Synthesis Of Polypeptide Nanogel And Its Application In Intravesical Chemotherapy For Bladder Cancer

ObjectiveApproximately 75%of primary bladder tumors are non-muscle-invasive bladder cancer?NMIBC?.Intravesical chemotherapy has been recommended after the gold standard of transurethral resection of bladder tumor to prevent bladder cancer?BC?from local recurrence.However,due to rapid urine excretion and barrier protection of bladder wall,the clinical performance of chemotherapeutic drug is severely compromised.In the present work,a smart positively charged disulfide-crosslinked nanogel of oligoarginine-poly?ethyleneglycol?-poly?L-phenylalanine-co-L-cystine??R₉-PEG-P?LP-co-LC??was prepared to prolong the retention period and enhance the penetration capability of chemotherapeutic agent toward bladder wall.PEG significantly improved the aqueous dispersibility of the 10-hydroxycamptothecin?HCPT?-loaded R₉-PEG-P?LP-co-LC??i.e.,R₉NG/HCPT?,and enhanced the mucoadhesive capability by the nonspecific interaction between PEG chain and bladder mucosa accompanied with the electrostatic interaction between the cationic R₉ and negatively charged bladder mucosa.Besides,R₉ as a cell-penetrating peptide could efficiently penetrate through cell membrane and deliver carried cargo.The disulfide bond endowed the selective release behavior of HCPT triggered by the intracellular reductive microenvironment.As an advanced chemotherapeutic nanoformulation,the smart R₉NG/HCPT indicated a great potential in the clinical intravesical BC chemotherapy.MethodsPart 1:MI-PEG-P?LP-co-LC?was synthesized by the ring-opening polymerization?ROP?of L-phenylalanine N-carboxyanhydride?LP NCA?and L-cystine N-carboxyanhydride?LC NCA?.R₉-PEG-P?LP-co-LC?was synthesized by the decoration of R₉ at the end of PEG by sequential deprotection,amidation,and Michael addition reactions.PEG chain is hydrophilic.The outstanding mucoadhesiveness benefited from the nonspecific interaction between PEG chain and bladder mucosa.P?LP-co-LC?is hydrophobic and reduction-responsive,which can accurately release HCPT within tumor cells.Hydroxycamptothecin?HCPT?was applied as a model drug and encapsulated into the core of R₉-PEG-P?LP-co-LC?and MI-PEG-P?LP-co-LC??i.e.,NG/HCPT negative control?.The physicochemical properties of R₉NG/HCPT and NG/HCPT,such as the drug loading content?DLC?,the hydrodynamic radius?R_h?,the zeta potential and the release behavior,were systematically characterized.Part 2:A Confocal laser scanning microscopy?CLSM?was performed to qualitatively evaluate the cell uptake and intracellular release behaviors of R₉NG/HCPT.The cell uptake and intracellular release behaviors were further confirmed quantitatively by microplate reader.A standard MTT assay was carried out to assess the potential cytotoxicity of R₉NG/HCPT.The R₉NG/HCPT-induced apoptosis of human BC 5637 cells was assessed by flow cytometry analysis?FCM?.Part 3:CLSM was employed to demonstrate the mucoadhesiveness and permeability of R₉NG/HCPT.The bladder accumulation of R₉NG/HCPT was verified by high-performance liquid chromatography?HPLC?.The antitumor efficacy of R₉NG/HCPT was assessed both on orthotopic BC in C57Bl/6 mice and SD rats.To define the development of BC,especially the tumor size,the cystography was performed.In order to evaluate the systemic toxicity of R₉NG/HCPT,the body weight of the experimental animals was monitored.To further accurately evaluate the antitumor activity of R₉NG/HCPT,the histopathological examination and immunohistochemical staining of the bladders was performed after the completion of intravesical chemotherapy.ResultsPart 1:The hydrodynamic radius?R_h?of R₉NG/HCPT in phosphate-buffered saline?PBS?at pH 7.4 was 48.9±0.7 nm,while that of NG/HCPT was detected to be 44.8±1.3nm.The zeta potential of NG/HCPT was-17.27±0.8 mV,while that of R₉NG/HCPT was26.9±1.2 mV.The positive charge of R₉NG/HCPT further demonstrated the successful conjugation of R₉ onto the surface of nanogel,which could enhance the interaction between R₉NG/HCPT and negatively charged cell membrane and subsequently upregulated cell internalization.R₉NG/HCPT possesses excellent stability under physiological conditions.However,the disulfide bond could be rapidly broken in the reductive microenvironment,resulting in the selective HCPT release in the cancer cells.Part 2:The CLSM images presented that the cells treated with R₉NG/HCPT exhibited the strongest HCPT fluorescence than those of free HCPT or NG/HCPT.With the prolongation of co-incubation time,the endocytosis of R₉NG/HCPT increased significantly and a relatively higher content of HCPT was maintained as compared to that of free HCPT or NG/HCPT.The increased HCPT concentration in the cells of R₉NG/HCPT group was about 1.9 times compared with the cells treated with free HCPT,while was about 1.5 times as much as that in the NG/HCPT group at 6 h.The cytotoxicity of R₉NG/HCPT surpassed those of free HCPT and NG/HCPT against human BC 5637 cells.Importantly,the half maximal inhibitory concentration(IC₅₀)of R₉NG/HCPT was 2.4±0.1?g mL^-1 in comparison with free HCPT(i.e.,8.7±0.8?g mL^-1)and NG/HCPT(i.e.,4.7±0.2?g mL^-1).R₉NG/HCPT contributed most to an abundance of apoptotic cells.The total percentage of apoptotic cells was 22.5%.In contrast,the total percentage of cells displayed apoptosis was reduced to 12.2%and 18.7%after incubation with free HCPT and NG/HCPT,respectively.Part 3:R₉NG/HCPT possesses high adhesion and retention toward the urothelial surface.On the contrary,the fluorescence signal in the bladders exposed to free HCPT decreased rapidly,which was due to the continuous excretion of urine.The fluorescence intensity of R₉NG/HCPT was 1.6 times higher as compared with that of free HCPT at 0.5 h.Inspiringly,the fluorescence intensity increased from 2.2 to 4.8 times when the retention time was prolonged from 2 to 6 h.The fluorescence intensity of R₉NG/HCPT gradually spread throughout the whole bladder tissue over time,and remained a comparatively higher HCPT content.HCPT,released from R₉NG/HCPT,was mainly accumulated in the bladder,which was 2.2 times higher as compared with free HCPT.The largest space-occupying lesion was identified in the bladder exposed to PBS,while there was no significant abnormality recognized in the bladder administrated with R₉NG/HCPT based on the smooth margin.The superior antitumor efficacy of R₉NG/HCPT was intuitionally approved by cystography.The experimental animals administrated with R₉NG/HCPT showed a stable increase in body weight during the treatment,indicating a negligible systemic toxicity of R₉NG/HCPT.Large amounts of cancer cells with spherical or spindle nuclei and obvious atypia were observed in the bladder section of the PBS group.Fortunately,the tumor cells showed karyopyknosis,chromatic agglutination,apoptotic body,and ill-defined morphology in the bladder administrated with R₉NG/HCPT,indicating the predominant contribution of R₉NG/HCPT in tumor suppression.The immunohistochemical staining for caspase-3 indicated that a large number of cells were undergoing apoptosis in the R₉NG/HCPT group.The expression of Ki-67 demonstrated that the growth of BC cells was obviously suppressed by R₉NG/HCPT.Conclusion1.A cationic R₉-decorated disulfide-crosslinked R₉NG/HCPT with enhanced adhesion and penetration toward bladder mucosa was designed and developed for upregulated BC chemotherapy.2.PEG and R₉ endowed R₉NG/HCPT with upregulated mucoadhesiveness,and R₉further promoted the permeability of laden nanogel within bladder wall.3.The reduction-responsive property enabled R₉NG/HCPT to release the cargo specifically in cancer cells triggered by the intracellular reductive microenvironment.R₉NG/HCPT may achieve the same intracellular drug concentration as clinical chemotherapeutic agents,but the administered quantity of R₉NG/HCPT will decrease.4.R₉NG/HCPT showed superior anitumor efficacy.