ABSTRACT
The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH-dependent conformational change and pH-dependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes. Both polymers are relatively non-toxic, and moreover ISA 23 has the beneficial property in vivo, of being non hepatotropic when administered intravenously. Although ISA 23 and ISA 1 demonstrate ability to transfect cells, ISA 1 is also able to promote intracellular delivery of non-permeant toxins. The aim of this study was to synthesise random and block copolymers of ISA 1 and ISA 23 and investigate whether these second generation hybrids would allow optimisation of PAA biological characteristics. Random and block copolymers of ISA 1 and ISA 23 were synthesised by hydrogen transfer polyaddition to generate a library of PAAs with an ISA 23:ISA 1 molar ratios of 2:1 to 4:1. The resultant polymers have a pI slightly below 7.4 and a M(w) of 19,900-49,000 g/mol and a M(n) of 13,100-24,100 g/mol. Whereas none of the random or block copolymers were haemolytic at pH 7.4 all demonstrated pH-dependent membrane activity. At pH 5.5 they caused 50-60% haemoglobin (Hb) release over 1 h. This was slightly less than that seen for ISA 23 (80% Hb release). None of the copolymers were cytotoxic against B16F10 cells during a 72 h incubation (IC(50) > 2 mg/ml; MTT assay). The ability of the random and block copolymer PAAs to deliver the toxin gelonin was also examined, but only ISA 1 and the block copolymer B2 (ISA 23:ISA 1 at a 2:1 molar ratio) were able to promote intracellular delivery, as measured by cytotoxic activity. It would be interesting to study the body distribution of B2 and determine whether this toxin-delivering PAA is able to escape liver capture.
Subject(s)
Biocompatible Materials/chemistry , Polyamines/chemistry , Polymers/chemistry , Animals , Drug Carriers/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Melanoma, Experimental , Mice , Models, Chemical , Plant Proteins/chemistry , Ribosome Inactivating Proteins, Type 1 , Temperature , Tetrazolium Salts/pharmacology , Thiazoles/pharmacologyABSTRACT
On exposure to an acidic pH, linear poly(amidoamine)s (PAAs) cause membrane perturbation and consequently have potential as endosomolytic polymers for the intracellular delivery of genes and toxins. Previous studies used PAAs in the hydrochloride form only. The aim of this study was to investigate systematically the effect of the PAA counterion on pH-dependent membrane activity, general cytotoxicity, and PAA solution properties to help guide optimization of PAA structure for further development of PAA-protein conjugates. PAAs (ISA 1, 4, 22, and 23; M(w) 10000-50000 g/mol) were synthesized to provide a library of PAAs having different counterions including the acetate, citrate, hydrochloride, lactate, phosphate, and sulfate salts. pH-Dependent membrane activity was assessed using a rat red blood cell haemolysis assay (conducted at a starting pH of 7.4, 6.5, or 5.5; 1 mg/mL; 1 h), and general cytotoxicity was investigated using a murine melanoma cell line (B16F10) and a human bladder endothelial-like cell line (ECV-304). Whereas poly(ethyleneimine) was haemolytic at the starting pH of 7.4 at 1 h [ approximately 50% haemoglobin (Hb) release], none of the PAA salts were haemolytic at a starting pH of 7.4 or 6.5. Although PAA acetate, citrate, and lactate were also non-haemolytic at the starting pH of 5.5, the sulfate and hydrochloride forms caused significant haemolysis (up to 80% Hb release) and ISA 22 and 23 phosphate were also markedly haemolytic ( approximately 70% Hb release). These counterion-specific differences were also clearly visible using scanning electron microscopy, which was used to visualize the red blood cell morphology. All PAAs were relatively nontoxic (IC(50) >or= 300-5000 microg/mL) compared to poly-l-lysine (IC(50) = 2-10 microg/mL), the PAA hydrochloride salts produced the greatest cytotoxicity, and the B16F10 cells were more sensitive than the ECV-304 cells. Small-angle neutron scattering suggested that ISA 23 hydrochloride had a larger hydrodynamic radius (5.1 +/- 0.2 nm) than the citrate salt (3.1 +/- 0.2 nm). These results provide indirect evidence for the salt- and pH-dependent changes in the conformation of the polymer coil. This study clearly demonstrates the importance of optimization of the counterion form when developing endosomolytic polymers designed to mediate pH-dependent membrane permeabilization.
