Preparation And Investigation Of Multifunctional Nanodrug For Bladder Cancer Treatment

Background:Bladder cancer is the 10^thmost common malignancy worldwide,accounting for3%of all new cases and killing approximately 210,000 people each year.In addition,early disgnosis,early treatment and regular mornitoring of bladder cancer are of clinical importance due to that bladder cancer is highly prone to recurrence and progression.Although traditional diagnosis and treatments for bladder cancer can have a definite clinical impact,they are also criticized for their tremendous and indiscriminate damage to normal cells and tissues.Thus,the precise diagnosis and treatments for bladder cancer have been attracting a lot of interest from researchers in recent years.According to the literatures,overexpression of fibroblast growth factor receptor3 can occur in 75%of non-muscle-invasive bladder cancers.Mutations of this receptor could lead to the development of non-ligand-dependent dimers and activation of downstream signalling pathways,eventually leading to the development of bladder cancer.As a result,a large number of studies have been conducted to explore the use of fibroblast growth factor receptor inhibitors for the targeted treatment of bladder cancer.Erdafitinib is the first fibroblast growth factor receptor inhibitor to be approved by the US Food and Drug Administration for the treatment of progressive or metastatic bladder cancer.Although erdafitinib has been shown to be effective in the treatment of bladder cancer,the high frequency of administration and the occurrence of multiple side effects have significantly limited its clinical application.Therefore,the development of a safer and more effective drug platform for the delivery of erdafitinib is of great clinical significance.Nanodrugs hold great promise in drug loading and functionalization,and nanodrugs could be afforded with new functions through precise design,including prolonged in-vivo half-life,reduced cytotoxicity and imaging capabilities etc.However,the clinical application of nanodrugs constructed by organic or inorganic materials remains restricted for their strong cytotoxicity and poor biocompatibility.Furthermore,the effective production of nanodrugs cannot be reliably guaranteed because of the cumbersome preparation process and wide particle size distribution.Therefore,it’s imperative to develop multifunctional nanodrugs using materials with good biocompatibility and low cytotoxicity,which will broaden the clinical application of nanodrugs and increase the clinical beneficiaries.Elastin-like polypeptide is an artificial peptide originated from the hydrophobic region of elastin,which exhibits a series of properties that are difficult to achieve using polymers.First of all,elastin-like polypeptide is biocompatible and biodegradable,which can be degarded into harmless amino acids.In addition,it is possible to afford elastin-like polypeptide or elastin-like polypeptide based fusion proteins with specific functions through method of genetic engineering.Interestingly,elastin-like polypeptides also exhibit temperature responsiveness due to their characteristic of low critical solution temperature,leading to a more simplified purification process.As a result,elastin-like polypeptides are widely applied in the fields of drug loading.Based on the previous research of our group,the nanocarriers constructed with recombinant elastin-like polypeptide are characterised by high drug loading capacity,low toxicity,long-lasting effect and good biocompatibility.In addition,the fusion protein constructed by monomeric near-infrared fluorescent proteins and elastin-like polypeptide not only shows the above advantages of elastin-like polypeptide,but also exhibits high-resolution imaging capability for deep tissue,making it a perfect choice for the development of theranostic platform.Objectives:This work developed a multifunctional nanodrug by ultilizing biocompatible materials for the bladder tumor therapy.Furthermore,we explored the morphology,the in-vitro and the in-vivo tumor suppression effect,side effects,pharmacokinetics parameters,intracellular distribution and imaging capability of the prepared multifunctional nanodrug.Methods:1.First of all,we developed m IFP-K72 recombinant protein through the method of genetic engineering.Based on the prepared m IFP-K72 recombinant protein,carboxylated polyethylene glycol and erdafitinib,we further developed a multifunctional nanodrug through self-assembly strategy.The size and morphology of the nanodrug and nanocarriers were characterized by transmission electron microscopy and dynamic light scattering,respectively.2.The in-vitro anti-tumor efficacy was further assessed by cell cytotoxicity assay and live/dead staining assay,and the IC50 of the applied drugs was compared.At different time point after injection,mice plasma was collected and the content of erdafitinib was further measured by high performance liquid chromatography.Furthermore,the in-vivo half-life and bioavailability of nanodrug was calculated using the result above.In addition,the intracellular distribution of nanodrug and erdafitinib were observed by confocal microscope,and the intracellular mechanism of the nanodrug was analysed.3.A heterogeneous subcutaneous bladder tumour model was prepared and its tumour growth was recorded during the treatment.The tumor suppression effect and side effects of each drugs were further evaluated.The isolated tumor tissues were applied for the histological analysis and evaluation of the expressed cell proliferation related antigen.Lastly,the real-time imaging capability of nanodrug was assessed by the Maestro EX fluorescence imaging system.Results:1.Uniform nanoparticles were prepared by self-assembly strategy,where the diameter of the nanodrug and the vehicle was 161.8±45.6 nm and 120.8±36.1 nm,respectively.The hollow spherical structure of vehicle was highly suitable for drug loading,exhibiting a loading efficiency and encapsulation efficiency of 33.6%and43.7%,respectively.2.The erdafitinib exhibited stronger in-vitro anti-tumor effect to the FGFR overexpressed RT4 cell than the FGFR low-expressed T24 cell,verifying the selectivity of erdafitinib.In addition,the nanodrug showed stronger anti-tumor effect in both cells when compared with pristine erdafitinib.Notably,the vehicle was non-toxic to both cells.The IC₅₀of nanodrug and erdafitinib for RT4 cell and T24 cell were 14.0 nmol/L and 29.9 nmol/L,25.5μmol/L and 35.4μmol/L,respectively.Interestingly,the Live/dead staining assay came out a similar result,where vehicle was harmless while naodrug and erdafitinib showed close anti-tumor effect.3.The in-vivo half-life and bioavailability of nanodrug were 8.3 times（14.4 h）and 5.0 times（8.0μg/m L·h）that of the pristine erdafitinib.In addition,erdafitinib and nanordrug were mainly distributed in the lysosome after transformed into the cells.4.By recording tumor growth of heterogeneous subcutaneous tumor model in each group,it was found out that all nanodrug group and erdafitinib group showed significant tumor growth inhibition effect and the average tumor volumes in the high,medium and low nanodrug group and erdafitinib group were 52.8±9.3 mm³,52.7±15.6 mm³,70.6±13.0 mm³and 48.8±3.4 mm³,respectively.The corresponding tumor growth inhibition rates of these four groups were 69.1%,69.1%,58.6%and71.4%,respectively.In contrast,the tumor volume of PBS group and vehicle group increase to 170.7±14.4 mm³and 160.5±6.7 mm³,respectively.5.HE staining and immunohistochemistry analysis also revealed a significant tumor growth inhibition effect of nanodrug.The proportion of tumor cells in the nanodrug or erdafitinib group was significantly lower than that of the control group.Scattered calcification could be observed in the nanodrug group while the control group showed significant nuclear heterogeneity and high nucleoplasmic ratio.Furthermore,the nanodrug group and erdafitinib group exhibited significantly lower Ki67 expression when compared with that of the control group.After quantification,Ki67 expression in the high-dose,medium-dose nanodrug groups and the erdafitinib group was found to be lower than the other three groups and there is a significant difference,while Ki67 expression in the low-dose nanodrug group was lower than that in the PBS and vehicle group and there is also a significant difference.Notably,the blood phosphorus content of the erdafitinib group after treatment were significantly higher than those of the other five groups with an average concentration of 3.8±0.3 mmol/L.6.Nanodrug exhibited high-resolution targeted imaging capability.16 h after injection,significant fluorescent signals were found locally in the tumor in the nanodrug group,while no fluorescent signals were found in the PBS group.Conclusion:1.This study proposed a general assembly strategy for nanodrug preparation and prepared a multifunctional nandodrug,which achieved the efficient loading,targeted delivery and slow release of erdafitinib.2.The multifunctional nanodrugs effectively prolonged the in vivo half-life of erdafitinib and alleviated the side effects caused by erdafitinib treatment,thus accomplishing a long-lasting,potent and safe bladder tumor treatment.3.This multifunctional nanodrug demonstrates high-resolution imaging capabilities,thus achieving simultaneous bladder tumor treatment and imaging.