Synthesis Of NRP-1 Targeted Compound Exosomes And Their Imaging And Therapy Effect On Glioma

Aim Glioma is the most common intracranial primary malignant tumor,the incidence of which is rising year by year,with high disability and mortality rate.It is urgent to improve the diagnosis and treatment level.The main intractable problems are as follow: 1)the existing imaging methods are difficult to diagnose glioma in the early stage of tumor onset or recurrence,so patients lose the opportunity of radical operation and radiotherapy;2)the blood-brain barrier(BBB)hinders most of the antitumor drugs into the tumor tissue,leading to fail to reach the effective concentration,and the effect is a empty talk.The safe and BBB-crossing drug delivery vehicles(DDVs)hold great promise to overcome the above difficulties.Exosomes(Exos)has innate strong loading and BBB-crossing ability.It is a potential new type of brain targeting DDVs.In our study,the Exos derived from Raw264.7(a mouse macrophages cell line)were used as a carrier to modified with the neuropilin-1(NRP-1)targeted peptide(RGE)and loaded with curcumin(Cur)and superparamagnetic iron oxide nanoparticles(SPION).Thus,the glioma targeted engineered Exos named RGE-Exo-SPION/Cur was designed and prepared.This engineered Exos can cross the BBB and realize the targeted imaging and therapy of glioma.It can inhibit the growth of glioma through magnetic fluid hyperthermia(MFH),drug therapy and the synergistic effect of the two.This study verified the above hypothesis by experiments in vitro and in vivo,which provide new perspective and experimental basis for the diagnosis and treatment of glioma also for the application of Exos in tumor theranostics.Methods 1.Exos extraction and characterization Raw264.7 cell supernatant was collected as the source of Exos,using the sequential method of "ultrafiltration then differential ultracentrifugation" to extract the Exos.The extract was characterized by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),bicinchoninic acid(BCA),western blot(WB),and laser confocal microscopy(LCM).The shape,size,concentration,zeta potential,total protein content and characteristic membrane proteins were measured.The effects of p H value,storage temperature,freezing thawing times,storage time on Exos were observed by NTA.High throughput sequencing and affinity purification mass spectrometry were used to describe the profile of RNAs and proteins in this Exos.2.Preparation and characterization of RGE-Exo-SPION/Cur The preparation steps are as follows: 1)the sequential method of electroporation,click chemistry(azide cycloaddition reaction),RGE-Exo-SPION/Cur were prepared by method of “electroporation sequential click chemistry(azide cycloaddition reaction)”.Fluorescence microscope(FM),LCM,and flow cytometry(FCM)were used to verify the success of RGE peptide conjugation.The amount and concentration of the RGE polypeptide on the surface of the engineered Exos were calculated according to the absorbance of FITC(linked with RGE peptide with a fixed ratio).Then SPION was observed by TEM observation and Prussian blue staining(PLS).Cur was observed by LCM,to prove the successful loading of SPION and Cur.After that,the concentration of SPION and Cur was determined by phenanthroline spectrophotometry and UVS respectively.3.RGE-Exo-SPION/Cur evaluation of the targeting ability of glioma First,the expression of NRP-1 protein on U251 cells and Bel-7404 cells was observed by FM and WB.The target ability to glioma cells of RGE-Exo-SPION/Cur was evaluated by LCM and FCM,while the targeting ability to orthotopic xenografts was observed with the living animal fluorescence imaging.4.Targeted imaging function of RGE-Exo-SPION/Cur to glioma At the cell level,U251 and Bel7404 were collected after incubated with RGE-Exo-SPION/Cur for magnetic resonance imaging(MRI)to evaluate the enhancement effect.At the glioma xenograft level,RGE-Exo-SPION/Cur were injected into the tumor bearing mice through the tail vein,then MRI were scanned to show the size,border and enhancement effect of tumors.The inhibit effect of RGE-Exo-SPION/Cur on glioma was evaluated by calculating cell ability with CCK-8 and measuring tumor size by MRI,then compared with other groups to evaluate the target effect.5.Targeted therapy function of RGE-Exo-SPION/Cur to glioma In vitro,the cell viability was measured by CCK-8.In vivo,tumor size measured by MRI was used to calculate the tumor volume.Ex vivo,histopathological tissue slices were made to observe the ability of RGE-Exo-SPION/Cur to enter the tumor tissue(4 hours after the first injection)and the inhibitory effect on glioma(10 days after treatment),thus give a comprehensive evaluation on the targeted therapeutic effect of RGE-Exo-SPION/Cur on glioma.6.Safety evaluation of RGE-Exo-SPION/Cur and survival observation of tumor bearing mice 3 of each group were randomly selected at fifteenth days after the beginning of treatment,and the serum index such as creatine kinase-MB(CK-MB),aspartate transaminase(AST)and serum creatinine(Scr)were examined to evaluate the functional changes of the main organs(heart,liver and kidney),which indicate the safety of RGE-Exo-SPION/Cur in vivo.The survival period of the tumor bearing mice were recorded,and compared with other groups to evaluate the survival benefit of the RGE-Exo-SPION/Cur.Results 1.Under TEM,the extract contained round-like and cup-shaped vesicles of 50nm-150 nm(most are around 70 nm),with clear membrane structures and homogeneous size distribution.These characteristics were in line with Exos reported in the literature.WB analysis showed that the obtained precipitate expressed characteristic Exos membrane proteins,CD63 and CD81,NTA showed that the size distribution peak of Exos was 113.2 ± 4.2nm,the average zeta potential was-28.1 ± 3.6 mv,and the concentration was 4.4 x 1012/ m L.Approximately 1 mg of Exos(total Exos protein measured by BCA)could have been isolated from every 5×108 Raw264.7 cells.The fluorescence signal of CD47 antibody was detected by LCM,Flow cytometry also confirmed the expression of CD47 protein on the Exos.The evaluation of p H value,storage temperature,storage time and freezing-thawing cycles of suspension found that after storage of 24 h,Exos in PBS solution of p H=7.4 and 4 C was the most stable respectively.Exos concentration gradually decreased with the increase of freezing-thawing cycles and storage time,and decreased dramatically after more than 3 times,4 weeks later,the Exos suspended in p H=7.4 PBS solution and storaged at-80? have a minimum decline of concentration.The analysis of RNA omics and proteomics firstly verified the purity of Raw264.7 cells derived exosomes,furthermore,the results showed that the exosomes could be used as a drug carrier,which was in line with previous reports.However,it needs rigorous experimental design and careful observation on the possible biological side effect.2.RGE-Exo-SPION/Cur still had a round or round-like membrane structure,with a relative uniformity of size distribution,and SPION particles could be seen inside the Exos under TEM.There was CD63 and CD81 protein expression on the surface of RGE-Exo-SPION/Cur.The peak value of Exos size was 122.7 ± 6 nm,the zeta potential was-26.1 ± 3.2 mv,and the concentration was 1 x 1012/m L.The conjugation of RGE peptide and the loading of SPION/Cur were all verified,The concentration of RGE conjugated on the Exos was detected as 98 n M,and the number of RGE peptide connected to each Exos was about 52.The concentration of SPION was 4000 ?g ± 20 ?g/m L(1000 ?g Exos),and the concentration of Cur was 4000 ?g ± 35 ?g/m L.The gross morphology,quantity,size,membrane surface protein of Exos and concentration of SPION/Cur did not change significantly during the observation period(4 weeks).3.Both WB and FM showed that there was a high level of expression in U251 cells,while nearly no expression in Bel-7404.RGE-Exo can enter U251 cells more than free-Exo,rather than Bel-7404 cells.CM-Di I,free-Exo and CM-Di I labeled RGE-Exo were injected into the tumor bearing mice via tail vein respectively,then,We found that RGE-Exo was earlier(< 1h),longer(> 8h),and more(P < 0.05)gathered in the tumor,but the normal organs such as liver and spleen were less distributed and faster eliminated.Compared with free-Exo,the targeting ability of RGE-Exo to glioma was significantly enhanced.4.The negative contrast effect of MRI was enhanced with the increasing of intracellular SPION concentration in a certain range.Among all the treatment groups,RGE-Exo-SPION exhibited the most obvious enhancement effect on U251 cells iv vitro.When applied in vivo,RGE-Exo-SPION also showed a stronger enhancement effect than nontarget groups,which clearly showed the size and boundary of the tumor thus significantly distinguished tumor with the surrounding normal tissues.Meanwhile,the same effect was observed in the RGE-Exo-SPION/Cur group.5.As to inhibition test,in MFH treatment,RGE-Exo-SPION could targeted inhibit the growth of U251 cells and the effect was close to free-SPION group(P > 0.05),which was obviously stronger than free-Exo-SPION group(P < 0.05).In Cur treatment,the results were similar to those of the MFH group;While in MFH/Cur combined treatment,SPION mediated MFH and Cur mediated drug treatment both inhibited the growth of U251 cells significantly,and synergistic effect was found when they combined.The most significant inhibitory effect of was observed in RGE-Exo-SPION/Cur group(P < 0.05),and similar effect was also found on glioma xenografts(P < 0.05).RGE-Exo had no effect on tumor growth.Although tumors in group of free-Exo-Cur,free-Exo-SPION,free-Exo-SPION/Cur,free-SPION and free-Cur were slightly smaller than those in PBS and RGE-Exo groups,while the tumor size was still significantly larger than those of before treatment(P < 0.05)..6.During the treatment,without significant abnormality of CK-MB AST and Scr in the serum of tumor bearing mice,the RGE-Exo-SPION/Cur was considered more tolerable.As for survival period,the longest time was observed in RGE-Exo-SPION/Cur group,while in other treatment groups,the survival time did not prolonged than the control groups(PBS and RGE-Exo)because of the failure to reach the effective concentration in tumor tissue and the side effects due to insufficient targeting ability(P > 0.05).Conclusions 1.Exosomes extracted by the procedure of "ultrafiltration sequential differential ultracentrifugation" have the typical characteristics as reported,the abundant yield and purity can meet the needs of the follow-up experiments.Compared with separate ultrafiltration or differential ultracentrifugation,the combination of the two is more efficient,makes it being a feasible and efficient extraction method for the study with higher quantity and concentration requirements.The study found that the p H value,storage temperature,storage time and freezing thawing times are factors affecting exosomes.There is no significant change in the concentration,size,morphology and membrane protein when stored at-80 ? in the PBS of p H=7.4 for 4 weeks,during which,it was not suitable for repeated freezing and thawing(< 3 times).2.The integrity and characteristics of Exos remains well maintained in the RGE-Exo-SPION/Cur,which synthesized by the method of electroporation sequential clicking chemistry.After optimized reaction conditions,RGE-Exo-SPION/Cur could conjugate sufficient RGE peptides,and load adequate SPION and Cur with suitable proportions,all of which laid the foundation for subsequent targeting ability,imaging and therapeutic functions.In addition,the engineered exosomes are characterized as stable and easy to store.3.RGE-Exo-SPION/Cur enhanced the MRI contrast effect of glioma cells and orthotopic xenografts,which facilitate the diagnosis earlier and more accurate.RGE-Exo itself has no effect on the growth of glioma,while after loading of SPION and Cur,the engineered Exos(RGE-Exo-SPION/Cur)can exert strong inhibition effect both in vitro and in vivo,and more effective than SPION/Cur without Exos encapsulation and target modification.What is more worth mentioning is that RGE-Exo-SPION/Cur have good safety in vivo thus the advantage of tumor inhibition can be translated into benefit of survival time.