Synthesis And Application Of Injectable Hydrogel For Bladder Cancer Phototherapy

Objectives:1.To synthesize and characterize rare earth-doped fluorescent upconversion-gold hybrid nanoparticles(UCNP-Au NPs)with near-infrared light response.Then we evaluate its photo-thermal conversion effect and peroxidase-like property,which are the basic characteristics for subsequent PTT and PDT research.2.To prepare the injectable DNA-UCNP-Au hydrogel compounded by UCNP-Au NPs and salmon sperm DNA(smDNA),and perform basic characterization.Explore its efficacy against bladder cancer(T24 cell line)with PTT and PDT upon 808 nm near-infrared light irradiation.3.To prepare the injectable UCNP-Au-Alg hydrogel composed of UCNP-Au NPs and sodium alginate(Alg),and perform basic characterization.To explore its PTT effect against bladder tumors in vitro and in vivo with 808 nm laser irradiation.At the same time,the rat tibia defect model is used to study its additional protection and repair of damaged bone caused by bone tumors.Methods:1.Upconversion nanoparticles(Na YF4:Yb,Er)doped with Yb,Er and Nd were prepared by high temperature oil solvent method.And the shell-core upconversion nanoparticles(Na YF4:Yb,Er@Na YF4:Nd,UCNP)were synthesized through epitaxial growth.Then the obtained UCNPs could be excited by 808 nm laser.Subsequently,the oleic acid molecules on the surface of UCNP were protonated with H⁺.Then the oil-soluble UCNP became water-soluble.UCNP@PEI was prepared by mixing polyethyleneimine(PEI)with ligand-free UCNPs.Finally,ultra-small gold nanoparticles were grown in situ on the surface of UCNP@PEI through oxidation-reduction reaction to obtain UCNP-Au NPs.The morphological characteristics were described by transmission electron microscope and hydrated particle size.The surface potential was measured by Zetasizer Nano.The UV absorption spectrum and 808 nm laser excitation spectrum were done to analyze its absorption and light conversion characteristics.Photothermal conversion efficiency of UCNP-Au NPs was calculated.Using 3,3',5,5'-tetramethylbenzidine(TMB)and p-benzoic acid(TA)as ROS probes to analyze the peroxidase-like property of UCNP-Au NPs with or without 808 nm laser irradiation.Using2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)as a probe to test the intracellular ROS production during 808 nm laser irradiation.2.The long-stranded smDNA was heated to 90?to cause thermal cracking between the double strands.In the subsequent annealing process,mismatches were randomly formed to obtain first step of cross-linking.Then,DNA-UCNP-Au hydrogel was prepared by mix positive UCNP-Au NPs with the heated smDNA gel.The changes in the mechanical properties of the DNA-UCNP-Au hydrogel were realized by adjusting the content of DNA and UCNP-Au NPs in the system.And the mechanical property was analyzed by a rheometer.The photothermal conversion efficiency of DNA-UCNP-Au hydrogel was calculated with 808 nm laser irradiation.Then the inhibition to T24 cell line caused by irradiation was tested with MTT assay.Subsequently,the DNA-UCNP-Au hydrogel was injected into mice to study its biocompatibility and photothermal conversion effect in vivo.Finally,T24tumor-bearing nude mice was used to study the anti-tumor effects of pristine UCNP-Au NPs and DNA-UCNP-Au hydrogel upon 808 nm laser irradiation.The degradation and excretion of DNA-UCNP-Au hydrogel in bladder were simulated in vitro.3.Na Gd F4:Yb,Er@Na Gd F4:Yb,Nd were prepared as before.Then,UCNP-Au NPs with higher relative density of Au NPs were prepared.The injectable UCNP-Au-Alg hydrogel is obtained by mixing negatively charged sodium alginate and positive UCNP-Au NPs.The elastic UCNP-Au-Alg-Ca²⁺ hydrogel was prepared by secondary cross-linking with Ca²⁺.The mechanical properties were measured through the tensile test,compression test and rheometer analysis.The photo-thermal effect of UCNP-Au-Alg hydrogel and UCNP-Au-Alg-Ca²⁺ hydrogel under 808 nm laser irradiation was verified in vitro.MTT,live/dead cell staining,and flow cytometry were used to study the influence to UCNP-Au-Alg-Ca²⁺ hydrogel cultured T24 cells.The UCNP-Au-Alg-Ca²⁺ hydrogel in vivo was obtained by injecting UCNP-Au-Alg hydrogel into tissues directly.Then the stability and photothermal conversion effect were further analyzed.The in vivo anti-tumor study of UCNP-Au-Alg-Ca²⁺hydrogel under 808 nm laser irradiation was observed in T24 tumor-bearing nude mice.The tumor slices were stained with H&E,Ki67,and TUNEL to explored the mechanism.Rat tibia defect model was used to explore the support and protection effect of UCNP-Au-Alg-Ca²⁺hydrogel on damaged bone.Results:1.The average particle size of the synthesized UCNP-Au NPs was 32.4±1.4 nm,the hydrated particle size was 247.9 nm,and the surface potential was 25.4 m V.UCNP-Au NPs had a surface plasmon resonance absorption peak at the wavelength of 540 nm,which overlapped with the emission peak of UCNP upon 808 nm laser irradiation.The photo-thermal conversion efficiency of pristine UCNP-Au NPs aqueous solution was 32.44%.It could induce the decomposition of H₂O₂ in vitro and oxidize TMB and TA.This reaction was enhanced upon 808 nm laser irradiation.As the UCNP-Au NP was absorbed by T24 cells,it would induce the production of intracellular ROS under 808 nm laser irradiation.2.The composite DNA-UCNP-Au hydrogel with the characteristics of shear thinning could be injected through fine needle.The photothermal conversion efficiency was 42.67%.Compared with the pristine UCNP-Au NPs aqueous solution,the duration time of DNA-UCNP-Au hydrogel in vivo could be extended to 2 days.The tissues injected with UCNP-Au NPs could reach to 56.8? under 808 nm laser irradiation for 3 minutes,while tissues injected with DNA-UCNP-Au hydrogel could reach to 62.2?.The anti-tumor study with T24 tumor-bearing mice confirmed that DNA-UCNP-Au hydrogel had a better anti-tumor effect with 808 nm laser irradiation(1 W/cm²,for 3 min),and no tumor recurrence was seen during the 21-day observation period.However,the tumors treated with pristine UCNP-Au NPs recurred in 21 days.Experiments in vitro successfully simulated the procedure of degradation and excretion of DNA-UCNP-Au hydrogel after injection into the bladder.3.The UCNP-Au-Alg-Ca²⁺ hydrogel was obtained by soaking UCNP-Au-Alg hydrogel into calcium chloride.The average tensile strength of UCNP-Au-Alg-Ca²⁺hydrogel was 1.29±0.18 MPa,and the compressive strength was 1.17±0.09 MPa.The photothermal conversion efficiency of UCNP-Au-Alg hydrogel was 36.7%.As the introduction of Ca²⁺,the penetration of 808 nm laser in hydrogel was decreased,and the temperature rose unevenly upon irradiation.After 3 minutes of irradiation,the surface temperature could reach to 111 ?.When the UCNP-Au-Alg hydrogel was injected into tissues,the Ca²⁺ in extracellular fluid could induce the in situ formation of elastic UCNP-Au-Alg-Ca²⁺ hydrogel.The compact network maintained complete in tissues for at least two weeks.Because the UCNP-Au NPs were locked in the network,the irradiation could be repeated at one week after irradiation,and the local temperature could still be more than 50?.As a result,all the cancer cells were eliminated.Due to the filling of UCNP-Au-Alg-Ca²⁺ hydrogel in the bone defect,the damaged bone was protected from the occurrence of complete fractures.Conclusions:In this study,the novel NIR-light-responsive UCNP-Au NPs are successfully prepared.In addition to high photothermal conversion efficiency and light stability,it can induce the production of ROS under 808 nm laser irradiation in vitro or in vivo.The cationic UCNP-Au NPs are easier to compound with negative charged biological molecules.The obtained injectable DNA-UCNP-Au hydrogel has stronger photothermal conversion efficiency and biological stability than pristine UCNP-Au NPs.Combined with external 808 nm laser irradiation,it can be applied to the infusion treatment for NMIBC(non-muscular invasive bladder cancer)and the percutaneous treatment for MIBC(muscular invasive bladder cancer).The remarkable material will provide a new platform for clinical bladder cancer treatment.The UCNP-Au-Alg hydrogel can be further strengthened with the Ca²⁺in tissues.The obtained UCNP-Au-Alg-Ca²⁺ hydrogel with good biocompatibility will delay the metabolism of UCNP-Au NPs.Thereby,repeated irradiation can be achieved through once injection to eliminate all the cancer cells.For the bone metastases,the obtained elastic UCNP-Au-Alg-Ca²⁺ hydrogel filling in the damaged bone can stabilize the bone structure and avoid the occurrence of complete fractures.