Synthesis And Application Of Targeted Nanophotosensitizers For Tumor Photodynamic Therapy

Objectives:1.To synthesize and characterize novel targeted nanophotosensitizers scFv-Z@FRT and HANP/Ce6 complexes.They are of different particle sizes.And then we evaluate their stability,water solubility and ROS generation capacity respectively to offer a basis for further research and application.2.To evaluate the binding capacity of scFv-Z@FRT to different tumor tissue.Clearance of CAF and collagen of scFv-Z@FRT mediated PDT in bilateral 4T1 tumor models is discussed,and the impact of accumulation and penetration of nanoparticles with different partical sizes in tumors is evaluated after PDT.3.To investigate the cytotoxicity and targetability of HANP/Ce6 complexes,and their distribution in whole body.To evaluate the antitumor efficacy of HANP/Ce6complexes mediated PDT on HT29 tumor-bearing mice,and to explore the value of¹⁸F-FDG PET in its efficacy evaluation.Methods:1.Polymerase chain reaction?PCR?was used to amplify the FRT DNA segment from the cDNA.The PCR product was ligated to the pRSF plasmid,and the product was used to transform competent cells of E.coli XL1-Blue.The selected pRSF/FRT plasmid was used to transform E.coli BL21?DE3?,and then the production of ferritin was induced and purified.In order to obtain anti-FAP scFv,the DNA coding sequence was inserted into a pOPE101 plasmid and transformed into E.coli JM109 with ampicillin-resistance.A PelB signal peptide sequence was inserted into the N-terminus of the scFv,and anti-FAP scFv was induced and purified.The pH of a FRT solution was reduced to².0 to break the interaction between adjacent ferritin subunits.Then we added ZnF₁₆Pc in DMSO into the solution,and then we slowly turned the pH back to neutral to induce nanocage reconstitution.The resulting Z@FRT was then purified.The anti-FAP scFv was coupled to the surface of Z@FRT using bis?sulfosuccinimidyl?suberate?BS3?as a crosslinker,the raw product was purified and then we obtained scFv-Z@FRT.DLS was used to analyse FRT,Z@FRT and scFv-Z@FRT.The final product scFv-Z@FRT was placed into PBS for one week to observe the dissolution and stability.A 671 nm laser at 0.1 W/cm² was used to irradiate the Z@FRT and free ZnF₁₆Pc solutions at 1,3,5,10,and 15 min respectively,and the 525 nm fluorescence was recorded and the singlet oxygen generation was compared between Z@FRT and free ZnF₁₆Pc.2.scFv-Z@FRT was labeled with IRDye800CW?ex/em=780/800 nm?,and then scFv-Z@FRT was incubated with tissue sections from primary 4T1 tumor,LL/2metastasis in liver and lung to verify scFv-Z@FRT binding ability to tumors of different types.In order to verify its targeting,we added blocking groups in different types of tumors,that is,free scFv?30×?was applied simultaneously for blocking experiments.To assess the clearance of CAF and collagen,we injected¹0⁶ 4T1 cells per site to both left and right hind limbs of each mouse to establish a bilateral 4T1tumor model in nude mice.When the tumor size reached¹00 mm³,scFv-Z@FRT was injected intravenously into the mice?n=3?.After 24 hours,the right-hand-sided tumor was irradiated with a 671 nm laser?300 mW/cm² for 15min?,while the left-hand-sided tumor was covered with aluminum foil.Mice were euthanized 2 days after the treatment,tumor sections were made and stained with anti-?-smooth muscle actin??-SMA?antibody to detect the clearance of CAF.In addition,the content of collagen in tumor ECM was detected by Mason trichrome staining.In order to investigate the impact of PDT on tumor accumulation and penetration of nanoparticles,we also used the above animal model.48 h after irradiation,mice were intravenously injected with 1nmol IRDye800CW?ex/em=780/800 nm?-labeled BSA?5 mg/kg?,10nm PEGylated CdSSe/ZnS QDs or 50 nm PEGylated QDs respectively?n=5 per group?.In vivo imaging was performed at 5 min,10 min,30 min,1 h,4 h,and 24 h after injection.After the 24 h imaging,the mice were euthanized and the tumors were harvested to perform ex vivo imaging.Tumor specimens were incubated with phycoerythrin-labeled anti-CD31 antibody for immunofluorescence staining analysis to observe the extravasation and diffusion of BSA and QDs.3.The HANP/Ce6 complexes were prepared using a high-pressure homogenizer.HA and 5?-cholanoic acid conjugate HA-5?-CA were synthesized in the presence of EDC and NHS.A Ce6 solution was dissolved in DMSO with sonication for 15min,and then slowly added into an HANP solution and homogenized for 10 min,then we removed free Ce6 to obtain HANP/Ce6 complexes.The samples were analyzed based on a standard curve of free Ce6.We observed a linear relationship between the concentration of Ce6 and its absorbance to calculate Ce6 loading efficiency.The absorbance spectrum of HANP/Ce6 complexes was scanned and compared with that of free Ce6.The fluorescence intensity of the HANP/Ce6 complexes and free Ce6was measured with a fluorescence spectrophotometer using an excitation wavelength of 403 nm.Dynamic light scattering was used to measure the size distribution of HANP/Ce6 complexes.The stability test of HANP/Ce6 complexes and free Ce6 were conducted in fetal bovine serum?FBS?,cell medium containing 10%FBS,PBS,and water for 7 days respectively.We compared the release of Ce6 from HANPCe6 with or without hyaluronidase.To investigate the relationship between the presence or absence of hyaluronidase and ROS generation,we used a DCFH-DA kit to detect ROS generation in free Ce6 and HANP/Ce6 complexes with or without hyaluronidase.4.To investigate the targeting of HANP/Ce6 complexes,HT29 cells and NIH3T3cells were selected and divided into an HANP/Ce6 group and a free Ce6 group.HANP/Ce6 complexes and free Ce6?20?g/mL?were added respectively.To verify the specific binding of HA to CD44,we added excess amount of HA to the HT29 cells to set up a blocking group.We used laser scanning confocal fluorescence microscope to observe the result after staining with DAPI.HT29 cells were cultured and divided into an HANP/Ce6 group and a free Ce6 group.Laser irradiation was given after incubation to detect the ROS generation capacity.To investigate the cytotoxicity,we set up an HT29 group and a NIH3T3 group,and treated with different concentrations of HANP/Ce6 complexes and free Ce6?40?g/mL,20?g/m L,10?g/mL,5?g/mL and2.5?g/mL?respectively.Cells were then treated with or without laser irradiation.Cell viability was assessed by MTT assay.To further confirm the PDT-induced cancer cell death,calcein-AM/PI co-staining was performed.A nude mouse model of HT29tumor was constructed.When the tumor grew to about 60 mm³,mice were randomly divided into an HANP/Ce6 group and a Ce6 group?n=3 per group?.HANP/Ce6complexes?5 mg/kg of equivalent amount of Ce6?and Ce6 were administered to the mice via tail vein injection,respectively.The fluorescent signals were observed using Carestream FX Pro at time points of 1,2,4,6,12,and 24 h after intravenous injection.Mice were sacrificed at 4 h post-injection?p.i.?,tumors and major organs,including heart,liver,spleen,kidney,and muscle were collected for imaging using Carestream FX Pro.We also used the nude mouse model of HT29 tumor,the tumor-bearing mice were randomly divided into the following five groups?n=5 per group?:?1?Saline,?2?free Ce6 with laser,?3?free Ce6 without laser,?4?HANP/Ce6 with laser,?5?HANP/Ce6 without laser.Tumor size,mouse weight were monitored,and PET scans and image analysis were performed.Mice were sacrificed two weeks after the treatments,and tumors and major organs in each group were taken and analyzed using HE staining.Results:1.Successfully synthesized novel targeted nanophotosensitizers scFv-Z@FRT and HANP/Ce6 complexes.They were of different particle sizes and both of them had good stability,water solubility and ROS generation capability.2.scFv-Z@FRT was able to bind to different types of tumors?primary 4T1tumor as well as LL/2 metastasis in liver and lung?through scFv-FAP interactions,and it could be blocked by free scFv.In the tumors of bilateral 4T1 tumor models,the level of positive?-SMA staining was dramatically decreased after PDT treatment,and the Mason trichrome staining showed that the contents of collagen were reduced significantly compared to the un-irradiated tumors,indicating that CAF and collagen were eradicated due to the scFv-Z@FRT mediated PDT.After PDT treatment,different partical sizes of the macromolecules/nanoparticles?BSA,10 nm QDs,50 nm QDs?were given,the accumulation and penetration of nanoparticles with different partical sizes in the tumor were observed to be improved.Immunofluorescence staining showed that all the nanoparticles with different partical sizes showed vascular extravasation.However,many of the relatively large nanoparticles?50 nm QDs?accumulated at the tumor peripheral region.This suggests for relatively bulky nanoparticles,diffusion in tumors is still limited despite of PDT-induced ECM breakdown.3.Degradation of HANP with hyaluronidase made it possible to detect fluorescence after the release of Ce6 in the HANP/Ce6 complexes,and the amount of Ce6 released after using hyaluronidase increase significantly compared with the group without hyaluronidase.Compared with Ce6 encapsulated in HANP,more ROS generated after its released from HANP.In the HANP/Ce6 targeting investigation,CD44-positive cells were able to take up a large amount of HANP/Ce6 complexes,and could be blocked by HA,indicating its good CD44 targeting.In the investigation of ROS gengration capacity,the amount of ROS generation continued to increase with the increase of Ce6 concentration in the HT29 cells treated with free Ce6 or HANP/Ce6 complexes.But the ROS generation ability of HANP/Ce6 complexes was significantly better than free Ce6.HANP/Ce6 had good cytotoxicity,when the cells in the HANP/Ce6 group were irradiated with laser,the cell viability decreased with the increase of HANP/Ce6 concentration.Only 13.05%±1.43%of the cells in the HANP/Ce6 with laser group survived at a concentration of 40?g/mL?the equivalent amount of Ce6?.After intravenous injection to mice,fluorescence imaging showed that HANP/Ce6 comlpexes accumulated in the tumor area and the liver,and the fluorescence signal reached a peak at 4 hours p.i.,but there was no obvious fluorescence signal observed in other major organs and tissues.After PDT on the HT29 tumor-bearing mice,compared with the control group,tumor growth in mice of the HANP/Ce6 with laser group was significantly suppressed,and no detectable side effects were observed,which was also confirmed in histological analysis.In terms of efficacy monitoring,changes in tumor size were consistent with changes in ¹⁸F-FDG uptake.PDT-induced cell death occured shortly after the initiation of treatment,and the ¹⁸F-FDG PET effectively monitored PDT tumor responses before changes in tumor size were observed.Conclusions:In this study,two kinds of novel targeted nanophotosensitizers,scFv-Z@FRT and HANP/Ce6 complexes,of different particle sizes are successfully synthesized,both of which have good stability,water solubility,biocompatibility,and ROS generation ability.scFv-Z@FRT uses ferritin as a carrier,which not only encapsulates photosensitizer ZnF₁₆Pc,but also has good biocompatibility.scFv has CAF targeting by specifically binding to FAP,which can help eradicate CAF effectively by PDT in order to improve the accumulation and penetration of nanoparticles in tumors.With its good properties,ferritin has shown broad research prospects as a nano-platform in drug and imaging agent delivery.In addition,tumor stroma targeting also provides new ideas for the design of tumor-targeted nanomedicines and PDT.Since the FAP is highly expressed in most solid tumors,this method is expected to work effectively in other types of tumors.This kind of PDT,which improves the accumulation and extravasation of nanoparticles,is expected to be used as a pre-treatment application for other nanomedicines and has potential research value.On the other hand,HANP/Ce6 complexes accumulates in tumors through the active and passive targeting effect,that is,it accumulates in tumors through CD44 targeting and enhanced permeability and retention?EPR?effect.HANP/Ce6 mediated PDT can reduce the dosage and improve the efficacy.HANP has potential value as a drug delivery platform in improving the performance of other hydrophobic drugs.At the same time,compared with the traditional monitoring method based on chenges in tumor size,¹⁸F-FDG PET could be used for predicting and monitoring PDT therapeutic effect,in order to monitor the tumor responses earlier,so as to be used as a guide in the clinical adjustment of the dosage and therapeutic schedule in time.