Targeted And Responsive Nano Photosensitizers Based On Self-Assembly Of Lactose-Conjugated BODIPY For Anti-Tumor Research

A carrier-free multifunctional nano photosensitizer(CFM NPS)based on self-assembly of single DPT photosensitizer-containing amphiphilic small molecules(CFM-DPT NPS)combines targeted delivery,tumor microenvironment(TME)responsive release(TDTR),and DPT with a single photoactivable motif under a single irradiation(denoted as CF-TDTR-DPT NPS).Obviously,such CF-TDTR-DPT NPSs hold great potential to enhance efficacy,safety,and accuracy of cancer treatment as well as facilitate clinical translation.Nevertheless,their fabrication remains a great challenge as considerations must be given to the rational integration of multi-functions,especially targeting specificity and controlled release,and the balance between hydrophilicity and hydrophobicity,which is very important for the self-assembly and stability of the nanostructures.Moreover,in the process of transforming phototherapy to clinical,it still faces the inherent limitations of phototherapy.For example,the hypoxic microenvironment of the tumor will affect the effect of photodynamic therapy,and the high expression of heat shock protein during photothermal therapy will cause heat resistance.In this study,based on the structural modification of the hydrophobic BODIPY with the characteristic of combined photodynamic and photothermal therapy,we synthesized two amphipathic small molecules BSL and LSB with both carbohydrate targeting and tumor microenvironment responsive motifs.BSL NPS was constructed with self-assembly of BSL,and phototherapy research has been carried out in the cell and animal experiments.LSB was co-assembled with the hypoxia inhibitor atovaquone and the heat shock protein inhibitor aspiramycin hydrochloride to construct a dual drug delivery system,and phototherapy research has been carried out in the cell and animal experiments.The main research contents are as follows:1.Synthesis and characterization of lactose-modified amphiphilic small molecules BSL and LSB based on BODIPYBSL and LSB are amphiphilic molecules composed of hydrophobic BODIPY and hydrophilic lactose moieties linked by GSH-responsive disulfide bonds.First,compounds 1(BODIPY),2(mono-acetylene substituted 2,2'-dithiodiethanol)and 3(a lactose derivative containing an azido group)were synthesized according to the reported procedures.Compound4,a BODIPY derivative containing two disulfide bonds and two alkyne functional groups,was synthesized through reactions of 1 and 2,with a yield of 37.6%.Compound 9,a BODIPY derivative containing a disulfide bond and an alkyne functional group,was synthesized by reactions of 1 and 2,with a yield of 44.2%.Finally,BSL was synthesized with a yield of 21.3%through Cu AAC reactions of 3 and 4,and LSB was synthesized with a yield of 76.5%through the click reactions of 9 and 4.The chemical structures of the synthesized compounds was characterized by ¹H nuclear magnetic resonance(NMR),¹³C NMR,and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS).1.Research on the construction of BSL NPS and its anti-tumor phototherapyBSL is an amphiphilic molecule composed of hydrophobic BODIPY and hydrophilic lactose moieties linked by GSH-responsive disulfide bonds.First,compounds 1(BODIPY),2(mono-acetylene substituted 2,2'-dithiodiethanol)and 3(a lactose derivative containing an azido group)were synthesized according to the reported procedures.Compound 4,a BODIPY derivative containing two disulfide bonds and two alkyne functional groups,was synthesized through reactions of 1 and 2,with a yield of 37.6%.Finally,BSL was synthesized with a yield of 21.3%through Cu AAC reactions of 3 and 4.With self-assembly of BSL,a nano photosensitizer BSL NPS with carbohydrate targeting and tumor microenvironment responsive motifs was constructed.Its critical aggregation concentration was determined by surface tension to be 19.7?M,and then its morphology was characterized by scanning electron microscopy,transmission electron microscopy and dynamic light scattering particle size analyzer.After targeted uptake by liver cancer cells,BSL will be reduced by high concentrations of GSH to release BODIPY.Under irradiation,ROS and heat are generated,which could damage mitochondria and cause apoptosis of cancer cells.Without irradiation,BSL NPS has good biocompatibility and would not cause damage to cells.Finally,the in vivo experiments demonstrated that the experimental group with twice administrations showed good tumor suppression effects.At the same time,the pathological section,blood routine and various biochemical index studies all showed that the system have good anti-tumor effects and biological safety.2.The dual-drug nano-delivery system constructed via self-assembly of LSB and its anti-tumor researchIt is difficult to synthesize and purify BSL,so that we synthesized the BODIPY derivative LSB for dual-drug delivery.LSB is an amphiphilic molecule composed of hydrophobic BODIPY and hydrophilic lactose moieties linked by GSH-responsive disulfide bond.Compound 9,a BODIPY derivative containing a disulfide bond and an alkyne functional group,was synthesized by reactions of 1 and 2,with a yield of 44.2%.Finally,LSB was synthesized with a yield of 76.5%through the click reactions of 9 and 3.We synthesized LSB for atovaquone(ATO)and aspiramycin hydrochloride(17-DMAG)loading study to overcome the inherent difficulty of the hypoxic microenvironment in phototherapy,which affects the effect of photodynamic therapy and the thermal resistance of tumor cells.We constructed LSB NPS,AB NPS loaded with single-agent ATO,and DB NPS with single-agent 17-DMAG as control groups.The dual-drug loading system ADB NPS has good assembly performance.The loading capicity of ATO and 17-DMAG are 20.66%and 5.40%,respectively.BODIPY released with response to high concentrations of GSH exhibits enhanced singlet oxygen generation and photothermal conversion.In cell experiments,ADB NPS exhibited good ROS generation performance under hypoxia or normoxia,and enhanced ability to induce apoptosis.Finally,in the in vivo experiments,the biological safety and anti-tumor characteristics of this system were studied via the analysis of tissue and organ distribution,thermal imaging,pathological section,blood routine,biochemical indicators,and tumor volume and weight.The ADB NPS with irradiation group can completely eliminate tumor tissues,and would not cause damage to the body.The results demonstrated that the dual-drug loading system has good anti-tumor effects and biological safety.In summary,our research is based on BODIPY motif which has dual phototherapy properties,and we synthesized two amphiphilic derivatives through glycosylation.Based on the derivatives,two safe and effective nano photosensitizer anti-tumor systems are constructed,which can achieve the targeting,responsive,photodynamic and photothermal synergistic anti-tumor effects,especially the second system,through the loading of two drugs,which can successfully break the inherent barriers of phototherapy such as hypoxia and thermal resistance,providing a new strategy for clinical anti-tumor treatment.