than that of the Bio-Gel MA@EXs hydrogels group in vitro.2.The number of released EXs in the Bio-Gel MA@Bio-EXs（2:1）hydrogels group were significantly lower than that in the control group in vitro at days 1 and 4.3.Compared with the control group,the signal intensity increased significantly in the Bio-Gel MA@Bio-EXs（2:1）hydrogels group at 1,4,7 and 28 days post-surgery in vivo.4.Following implantation in mice for 7 days,in vivo MSC-EXs released from the Bio-Gel MA@EXs or Bio-Gel MA@Bio EXs hydrogels was phagocytosed primarily by macrophages.5.The representative images of HE staining showed the presence of implanted hydrogels with cell infiltration.Although fibrous tissues surrounding the implants could also be observed,no signs of systemic toxicity in the major organs of mice could be observed.[Conclusion]The biotin-avidin system could be used to prolong the release of MSC-EXs and enhance the retention of MSC-EXs when the molar ratio of Bio-EXs and avidin was 2:1.Moreover,the biotin–avidin system did not influence the biocompatibility of Gel MA.PartⅣRepair-Promoting Function of Bio-Gel MA@Bio-EXs Hydrogels with Sustained Delivery of MSC-EXs in Periodontal Defects[Purpose]Periodontal multi-tissue defects were established to evaluate the repair-promoting function of Bio-Gel MA@Bio-EXs hydrogels with sustained delivery of MSC-EXs to determine whether biotin-avidin system could enhance the therapeutic effect of MSC-EXs.[Methods]1.The periodontal multi-tissue defect model was established.The rats were divided randomly into different groups and treated as follows:（1）Blank control group;（2）Bio-Gel MA;（3）Bio-Gel MA@EXs hydrogels（containing 50μg of MSC-EXs）;（4）Bio-Gel MA@Bio-EXs hydrogels（containing 50μg of Bio-EXs）.2.The newly formed hybrid periodontal tissues including alveolar bone,periodontal ligament,and cementum in the periodontal multi-tissue defects at 7 and 28 days post-surgery were analyzed based on Micro-CT scanning,HE staining,and Masson staining.3.The polarization states of macrophages in the periodontal multi-tissue defects were analyzed by immunofluorescent staining at 7 days post-surgery.[Results]1.Periodontal multi-tissue defects were successfully established.Little bone formation could be observed in the periodontal multi-tissue defects at 1 week post-surgery.2.Compared with the defects without any implantation or the defects receiving Bio-Gel MA hydrogels after 28 days post-surgery,defects that received Bio-Gel MA@EXs hydrogels showed newly formed bone,decreased trabecular bone separation（Tb.Sp）,increased volume of bone/tissue（BV/TV）as well as trabecular bone thickness（Tb.Th）.Compared with Bio-Gel MA@EXs hydrogels,the BV/TV and buccal plate thickness（BP.T）in the Bio-Gel MA@Bio-EX group were higher and the highest among all tested groups.3.At 28 days post surgery,little bone formation and few newly oriented PDL could be observed in the defects without any implantation or the defects receiving Bio-Gel MA hydrogels,and the defect area was filled with connective tissue.Compared with the Blank and Bio-Gel MA groups,the Bio-Gel MA@EXs group showed newly formed alveolar bone and PDL.However,within the defects receiving Bio-Gel MA@EXs hydrogels,the newly formed alveolar bone surfaces were rough and serrated,and the PDL were disorganized.In contrast,more newly formed bone could be observed in the Bio-Gel MA@Bio-EXs group,and the surface of newly formed alveolar bone was relatively continuous and smooth compared to the Bio-Gel MA@EXs group.4.Compared with the defects without implantation or defects receiving Bio-Gel MA hydrogels,fewer numbers of CCR7+/CD68+or i NOS+/CD68+cells（M1 macrophages）were presented while cells positively stained for CD163+/CD68+or CD206+/CD68+cells（M2 macrophages）were frequently found in the defects receiving Bio-Gel MA@EXs or Bio-Gel MA@Bio-EXs hydrogels.Compared with the Bio-Gel MA@EXs hydrogels,the ratio of i NOS-positive M1 macrophages in Bio-Gel MA@Bio-EXs group was the lower and the lowest among the four groups,while the ratio of CD206-and CD163-positive M2macrophages in Bio-Gel MA@Bio-EXs group were higher and the highest among the four groups.[Conclusion]These findings strongly suggest that MSC-EXs exert immunomodulatory and regenerative effects across healing cascades and that the biotin–avidin system enhances their immunomodulatory and regenerative functions via the sustained delivery of MSC-EXs.