Basic Investigation On The Tumor Microenvironment-Responsive Therapeutic And Imaging System Based On Manganese Nanomaterials

Nanotechnology has promising prospect for tumor therapy and tumor imaging.Developing smart nano drug system with the concept of taking advantage of the tumor microenvironment is one way of amplifying the imaging signal,improving the therapeutic effect and weakening the unwanted side effect.Photodynamic therapy(PDT)and photothermal therapy(PTT)were paid more attention for cancer treatment in recent years.Increasing the oxygen level in the tumor region and removing barriers blocking the action of reactive oxygen species(ROS)are new alternatives to improve the therapeutic effect of PDT.For the combination of PDT and PTT,underlying the potential mechanisms of ROS and heat effect would be useful ways for chosing reasonable combination strategies with enhanced phototherapy.In this study,poly(lactic-co-glycolic acid)(PLGA)nanomaterial by surface decoration with MnO2 shells and amorphous porous manganese phosphate nanomaterial were prepared for constructing three kinds of tumor microenvironment responsive magnetic resonance imaging(MRI)and combination therapy systems.On one hand,the photodynamic effect or phototherapy was enhanced by tapping new sources of supply and reducing consumption of ROS relying on MnO2,the action of ACF in inhibition of hypoxia-inducible factor 1a/vascular endothelial growth factor(HIF-1a/VEGF)pathway which promoted treatment escape,and the multi-mechanisms of mTOR inhibitor rapamycin,respectively.On the other hand,they could achieve tumor microenvironment triggered MRI and therapeutic agents release.The main contents are as follows:1.A photodynamic system enabling tapping sources of supply and reducing consumption of ROS for enhancing tumor photodynamic therapy and magnetic resonance imagingPhotodynamic therapy(PDT)is a non-invasive treatment approach that employs a photosensitizer,an appropriate excitation light,and oxygen(O2)molecules through generation of cytotoxic ROS.Apart from increasing drug accumulation at the tumor site with the help of the nano-targeting technology,some nanomaterials could interact with the tumor microenvironment,thus alleviating tumor hypoxia and scavenging the glutathione level that consumes the ROS.In a word,tapping new sources of supply and reducing consumption of ROS is a strategy for enhancing the therapeutic effect of PDT.In this study,the well-designed hematoporphyrin monomethyl ether(HMME)-loaded PLGA nanoparticles by surface decoration with MnO2 shells were constructed in an attempt to enhance PDT and MRI.Firstly,HMME-loaded PLGA nanoparticles were prepared,and layered manganese oxide was formed on the surface of PLGA/HMME nanoparticles by oxidation-reduction method.The coated MnO2 nanoshells could catalyse the abundant H2O2 in the tumor site to produce many oxygen molecules,which could be favourable to ROS generation by HMME under light irradiation.Besides,the MnO2 shells could be reduced into Mn2+ by glutathione(GSH)for enhanced MRI while the GSH level decreased obviously.This process ultimately enhanced the PDT effect.The characteristics of the formulation showed that the mean size of the nanoparticles was 209.8 mnm and the mean zeta potential was-4.4 mV,which were beneficial for the good stability.After HMME being loaded into the PLGA nanoparticles with an entrapment efficiency of 75.0%,the ROS generation efficiency of HMME was not affected.The PLGA/HMME@MnO2 NPs released only approximately 56.9%of HMME in pH 7.4 PBS.When the released medium was added with GSH,promoted HMME release with a percentage of 70.%was measured.The in vitro release showed that the MnO2 coating could delay the HMMM release.The ROS generation content was determined by the flow cytometry.The positive cell percentage of HMME + 532 nm irradiation group,PLGA/HMME + 532 nm irradiation group and PLGA/HMME@MnO2 + 532 nm irradiation group was 14.9%,27.5%and 35,8%,respectively.Besides,the GSH level within the cells treated with PLGA/HMME@MnO2 decreased obviously compared to that of blank group.The pharmacokinetics study showed that the AUC of PLGA/HMME@Mn02 was 115.26 ?g/(mL·h)and the t1/2 was 15.29 h,indicating that this system could prolong the circulation time.The in vivo antitumor therapy effect showed that compared with the relative tumor volume of mice in HMME + 532 nm irradiation and PLGA/HMME+532 nm irradiation groups,that of PLGA/HMME@MnO2 + 532 nm irradiation group was 1.5 ± 0.4,indicating an obvious difference.Hyproxyprobe was used for tumor staining of hypoxia.The result showed that the Mn02 could ameliorate the hypoxia condition.The in vitro and in vivo MRI results confirmed that this system could enhance the T1-weighted MRI effect with a higher relaxivity value more than 1 mM-1 s-1.In summary,this organic and inorganic hybrid nanomaterial paved a way for enhancing PDT and MRI.2.Ultra pH-activatable manganese phosphate nanoplatform for cascade photodynamic cancer therapy and magnetic resonance imagingThe oxygen consumption induced by the PDT could aggravate the hypoxia environment,which resulted into the therapeutic resistance or treatment escape.HIF-1?/VEGF is one prime example of tumor signalling pathways that promote PDT treatment escape.In the current study,firstly,amorphous porous manganese phosphate nanomaterial was synthesized by one pot solvothermal reaction.The ultra-sensitive pH-responsive property was achieved due to the amorphous feature,which could be applied for the controlled release of the therapeutic agents.Importantly,the released Mn2+ after the disintegration of the nanomaterial could be used as the T1-weighted contrast agents.The porous feature provided space for loading the photosensitizer chlorin e6(Ce6)and HIF-1?/VEGF inhibitor acriflavine(ACF).Finally,carboxymethyl dextran(CMD),a hydrophilic polymer,was chemically anchored on the surface of porous manganese phosphate through the pH-responsive boronate esters.The CMD modification could not only reduce the leakage of the loaded drug during the circulation process in vivo,but also afford the reappearance of phenylboronic acid under the tumor microenvironment,which is favourable for the binding with the sialic acid expressed on the tumor cells in order to promote the cellular uptake behaviour.After the(3-Aminopropyl)trimethoxysilane(APTMS)modification,the PMP NPs with hydrophobic surface property were converted to the amine PMP NPs with well-dispersed property in water.The results showed that the hydrodynamic size of C-PMP/Ce6/ACF NPs was around 180.7 nm and the PDI of them was about 0.21.The zeta potential was about-22.6 mV,which was beneficial to make the NPs have good stability in vitro and in vivo.The release percentages of ACF and Ce6 for C-PMP/Ce6/ACF NPs after incubation for 12 h at pH 7.4 were 60.89%and 52.70%,respectively.However,the release percentages of ACF and Ce6 for C-PMP/Ce6/ACF NPs after incubation for 12 h at pH 6.0 were 89.84%and 96.87%,respectively.Therefore,thein vitro drug release experiment showed the obvious pH-responsive drug release profile.The r,relativity value of this system was measured to be 1.9 mM-1 s-1 and 4.8 mM-1 s-1 at pH 7.4 and pH 6.0,respectively.Under the 660 nm irradiation,C-PMP/Ce6/ACF could obviously inhibit the growth of SMMC-7721 cells,which could be attributed to the abundant ROS generation for apoptosis induction as well as DNA damage.The apoptosis percentage of the combined therapeutic group reached up to 92.4%.The RT-PCR and ELISA assays demonstrated that ACF could reduce the expression of VEGF mRNA and VEGF,respectively.The in vivo results showed that under the 660 nm irradiation,the relative tumor volume of mice from C-PMP/Ce6 and C-PMP/Ce6/ACF group was 3.7 ± 0.4 and 2.7 ± 0.3.The MRI in vivo showed that the appreciable whitening phenomenon at the tumor site could be easily observed at the tumor site from both the coronal plane and the cross section.Consequently,the C-PMP/Ce6/ACF NPs could accumulate at the tumor site to some extent and could provide information about the tumor when it was used as a T1 MRI contrast agent.In summary,the C-PMP/Ce6/ACF NPs exhibited great potential in enhancing the therapeutic effect,restricting the treatment escape and achieving tumor microenvironment triggered imaging.3.Co-delivery of rapamycin and indocyanine green for multi-mechanism tumor therapy by the porous manganese phosphate nanoparticlesThe combination of the therapeutic agent increasing the autophagy and indocyanine green(ICG)has the potential to enhance the anticancer effect due to the autophagy effect of ROS and heat effect of ICG under irradiation.The mTOR inhibitor rapamycin(RAPA)could not only decrease the expression of HIF-1?,but also promote the autophagy process.Poor aqueous stability and rapid clearance from the blood limit ICG use.Besides,RAPA also showed poor aqueous stability.Therefore,it is reasonable to use a carrier with high drug loading efficiency for improving the drug stability and the combination application.In this study,the porous manganese phosphate nanoparticles with high drug loading efficiency,ultra pH responsive drug release and MRI properties were further used for delivering RAPA and ICG in order to achieve the comprehensive tumor therapy with multi mechanisms.Poly glutamic acid(PGA)was chemically anchored on the surface of porous manganese phosphate and could not only increase the stability of the system,but also afford the enhanced cellular uptake of the nanoparticles mediated by the glutamyl transpeptidase.For the MCF-7 cells,the ROS and heat effect of phototherapy induced by ICG triggered autophagic death and apoptosis while the mTOR inhibitor rapamycin promoted the autophagic process and inhibited the HIF-1?/VEGF pathway,which ultimately resulted into the tumor growth inhibition with multi mechanisms.The formulation characterization showed that the entrapment efficiency of ICG and RAPA was 44.3%and 44.0%,respectively.The stability of ICG was improved obviously because of the entrapment of it in the pores of the manganese phosphate nanoparticles in an amorphous state.Importantly,this process also promoted the heating generation ability.Under the same concentration,the temperature increased by 18.1 ? and 10.0 ? for the PGA/PMP/RAPA/ICG and ICG within 3.5 min,respectively.The western blotting result showed that the autophagic marker LC3I was converted to LC3 II for cells treated with ICG phototherapy or RAPA treatment.The autophagic flux assay was measured by the mCherry-EGFP-LC3 fusion protein.Besides,the ultrastructure observation of the treated cells further confirmed that the combination of phototherapy and RAPA promoted the autophagy process.Therefore,the inhibition of MCF-7 cells related to the ROS accumulation,decrease of the mitochondrial membrane.qPCR showed that the HIF-la and VEGF mRNA were decreased when the cells were treated with RAPA.For the tumor-bearing mice injected with PGA/PMP/ICG/RAPA,the temperature of the tumor site increased from 35.1 ? to 43.2 ? when the tumor site was irradiated by an 808 nm laser for 1.5 min.The tumor volume of the mice in the PGA/PMP/ICG/RAPA group was inhibited with a relative tumor volume of 1.8 ± 0.4.Compared to the single phototherapy group,PGA/PMP/ICG/RAPA system could decrease the level of HIF-la expression and VEGF simultaneously,which finally decreased the vessels density in the tumor site.In summary,phototherapy and mTOR inhibitor RAPA afforded the comprehensive tumor therapy with multi mechanisms as well as improved drug stability.