Tumor-triggered Personalized MiRNA Cocktail Therapy For Hepatocellular Carcinoma

Background:Given its obscure initiation,complexed heterogeneity,malignant potential and frequent drug resistance,hepatocellular carcinoma?HCC?is one of the most malignant primary cancer worldwide.Existing systemic therapies show limited benefit on clinical outcomes of advanced and unresectable HCC.It still lacks an ideal radical therapeutic regime to date.Aberrant expression of mi RNA in HCC has been well established and known to functionally modulate various biological processes.mi RNAs acting as tumor suppressors or oncogenes could be regulated by exogenous oligonucleotide therapeutics.A feasible approach for targeting delivery of mi RNA-based therapeutics is introducing advanced nanoscale systems responsive to a specific tumoral circumstance.Given HCC is a heterogenous disease caused by simultaneous disorder of functional network composed of multiple genes,and deregulation of specific mi RNA is not consistent in all patients,synergistic regulation plus personalized regimen could maximize mi RNA's efficiencyAim:We aimed to synthesize a p H-responsive PEI-?CD@Ad-CDM?DLink_m?-PEG?PCACP?nanoparticle featuring PEGylation detachment and size transformation at tumor site for mi RNA tumor-targeting delivery.We then proposed a cocktail mi RNA therapy?mi R-cocktail?mediated by PCACP,which is composed of mi R-199a/b-3p mimics?mi R199?and antisense-mi R-10b?antimi R10b?,and aimed to demonstrate its synergistic antitumor effect.We also expect to fulfill the personalized mi R-cocktail therapy based on individual mi RNA expression profiling.Methods:Section1:Polyethyleneimine crosslinked cyclodextrin?PEI-?CD?was adopted as the parent gene vector,and we synthesized a triblock copolymer Ad-CDM-PEG by connecting the p H-responsive linker CDM with PEG and adamantylamine.The Ad-CDM-PEG could further self-assemble with PEI-?CD by host-guest interaction between cyclodextrin and adamantane,constituting a novel mi RNA delivery vector PEI-?CD@Ad-CDM-PEG?PCACP?.¹H NMR spectroscopy was performed to validate the structures of chemical compounds.The electrophoretic mobility of PCACP/mi RNA system was tested by gel electrophoresis,according to which the applicable nitrogen to phosphorus?N/P?molar ratios were decided.The nucleotide-loaded nanoparticles were observed by TEM,size and Zeta???potential were decided by DLS analysis.We further conducted investigations of the p H-responsiveness,mi RNA release behavior,cellular uptake and transfection efficacy of PCACP.Tumor inhibition ability of PCACP/mi R199system was validated on Huh-7 cell lines and Huh-7 derived xenograft.After treatment,the expression level of mi R-199a/b-3p was quantified by q RT-PCR,and levels of mi R-199a/b-3p targeted proteins were measured by Western blot and immunohistochemistry.Section2:PCACP was employed as the gene vector for mi RNA delivery.CCK-8test was used to study the inhibitory effect of mir199 and antimir10b on Huh-7 viability,and drug combination index?CI?was calculated.PCACP/mi R-cocktail was applied on Huh-7 cells and Huh-7 xenograft models to confirm the synergistic effect on tumor proliferation and metastasis by mir199 and antimir10b.q RT-PCR was conducted to quantify the expression of mi RNA after treatment.Western blot,immunohistochemistry and Transwell assays were used to verify the molecular mechanism of synergism.PDX model was established,mi RNA expression profiling was obtained by mi RNA sequencing.According to the individual mi RNA expression characteristics,personalized mi RNA adjustment was given.The therapeutic effect was assessed by tumor growth curve and AFP level.Western blot and mi RNA sequencing of tumor tissues revealed the tumor inhibiting mechanism.Results:Section1:PCACP exhibited good ability to compact micro RNA into uniform spherical,core-shell-like nanoparticles,with average size of 147.4±0.90 nm and?potential of positive 19.83±1.36 m V at N/P ratio from 3.DLS analysis and TEM observation revealed PCACP/mi RNA underwent mild size decrease after acidic stimulation,achieved average size of 42.45±0.32 nm at 24 h,while the size and microstructure of the nanoparticles remained static in neutral solution.Drug release kinetics of PCACP/mi RNA showed a robust quickening release of mi R199 at p H 6.5,with a first-order half-life of 4.2 h and a maximum release percentage of 85.65%,significantly higher than at p H 7.4.Laser confocal microscope and flow cytometry showed elevated cellular internalization of PCACP/mi RNA after p H 6.5 pretreatment.Real-time distribution of Cy5-labeled PCACP/mi RNA validated tumor targeting and accumulating effect of this nanoparticle.In vitro and in vivo experiments confirmed effective regulation of mi R-199a/b-3p and its target proteins expression by PCACP/mi R199 system,which significantly constrained HCC proliferation.Section2:Remarkable cell proliferation inhibiting effect was achieved by PCACP/mi R-cocktail as compared to monotherapy(IC₅₀=2.66?g/m L,CI=0.26),indicating the synergic therapeutic effect.PCACP/mi R-cocktail treated cells and tumor xenograft showed synergistic HCC suppression by effectively regulating mi R-199a/b-3p and mi R-10b level,and inhibiting downstream PAK4/Raf/MEK/ERK and m TOR/AKT pathways,as well as inhibition of EMT by increased E-cadherin and attenuated Vimentin expression.Significant migrating inhibitory effect was observed in cells treated with pre-activated PCACP/mi R-cocktail system by Transwell and wound healing assays.We established a HCC PDX model in which expression of mi R-199a/b-3p was markedly decreased and mi R-10b was upregulated according to mi RNA sequencing,treated PDX model with PCACP/mi R-cocktail with personalized regime,and achieved synergistic tumor inhibiting effect.Conclusion:In this study,we successfully developed a tumor-acidity-cleavable,size transformable nanoparticle PCACP with high transfection efficacy for mi RNA delivery.The simultaneous transfection of mi R199 and antimi R10b was verified to have a cumulative effect in restraining HCC tumor progression and improving prognostic outcomes both in vitro and in vivo.PCACP/mi R-cocktail therapy is a promising personalized therapeutic strategy for future adjuvant therapy of HCC treatment clinically.