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Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pHdependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of 125 I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3 h) and ISA1 dose (0.04-100 mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed~60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10 mg/kg to 100 mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1 h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

The pH-responsive poly(amidoamine)s (PAAs) have been previously described. Whereas ISA23 enhances transfection in vitro and ISA1 promotes the cytosolic delivery of the non-permeant toxins this process shows poor efficiency. The aim of this study was to prepare and evaluate PAA conjugates containing the membrane disrupting peptide melittin (MLT). It was hypothesised that PAA conjugation would reduce the haemolytic activity of MLT at pH 7.4, however, upon delivery to tumours by the EPR effect, the polymer would uncoil in an acidic environment exposing MLT and allowing it to interact with membranes. PAA-MLT conjugates were prepared using MLT as a comonomer together with bis-acryloylpiperazine, 2-methylpiperazine and bis-hydroxyethylethylenediamine (ISA1-like), or bis-acrylamidoacetic acid and 2-methylpiperazine (ISA23-like). The melittin content of the conjugates was 6-19% (w/w). Although ISA1-MLT improved gelonin delivery compared to the parent polymer ISA1 (␣ 13-fold increase) and showed pH-dependent haemolytic activity at a polymer concentration of 0.05 mg/ml, this conjugate also displayed high haemolytic activity at pH 7.4. In contrast, ISA23-MLT like the parent compound ISA23 did not deliver gelonin. However, this conjugate could have potential as a novel polymeric anticancer conjugate due to its lack of haemolytic activity at pH 7.4 and retention of cytotoxicity.

Poly(amidoamine)s with amino pendant groups were prepared by hydrogen‐transfer polyaddition of primary and secondary amines to bis‐acrylamines. Dansyl cadaverine (DC) doxorubicin (Dox) were bound to the polymers via a cis‐aconityl spacer to give conjugates containing 3 µg of DC per mg of polymer and 28 to 35 µg of Dox per mg of polymer. Release of DC and Dox at physiological and acidic pH varied from 0 to 35% over 48 h and was pH dependent. Although the ISA1Dox conjugate (IC50 = 6 µg Dox · mL−1) presented similar toxicity as the parent polymer without Dox, ISA23Dox showed increased toxicity (IC50 = 10 µg Dox · mL−1). These results suggest that ISA23Dox is able to release biologically active Dox in vitro and that this conjugate might be suitable for further development.magnified image

Summary: 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 $\overline M _{\rm w}$ of 19 900–49 000 g/mol and a $\overline M _{\rm n}$ 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 (IC50 > 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. magnified image