RGD-L-TRAIL-labeled Magnetic Microbubbles For Colon Cancer Molecular Imaging And Treatment

In recent years,magnetic nanoparticles(MNPs)and microbubbles(MBs)have come to be used as effective imaging contrast agents and drug carriers,respectively.MBs loaded with MNPs can act as novel carrier materials that exhibit the strengths of both MBs and MNPs.Given that they have both excellent magnetic and acoustic properties,they are increasingly being used as an integrated multifunctional platform for dual-mode ultrasound(US)/magnetic resonance imaging(MRI)as well as the targeted delivery of genes and various drugs.The accurately and efficiently targeted delivery of therapeutic/diagnostic agents into tumor areas in a controllable fashion remains a big challenge.Herein,a novel cancer targeting magnetic microbubbles were elaborately fabricated.Firstly,the ?-Fe2O3 magnetic iron oxide nanoparticles(MNPs)were optimized to chemically conjugate on the surface of polymer microbubbles.Then,Arginine-Glycine-Aspartic acid(RGD)-L-Tumor necrosis factor-Related Apoptosis-Inducing Ligand(RGD-L-TRAIL),antitumor targeting fusion protein,were precisely conjugated with magnetic nanoparticles of microbubbles to construct RGD molecularly targeted magnetic microbubble(MMB),which was defined as RGD-L-TRAIL@MMB s.Such RGD-L-TRAIL@MMB s have been endowed with the multi-gradient cascade targeting ability following by magnetic targeting,RGD as well as enhanced permeability and retention effect regulated targeting to result in the highly cancerous tissue targeting efficiency.Due to the highly specific accumulation of RGD-L-TRAIL@MMBs in tumor,the accurate diagnostic information of tumor can be obtained by dual ultrasound and magnetic resonance imaging.After imaging,the TRAIL molecules as anticancer agent also get right into the cancer cells by nanoparticle-and RGD-mediated endocytosis to effectively induce the tumor cell apoptosis.Therefore,RGD-L-TRAIL conjugated magnetic microbubbles could be developed as molecularly targeted multimodality imaging delivery system with the addition of chemotherapeutic cargoes to improve cancer diagnosis and therapy.The following points are mainly involved in the dissertation:1.First,PVA was modified to obtain telechelic PVA with carboxylic groups at the chain ends.The modified PVA was used to fabricate MBs present carboxyl on their surface,thus not only improving the stability of MBs,but also creating the opportunity for MBs to couple with other active substances.Next,The particle size and stability of MBs are regulated through adjustments the three factors,i.e.the concentration of PVA aqueous solution,the water-oil ratio of the first emulsification and the rate of the second emulsification during the preparation process.In vitro/vivo US imaging experiment was performed on the optimized MBs,which have a average particle size of 1.38?m,uniform particle size distribution and good stability.This study lay a good foundation for the application of MBs in vivo.2.The MMBs were fabricated by assembling MNPs in different concentrations on the surfaces of MBs by the "surface-coating" approach.The relationships between the structure,magnetic properties,stability of the MMBs,and their use in magnetic resonance/ultrasound(MR/US)dual imaging applications were determined.An analysis on the underlying mechanism showed that the synergistic effects of covalent coupling,electrostatic adsorption,and aggregation of the MNPs allowed them to be unevenly assembled in large amounts on the surfaces of the MBs.In vitro MR/US imaging experiments showed that with an increase in the number of MNPs on the surfaces of the MBs,the MMBs exhibited better T2 MR imaging contrast,as well as an increase in the US contrast for longer durations.In vivo experiments also showed that,by optimizing the structure of the MMBs,enhanced MR/US dual-modality image signals could be obtained for mice tumors.3.Molecularly targeted microbubbles for cancer theranostics was elaborately prepared by conjugating the Arginine-Glycine-Aspartic acid(RGD)-L-Tumor necrosis factor-Related Apoptosis-Inducing Ligand(RGD-L-TRAIL)protein on the surface of SPIO loaded magnetic microbubbles.The particle size,hysteresis loop,microscopic structure and stability of RGD-L-TRAIL-labeled magnetic microbubbles were investigated,the optimal amount of RGD-L-TRAIL protein conjugated on the MMBs was detailed to study to determine the most suitable protein coupling.4.In vitro US and in vitro MR imaging were carried out using RGD-L-TRAIL@MMBs,it is concluded that RGD-L-TRAIL@MMBs enhanced US imaging for approximately 5 min in vitro experiment.The decreased T2 signal value demonstrated the RGD-L-TRAIL@MMBs remained its suitability as a contrast agent for T2 enhancement of MRI.It was confirmed hat RGD-L-TRAIL magnetic microbubbles molecular probe can enhance ultrasound/magnetic resonance imaging.5.The in vitro tumor cell targeting and apoptosis-inducing capabilities of RGD-L-TRAIL@MMBs were confirmed using COLO-205 colon cancer cells treated with MMBs,RGD-L-TRAIL,or RGD-L-TRAIL@MMBs respectively.The result demonstrated that apoptosis from RGD-L-TRAIL@MMBs group is comparable to the RGD-L-TRAIL group.This evidence further indicated that after conjugation of RGD-L-TRAIL on the structure of MMBs,the biological activity of RGD-L-TRAIL has been maintained.6.To evaluate the in vivo US targeting imaging effect,colon tumor bearing nude mice were injected intratumorally or via the caudal vein with MMBs or RGD-L-TRAIL@MMBs and then imaged by US.Although there is difference between intratumoral and intravenous injection,the RGD-L-TRAIL@MMBs maintaining the US enhancement for more than 10 min exhibited the good targeting efficiency of RGD-L-TRAIL@MMBs to tumor neovasculature.Similarly,the in vivo MR targeting imaging enhancement was verified.Regardless of the mode of injection,changes in T2 signal strength were more pronounced by injection of RGD-L-TRAIL@MMBs as compared to MMBs.These MRI results are consistent with those obtained by US imaging and confirmed that RGD-L-TRAIL@MMBs are also an effective MRI contrast agent.In addition,the distribution of RGD-L-TRAIL@MMBs in tumor tissue was assessed by staining tumor tissue sections with Prussian blue to localize SPIO and by immunohistochemical(IHC)detection of TRAIL expression.This result also indirectly proved the effective coupling of RGD-L-TRAIL and SPIO in the structure of microbubbles and the tumor targeting of RGD-L-TRAIL@MMBs.7.At last,the tumor therapeutic efficacy was tested to evaluate the in vioo therapeutic effects of RGD-L-TRAIL@MMBs on colon cancer.The results clearly demonstrated that injection of RGD-L-TRAIL or RGD-L-TRAIL@MMBs could inhibit the growth of colon cancer xenografts.Whereas MMB-treated tumors did not differ significantly from the control group.The results are in agreement with the tumor cell apoptosis in each group with the TUNEL assay.