Preparation Of PH-responsive Doxorubicin-layered Double Hydroxide Controlled Release System For Tumor Therapy

In recent years,the unique characteristics of tumor microenvironment provide conditions for engineering various intelligent nanoscale drug delivery systems with related stimuli-responsive switches,in which the acidic microenvironment of tumor tissue is used to design drug delivery systems with pH-sensitive targeting.In this study,the anticancer drug doxorubicin（Dox）was modified to prepare a novel pH responsive doxorubicin complex（Hydrazone-Dox）,which not only has an acid sensitive hydrazone bond,but also has a carboxyl group for convenient encapsulation into layered double hydroxide nanoparticles（LDHs）.Finally,the nanoscale drug delivery system of pH-responsive Hydrazone-Dox complexes encapsulated LDHs（Hydrazone-Dox-LDHs）were successfully engineered by ion-exchange method.Various experiments have been carried out including drug loading performance,physicochemical properties,the release behavior,enhancement of anti-tumor effects and mechanisms in vitro,which are summarized as follows:Preparation and characterization of LDHs:Initially,we have synthesized LDHs containing Mg²⁺,Al³⁺and NO₃^-using the hydrothermal-assisted co-precipitation method.Then,the effects of molar ratio of metal ions and hydrothermal temperature on the morphology,particle size,layer charge properties,crystallinity,and layer spacing of LDHs were investigated.The results of transmission electron microscope（TEM）and X-ray diffraction（XRD）showed that the crystallinity and particle size of LDHs increased and the degree of aggregation decreased with the rising hydrothermal temperature.Notably,the lamellae of fabricated LDHs were hexagonal.In addition,the zeta potential values of all LDH-based samples were more than+35 mV.It should be noted that the greater metal ion molar ratio of Mg²⁺and Al³⁺,the smaller the potential values of the LDHs.However,the influence of hydrothermal temperature and molar ratio of metal ions on LDHs layer spacing was not so obvious.When the hydrothermal temperature as well as the molar ratio of metal ions were set at 100℃and 3,respectively,the LDHs had shown the largest layer spacing and better crystallinity,resulting in the particle size of 104.33 nm±14.15 nm.Therefore,the LDHs synthesized under this condition were chosen as optimal for subsequent experiments.Preparation and characterization of Hydrazone-Dox-LDHs-based drug delivery system:the pH responsive hydrazone complexes of Dox were synthesized by the reaction of methyl carbazate and Dox,and the yield was calculated as 81.2%±2.14%.Its chemical structure was identified by nuclear magnetic resonance spectroscopy（¹H-NMR）.The influences of the mass ratio of drug and carrier on drug loading and entrapment efficiency were investigated.The results showed that with the increase of dosage,the drug loading gradually increased,and when the dosage ratio was more than1/5,the drug loading was finally stable at about 4.22%.In such circumstances,the encapsulation efficiency increased first and then decreased with the increase of the dosage ratio.Further,the designed nanoconjugates were analyzed by Fourier transform infrared（FTIR）spectra,X-ray diffraction（XRD）,thermal analysis（TG-DTA）,and ultraviolet-visible（UV-Vis）spectroscopic techniques,which systematically proved the designed Hydrazone-Dox complexes were successfully loaded into the gallery spaces of LDHs.The release behavior in vitro of durg from the LDHs was investigated in PBS at various pH values（5.0 and 7.4）mimicking the tumor microenvironment and physiological fluids.It has shown a better release of Dox from the LDHs gallery in pH5.0 compared to that of 7.4.Another interesting feature is that the release and dismantling of Dox in pH 5.0 were continued for more than 120 h,demonstrating the efficient sustainability of drug in the acid tumor microenvironment facilitating its ultra-slow release.In vitro anti-tumor efficacy:Human breast cancer cells（MCF-7）and human cervical cancer cells（HeLa）were used as cell models to demonstrate the anti-tumor effect.MTT and hemolysis experiments showed LDHs had excellent biocompatibility.On the other hand,the Hydrazone-Dox complexes had the same toxicity to cancer cells as pure Dox,while the eventual Hydrazone-Dox-LDHs formulation exhibited higher cytotoxicity in both the cells than free Dox,which was consistent with the results of flow cytometry apoptosis.Moreover,the pH-responsive controlled release system also showed a significant effect on cells cycle,it could arrest the HeLa cells in S and G2/M phase.Mechanism of the pH-responsive controlled release:The cellular internalization,as well as intracellular delivery of Dox from layered double hydroxide nanocomposites,were investigated by confocal laser scanning microscope（CLSM）investigations after incubation with HeLa cells.The experimental results demonstrated that the Dox species entered cells through passive diffusion and then diffused to the nucleus.While Hydrazone-Dox-LDHs composites internalized cells by endocytosis were and delivered the Dox in the cytosol through subsequent pH-triggered pathway and lysosomal escape.In addition,the results of AO staining showed that Hydrazone-Dox-LDHs composites could extinguish the permeability of lysosomal membrane of HeLa cells.Simialarly,the DCFH-DA probe was used to investigate the ROS generation ability of Hydrazone-Dox-LDHs composites.After incubation with HeLa cells for 24h,the amounts of ROS produced by Hydrazone-Dox-LDHs composites in cells were about 3-fold higher to that of the free Dox species.Moreover,using LDHs to deliver Dox could improve the ROS generation ability.In summary,there are two ways for Hydrazone-Dox-LDHs composites to induce apoptosis.On the one hand,Hydrazone-Dox-LDHs composites in lysosome could cause proton sponge effect,and the lysosomal hydrolases such as cathepsins could be released into cytosol through a process of lysosome membrane permeabilization（LMP）pathway,inducing cancer cell apoptosis.On the other hand,Dox could generate ROS to destroy the structure of cell membrane and DNA,leading to the cancer cell apoptosis.In conclusion,Hydrazone-Dox-LDHs composites have shown extended release specifically in the tumour environment.Moreover,the utilization of pH-responsive complexes led to enhance cytotoxicity of the Dox and it is anticipated to become one of the efficient delivery systems of anticancer drugs.