| Photodynamic therapy?PDT?is a new method for the treatment of various diseases by using photosensitizers to produce reactive oxygen species after photoactivation at specific wavelengths to kill tumors or specific cells,especially in the treatment of skin diseases and superficial tumors.As a typical representative of second-generation photosensitizers,chlorin-based photosensitizers have become the hotspot in the research of new photosensitizers because of their advantages of large absorption wavelength,high absorption coefficient,fast removal rate,and diverse structure.In the study of photodynamic therapy for tumors,it was found that the activity of histone deacetylase?HDAC?in tumor cells was significantly increased after photodynamic therapy,while the level of histone deacetylation was decreased,which may cause tumor cells rapid proliferation and tumor recurrence and metastasis.Recurrence and metastasis are one of the most important problems in clinical photodynamic therapy.If HDAC inhibitors and photosensitizers can be administered together by drug molecular design or new drug delivery system,the different mechanisms of HDACis and photodynamic therapy can be combined,which is expected to enhance the anti-tumor activity of single treatment mode,play a synergistic role and achieve better therapeutic effect.The work of this thesis is divided into two parts:Firstly,we attempt to synthesize a novel dual-mode small molecule drug by covalent crosslinking of pyropheophorbide a with classical HDACIS suberaniline hydroxamic?SAHA?in order to achieve the synergistic effect of the drug with multi-effect and different anti-tumor mechanisms,and preliminarily study had been done of its killing effect on cancer cells;Second,in order to overcome the shortcomings of poor solubility of photosensitizer and HDACis,short half-life in vivo and to improve their co-administration efficiency and enhance their targeting,we designed and synthesized a self-assembled nanomicelle drugs delivery system with folate targeting,pH-sensitive release in tumor sites by covalent binding of HDACis?chidmide?.The combined delivery system realizes the co-loading of the photosensitizer pyropheophorbide a by the hydrophobic interaction inside the micelle at the time during micelle formation,and finally achieves the targeted co-administration of the photosensitizer and HDACis,synergistic effect,etc.The anti-tumor effect of the combined delivery system was verified by cell experiments in vitro and tumor-bearing mice in vivo experiments.1 Synthesis,characterization,and activity of small molecule dual-mode antitumor drugsIn this part,the chlorophyll a was used as the raw material to synthesize the chlorin-based photosensitizer pyropheophorbide a by two-step acid degradation reaction,and then the photosensitizer and HDACis?SAHA?are covalently crosslinked with cystamine containing a redox-responsive disulfide bond.A novel dual-mode small molecule chlorin-SS-HDACis compound with both anti-tumor activitys of light and chemotherapy was synthesized.Compared with the ultraviolet absorption spectrum of the original photosensitizer Pha,the synthesized Pha-SS-SAHA compound has 9 nm blue shift with the maximum absorption wavelength,which lead to a decrease in tissue penetration.The results of anti-tumor experiments in vitro showed that Pha-SS-SAHA could effectively kill mouse melanoma cells B16-F10,and the IC50 of Pha-SS-SAHA and Pha under unilluminated conditions were 9.505±0.540?M,7.171±0.400?M and 4.576±0.609?M calculated according to CCK-8 method.Compared with the original photosensitizer,the dark toxicity of Pha-SS-SAHA slightly decreased.The IC50 values of Pha-SS-SAHA and Pha under light conditions with 10 J/cm2 were 0.821±0.051?M and 0.499±0.018?M respectively.When 1?M Pha-SS-SAHA,Pha or SAHA were administered,the apoptosis of B16-F10 cells was detected at 10 J/cm2 illumination after incubation for24 h.The proportion of cell necrosis was 65.54%,92.01%,17.12%,respectively.The Pha-SS-SAHA has lower phototoxicity to tumor cells than photosensitizer Pha,which may be related to the steric hindrance of SAHA to the porphyrin ring of Pha inhibiting the formation of ROS.It is more likely to be cross-linking of covalent bonds affecting the effective release of photosensitizer molecules,and this strategy or cross-linking method is less suitable for co-administration of the two drugs.Therefore,this is also an important reason for us to carry out the second part of this paper.2 Synthesis,characterization,and activity of pH-responsive polymer dual-mode anti-tumor micelles targeted by folate.Chidamide is a benzamide HDACis,which is the first original innovative drug with independent intellectual property rights in China.In this part of the study,we designed a pH-responsive co-loaded micelle Pyropheophorbidea@Folate-Polymer polyethylene glycol-b-poly?aspartic acid?-Chidamide?Pha@FPPC?.Firstly,chidamide was grafted onto the pH-responsive block polymer PEG-b-PAsp.At the same time,folate?FA?was conjugated on the head of the polymer as a target to increase the targeting of drug delivery system.When the amphiphilic block copolymer FPPC forms a micelle by self-assembly,the poorly soluble photosensitizer Pyropheophorbide a can be encapsulated inside the micelle by hydrophobic interaction to form a co-loaded nanomicelle Pha@FPPC.The co-loaded micelle can achieve the co-targeting drug delivery of photosensitizers and chidamide and the responsive release of the tumor site,which is expected to play a synergistic role in different anti-tumor mechanisms.?1?Using L-aspartic acid?-benzyl ester?H-Asp?OBzl?-OH?as raw material,FA-PEG-Pasp was synthesized by intramolecular cyclization,intermolecular polymerization,and hydrolysis.Then the amphiphilic block polymer FPPC was synthesized by covalently bonding cedarbenzidine with 2-?7-oxo-benzotriazole?-N,N',N',N'-tetramethylurea hexafluorophosphate?HATU?as condensation agent.The 1H-NMR characterization indicated that FPPC contained both the characteristic absorption peaks of chidamide and FA-PEG-Pasp,which proved the successful construction of polymer materials.The polymer material FPPC can form micelles by self-assembly in PBS solution,and Pha can be encapsulated in the micelle by hydrophobic interaction in this process.Then double-mode anti-tumor micelle Pha@FPPC with folate targeting and pH responsive release can be constructed.?2?Drug loading,particle size were detected for the drug-loaded micelles and blank micelles,and pH response release of Pha were detected for the drug-loaded micelles,respectively.The drug loading of Pha in the micelles was 4.67%.The microscopic morphology of the blank micelles and the photosensitizer-loaded micelles was observed by transmission electron microscopy.The particle size of the blank micelles was about 83 nm,and the particle size of the drug-loaded micelles was around 91 nm,all of which had regular spherical morphology.At 72 h,the release rate of the drug-loaded micelles at pH 5.2 and 7.4 was 60.70%and 47.75%,respectively,which showed good pH responsiveness.?3?The photodynamic effects of Pha@FPPC on A2780,B16-F10,and HUVEC cells in vitro were determined by cyctotoxicity test.The results showed that Pha@FPPC had lower dark toxicity and higher phototoxicity than the model photosensitizer Pha.(In the B16-F10 cell line,the IC50 value of phototoxicity decreased from 0.186?M to 0.169?M,the IC50 value of dark toxicity increased from 4.722?M to 34.614?M,and the dark toxicity/phototoxicity increased by 8 times).The in vitro targeting and cell uptake capacity of Pha@FPPC on B16-F10 cells was examined at the cellular level.Using Pha's own red fluorescence,the tumor cells incubated with Pha@FPPC,Pha@PPC,and Pha at the same Pha concentration were observed by laser confocal microscopy,and the targeting and uptake ability was determined according to the fluorescence intensity of red.The results showed that Pha@FPPC>Pha@PPC>Pha.Flow cytometry was used to evaluate the anti-tumor activity and mechanism of Pha@FPPC from three aspects:apoptosis level,ROS yield,and cycle arrest effect.Under irradiation,when the dose concentration of the drug?calculated as Pha?was 0.1?M,the percentage of B16-F10 cell necrosis caused by Pha@FPPC reached 95.45%,the average ROS yield was 550.16,while that induced by Pha alone was only 52.36%,and the average ROS yield was 330.35.Compared with the model drug Pha,the Photodynamic Killing Effect of Pha@FPPC was nearly doubled.The blank micelle FPPC blocked the G1/G0 phase of tumor cells.Free Pha mainly blocked tumor cells in G2 phase,and Pha@FPPC blocked both G1/G0 and G2 phases of tumor cells.It showed the cell cycle arrest characteristics of Pha and chidamide,indicating that the co-loaded micelles exerted a dual anti-tumor effect of PDT and HDACis.?4?At the animal level,the photodynamic anti-tumor effect of Pha@FPPC was further investigated.C57 mice were used to establish a mouse tumor-bearing model by inoculated with 1×105 B16-F10 cells in the right hind limb of each mouse.Five groups included normal saline group and experimental group?Pha@FPPC group,Pha@PPC group,Pha group,and FPPC group?were established,with 5 mice in each group.Each experimental group was injected into the tail vein Pha at a concentration of 1 mg/kg on the 8th day after tumor inoculation.After 45minutes,the tumor was irradiated with the laser for 360 s?the total light intensity was 90 J/cm2?.The tumor volume and survival of mice in each treatment group were measured.The results showed that the mice treated with Pha@FPPC had the smallest tumor volume and the longest survival time,which were significantly different from the control?p<0.001?,the Pha group?p<0.01?and the FPPC group?p<0.05?.At the same time,the lung tissue sections of the dead mice in each group were observed by H&E staining.It showed that there was obvious melanoma lung metastasis in the saline group and the Pha group,but no metastasis was found in the other treatment group grafted with chidamide.It is proved that chidamide has a strong inhibitory effect on the metastasis of B16-F10cells.In this study,a redox-responsive small molecule dual-mode antitumor drug Pha-SS-SAHA was successfully synthesized,which showed certain anti-tumor activity,but the photodynamic activity of the novel photosensitizer molecule was decreased.At the same time,a pH-responsive co-loaded nanomicelle Pha@FPPC was successfully constructed.The Pha and chidamide co-loaded micelles not only showed good pH responsiveness and targeting,but also showed good resistance tumor activity.The co-loading system achieves the"space-time"release of the tumor site,reduces the toxic side effects of the photosensitizer and chidamide,as well as prolongs the half-life of the chidamide and improved its bioavailability.In this study,we expiored a new model of PDT combined with drugs,showed that Pha@FPPC micelles have the potential to inhibit tumor recurrence and metastasis,and provides an important reference for clinical resolving tumor recurrence and metastasis after PDT. |
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