Poly(amidoamine)-Cholesterol Conjugate Nanoparticles Obtained by Electrospraying as Novel Tamoxifen Delivery System.

A new poly(amidoamine)-cholesterol (PAA-cholesterol) conjugate was synthesized, characterized and used to produce nanoparticles by the electrospraying technique. The electrospraying is a method of liquid atomization that consists in the dispersion of a solution into small charged droplets by an electric field. Tuning the electrospraying process parameters spherical PAA-chol nanoparticles formed. The PAA-cholesterol nanoparticles showed sizes lower than 500 nm and spherical shape. The drug incorporation capacity was investigated using tamoxifen, a lipophilic anticancer drug, as model drug. The incorporation of the tamoxifen did not affect the shape and sizes of nanoparticles showing a drug loading of 40%. Tamoxifen-loaded nanoparticles exhibited a higher dose-dependent cytotoxicity than free tamoxifen, while blank nanoparticles did not show any cytotoxic effect at the same concentrations. The electrospray technique might be proposed to produce tamoxifen-loaded PAA-chol nanoparticle in powder form without any excipient in a single step.

Structural characterisation of poly(amidoamine) networks via high-resolution magic angle spinning NMR.

A comprehensive structural characterisation of cross-linked insoluble poly(amidoamine) (PAA) networks was performed by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. Model samples with 20%, 40% and 80% cross-linking degrees were prepared and the best conditions to obtain high-resolution spectra in the gel phase determined. Whereas the samples with 20% and 40% cross-linking degrees could be exhaustively resolved and described, the sample with 80% cross-linking degree could not be characterised by this technique owing to insufficient mobility of the polymer segments. Even with this limitation, the method developed in this study can be reasonably considered as a general one, which enables exhaustive characterisation of cross-linked PAA networks of biomedical interest.

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.

Synthesis and properties of novel block copolymers containing poly(lactic-glycolic acid) and poly(ethyleneglycol) segments.

A synthetic process for obtaining high-molecular-weight block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments has been established. This process involves the reaction of poly(ethylene glycols) with phosgene, followed by polycondensation of the resulting alpha, omega-bis(chloroformates) with poly(lactic-glycolic acid) oligomers. The copolymers have been characterized for their molecular weight, solubility properties, water absorption and preliminarily thermal behaviour. All evidence points to the conclusion that the process described is a general one, enabling biodegradable polymers to be obtained tailor-made according to specific requirements.

Degradation behaviour of ionic stepwise polyaddition polymers of medical interest.

The aim of this presentation is to review some of our recent work mostly on poly(amidoamine)s (PAAs) and some other families of polymers structurally related to PAAs of medical interest. PAAs are obtained by stepwise polyaddition of primary monoamines, or bis secondary amines, to bisacrylamides. There are several other ter-amino polymers structurally related to PAAs, such poly(amido phosphine)s (PAPs), poly(ester-amine)s (PEAs), poly(ketone-amine)s (PKAs), poly(amidothioeteramine)s (PATAs) poly(esterthioether amine)s (PTEAs), and poly(sulphone thioetheramine)s (PSTAs). Most of the PAAs exhibit heparin complexing ability. PAAs are also being considered as soluble carriers for delivering anti-cancer drugs. Some of these polymers have been studied as antimicrobial agents. PAAs with different structures degrade at different rates under physiological conditions. The degradation rate is also strongly influenced by pH. The quaternarized PATAs and PTEAs are reasonably stable over a period of some days, but ultimately degrade to oligomeric products, while the quaternized PAAs do rapidly degrade.

Recent results on functional polymers and macromonomers of interest as biomaterials or for biomaterial modification.

Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.

The ischaemia-hyperpnea test in the evaluation of neuronal hyperexcitability syndrome.

Electromyography (EMG) identifies abnormalities in the neuronal hyperexcitability syndrome (NHS) with high frequencies of false positive results. We examined 25 subjects with autonomic, psychic and neuromuscular symptoms of NHS. They underwent the ischaemia-hyperpnea test using stringent time criteria to study their symptoms as well as their electrolytical balance. The Kaplan-Meier analysis showed a significantly different incidence of repetitive discharges in the two groups in the post-ischaemic period (p < 0.004) and during hyperpnea (p < 0.001). The ischaemia-hyperpnea EMG test, when evaluated in relation to the the duration of activation procedure, was able to differentiate NHS patients from controls only within 4-6 minutes of ischaemia or hyperpnea. When restricted to this period, EMG observation with the triggering of Ischaemia-hyperpnea identified NHS with a high degree of sensitivity and specificity.

A comparison between the hemolytic and antibacterial activities of new quaternary ammonium polymers.

New quaternary ammonium polymers, which in a previous work had shown relevant antibacterial properties, have been investigated as regards to their hemolytic activity (HA) in comparison with a low molecular weight commercial antibacterial agent, Steramine G (SG). All polymers exhibit negligible, or at most modest, HA at dosages and contact times at which SG is strongly hemolytic.

A polymer-Triton X-100 conjugate capable of PH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments.

Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml).

On the suitability of urethane bonds between the carrier and the drug moiety in poly(ethyleneglycol)-based oligomeric prodrugs.

Some poly(ethyleneglycol) derivatives of propranolol were prepared, in which the drug molecule was linked to the oligomeric carrier via urethane bonds in order to study the suitability of this bond as a linkage between the carrier and the drug moiety in oligomeric prodrugs. For comparison purposes also ethanol- and butanol-based prodrugs of propranolol were prepared, with the same linkage between drug and promoiety. The urethane compounds were obtained by condensation via N,N'-carbonyldiimidazole. None of these compounds gave rise to appreciable blood concentrations of propranolol after oral administration, thus suggesting in this particular case, a relative in vivo resistance of urethane bonds towards cleavage.

Pharmacokinetic results on naproxen prodrugs based on poly(ethyleneglycol)s.

Five prodrugs of S(+)-2-(6-methoxy-2-naphthyl)propionic acid (naproxen), in which the drug was bound by ester linkages to diethyleneglycol (I), triethyleneglycol (II), octanediol (III), butyl-triethyleneglycol (IV), and butyl-tetraethyleneglycol (V), respectively, were prepared and tested for their pharmacokinetic properties after oral administration. It was found that bioavailabilities decreased in the order, and in all cases were lower than that of the free drug.

New quaternary ammonium polymers as antimicrobial agents. Part II. Alkylation products of linear aliphatic poly (aminodisulphides).

Two new polymeric disulphides containing t-amino groups in their main chain, namely poly[1,8-(3,6-dimethyl-3,6-diaza) octaine diyl disulphide] (5) and poly[1,8-(1,12-(3-10-dimethyl-3,10-diaza) dodecane diyl disulphide] (6) were prepared by the polyoxidation of 3,6-dimethyl-3,6-diazaoctane-1,8-dithiol (3) and 3,10-dimethyl-3,10-diazadodecane-1,12-dithiol (4), respectively. They were quaternized with methyl iodide and benzyl bromide, and the resulting quaternary ammonium polymers were preliminarily tested for antimicrobial activity against Escherichia coli K12, Pseudomonas aeruginosa, and Staphylococcus aureus. All the quaternized products showed interesting killing potency against P. aeruginosa. The benzylated products, besides being more active against P. aeruginosa, showed fair activity also against the other bacterial strains tested.

Poly(amidoamine)s with potential as drug carriers: degradation and cellular toxicity.

Poly(amidoamine)s were synthesized by polyaddition reaction: to bis-acryloylpiperazine of piperazine (1), or N,N'-bis(2-hydroxyethyl)ethylenediamine (2), and to 2,2-bis(acrylamido)acetic acid of piperazine (3). Compound 2 was also end-capped with 4-hydroxythiophenol, thus introducing a terminal moiety suitable for radio-iodination using the chloramine T method (4). Such polymers behave as bases in aqueous solution, and their net average charge alters considerably as the pH changes from 7.4 to 5.5. This results in a change in polymer conformation which may prove useful in the design of polymeric drug delivery systems. However, their suitability for use in the organism will depend on polymer toxicity and also on their rate of biodegradation. Here we studied the biological properties of the above poly(amidoamine)s with a view to optimizing the synthesis of novel drug carriers. The general cytotoxicity of compounds 1, 2, 3, and 4 was examined in vitro using two human cell lines, hepatoma (HepG2) and a lymphoblastoid leukaemia (CCRF). Several different methods [the tetrazolium (MTT) test, [3H]leucine or [3H]thymidine incorporation, or counting cell numbers] were used to measure cell viability. Compounds 1, 2, and 4 were much less toxic to both cell lines than equivalent concentrations of the polycationic poly-L-lysine, and in no case did viability fall below 50% (concentrations up to 2 mg/ml). Although compound 2 was not markedly toxic to HepG2 cells, concentration-dependent toxicity was observed against CCRF cells. In this case, the polymer concentration decreasing viability by 59% (ID50) was approximately 50 micrograms/ml for compound 2 compared with an ID50 of approximately 10 micrograms/ml for poly-L-lysine. The rate of hydrolytic degradation of compound 2 was examined using viscometric measurements and gel permeation chromatography (GPC). After incubation at pH 7.5 and 8.0 for 24 h, polymer intrinsic viscosity was decreased by approximately 50% and GPC elution profiles showed a simultaneous increase in polymer retention time, indicating a fall in molecular weight. Hydrolytic degradation progressed much more slowly at pH 5.5. Compound 4 was also incubated with a mixture of isolated rat liver lysosomal enzymes (tritosomes) at pH 5.5, but no increase in the rate of degradation was observed.