Investigation On The Anti-tumor Activity Of MLN4924 Nano Drug Delivery System Based On Platelet Membrane Bionics

NEDD8 is a ubiquitin-like protein(UBL),that mediates Neddylation-like ubiquitin modification.Like ubiquitin,NEDD8 plays an important role in apoptosis,DNA damage and signal transduction.NEDD8 must be catalyzed by NEDD8 activating enzyme(NAE)to produce activity.MLN4924 is a small molecular inhibitor targeting NAE.A number of phase I/II clinical studies have shown that MLN4924 can effectively promote the apoptosis of human tumor cells.MLN4924 is a hydrophobic drug with poor water solubility and low bioavailability.At the same time,MLN4924 lacks tumor targeting ability and is easy to cause serious side effects.These problems hinder the clinical application of MLN4924.Based on these problems,we strategically designed a nano-delivery system that can actively target the tumor site and responsively release drugs,which is helpful to achieve accurate delivery,attenuated toxicity and synergistic effect of small molecular inhibitor MLN4924.Here,this research reports on a platelet membrane–coated polyamidoamine dendrimer(PAMAM)nano-delivery platform(P-PAM/MLN)for active targeting of tumors and p H-responsive release of MLN4924.The platelet membrane-coated shell provides a natural means of tumor targeting.The PAMAM core can carry drugs through hydrophobic interaction.At the same time,its p H-sensitivity enables lysosomes disruption upon cellular uptake and promote the release of encapsulated MLN4924,which relies on the proton sponge effect.Finally induce tumor cells apoptosis.The details are as follows:1.Preparation and characterization of P-PAM/MLN.PAM/MLN,was synthesized by thin film dispersion method.Platelet membrane was extracted from platelets,and platelet membrane vesicles(PMV)were prepared by ultrasound;PMV was used to disguise PAM/MLN to prepare P-PAM/MLN.By means of transmission electron microscope(TEM),dynamic light scattering(DLS)and confocal laser scanning microscope(CLSM),they proved that PAM/MLN and P-PAM/MLN were successfully prepared,and their particle sizes were about 15 nm and 160 nm,respectively.After membrane camouflage,the potential decreased from 30 m V to-19 m V.The morphology of P-PAM/MLN was spherical,with obvious shell-core morphology,and the drug loading efficiency was about 4.77%.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western Blot confirmed that P-PAM/MLN carried platelet membrane.The results of TEM,DLS and drug release experiments under different p H conditions show that P-PAM/MLN has the ability of p H response to release drugs.Under the condition of p H 7.4,the nanoparticles had a stable spherical structure,and the release rate of MLN4924 was only 15.7%.The p H decreased to 5.0 to simulate the environment of intracellular lysosomes,and the shell deformation and rupture of P-PAM/MLN was observed.the particle size measured by DLS was more than 1000 nm.MLN4924 was released and the cumulative release rate of 48 h was about 67.3%.These simulation experiments have laid the foundation for MLN4924 in the release of tumor cells.2.Study on the antitumor activity of MLN4924 in vitro.In this study,human breast cancer cells(MCF-7)were used as model cells to evaluate the anti-tumor activity of free drug MLN4924.The survival rate of MCF-7 cells treated with MLN4924 for different time was detected by CCK-8 assay.After treatment with MLN4924 for 48 h and 72 h,the cell survival rates decreased significantly in a dose-dependent and time-dependent manner,with IC50 values of 0.92 μM and 0.09μM,respectively.MLN4924 can also inhibit cell clone formation and significantly reduce the number of cell colonies.The results of Western Blot assay showed that MLN4924 could inhibit Neddylation ubiquitin modification and down-regulate the expression of cullin-NEDD8 protein in MCF-7 cells.After MLN4924 treatment,the expression of γ-H2 AX protein,a marker of DNA damage,was significantly increased,and the expression of apoptotic protein cleaved caspase 3 was also increased.3.Study on the antitumor activity of P-PAM/MLN in vitro.Doxorubicin(DOX)was used as a fluorescence signal to visualize the cell targeting and p H-sensitive release behavior of P-PAM/MLN.The result of cell uptake assay showed that P-PAM/MLN could be effectively uptake by MCF-7 cells,and the uptake behavior was time-dependent.Compared with the exposed PAM/MLN nanoparticles,the nanoparticles coated with the membrane significantly reduced the uptake of RAW264.7 macrophages and had the ability of immune escape.Tumor targeting experiment showed that compared with R-PAM/MLN which coated on erythrocytes membrane without tumor targeting ability,the uptake of P-PAM/MLN by MCF-7cells was significantly increased after platelets coating.The biological distribution of the P-PAM/MLN in MCF-7 cells was investigated by CLSM.When incubated for 2 h,P-PAM/MLN could be quickly absorbed by MCF-7 cells and most of them were captured by lysosome.When the incubation time was 4 h,the co-localization rate with lysosome was still high,indicating that the number of P-PAM/MLN entering lysosomes increased.However,when the incubation time was extended to 6 h,the lysosomes broke and the co-localization rate decreased sharply,indicating that the proton sponge effect of PAMAM could promote the lysosome escape and drug release of P-PAM/MLN.The cytotoxicity of blank vector P-PAM was investigated by CCK-8 method.After co-incubation with cells for 72 h,the cell viability was still more than 80%,which proved that the vector P-PAM had good safety in vitro.The results of cell inhibition by P-PAM/MLN showed that the cell survival rate was about31% after incubation with P-PAM/MLN for 72 h.Compared with MLN4924 and R-PAM/MLN,P-PAM/MLLN has more significant inhibitory effect on MCF-7 cells,which proves that platelet membrane encapsulation can effectively promote the uptake of tumor cells,so it has better anti-tumor activity in vitro.In summary,the biomimetic nano-drug delivery system constructed in this paper provides a means to realize the effective loading of small molecule inhibitor MLN4924,targeted delivery in vitro and responsive release of p H,so as to achieve the purpose of reducing toxicity and increasing efficiency in tumor therapy,and can be used to expand the application of MLN4924 nano-delivery system in vivo in the future.