Journal of Bioactive and Compatible Polymers

Highly reactive hydroxyl radical (OH) is generated during reaction of Cu,Zn-SOD (SOD)¹ with H₂0₂, its own enzymatic reaction product (SOD plus 0;-). During inactivation of Cu,Zn-SOD with H₂0₂, Cu²+ liberated from the SOD catalyzes the formation of OH from H₂0₂. The OH thus generated will react with a,-protease inhibitor (a,-PI) if it is available in the milieu. To circumvent OH generation, we prepared eight different polymer-SOD conjugates and compared their stability against H₂0₂ treatment. Furthermore, the effect of each polymer-SOD conjugate on a,-PI in the presence of H₂0₂ was tested. Most polymer-SOD conjugates, except poly(styrene-co-maleic acid butylate; SMA)-SOD and poly(ethylene glycol; PEG)-SOD, showed higher stability than native SOD against inactivation by H₂0₂. The residual activity of divinylether maleic acid/anhydride copolymer (pyran copolymer)-conjugated SOD was 87% of the initial activity after incubation with 0.1 mM H₂0₂ for 2 h, whereas that of native SOD was 49%. Antiprotease activity of ca,-PI was completely abrogated when the inhibitor was treated with native SOD and 3.0 mM H₂0₂, but there was no change with pyran copolymer-SOD after 3 h of incubation with H₂0₂. Our results suggest that pyran copolymer captured loosely bound Cu²+ during the reaction of SOD with H₂0₂, thus resulting in suppression of both 0OH-mediated inactivation of SOD and suppression of activity of a,-PI. PEG showed little Cu²+-binding activity; no appreciable protection of SOD and a,-PI against OH generation was observed when Cu,Zn-SOD was conjugated with PEG. Circular dichroism and electron spin resonance studies of Cu,Zn-SOD treated with H₂0₂ indicated that the peptide conformation as well as the copper ligand of Cu,Zn-SOD can be stabilized not only by pyran copolymer per se but also by conjugation of Cu,Zn-SOD with some copolymers such as pyran.

Transplantable human squamous cell carcinoma cells were coincubated with 1,2-dimyristoylamido-1,2-deoxyphosphatidyl-choline (D14DPC) liposomes. These proteoliposomes and disrupted tumor cell suspensions were injected subcutaneously into two groups of nude mice, respectively, while the third group was untreated. Ten days previously, tumor had been implanted into both of the mice forefeet. In the seven-week old mice pretreated with the proteoliposomes, one of the tumors disappeared, while another tumor remained unchanged in size and the remaining two tumors regrew. Regrowth of the tumors was observed in both control groups. Their size and weight were significantly larger (p < 0.05) than those in the proteoliposome group. In all the 5-week old mice, the tumor masses grew but the size and weight of the tumor in the proteoliposome treated group were significantly smaller (p < 0.05) than those in the other two groups. The results showed the effectiveness of the proteoliposome treatment as a possible clinical application.

In order to hydrophobize heparin, dodecanal, cholic acid and stearic acid were conjugated, respectively, to partially N-desulfated heparin with the formation of imine bonds in the case of dodecanal and amide bonds in the case of the two latter compounds. It was found that the three conjugates were aggregated in aqueous solution with the formation of-300 nm aggregates. The conjugates retained the anticoagulant activity of the parent heparin according to in vitro tests. In vivo, slightly prolonged activity was observed after s.c. injection. The activity lasted for at least 2 hours after i.v. injection suggesting that aggregates of hydrophobized heparin were not captured by the reticuloendothelial system. These new water dispersed aggregates are of potential interest for the transport and slow release of hydrophobic drugs in blood either as macromolecular prodrugs or after molecular microencapsulation, and for oral administration.

Some thermally derived proteinoids are known to form hollow microspheres under specific conditions. In this study, oligomers consisting of L-aspartic acid, L-glutamic acid, L-phenylalanine, and L-tyrosine were prepared through the corresponding a-amino acid-N carboxyanhydrides (NCAs). The oligo(amino acids) synthesized by NCA method had better control on the composition as well as molecular weight. These oligo(amino acids) were found to spontaneously form microspheres under acidic conditions. It was also found that is possible to encapsulate a variety of drug entities within these unique self-assembling systems. The microspheres which form spontaneously at low pH's also dissolve in water at neutral pH and consequently have the potential to be used as oral drug delivery systems.

Oligomerizations of various amino acid N-carboxyanhydrides (NCAs) were prepared in the presence of L-tyrosine methyl ester (Tyr.Me). Tyr.Me was found to be an efficient initiator to form oligopeptides consisting of L-aspartic acid, L-glutamic acid, L-phenylalanine, and L-tyrosine with random arrangements of amino acids. In acidic solution, the oligopeptides formed microspheres which were capable of encapsulating macromolecular drugs such as insulin and heparin. These microspheres were considered to be potential oral drug delivery systems.