Preparation,Characterization And In Vitro-in Vivo Evaluation Of Bortezomib Supermolecular Aggregation Nanovehicles

Backgrounds:Intolerable toxicity and unsatisfactory therapeutic effects are still big problems retarding the use of chemotherapy against cancer.Nano-drug delivery system promised a lot in increasing the patients'compliance and therapeutic efficacy.As an unique nano-carrier,supermolecular aggregation nanovehicle has attracted increasing interests due to the following advantages:announcing drug loading efficacy,pronouncing in vivo performance and simplified production process.Methods:1.In this study,the supramolecular aggregation nanovehicle BTZ-NP was simply prepared by combining bortezomib(BTZ),tannic acid(TA),polyvinyl pyrrolidone(PVP)for the treatment of breast cancer.The particle size,morphology,surface zeta potential and stability were characterized by Zetasizer Nano(Malvern),transmission electron microscope(TEM)and scanning electron microscope(SEM).2.The mechanism of supramolecular nano BTZ-NP synthesis was characterized by hydrogen nuclear magnetic resonance spectroscopy(~1H-NMR),infrared absorption spectroscopy(IR),isothermal titration calorimetry(ITC)experiments.In the meantime,the interaction between BTZ and the other two components was further explored.3.The drug release of BTZ-NP was tested in phosphate buffer solutions(PBS)with different pH values(pH 7.4,pH 6.5).4.A serial concentrations of BTZ-NP solution,with PBS and deionized water as negative and positive control,was tested to calculate the hemolysis rate in vitro to verify its safety.5.Using blank supramolecular nanocarriers and free BTZ as controls,through cell uptake experiment,MTT experiment,transmission electron microscope experiment,Calcein-AM/PI live-dead cell double staining experiment,and Caspase-3 expression level the uptake effect of breast cancer4T1 cells on BTZ-NP,the antitumor effect of BTZ-NP on the cellular level and their biocompatibility were evaluated respectively.6.Construct a Balb/c mouse breast cancer xenotransplantation model,and use the near-infrared fluorescence IVIS?Lumina III small animal in vivo imaging system and inductively coupled plasma mass spectrometry(ICP-MS)method to determine the distribution of boron in the body,thereby indirectly reacting BTZ-The distribution of NP in the body.7.A breast cancer xenograft model of Balb/c mice was constructed,and its therapeutic effect was evaluated by weight and tumor growth curve,tumor weight and its pictures,Ki-67 protein and proliferating cell nuclear antigen(PCNA)expression in tumor tissues.The biocompatibility was evaluated by blood cell analysis,weight change,and hematoxylin-eosin(HE)staining.Results:1.The typical light-blue opalescence supermolecule BTZ-NP showed spherical morphology in both TEM and SEM images with the diameter of about 100±40 nm and,the surface zeta potential was about-20.0±8.5 mV.2.The boron hydroxyl group of BTZ and the phenolic hydroxyl group of TA form a boron ester,the phenolic hydroxyl group of TA and the carbonyl group of PVP form a hydrogen bond,that is,the intermolecular interaction between TA and the other two components promotes the formation of supramolecular BTZ-NP.3.BTZ-NP released faster in the slightly acid environment than in neutral conditions as the total amount of drug released from the former was about 70%,while the latter was only 35%.4.Hemolysis and agglutination reaction did not occur in each concentration gradient BTZ-NP.5.The uptake of BTZ-NP by breast cancer cells is time-dependent.In the MTT experiment,the free anti-tumor effect is the strongest with free BTZ,followed by BTZ-NP,both of which increase with the increase of BTZ concentration.Calcein-AM/PI live cell-dead cell double staining experiment and Caspase-3 expression results showed that compared with the free BTZ group,the apoptosis in the BTZ-NP group was slightly more obvious.6.In vivo imaging results of small animals showed that after 2 hours,BTZ-NP began to gradually enrich in tumor tissues and reached a maximum value in the following24 hours.ICP-MS experiments show that BTZ-NP is enriched in the tumor site and the distribution of drugs in the heart is reduced.7.The mice in the BTZ-NP treatment group had no significant changes in body weight and did not produce long-term toxicity,demonstrating their good safety and biocompatibility.In terms of anti-tumor effect,the tumor-bearing mice treated with PBS grew rapidly,and the BTZ solution helped to slow the growth of the tumor.In contrast,the BTZ-NP treatment group produced the most satisfactory anti-tumor effect.Conclusions:Due to the simple preparation,stable structure,pH and tumor microenvironment sensitivity,and promising tumor targeting ability of the BTZ nanovehicles,the supermolecule displayed promising tumor curing effects and negligible systemic toxicity.