Paolo Ferruti

Poly(amidoamine)s as Potential Endosomolytic Polymers: Evaluation In Vitro and Body Distribution in Normal and Tumour-Bearing Animals

Fusogenic peptides derived from viral coat proteins cause perturbation of the endosomal membrane and are often used to improve the transfection efficiency of non-viral vectors in vitro. However, fusogenic peptides have limited potential for use in vivo due to their inherent immunogenicity. Totally synthetic polymers that are endosomolytic should circumvent this problem and could be useful as components of non-viral delivery systems as long as they do not immediately localise in the liver after intravenous (i.v.) injection. Linear poly(amidoamine) polymers (PAAs) having amido- and tertiary amino-groups along the main polymer undergo pH-dependent conformational change and thus provide an ideal opportunity for design of polymers that display membrane activity at low pH. Here we describe four PAAs, ISA 1 (Mn = 6900 Da) and ISA 23 (Mn = 10,500 Da) and their analogues ISA 4 and ISA 22 (Mn approximately 8000 Da) containing approximately 1 mol% 2-p-hydroxyphenyl ethylamine to allow radioiodination and thus monitoring of their biodistribution. In vitro cytotoxicity was assessed by MTT assay after incubation of PAAs with B16F10 and Mewo cell lines. The IC50 values observed for all PAAs were > 2 mg/mL in comparison with poly(L-lysine) which displayed an IC50 in the range 0.01-0.1 mg/mL. At pH 7.4 none of the PAAs studied was haemolytic at 1 h at concentrations below 3 mg/mL. PAAs were subsequently incubated with rat red blood cells for 24 h (1 mg/mL) at different pHs. In contrast to poly(L-lysine) which was haemolytic at pH 7.4, 6.5 and 5.5, none of the PAAs was lytic at pH 7.4, but they became membrane active at lower pH (approximately 45% for ISA 4, 50% for ISA 22 and 90% for ISA 23). These observations were substantiated by SEM and confirm the pH-dependence of membrane activity. After i.v. injection to rats 125I-labelled ISA 4 was immediately taken up by the liver (> 80% recovered dose at 1 h) whereas 125I-labelled ISA 22 was not (liver uptake was < 10% recovered dose at 5 h). Furthermore, biodistribution studies in mice bearing subcutaneous B16F10 melanoma showed that 125I-labelled ISA 22 was still accumulating in tumour tissue after 5 h (2.5% dose/g). PAAs have potential as endosomolytic agents and quantitation of the endosome to cytoplasm transfer is warranted after i.v. administration.

Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine)

Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa). The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity. On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection. This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site. The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed. Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase. Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates. Unconjugated uricase was rapidly removed from circulation. All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Degradation behaviour of block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments

The degradation behaviour of a new class of multi-block copolymers containing poly(D,L-lactic-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) segments was studied under conditions mimicking those found in biological fluids. The dissolution times of the new PLGA-PEG multi-block copolymers mainly depend on the length of the PEG segments present in their structure, i.e., on their PEG content on a weight/weight basis. At higher PEG contents, partially degraded PLGA segments are brought in solution by attached PEG segments. In all cases, the dissolution rates decrease by increasing the total surface area of the specimens tested.

Poly(amidoamine)-mediated intracytoplasmic delivery of ricin A-chain and gelonin.

Poly(amidoamine)s (PAAs) are water-soluble synthetic polymers designed to be biodegradable and biocompatible. Moreover, they display membrane disruptive properties in response to a decrease in pH. This attribute confers PAAs with endosomolytic properties in vitro and in vivo. A model system was developed to quantify their ability to promote the endosomal escape of macromolecules that may be interesting as therapeutic agents. Here, two PAAs (ISA 1 and 4) were incubated with B16F10 cells in vitro together with two non-permeant toxins: either ricin A-chain (RTA) or gelonin. The relatively non-toxic PAAs ISA 1 and 4 (IC50>1.5 mg/ml) restored activity to the inherently inert toxins. The IC50 values for the ISA 1/RTA and ISA 1/gelonin combinations were 0.65+/-0.05 and 0.55+/-0.12 mg/ml, respectively. Similarly, when ISA 4 was incubated with a non-toxic combination of RTA and gelonin the IC50 value decreased to 0.57+/-0.03 and 0.43+/-0.26 mg/ml, respectively. In contrast, the neutral polymer dextran and the PAA ISA 22 were unable to mediate this effect. These observations suggest that specific PAA-toxin combinations warrant further development as novel therapeutics.

Linear amino polymers: Synthesis, protonation and complex formation

Linear amino polymers containing basic nitrogen atoms are critically reviewed with regard to their synthesis, protonation and complex formation in solution with metal ions. Cross linked resins having essentially the same structure as linear polymers, are also mentioned. As far as the protonation is concerned, special care has been given to thermodynamic aspects, and to the most probable protonation mechanism. Complexing abilities of these polymers have been evaluated through stability constants and spectroscopic parameters. Practical implications of the properties have been considered.

Poly(amidoamine) Conjugates Containing Doxorubicin Bound via an Acid-Sensitive Linker

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 microg of DC per mg of polymer and 28 to 35 microg 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 (IC(50) = 6 microg Dox x mL(-1)) presented similar toxicity as the parent polymer without Dox, ISA23Dox showed increased toxicity (IC(50) = 10 microg Dox x 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.

Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent 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 (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 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 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. 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.

Novel agmatine-containing poly(amidoamine) hydrogels as scaffolds for tissue engineering

Novel biocompatible and biodegradable amphoteric poly(amidoamine) (PAA) hydrogels were designed for applications as scaffolds for tissue engineering. These hydrogels (PAA-AG1 and PAA-AG2) were obtained by polyaddition of 2,2-bisacrylamidoacetic acid with 2-methylpiperazine and 4-aminobutyl guanidine, a bioactive molecule with a known ability to induce adhesion to cell membranes. They contain carboxylic functions in their main chain and interchain connections deriving from two different cross-linking agents: for PAA-AG1, a multifunctional primary amine, that is, 1,10-decanediamine; for PAA-AG2, a purposely synthesized PAA (PAA-NH(2)) containing pendant NH(2). Both PAA-AG1 and PAA-AG2 proved noncytotoxic and adhesive to cell membranes, as ascertained by means of cytotoxicity and proliferation tests carried out on fibroblast cell lines. Good apparent mechanical strength was also observed in the case of PAA-AG2, cross-linked with the PAA-NH(2). Both PAA-AG1 and PAA-AG2 underwent degradation tests under controlled conditions simulating the biological environments, that is, Dulbecco medium at pH 7.4 and 37 degrees C. They completely dissolved within 10 and about 40 days, respectively. In both cases, the degradation products were completely noncytotoxic. All the results of this paper point to the conclusion that agmatine-based PAA hydrogels are excellent substrates for cell proliferation.

Enhanced antiviral activity of Acyclovir loaded into β-cyclodextrin-poly(4-acryloylmorpholine) conjugate nanoparticles

Novel polymeric nanoparticles based on a beta-cyclodextrin-poly(4-acryloylmorpholine) mono-conjugate (beta-CD-PACM), a tadpole-shaped polymer in which the beta-CD ring is the hydrophilic head and the PACM chain the amphiphilic tail, were prepared by the solvent injection technique. Acyclovir-loaded nanoparticles were prepared from inclusion complexes of Acyclovir with beta-CD-PACM. Both unloaded and drug-loaded nanoparticles were characterized in terms of particle size distribution, morphology, zeta potential, drug loading and in vitro drug release rate. The antiviral activity of Acyclovir loaded into beta-CD-PACM nanoparticles against two clinical isolates of HSV-1 was evaluated and found to be remarkably superior compared with that of both the free drug and a soluble beta-CD-PACM complex reported in a previous paper. Fluorescent nanoparticles loaded with coumarin 6 were also prepared in order to investigate the nanoparticle cell uptake by confocal laser microscopy. It was found that the nanoparticles are internalized in cells and locate in the perinuclear compartment.

A novel modification of poly(L-lysine) leading to a soluble cationic polymer with reduced toxicity and with potential as a transfection agent

Chemical modification of poly(L-lysine) by hydrogen transfer addition of N,N-dimethylacrylamide to the primary amino groups results in new water soluble derivatives with reduced cell toxicity which maintain basic properties. Kinetic studies on the addition reaction were performed using model compounds, and a reliable kinetic model was obtained which proved to be valuable also in the case of poly(L-lysine). A peculiar feature of the addition reaction of poly(L-lysine) on N,N-dimethylacrylamide is that, starting from partially protonated poly(L-lysine), the reaction stops after that all the unprotonated units originally present have reacted. Toxicological studies on modified poly(L-lysine) in comparsion with parent poly(L-lysine) showed that the modifying reaction leads to a reduction in toxicity.