Anionic Polyamidoamine Dendrimers Research Articles

The Nature of the Inhibitory Action of Anionic Polyamidoamine Dendrimers of Generation 1.5–3.5 on the Activity of the Fibrinolytic System

Polyamidoamine (PAMAM) dendrimers are used in medicine for systemic drug delivery. The safety assessment of biomaterials, which come into contact with blood components and tissues is especially important. For example, PAMAM-NH2 dendrimers G4–G7 cause platelet and fibrinogen aggregation. We have studied the effect of the PAMAM-COOH dendrimers G1.5–G3.5 on key components of the fibrinolysis system. The dendrimers have not affected the plasmin activity. However, the amidolytic activities of tissue activators of plasminogen (tPA) and urokinase (uPA) have decreased markedly with increasing the dendrimer concentration and generation. The peak intensity at 340 nm of Glu-plasminogen fluorescence in the absence and presence of 0.15 M NaCl has decreased with increasing the dendrimer concentration, thus indicating the change in the microenvironment of the Trp residues and molecular conformation of the open and closed Glu-plasminogen forms. A comparison of the fluorescence quenching constants suggests that the anionic dendrimer and chloride ion simultaneously bind to Glu-plasminogen and each of these ligands exerts its independent effects through the separate binding sites. With increasing the dendrimer concentration, the rate of the tPA- and uPA-induced activation of Glu-plasminogen has dramatically decreased due probably to the formation of dendrimer*plasminogen complexes with the increasing dendrimer content, which makes the plasminogen bond increasingly unavailable for the cleavage by the activators. The rate of tPA- and uPA-induced lysis of the plasma clot in plasma has moderately decreased with increasing the dendrimer concentration due probably to the reduced effect of the dendrimer on the fibrin-bound plasminogen. Thus, the nature of the inhibitory action of the anionic PAMAM dendrimers on the Glu-plasminogen activation and tPA- and uPA-induced thrombolysis consists of a noticeable inactivation of the activators and a significant change in plasminogen conformation. The PAMAM-COOH dendrimers in circulation can affect other physiological and pathological processes, which are associated with an important role of the plasminogen/plasmin system.

Influence cationic and anionic PAMAM dendrimers of low generation on selected hemostatic parameters in vitro.

Polyamidoamine (PAMAM) dendrimers are a new class of monodisperse polymers that are used for drug delivery in systemic administrations. The influence of PAMAM dendrimers on components of the blood coagulation system has been extensively studied, but their effect on the activity of the fibrinolysis system has not been studied to date. The effect of cationic (G1-G3) and anionic (G1.5-G3.5) PAMAM dendrimers on the conformation and function of the main components of the coagulation and fibrinolysis systems was comparatively studied. Changes in overall plasma hemostatic potential, thrombin generation, prothrombin time, thrombin and tPA activities, the fluorescence of fibrinogen and plasminogen, zeta potential, polymerization of fibrinogen, and activation of plasminogen were analyzed to assess coagulofibrinolytic mechanisms of influence of the charge of the dendrimers. Cationic dendrimers increased prothrombin time, suppressed thrombin generation in plasma, and changed the conformation and coagulability of fibrinogen, while anionic dendrimers did not have such effects. Anionic dendrimers slightly reduced tPA activity and altered plasminogen conformation much more strongly than the cationic dendrimers. Plasminogen activation by tPA was strongly inhibited by anionic dendrimers and weakly stimulated by cationic dendrimers. All these effects were enhanced with increasing generation and concentration of the dendrimers. PAMAM-NH2 dendrimers inhibit the extrinsic activation pathway of the coagulation system and alter the conformation and function of fibrinogen. PAMAM-COOH dendrimers change the conformation of plasminogen and inhibit its activation by tPA. This study gives new insight into the effect of anionic PAMAM dendrimers on the activity of the fibrinolytic system. For intravenous applications, the antifibrinolytic effect of anionic PAMAM dendrimers of generation ≥G2.5 should be considered.

Properties of Bioconjugates of Streptokinase with Anionic Polyamidoamine Dendrimers of Various Generations

Covalent conjugates of streptokinase (SK) with polyamidoamine (PAMAM) dendrimers G1.5, G2.5, and G3.5 (SK–G1.5, SK–G2.5, and SK–G3.5) with the protein–polymer molar ratios of (1: 1), (1: 5), and (1: 10) were obtained and their properties were studied as compared to the properties of free SK. It was shown that the initial rates of formation of the modified Pm. SK complex, activation of plasminogen, and lysis of the plasma clot under the action of SK–dendrimer conjugates decreased with increasing number of bound dendrimers (from 1 to 10) and increased with increasing dendrimer generation (from G1.5 up to G3.5). Conjugates SK–G3.5 (1: 1) and (1: 5) were the most active compared to other conjugates. It was found that the catalytic efficiency of plasminogen activation (kPg/KPg) by conjugates SK–G3.5 (1: 1) (0.15 μM–1 min–1) and SK–G3.5 (1: 5) (0.12 μM–1 min–1) was comparable to the efficiency of free SK (0.18 μM–1 min–1). Probably, small in size, soft, and easily deformable dendrimers G1.5 and G2.5 are able to penetrate into the internal shielded cavities of the native SK molecule and there modify amino groups that are important for the effective formation of the Pm · SK complex. By contrast, the larger and more rigid molecule of dendrimer G3.5 modifies, mainly, exposed lysine residues in the SK molecule, without affecting the latent internal lysines. Conjugates SK–G3.5 (1: 1) and (1: 5), which had the maximum activator activity, retained up to 85% of thrombolytic activity compared to the activity of free SK. In addition, due to modification of the exposed lysines—most sensitive to proteolysis in the SK molecule—with dendrimer G3.5, which has the highest density of negative charge on its surface, SK–G3.5 (1: 1) and (1: 5) conjugates were more stable in plasma and caused less exhaustion of plasma levels of plasminogen, α2-antiplasmin, and fibrinogen than free SK in vitro. Thus, thrombolytic activity of the SK–dendrimer conjugates depends on the degree of modification of the amino groups of SK, size, stiffness, and density of the negative charge on the surface of the PAMAM dendrimer. Conjugates SK–G3.5 (1: 1) and (1: 5) are potential candidates for the development of a new thrombolytic agent.

Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines

Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96h and human cancer cell lines for 24h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, with EC50 values ranging from 0.16 to just below 1.7μM at 24 and 48hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values≥402μM (PAMAMs) and ≤240μM (PPIs) for HepG2 and ≤13.24μM (PAMAMs) and ≤12.84μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos.

Cationic nanoparticles directly bind angiotensin-converting enzyme 2 and induce acute lung injury in mice.

BackgroundNanoparticles have become a key technology in multiple industries. However, there are growing reports of the toxicity of nanomaterials to humans. In particular, nanomaterials have been linked to lung diseases. The molecular mechanisms of nanoparticle toxicity are largely unexplored.MethodsAcute lung injury was induced in wild-type mice and angiotensin-coverting enzyme 2 (ACE2) knockout mice by the intratracheal instillation of cationic polyamidoamine dendrimer (PAMAM) nanoparticles. For rescue experiments, losartan (15 mg/kg in PBS) was injected intraperitoneally 30 min before nanoparticle administration.ResultsSome PAMAM nanoparticles, but not anionic PAMAM nanoparticles or carbon nanotubes, triggered acute lung failure in mice. Mechanistically, cationic nanoparticles can directly bind ACE2, decrease its activity and down-regulate its expression level in lung tissue, resulting in deregulation of the renin-angiotensin system. Gene inactivation of Ace2 can exacerbate lung injury. Importantly, the administration of losartan, which is an angiotensin II type I receptor antagonist, can ameliorate PAMAM nanoparticle-induced lung injury.ConclusionsOur data provide molecular insight into PAMAM nanoparticle-induced lung injury and suggest potential therapeutic and screening strategies to address the safety of nanomaterials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-015-0080-x) contains supplementary material, which is available to authorized users.

Inhibition of PI3K contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity (INC6P.340)

Abstract Nanoparticles are increasingly used in biomedical applications due to their ability to change biodistribution, increase safety and improve efficacy of drugs. However, application of engineered nanomaterials in medicine is often halted by safety concerns. Endotoxin contamination is very common in nanomaterials and there is an increasing number of studies reporting that certain nanoparticles can exaggerate endotoxin-mediated inflammation. We studied the relationship between physicochemical properties of polyamidoamine (PAMAM) dendrimers and their ability to exaggerate LPS-induced procoagulant activity (PCA) of human leukocytes in vitro. The PCA is phospholipid-protein complex composed of tissue factor (TF) and phosphotidylserine (PS). We found that cationic but not anionic or neutral PAMAM dendrimers, increase LPS-induced leukocyte PCA. Cationic PAMAM dendrimers enhanced both LPS-induced TF expression and PS exposure on the cell surface. The mechanism of LPS-induced PCA exaggeration by PAMAM dendrimers is not mediated by the electrostatic interaction between cationic dendrimers and anionic LPS, calcium influx and secondary messengers released by dendrimer activated platelets. We hypothesize that dendrimers exaggerate LPS-mediated PCA through the inhibition of negative regulation of inflammation. Specifically, we demonstrate that cationic PAMAM dendrimers inhibit phosphoinositol-3-kinase (PI3K), a negative regulator of TF expression in response to endotoxin.

The influence of maltose modified poly(propylene imine) dendrimers on hen egg white lysozyme structure and thermal stability

In this study the influence of dendrimers’ surface modification upon the strength of interaction with proteins was examined. Unmodified, cationic poly(propylene imine) dendrimer of the fourth generation (PPI G4), two PPI G4 dendrimers, partially and fully coated with maltose residues, and anionic polyamidoamine dendrimer of the third and a half generation (PAMAM G3.5 dendrimer), were used in the study. Hen egg white lysozyme, which possesses a cationic net charge under physiological conditions, was chosen as a model protein. The influence of dendrimers on the thermal stability of lysozyme was studied using differential scanning calorimetry (DSC) and circular dichroism (CD) methods. Additionally, the effect of dendrimers on the availability of lysozyme tryptophan residues to fluorescence quenchers was examined. It was shown that modification of dendrimer surface with maltose reduced its influence on lysozyme properties. However, even full surface modification, resulting in a neutral surface charge, did not deprive dendrimer of the ability to interact with the protein. It was probably caused by the introduction of a large number of hydroxyl groups from maltose residues on the surface of the dendrimer. In the study a comparable strength of influence exerted on lysozyme by cationic PPI dendrimer and anionic PAMAM G3.5 dendrimer was observed. The possible explanation of this fact is the presence of both positively and negatively charged areas on the surface of lysozyme. Such areas allow dendrimers possessing opposite surface charges to interact with lysozyme.

Kinetic and thermodynamic study of the transfer of anionic polyamidoamine dendrimers across two immiscible liquids

The kinetics and thermodynamics for the phase transfer of carboxyl-terminated polyamidoamine (PAMAM) dendrimers across the water/dichloroethane interface were analyzed by cyclic voltammetry and electrochemical impedance spectroscopy. A three phase junction was employed by inserting a cylindrical gold electrode through the liquid–liquid interface. The reversible redox species decamethylferrocene (DMFc) was used in the organic phase in order to promote dendrimer transfer. It was found that the electrochemical behaviour of DMFc at the gold/dichloroethane interface depends on the generation and concentration of the dendrimer species in the aqueous phase. In addition, it was observed that the electrochemically driven transfer of these macromolecules corresponds to a quasi-reversible process. The data obtained from thermodynamic studies indicate that dendrimers are transferred between the two phases under study by an entropy controlled process.

Blood Compatibility of Polyamidoamine Dendrimers and Erythrocyte Protection

Dendrimers are global macromolecules with promising biomedical application. In order to investigate their blood compatibility, different generations of polyamidoamine (PAMAM) dendrimers terminated with different types of surface functional groups were incubated with red blood cells (RBC) for time course of haemolysis assay and RBC morphology observation. Cationic PAMAM dendrimers demonstrated obvious generation, concentration and time dependent haemolysis while anionic and neutral PAMAM dendrimers were much less hemolytic. The surface amino groups and the polymeric structure of cationic PAMAM dendrimers played pivotal roles in haemolysis induction; meanwhile the basic pH of cationic PAMAM solutions was an inneglectable factor. Due to the presence of plasma proteins in abundance, whole blood was found tolerable to haemolysis induced by dendrimers, possibly through complex formation of plasma proteins with cationic PAMAM dendrimers. It was deduced that poly-carboxylic acids may have the similar effect as that of plasma proteins. So by means of pH adjustment to the physical 7.4, reduction of administration dose and the combined use of plasma protein (or other protective additives such as negatively charged molecules), the blood compatibility and even biocompatibility of cationic dendrimers may be enhanced to "safe" level.

Direct Control of the Magnetic Interaction between Iron Oxide Nanoparticles through Dendrimer-Mediated Self-Assembly

Cationic superparamagnetic iron oxide nanoparticles were assembled using a series of anionic polyamidoamine dendrimers. The resulting assemblies featured systematically increasing average interparticle spacing over a 2.4 nm range with increasing dendrimer generation. This increase in spacing modulated the collective magnetic behavior by effective lowering of the dipolar coupling between particles. The results obtained in these studies deviate from the predicted dependence of collective behavior on interparticle spacing, suggesting that a dense assembly of magnetically "free" particles can exist with a surprisingly small space between particles.

Extended release of a novel antidepressant, venlafaxine, based on anionic polyamidoamine dendrimers and poly(ethylene glycol)‐containing semi‐interpenetrating networks

The multiple daily administration of venlafaxine, a novel third-generation antidepressant, was reduced based on polyamidoamine and polyethylene glycol (PEG)-containing semi-interpenetrating network (IPN), respectively. Venlafaxine was covalently linked to water-soluble G2.5 anionic polyamidoamine dendrimer via a hydrolyzable ester bond. Semi-IPN hydrogels were prepared by crosslinking acrylamide in the presence of PEG, and venlafaxine with predetermined amounts was loaded in situ. Dendrimer-venlafaxine conjugate and semi-IPNs were characterized by proton nuclear magnetic resonance and Fourier transform infrared, respectively. The effect of PEG concentration and molecular weight was studied and discussed for an optimal controlled release.

Effect of Anionic Polyamidoamine Dendrimers on the Crystallization of Calcium Carbonate by Delayed Addition Method

Abstract We examined the crystallization of CaCO3 by changing the time of addition of the G1.5 and G3.5 poly(amidoamine) dendrimers to a calcium carbonate solution after incubation for several minutes (1, 3, 20, or 60 min) at 30 °C. Based on the fact that the crystal phase obtained in the absence of the dendrimer was calcite, the dendrimers effectively stabilized the most unstable vaterite particles, even delaying the addition time of the dendrimer for 60 min. As the delayed addition time of the G1.5 dendrimer increased from 0 to 60 min, the particle size increased from 1.2 ± 0.5 to 4.7 ± 0.7 μm. As the generation number of the dendrimer increased from G1.5 to G3.5, the particle size of the spherical vaterite decreased under the same condition. The present results suggest that the anionic PAMAM dendrimers effectively modified the metastable vaterite surface and inhibited the further growth of vaterite particles by changing the addition time.

Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system?

To investigate systematically the effect of polyamidoamine (PAMAM) dendrimer size, charge, and concentration on uptake and transport across the adult rat intestine in vitro using the everted rat intestinal sac system. Cationic PAMAM dendrimers (generations 3 and 4) and anionic PAMAM dendrimers (generations 2.5, 3.5, and 5.5) that were modified to include on average a single pendant amino group were radioiodinated using the Bolton and Hunter Reagent. 125I-Labelled dendrimers were incubated with everted sacs in vitro and the transfer of radioactivity into the tissue and serosal fluid was followed with time. The serosal transfer rates seen for all anionic generations were extremely high with Endocytic Indices (EI) in the range 3.4-4.4 microL/mg protein/h. The concentration-dependence of serosal transfer was linear over the dendrimer concentration range 10-100 microg/mL. For 125I-labelled generation 5.5 the rate of tissue uptake was higher (EI = 2.48+/-0.51 microL/mg protein/h) than seen for 125I-labelled generations 2.5 and 3.5 (0.6-0.7 microL/mg protein/h) (p < 0.05). The 125I-labelled cationic PAMAM dendrimers (generations 3 and 4) displayed a tissue uptake (EI = 3.3-4.8 microL/mg protein/h) which was higher (p < 0.05) than the rate of serosal transfer (EI = 2.3-2.7 microL/mg protein/h), probably due to nonspecific adsorption of cationic dendrimer to the mucosal surface. As the anionic PAMAM dendrimers displayed serosal transfer rates that were faster than observed for other synthetic and natural macromolecules (including tomato lectin) studied in the everted sac system, these interesting nanoscale structures may have potential for further development as oral drug delivery systems.