Mitoxantrone Pre-loaded Targeted Hybrid Nanoparticles For Cancer Therapy

Malignant tumors rank first in three leading causes of death,which seriously threaten people's health.Chemotherapy is the standard treatment for cancer.However,severe toxicity and low therapeutic effects limit their application.The nano drug delivery systems(nano-DDSs)have unique advantages in targeted delivery of antitumor drugs,but there are disadvantages such as no specific selective action of tumor cells and insufficient drug release of target sites.Therefore,designing nano drug delivery systems with active targeting function and effective controlling of drug release is of great significance for effective treatment of malignant tumors.In this study,mitoxantrone(MIT)was preloaded into amorphous calcium carbonate nanoparticles(ACC-MIT)by gas diffusion reaction.The obtained ACC-MIT was then modified with phospholipid(PL),during which shielding polymer polyethylene glycol(PEG)and targeting moiety folic acid(FA)were decorated to the as-prepared hybrid nanoparticles by solvent diffusion method to finally construct a targeted hybrid platform(PL/ACC-MIT).The particle size and zeta potential of PL/ACC-MIT were 100 ± 3.7 nm and-10.19 ± 0.74 mV.Transmission electron microscopy showed PL/ACC-MIT were uniform spherical shape and uniform distribution,also showed apparent membrane-core structure and stability under biological environment.As determined by high performance liquid chromatography,the drug loading of PL/ACC-MIT was 4.58%.The result of release experiments indicated that ACC-MIT under different pH PBS,all showed significant water-sensitive release characteristics,and the cumulative release of MIT was more than 60% at 8 h;while PL/ACC-MIT was slowly released in PBS at pH 5.5 and 7.4.At pH 7.4,the cumulative release percentage of MIT reached only 21% at 24 h.PL/ACC-MIT was released slowly due to the protective effect of surface phospholipids,which could keep it stable in the circulation system.When PL/ACC-MIT was taken up by tumor cells,the phospholipid layer on the surface was hydrolyzed by lipase in tumor cells with high expression of lipase,and the water release core was exposed in response to water,so the water-sensitive release property could be achieved.The results of in vitro targeting experiments in A549 cells and HeLa cells showed that the fluorescence signal gradually became stronger as a function of time,indicating that the intracellular uptake of the nanoparticles was time dependent and positively related to the incubation duration.It was noted that at all tested time intervals,the HeLa cells exerted stronger fluorescence intensity than the A549 cells indicating that FA-modified PL/ACC could be actively targeted to FA-overexpressing HeLa cell lines.Intracellular drug distribution experiments showed that after 2 h incubation,obvious red fluorescence was observed in the nucleus,and the fluorescence signal intensity increased with the incubation time,indicating it could be quickly disintegrated to achieve the intracellular drug-sensitive release effect.Intracellular colocalization experiments showed that endocytosis of PL/ACC-MIT was mainly regulated by lysosomes.Cytotoxicity results showed that the cytotoxicity of PL/ACC-MIT under the same conditions was comparable to that of free MIT,indicating that MIT could be effectively released and exerted its antitumor effect.The results of HeLa cell sphere experiments showed that PL/ACC-MIT promoted drug penetration into MCTS and more effectively inhibited MCTS proliferation compared with the free MIT administration group.Xenograft model co-bearing HeLa and A549 tumors were established.PL/ACC-MIT nanoparticles modified with FA showed preferable accumulation into the HeLa tumor over the A549 tumor,indicating FA-decorated nanoparticles had higher affinity for HeLa cells overexpressing FR.An ex vivo study of nanoparticle distribution in the main organs and tumor tissues revealed that the average fluorescence intensity of PL/ACC-MIT in HeLa tumor tissues was 1.62 times that of A549 tumor tissues.The distribution of nanoparticles from superficial to in-depth of the tumor tissue showed PL/ACC-MIT nanoparticles could deliver their drug cargo from the exterior to the interior of the solid tumor.Compared with the negative control group,the tumor growth inhibition rates of MIT and PL/ACC-MIT groups were 49.98% and 68.31%,respectively,indicating that PL/ACC-MIT inhibited the growth of tumors more strongly.PL/ACC-MIT had better tumor targeting and tendency to accumulation into over-expressed tumor tissues due to surface FA modification.Which could penetrate deeply into tumor tissues,achieve the killing of deep tumor cells,and further enhance the antitumor effect of the hybrid nanoparticles system.