Application Of Nanodrug Delivery System To Enhance The Therapeutic Effect Of Glioblastoma

Background and Objective:Glioblastoma multiforme(GBM)accounts for approximately 46% of all malignant brain tumors,and has a poor prognosis with a mere 5% of patients surviving beyond 5years.Immunotherapies with immune checkpoint inhibitors such as blockades of the PD-1(programmed cell death protein 1)/PD-L1(programmed cell death-ligand 1)pathway have produced promising results in patients with varying types of cancer,these treatments however did not show significant survival benefits in GBM patients.The blood-brain barrier(BBB),composed of cerebral vascular endothelial cells with tight junctions,capillary basement membrane,and foot processes of pericytes and astrocytes,separates the central nervous system(CNS)from blood components and prevents most substances from entering the brain parenchyma in the blood.Although the presence of immune cell infiltration in GBM suggests that the integrity of the BBB could be compromised by CNS tumor development,the BBB were found to still pose a major physiological hurdle for drug delivery into CNS tumors.Thus,strategies for enabling delivery of immune checkpoint inhibitors across the BBB are essential for successful immunotherapy against GBM.RNA interference by small interfering RNA(siRNA)is a powerful technology for targeted gene silencing and has been increasingly used in cancer immunotherapy.Free siRNAs are not suitable for therapeutic use because of their poor pharmacokinetics,inferior tumor-targeting and insufficient cellular uptake,and therefore,siRNAs are often complexed with carriers for cancer immunotherapy.As a suitable nanocarrier for siRNA delivery,cationic lipid-based nanoparticles(LNPs)exhibit excellent biocompatibility,biodegradability,and structural flexibility.Ionizable cationic lipids typically have an amine headgroup that is critical for effective siRNA encapsulation and intracellular delivery.Ionizable cationic nanoparticle usually composes an amine headgroup,which is crucial for siRNA encapsulation and delivery into cytoplasm.Despite the BBB is considered as a great limitation for nano drug carriers free of targeting ligands to treat brain cancers,several passive targeted nanocarriers have been developed for crossing the BBB in orthotopic brain glioma models.Recent studies showed that positive surface charge improves the BBB permeability of polymer nanoparticle drug carriers.However,lipid nanoparticles that can directly deliver siRNA to brain with effective BBB penetration remains rare while highly demanded for GBM immunotherapy.Gold nanoparticles are nano scale materials.Among many nano materials,gold nanoparticles have the advantages of low toxicity,easy control of particle size and shape,and local surface plasmon resonance.Therefore,the application of gold nanoparticles in tumor sensors,drug release agents and enhancers in plasma photohyperthermia has been widely studied.One of the current research hotspots is to prolong the circulation time in vivo and to give priority to tumor localization,which are the key characteristics of effective delivery of nanoparticles.In the first part of the study,we design a library of ionizable cationic LNPs with different amine headgroups for brain-targeted siRNA delivery.We found that the structure of the amine headgroup of LNPs is a critical factor in determining the efficacy of LNPs for siRNA delivery,BBB penetrating and brain tumor targeting capacity.Using in vitro and in vivo screening approaches,we identified a leading lipid,BAMPA-O16 B with p Ka value around 6.5 as an efficient carrier to deliver siRNAs into cytoplasm of GBM tumor cells in vitro and into intracranial tumor tissues in a mouse orthotopic GBM model.We further demonstrated the potential of using BAMPA-O16 B to deliver siRNAs for simultaneous silencing CD47 and PD-L1,two immune inhibitory molecules involved in tumor-induced immune suppression,in mice with established intracranial GBM.We showed that simultaneous knockdown of CD47 and PD-L1 expression is significantly more effective in activating antitumor immunity than targeting any of them alone for GBM treatment,highlighting the great potential of using brain-targeted liposomal siRNA delivery for the immunotherapy of GBM and other brain tumors.In the latter part of this paper,we applied the PEGylated Au nanotetrapods(NTP@PEG).Compared with gold nanospheres and gold nanorods of the same size,NTP@PEG has obvious protein adsorption resistance to glioblastoma-bearing mice,reduces its phagocytosis in reticuloendothelial system,low accumulation in liver and spleen,and high penetration ability to blood-brain barrier.On the other hand,NTP@PEG showed a longer half-life of blood circulation,which increased the accumulation in glioblastoma and could penetrate into the tumor deeply.Because of the near-infrared surface plasmon resonance effect of NTPs,it has been widely used.After systemic administration,photothermal therapy was used to treat in situ glioblastoma in tumor-bearing mice.It was found that photothermal therapy had obvious inhibitory effect on tumor growth.Therefore,NTPs provide an ideal platform for drug delivery and a broad prospect for the treatment of brain diseases.Methods:1.We designed a library of ionizable cationic LNPs with different amine headgroups,and verified the silencing efficiency of siRNA.We then compared LNPs with different gene silencing efficiency to clarify the mechanism of their differences of silencing efficiency.2.We selected the LNP BAMPA-O16B/siRNA with the highest efficiency for gene silencing and BBB penetration.The uptake of nanoparticles by tumor cells in vitro and in vivo,as well as the silencing efficiency of CD47 and PD-L1 was detected.3.We observed the effect of systemic administration of BAMPAO16B/siCD47/siPDL1 on the growth of GL261 tumor and observed the survival of tumor-bearing mice.The anti-tumor immune response mechanism of BAMPAO16B/siCD47/siPDL1 was detected by flow cytometry.4.We compared the adsorption capacity of different morphology of gold nanoparticles on serum protein,detected their ability to penetrate the blood-brain barrier and their distribution in tumor tissue.5.We observed the effect of NTP@PEG on tumor photothermal therapy in mice with intracranial tumor after systemic administration.Results:1.The silencing efficiency of LNP/si GFP containing different amine headgroups on the expression of GFP was different.Four kinds of LNPs with different amine headgroups were selected to detect the phagocytosis,endosomal escape,p Ka value and the ability of penetrate blood brain barrier model in vitro.It was found that the nanoparticles with the peak value of 6-7 had strong endosomal escape ability and bloodbrain barrier permeability.2.GL261 cells had strong phagocytosis ability toward fluorescence labeled BAMPA-O16B/siRNA co-incubated with GL261 cells.The expression of CD47 and PD-L1 was reduced in vitro by co-incubation with GL261 cells by BAMPA-O16 B LNPs containing CD47 siRNA and PD-L1 siRNA.The silence efficiency of BAMPAO16B/siCD47/siPDL1 was similar to that of commercial Lipofectamine 2000.3.The tumor bearing mice of intracranial glioblastoma were systematically administered with fluorescence labeled BAMPA-O16B/siRNA.It was found that BAMPA-O16B/Cy5-si NC was enriched in the cytoplasm of tumor tissue,but rarely in normal brain tissue.BAMPA-O16B/siCD47/siPDL1 can reduce the expression level of CD47 and PD-L1 in tumor tissue,thus having a high delivery efficiency in tumor.4.BAMPA-O16B/siCD47/siPDL1 was given systematically,which could inhibit the tumor growth of intracranial tumor bearing mice,prolong the survival time of tumor-bearing mice,and reduce the ratio of Ki-67+ cells.The immune response was caused by the increase of CD8+ T cell infiltration,and the decreased ratio of TAMs and M2 subtypes.5.Three kinds of gold nanoparticles,NTP@PEG,NS@PEG,and NR@PEG with different morphologies were synthesized.After co-incubation with serum protein,it was found that NTP@PEG has strong anti-protein adsorption ability and good bloodbrain barrier permeability.It has a strong tumor enrichment effect in the tumor-bearing mice of intracranial glioblastoma,but less in the liver and spleen.6.NTP@PEG inhibited the growth of tumor and prolonged the survival of mice by photothermal therapy.The mechanism was due to the apoptosis of tumor cells induced by photothermal effect.Conclusion:1.The silencing efficiency of LNPs containing different amine headgroups on target genes is different,and the efficiency of the LNPs penetrating BBB in vitro or in vivo is different,and the difference may be related with surface properties.2.The ionizable cationic lipids within LNP/siRNAs with high efficacy of target gene silencing had a p Ka of about 6.5,and those with a p Ka below 5.5 were very poor in target gene silencing.3.The fluorescence labeled BAMPA-O16B/siRNA can be effectively absorbed by tumor cells and tumor tissues in vitro and in vivo,and the expression level of CD47 and PD-L1 molecules is reduced.4.BAMPA-O16B/siCD47/siPDL1 can significantly inhibit the growth of GBM in mice and prolong the survival time of mice.The anti-tumor mechanism is achieved by increasing the infiltration of CD3+ T cells and activated CD8+ T cells in tumor tissue,and reducing the total number of TAMs and M2 subgroup.5.Compared with NS@PEG,and NR@PEG,NTP@PEG has strong resistance to serum protein adsorption.Meanwhile,NTP@PEG can penetrate the blood-brain barrier model in vitro,and enter the brain tissue through the blood-brain barrier at the site of intracranial tumor,achieving enrichment at the tumor site.6.Because NTP@PEG has a good photothermal conversion efficiency at 808 nm,near infrared laser irradiation can induce tumor cell apoptosis,slow down tumor growth and prolong the survival time of mice.