Tumor Theranostic Using Chemotherapeutic Drug Bearing Functionalized Nanodiamond

Unhealthy lifestyles and harsh environment have contributed to cancer becoming an invincible opponent to human beings.Among the therapeutic regimens for cancer,conventional chemotherapy,which conveys anticancer drugs systemically to patients sequentially to kill cancerous cells,is the prevailing most frequent treatment.However,anticancer drugs,which possess some drawbacks,such as,metabolic trapping,multidrug resistance,nonspecific targeting and severe toxicities including serious organ damage,have been greatly restricted.Nanotechnology,which conjugated anticancer drugs onto functionalized nanocarriers by physical or chemical force,and then delivered drugs targeted into tumor region by the properties of tumor and normal tissue,are expected to depress the toxic side effects and simultaneously enhance the therapeutic efficiency.Nanodiamonds(NDs)has recently attracted significant attention in drug and gene delivery owing to its least toxicity compared to other carbon-based nanomaterials,optical properties,chemical stability and ease of surface modification.However,there are still some defects,such as low drug loading,poor targeting and lower treatment efficiency.Taking into account the aboved obstacles,in this paper,we have developed high load and pH responsive,lysozyme-responsive and folate-mediated pH response targeting nanodrug system based on PEGylated NDs.Besides,the endocytosis mechanism,inhibition of tumor and in vitro drug release were studied using HepG2,HeLa and MCF-7 cells as models by confocal microscopy and flow cytometry.Subsequently,in vivo distribution,antitumor effect,toxic and side effects of nanodrug systems were also evaluated using HepG2 or HeLa tumor-bearing mice.The specific researches are as follows:1.Here,to ensure high drug capacity and low drug leakage in blood circulation,and especially to ensure that it is delivered to the tumor region,a smart pH-responsive drug delivery system is designed and prepared using DOX adsorbed onto PEGylated NDs in sodium citrate medium(ND-PEG/DOX,NPDC).The drug loading amount can reach as high as 170 ± 1.26 ?g/mg,which is more than five times that seen in deionized water.Besides,about only 7% of DOX was released at pH 7.4,even if the NPDC release time lasted up to 50 h,while the released DOX increased markedly to nearly 80% when the pH was decreased to 5.0.This means NPDC can enhance therapeutic effect and reduce side effects.Furthermore,NPDC can significantly inhibit cell migration and change the cell cycle.Excitingly,the NPDC system was very smart for target the tumor site through the EPR effect and the accumulation of the drug system at the tumor site is more than twice that of other important organs.Importantly,NPDC can effectively inhibit tumor growth and inhibition rate is more than 3 times of free DOX.Furthermore,NPDC had low toxicity by organ index,blood biochemical indices and histological examination in comparison with free DOX.2.So far,the nanodiamond drug system is mainly based on the physical force,but the shortcoming is that the systems are not stable enough to result in premature drug leakage in the normal tissue.In order to improve the stability of drug system and high curative effect for efficient cancer chemotherapy,herein,the nanodiamond drug system(ND-PEG-GLY-DOX,NPGD)with responsiveness to lysozyme was fabricated using glycidol(GLY)as a bridge,to couple DOX onto PEGylated NDs through ester linkage.In vitro experiments found that NPGD was difficult to hydrolyze(drug release was only 8% after 110 h)in the simulated blood circulation environment(pH 7.4),resulting almost no release of drug and the fluorescence was turn-off.However,in the simulated intracellular environment(pH 4.5 with lysozyme),the release rate was fast and reached 70%.Confocal laser scanning microscopy(CLSM)and flow cytometry studies revealed that the cellular uptake of NPGD is via caveolae-mediated mechanism,and it was located in the lysosome,meanwhile,the overexpressed lysozyme could break the ester bond to result GLY-DOX release into nucleus to kill tumor cells and fluorescence signal turn on,achieving the diagnosis of tumor while inhibiting tumor proliferation.More importantly,in vivo studies display that NPGD has enhanced anticancer effect beyond conventional DOX treatment when intravenously injected into tumor-bearing nude mice.Furthermore,NPGD had low toxicity by blood biochemical indices and histological examination in comparison with free DOX.3.Currently,traditional chemotherapy allows the drug expose to healthy tissue as well as the tumors,resulting in severe drug side effects.To overcome this problem,targeted nanodrug systems with enhanced targeting,high efficiency and low toxicity need to be developed urgently.Herein,a novel tumor targeting delivery system(ND-PEG-HYD-FA/-DOX NPHF/D)was synthesized using the difference of pH between tumor and normal tissue,combined with acid-sensitive hydrazone bond and folic acid with active targeting properties.In vitro drug release found that NPHF/D has almost no release in the simulate physiological environment(pH 7.4),while the intracellular endosomal acidic pH(5.0)in tumor cells causes cleavage of the hydrazone bond,resulting in the active drug sustain release.Such a result makes NPHF/D act as a direct OFF-ON signal messenger for activatable cancer therapy.The results of in vitro cell experiments showed that the cellular uptake of NPHF/D is via both clathrin-and caveolae-mediated endocytosis processes,and also undergo receptor-mediated uptake into cancer cells and the uptake was related to the expression of folate receptor on the cell surface.MTT assay and apoptosis indicated that folic acid-targeted nanodiamond drug system is more lethal to tumor cells than non-targeted nanodiamond drug systems and free drugs.Interestingly,in vivo experiments found that NPHF/D targeted to tumor cells through the synergistic effects of passive targeting and folic acid receptor proactively targeting,resulting in a significant increase in antitumor effect with a inhibition rate of 87%,which is more than 5.4 times of free DOX,while effectively reducing the toxic side effects.It can be seen that NPHF/D could achieve both the diagnosis and targeted treatment of tumor.So this study not only introduces simple and effective strategy to design promising drug delivery platform for realizing the diagnosis and treatment of tumor in the biomedical applications of the smart nanodiamond carriers,but also providing significant advances toward deeper understanding and exploration of theranostic drug-delivery systems.