Perfluorodecalin (PFD)-filled polypeptide capsules as artificial oxygen carriers

Artificial oxygen carriers (AOCs) based on perfluorocarbons (PFCs) have been studied and developed for about half a century. Since PFCs are not dissolvable in blood, they are often emulsified or encapsulated before intravenous administration. In this dissertation, it is attempted to prepare capsules from a synthetic protein as a membrane material. For that purpose, triblock polypeptides consisting of a hydrophilic aspartate block, a hydrophobic phenylalanine block, and a central cysteine block for cross-linking are synthesized to form spherical solid protein-like capsule walls around PFC droplets.

We first investigate the effect of hydrophilic chain length to hydrophobic chain length ratio on the size and morphology of PFC droplets. Three di-block polypeptides Asp40-Phen with different hydrophilic chain length to hydrophobic chain length ratios, including Asp40-Phe5, Asp40-Phe9, and Asp40-Phe14, are first synthesized and investigated for their ability to emulsify PFC. When the ratio is smaller than 3:1, polypeptides are hardly soluble in water, and there are a large number of self-assembled micelles in the emulsion, which are well dispersed around the PFC droplets. When the ratio is larger than 8: 1, polypeptides are well dissolved in water and are suitable to stabilize PFC in water emulsions.

In the second step, single block oligo- or poly-cysteine peptides are synthesized and their free thiol group contents were studied, to make sure that cysteine block can crosslink properly. Two differently protected cysteine units, including S-benzyl-L-cysteine (BnCys) and S-carbobenzoxy-L-cysteine (CbzCys), are used as starting materials. Both amino acids can be efficiently converted into activated monomers, including N-(phenoxycarbonyl)-S-benzyl-L-cysteine (NPBnCys) and N-(4-nitrophenoxycarbonyl)-S-carbobenzoxy-L-cysteine (NNPCbzCys). Polymerization of the first monomer gives oligo- or poly-BnCys with good yields (around 80%) and narrow molecular weight distribution (PDI=1.2). However, polymerization of NNPCbzCys gives oligo- or poly-CbzCys with smaller yields (around 30%) and broad molecular weight distribution (PDI=1.8). Around 90% of the protecting groups of both differently protected oligo- or poly-cysteine units can be efficiently removed by acidolysis, and around 20% of free thiol groups can be detected for all the resulting oligo- or poly-cysteine peptides. If 1,2-ethandithiol was used as carbon cation scavenger and reducing agent for the deprotection reaction, around 39% of free thiol groups can be detected. Given that the polymerization of monomer NPBnCys is more efficient than that of monomer NNPCbzCys, it is better to synthesize triblock polypeptide with monomer NPBnCys.

Finally, various triblock polypeptides with different block ratios and different initiators are synthesized, and their ability to encapsulate PFC are studied. All triblock oligo- or poly-peptides can stabilize PFC in water efficiently. The capsule dispersion can be easily redispersed by shaking even after 1 month. Most of the capsules exhibit a diameter that ranges from 100 nm to 1000 nm. When cysteine chain dominates the peptide composition, the capsule wall exhibits enough mechanical strength to maintain spherical shape after a drying process, which indicates an existence of crosslinking between cysteine residues. As observed by NMR spectroscopy, the capsule wall allows for fast gas exchange and the gas exchange is completely reversible. The capsules also exhibit a large self-diffusion constant as around 2.05*10-12 m2/s in aqueous solution, which is good for oxygen delivery in the blood circulation. The capsules also exhibit little cytotoxicity when tested on cell cultures.



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