Synthesis Of Hemoglobin-based Oxygen Carriers With Multiple Functions And Their Potential Applications In Tumor Therapy

The tumor tissue is composed of not only the malignant cells, but also several host stromal cells including endothelial cells, smooth-muscle cells, fibroblasts, macrophages, myofibroblasts, and so on. These cells, together with surrounding extracellular matrix constitute the unique tumor microenvironment. The pathophysiology of tumors governs tumor proliferation, invasion, and metastasis as well as response to various therapies.The microenvironment of solid tumors is universally characterized by a state of hypoxia, which is resulted from an inadequate supply of oxygen. Hypoxia in tumor is connected with low extracellular pH and high glycolysis, and plays a critical role in metastasis formation. In fact, tumors exhibiting extreme hypoxia tend to be more metastatic than oxygenated tumors. Furthermore, Hypoxic tumors have been proved to be more resistant to radiation, chemotherapy and other cancer treatment. Therefore, in the past decades, various artificial oxygen carriers have been developed to alleviate the hypoxia state of tumors or act as an adjuvant therapy of cancer by increasing its radio-sensitization and chemo-sensitization. However, these oxygen carriers alone can’t delay the growth of tumors, greatly limiting their application in cancer therapy.Recently, a variety of smart drug delivery systems aiming at tumor microenvironment have been developed, such as pH responsive or tumor-targeting nano-drug carriers. These strategies can be applied to design novel oxygen carriers for improving the therapeutic effects of cancer.Based on these analyses, three multifunctional oxygen carriers were reported in this thesis:(1) Thermo-sensitive hemoglobin-based oxygen carrier (HBOC)Hemoglobin is responsible for storage and transport of oxygen in vivo and has become a potential candidate as artificial oxygen carrier. However, Hemoglobin extracted from the blood cells couldn’t be used directly due to its renal toxicity. Besides, the oxygenated hemoglobin can be gradually oxidized in air and then unable to deliver oxygen to tissues. Therefore, hemoglobin has been conjugated or encapsulated in nanoparticle carriers to improve the stability and reduce the side effects. In this chapter, hemoglobin was conjugated to a thermo-sensitive copolymer of poly(N-isopropylacrylamide) grafted dextran (Dex-g-PNIPAAm). The conjugate can exhibit a reversible phase transition with temperature in aqueous solution, and the oxygen-carrying behavior of hemoglobin in different state has been investigated.In detail, the conjugate was prepared by a reaction between the lysine amino groups of Hb and the carboxyl groups of HOOC-Dex-g-PNIPAAm, which was synthesized by single electron transfer living radical polymerization (SET-LRP) and post-carboxylation. The conjugate was characterized by FTIR,1H NMR, DLS, TEM and fluorescence spectra. Results showed it had a relatively low critical micelle concentration (CMC) and could form stable spherical nanoparticles upon heating above the LCST. The redox activity and gas-binding capacity of the Hb conjugate were subsequently confirmed by UV-vis spectroscopy, indicating the retention of Hb bioactivity after conjugation. Furthermore, dextran with different numbers of PNIPAAm chains was synthesized for comparison. It revealed that at 37℃, the temperature above the LCST, the conjugation of Hb to the copolymer Dex-g-PNIPAAm could improve the stability of Hb which increased with the number of PNIPAAm chains.(2) Tumor-targeting hemoglobin based oxygen carrierMost tumor cells over-express glucose transporters 1 (GLUT-1) in order to satisfy their huge demands for energy. Therefore, GLUT-1 can be acted as a target and specifically bind glycosylated nano-drug carrier to improve the utilization of drug and reduce the side effects. In this chapter, a new glycosylated HBOC has been prepared, which can specifically recognize tumor cells, further increase the oxygenation of tumor tissues, and then improve the curative effect of chemotherapy and radiotherapy.A glycosylated block copolymer of PMAG-b-PMAA was synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT). And then vinyl-terminated polycaprolactone was grafted to PMAG-b-PMAA through thio-ene addition reaction. The grafted copolymer (PMAG-b-PMAA-g-PCL) could be self-assembled into micelles and conjugated with hemoglobin. The synthetic polymers and the hemoglobin conjugated glycosylated micelles (Hb-GlycoMs) were characterized by FTIR,1H NMR, DLS, TEM and fluorescence spectra. UV-vis spectra were employed to detect the oxygen-carrying capacity and anti-oxidation performance, which indicated the retention of Hb bioactivity after conjugation. The specific recognition of Hb-GlycoMs with Con A was proved by turbidimetry assay. In addition, compared with normal cells, Hb-GlycoMs exhibited a much higher affinity to tumor cells (Hela) and was more easily endocytosed into the nucleus and cytoplasm.(3) Co-load of photosensitizer in HBOC for photodynamic therapyRecently, photodynamic therapy (PDT) has been developed as an effective clinical treatment for many tumors. However, the hypoxic tumors tend to suppress the photosensitization and exhibit more resistant to PDT. Besides, the photochemical depletion of oxygen can also cause hypoxia and limit curative effect of PDT. In this chapter, a hemoglobin based oxygen carrier was co-loaded with hydrophobic photosensitizer. The system could alleviate tumor hypoxia as well as compensate the oxygen depletion, which improved curative effect of PDT.A well-defined triblock copolymer of PEG-b-PAA-b-PS was synthesized by atom transfer radical polymerization (ATRP). It could self-assemble to form polymeric micelles for covalent conjugation of Hb via carbodiimide chemistry. The PEG-b-PAA-b-PS and resultant micelles in aqueous solution were comprehensively characterized by means of FTIR,1H NMR, GPC, DLS, TEM and fluorescence spectra. And the HBOC was evaluated to possess good oxygen-binding capacity and anti-oxidative activity via UV-vis spectroscopy. And then Zinc phthalocyanine (ZnPc) was encapsulated into the hemoglobin conjugated PEG-b-PAA-b-PS micelles to prepare a novel oxygen-carrying polymeric PDT agent, In addition, compared with the control micelles without Hb, the Hb conjugated photosensitizer carrier was able to generate more single oxygen (1O2) and exert greater photocytotoxicity on Hela cells in vitro.