Novel poly(amidoamine)s (PAAs) containing disulfide linkages regularly arranged along their backbones were synthesized by the stepwise polyaddition of 2-methylpiperazine to N,NЈ-bis(acryloyl)cystamine (BACy1) or N,NЈ-bis(acryloyl)-(L)cystine (BACy2). Both bisacrylamides had, in turn, been obtained by the reaction of acryloyl chloride with the corresponding amines. All the products were characterized with 1 H and 13 C NMR spectroscopy, and the average molecular weights of the polymers were determined by size exclusion chromatography. Both PAAs showed different solubility properties. In particular, PAA-Cy1, derived from BACy1, was sparingly soluble in water, whereas PAA-Cy2, derived from BACy2, was very soluble in aqueous media. The polymerization rates were investigated with 1 H NMR spectroscopy. In both cases, the experimental data were consistent with pseudo-second-order kinetics. The calculated kinetic constants were 5.96 ϫ 10 Ϫ3 and 5.90 ϫ 10 Ϫ2 min Ϫ1 L mol Ϫ1 for the polyaddition of BACy1 and BACy2, respectively. The observed hydrolytic degradation rate of PAA-Cy2 in a pH 7.4 tris(hydroxymethyl)aminomethane (TRIS) buffer was comparable to that of conventional amphoteric PAAs, that is, PAAs containing carboxyl groups in their repeating unit. Degradation experiments carried out in the presence of 2-mercaptoethanol with both PAAs demonstrated that the disulfide groups contained in its repeating units were susceptible to reductive cleavage in the presence of thiols.
A linear, amphoteric poly(amidoamine) nicknamed AGMA1, based on 4-aminobutylguanidine, or agmatine, was successfully prepared by Michael-type polyaddition of monoprotonated agmatine and 2,2-bis(acrylamido)acetic acid (BAC). Copolymers between AGMA1 and the biocompatible poly(amidoamine) ISA23 (deriving from the polyaddition of 2-methylpiperazine with BAC) were also prepared. Acid-base titrations gave for AGMA1 three acid dissociation constants, with pKa values of 2.25, 7.45, and >or=12.1, corresponding to a strong acid, a medium-weak base, and a strong base, respectively. The charge distribution profiles show that this polymer is prevailingly cationic at all physiological pH values, the positive net average charge per unit varying from about 0.5 at pH 7.4 to about 1.0 at pH 5, with an isoelectric point at pH approximately 10. Zeta-potential measurements confirmed this. Despite that, AGMA1 is nontoxic and nonhemolytic in vitro within all pH ranges tested (4-7.5). This is in contrast with the previously observed behavior of amphoteric PAAs, for instance ISA23, that are weakly hemolytic at pH 7.4 but highly hemolytic at pH 5/5.5. The lack of hemolytic activity of AGMA1 even at acidic pH values seems typical of the agmatine-BAC sequences and may be ascribed to their RGD-like structure. In fact, AGMA1-ISA23 copolymers behave in a way increasingly similar to that of ISA23; that is, they become hemolytic at low pH values as their ISA23 content increases.
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.
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.
A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).
Polyamidoamines (PAAs) represent a family of degradable polymers carrying tert-amine groups in the polymer backbone, which behave as polyelectrolytes in aqueous solutions. Many relevant properties of PAAs, including the ability to interact with components of the biological environments, such as nucleic acids, proteins, and living cells, are strongly dependent on their acid-base properties, hence on their ionization state in different biological districts. In this article, the protonation constants of a series of PAAs have been precisely determined by electrochemical techniques in order to build up a homogeneous library containing both the protonation constants and the average distribution of the charged species, hence the net average charge as a function of pH. Moreover, correlations between chemical and cytotoxicity, have been attempted.
Biodegradable and biocompatible amphoteric poly(amido-amine) (PAA)-based hydrogels, containing carboxyl groups along with amino groups in their repeating unit, were considered as scaffolds for tissue engineering applications. These hydrogels were obtained by co-polymerising 2,2-bisacrylamidoacetic acid with 2-methylpiperazine with or without the addition of different mono-acrylamides as modifiers, and in the presence of primary bis-amines as crosslinking agents. Hybrid PAA/albumin hydrogels were also prepared. The polymerisation reaction was a Michael-type polyaddition carried out in aqueous media. The PAA hydrogels were soft and swellable materials. Cytotoxicity tests were carried out by the direct contact method with fibroblast cell lines on the hydrogels both in their native state (that is, as free bases) and as salts with acids of different strength, namely hydrochloric, sulfuric, acetic and lactic acid. This was done in order to ascertain whether counterion-specific differences in cytotoxicity existed. It was found that all the amphoteric PAA hydrogels considered were cytobiocompatible both as free bases and salts. Selected hydrogels samples underwent degradation tests under controlled conditions simulating biological environments, i.e. Dulbecco medium at pH 7.4 and 37 degrees C. All samples degraded completely and dissolved within 10 d, with the exception of hybrid PAA/albumin hydrogels that did not dissolve even after eight months. The degradation products of all samples turned to be non-cytotoxic. All these results led us to conclude that PAA-based hydrogels have a definite potential as degradable matrices for biomedical applications.
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