Synthesis Of Manganese-based Nanoparticle System With Enhanced Tumor Microenvironment Responsivity And Their Theranostic Application In Glioblastoma

Background and ObjectiveGlioblastoma?GBM?is the highly vascularized malignant tumor of central nervous system,which is characterized by rapid progression,invasion and high recurrence rate.Recombinant human monoclonal antibody bevacizumab?BEV?is the first anti-angiogenesis drug for glioblastoma,targeting the vascular endothelial growth factor A?VEGFA?.However,clinical research found that the effect of anti-angiogenesis therapy was transient for GBM patients and it could easily result in the treatment resistance.There is increasing evidence that anoxic,acidic tumor microenvironment?TME?is responsible for the treatment resistance.Hence,the modulation of TME is crucial for overcoming treatment resistance and improving the effect of anti-angiogenesis therapy.With the development of nanomedicine,considering their high reactivity and specificity toward tumor endogenous H₂O₂ under acidic pH(MnO₂+H₂O₂+2H⁺?Mn²⁺+O₂+2H₂O),Mn?IV?-based oxides?manganese dioxide,MnO₂?have been widely explored as potent theranostic probes for both magnetic resonance imaging?MRI?and the modulation of TME to improve the anti-cancer effect.Despite these promising characteristics and tremendous application potential,the optimized reduction conditions of Mn?IV?are generally reported at pH 6.5 or lower,while the usual pH range in the TME is 6.5–7.0.The insufficient responsivity and slow MRI speed may hardly satisfy the clinical demands for rapid and sensitive cancer diagnosis.As a transition metal element,Mn has different valence states.The relatively unstable valence state Mn?III?,which has the intrinsic pH-dependent disproportionation reaction and can produce Mn²⁺ions in the weakly acidic environment(2Mn³⁺?Mn²⁺+Mn⁴⁺),has received much attention.To enhance the TME responsivity,the biomineralization approach was used to form the specific dual-valence nanoparticle system RGD-BMnNPs.Then,the Cy5 labeled VEGFA-siRNA was loaded onto the RGD-BMnNPs and to realize the enhanced TME responsivity MRI/fluorescence dual-modal imaging,overcome the drawbacks of the direct use of naked siRNA,such as non-specific biodistribution,decomposition and lower cellular uptake rate,and realize the efficient siRNA delivery in glioblastoma and enhanced anti-angiogenesis therapy with the modulation capacity of the TME.It provides a new approach for the design of clinical theranostic nanomedicine in glioblastoma.Study Methods1.RGD-BMnNPs were synthesized through a two-step method.First step,RGD peptide was bonded to the active sulfhydryl group?–SH?of BSA and to form RGD-BSA complex.Second step,RGD–BSA was employed as the template and stabilizer.The oxidation of the sequestered Mn?II?ions was carried out in alkaline conditions and aqueous phase through biomineralization at physiological temperature and to form Mn?III?and Mn?IV?dual-valence nanoparticle system RGD-BMnNPs.Evaluating the shape and size,and analyzing the Mn concentration of RGD-BMnNPs.DLS was applied to detect the hydrodynamic size of RGD-BMnNPs.2.Analysis of RGD-BMnNPs valence state and relaxation performance in different pH condition.XPS spectrum was used to analyze Mn valence state in tumor weakly acidic environment and physiological condition.7.0T small animal MRI was applied to test the r₁ of RGD-BMnNPs in pH=6.5-7.4 environment.3.Characterization of control group MnO₂ nanoparticles and comparison with RGD-BMnNPs.One-step method was used to form MnO₂ nanoparticles by using the reduction of KMnO₄ in aqueous environment.Evaluating the shape and size and analyzing the Mn concentration of nanoparticles.XRD was applied to verify the MnO₂ crystal.7.0T small animal MRI was applied to test the r₁ of MnO₂ nanoparticles in pH=6.5-7.4 environment,then,compared to the RGD-BMnNPs.4.RGD-BMnNPs loading VEGFA-siRNA-Cy5 efficiency and characterization.Agarose gel electrophoresis analysis was applied to detect the efficiency of RGD-BMnNPs loading VEGFA-siRNA-Cy5 and the appropriate ratio.Multiskan spectrum was used to analyze the UV-vis spectrum of RGD-BMnNPs loading VEGFA-siRNA-Cy5.In vivo fluorescence imaging and emission spectrum?Ex=650 nm?were applied to analyze the fluorescence signal of RGD-BMnNPs loading VEGFA-siRNA-Cy5.DLS was used to test the hydrodynamic size and zeta potential of RGD-BMnNPs loading VEGFA-siRNA-Cy5.5.In vitro cell experiment of RGD-BMnNPs/VEGFA-siRNA-Cy5.U87MG cells were incubated with RGD-BMnNPs,VEGFAsiRNA-Cy5,or RGD-BMnNPs/VEGFA-siRNA-Cy5in the dark.Then the results were observed by a confocal laser scanning microscopy.Cell viability of RGD-BMnNPs/VEGFA-siRNA-Cy5 was carried out through the standard CCK-8method.6.In vivo theranostic application of RGD-BMnNPs/VEGFA-siRNA-Cy5.Balb/c nude mice in SPF environment were used for the establishment of the glioma orthotopic model.7.0T small animal MRI was applied to test the T₁ signal change of tumor at different time point after the intravenous injection of nanocomposites.In vivo fluorescence imaging was used to test the fluorescence signal change of tumor in different time point.Fast frozen slices were applied to observe the existence of nanoparticles in tumor region.The therapeutic effect was confirmed by the intravenous injection of nanocomposites three times a week,then comparing the survival time and the representative proteins of TME and vessel in different time point between treated and control group.The H&E and immumohistochemical staining were also applied,and the Image Pro Plus 6.0 software was used to measure the microvessel area and diameter changes.Study Results1.MALDI-TOF-MS showed that the RGD peptide was bonded to the BSA and RGD-BSA was employed as the template to form RGD-BMnNPs through biomineralization approach.RGD-BMnNPs showed the irregular sheet-like structures with high-specific surface area and distribution uniformity.EDS result showed the nanoparticle system comprised of C,Mn and other elements.The hydrodynamic size of RGD-BMnNPs was 71.8±25.0 nm,corresponding to the SEM and TME results and Mn concentration of RGD-BMnNPs was 4m M through ICP-MS analysis.2.Mn?III?and Mn?IV?two valence state of RGD-BMnNPs existed in physiological pH=7.4 condition and the ratio was about 1:1.However,Mn?II?and Mn?IV?two valence state of RGD-BMnNPs existed in pH=6.5 weakly acidic environment and Mn?II?was the only existed state of RGD-BMnNPs after H₂O₂ added.The relaxation rate of RGD-BMnNPs was lower in physiological pH=7.4 condition and the r₁=0.81 mM^-1s^-1.But in acidic environment,the relaxation ability was enhanced and when p H was 6.9 or 6.5,r₁=1.53 mM^-1s^-1 or 2.25m M^-1s^-1.And the significant enhancement was observed when the H₂O₂ was added,the r₁was4.56 m M^-1s^-1.3.MnO₂ nanoparticles showed a spherical morphology,and the two diffraction peaks at37°and 66°?which correspond to the?101?and?002?basal reflections?indicated the successful synthesis of MnO₂.Compared with RGD-BMnNPs,MnO₂ nanoparticles couldn't respond to weakly acidic environment without H₂O₂ and generate Mn²⁺ions.And the relaxation rate of MnO₂ nanoparticles(r₁=1.33 mM^-1s^-1)was lower than the RGD-BMnNPs(r₁=4.56 mM^-1s^-1)at pH=6.5 with H₂O₂.4.Agarose gel electrophoresis assay showed that RGD-BMnNs were mixed with VEGFA-siRNACy5 at mass ratios 10:1 and the RGD-BMnNs could load entire siRNA molecules.UV-vis spectrum showed the intense absorption band at 600-700 nm range.The absorption value at 648 nm could be used to quantity the loaded siRNA concentration.The nanoparticle system showed the classical Cy5 fluorescent characteristics after loading siRNA.The hydrodynamic size of RGD-BMnNPs/VEGFA-siRNA-Cy5 was 80.5±30.1 nm,and the zeta potential was-7.16±0.6 mV.5.The U87MG cells incubated with RGD-BMnNPs/VEGFAsiRNA-Cy5 showed intense red fluorescence of Cy5 compared to cells incubated with free VEGFA-siRNA-Cy5 and RGD-BMnNPs.It proved the excellent cellular delivery performance of the RGD-BMnNPs.And CCK-8 viability assay showed negligible cytotoxicity to the U87MG cells in the tested Mn concentration ranges which represented the good biocompatibility of RGD-BMnNPs.6.With the special sensitive TME responsivity nanoparticle system design,the significant T₁ enhancement was observed only 30 min after injection in brain orthotopic glioblastoma and it lasted until 180 min.The specific red fluorescence was observed in the brain tumor through in vivo fluorescence imaging and it reached the peak after 30 min,then fell.The red fluorescence particles could be directly observed in the tumor fast frozen slices,which proved the efficient siRNA delivery in vivo.The survival time of treated group mice was longer than the control,and these representative proteins?HIF-1?,LDH-A,VEGF-A?expressions of treated group were lower than control group in different time point.The H&E and IHC results showed the treated group vessels'diameter and microvessel area were lower than the control,which exhibited a good anti-angiogenesis effect.Study Conclusion1.RGD-BMnNPs with dual-valence states and enhanced TME responsivity were successfully synthesized through a two-step method.This nanoparticle system can respond to the TME rapidly and compensate for the Mn?IV?nanoparticles imaging disadvantages in the weakly environment,which will promote its clinical translation.2.RGD-BMnNPs showed the sheet-like nanostructures with high-specific surface and excellent biocompatibility,which can load small molecules drugs through physical absorption and realize the efficient delivery of VEGFA-siRNA-Cy5 in vivo and in vitro.3.RGD-BMnNPs can realize the comprehensive modulation of TME,including alleviate hypoxia,reduce acidification level,which improve the tumor anti-angiogenesis siRNA effect and prolong the life time of tumor bearing mice.This nanoparticle system provides a new theranostic approach for clinical glioma treatment.