Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P?<?0.05) and paraquat (P?<?0.01) but not of PAMAMG4 dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66?±?3.95%, RFU: 17.87?±?1.34) as compared to NCI H157G cell line (63.07?±?6.8%, relative fluorescence units (RFU): 23.28?±?0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.     

Polyamidomine dendrimers: an excellent drug carrier for improving the solubility and bioavailability of puerarin

The potential of polyamidoamine (PAMAM) dendrimers as solubility enhancers and oral drug delivery system was well known. Herein, we investigated the possibility of PAMAM dendrimers for promoting the solubility and oral bioavailability of puerarin. In the present study, the effect of PAMAM dendrimers with different generations (G1.5, G2, G2.5, and G3) on the solubility of puerarin was evaluated at different concentrations and pH conditions. Further more, the puerarin–G2 dendrimer complex was conducted for the in vitro hemolytic toxicity studies and pharmacokinetics studies in rats. The solubility of puerarin was significantly higher in the presence of the full generation dendrimers (e.g. G2 and G3). No significant hemolysis was observed on erythrocytes (G2, 0–2.5?mg/mL) in the hemolytic toxicity studies. The pharmacokinetics parameters T_max, C_max, and AUC_{0–8 h} of puerarin suspension solution and puerarin–G2 dendrimer complex solution were 0.76?h, 1.50 µg/mL, 7.33 µg·h/mL and 0.33?h, 6.49 µg/mL, 14.02 µg·h/mL, respectively. These studies demonstrate that PAMAM dendrimers may be a promising strategy for peroral delivery of puerarin.     

Repeated intravenous injections in non-human primates demonstrate preclinical safety of an anti-inflammatory phosphorus-based dendrimer

Abstract

Dendrimers are nanosized hyperbranched polymers synthesized through an iterative step-by-step process; their size and structure are perfectly controlled, and they are widely used for biomedical purposes. Previously, we showed that a phosphorous-based dendrimer capped with anionic AzaBisPhosphonate groups (so-called ABP dendrimer) has immunomodulatory and anti-inflammatory properties toward the human immune system. It dramatically inhibits the onset and development of experimental arthritis in a mouse model relevant for human rheumatoid arthritis, a chronic inflammatory disease of auto-immune origin. In this article, we demonstrate in an unprecedented study that cynomolgus macaques repeatedly injected with the ABP dendrimer displayed no adverse response. Indeed, biochemical, haematological, clotting and immunological parameters remained with a normal physiological range during the study. Moreover, quantification of serum cytokines and histopathological analyses failed to reveal any noticeable lesion or noteworthy non-physiological occurrence. These results strengthen the potential of the ABP dendrimer as an innovative drug-candidate for the treatment of inflammatory diseases and favor the regulatory preclinical development of the molecule.     

Optimization and in vitro toxicity evaluation of G4 PAMAM dendrimer-risperidone complexes

Risperidone is an approved antipsychotic drug belonging to the chemical class of benzisoxazole. This drug has low solubility in aqueous medium and poor bioavailability due to extensive first-pass metabolism and high protein binding (>90%). As new strategies to improve treatments efficiency are needed, we have studied cationic G4 PAMAM dendrimers’ performance to act as efficient nanocarriers for this therapeutic drug. In this respect, we explored dendrimer-risperidone complexation dependence on solvent, temperature, pH and salt concentration, as well as in vitro cytotoxicity measured on L929 cell line and human red blood cells. The best dendrimer-risperidone incorporation was achieved when a mixture of 70:30 and 90:10 v/v chloroform:methanol was used, obtaining 17 and 32 risperidone molecules per dendrimer, respectively. No cytotoxicity on L929 cells was found when dendrimer concentration was below 3 × 10⁻² μM and risperidone concentration below 5.1 μM. Also, no significant hemolysis or morphological changes were observed on human red blood cells. Finally, attempting to obtain an efficient drug delivery system for risperidone, incorporation in G4 PAMAM dendrimers was optimized, improving drug solubility with low cytotoxicity.     

Novel cystamine-core dendrimer-formulation rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection by augmenting autophagy

Background: Cystic fibrosis (CF) is challenged with pathophysiological barriers for effective airway drug-delivery. Hence, we standardized the therapeutic efficacy of the novel dendrimer-based autophagy-inducing anti-oxidant drug, cysteamine.

Research design and methods: Human primary-CF epithelial-cells, CFBE41o-cells were used to standardize the efficacy of the dendrimer-cystamine in correcting impaired-autophagy, rescuing ΔF508-CFTR and Pseudomonas-aeruginosa (Pa) infection.

Results: We first designed a novel cystamine-core dendrimer formulation (G4-CYS) that significantly increases membrane-ΔF508CFTR expression in CFBE41o-cells (p < 0.05) by forming its reduced-form cysteamine, in vivo. Additionally, G4-CYS treatment corrects ΔF508-CFTR-mediated impaired-autophagy as observed by a significant decrease (p < 0.05) in Ub-LC3-positive aggresome-bodies. Next, we verified that in non-permeabilized CFBE41o-cells, G4-CYS significantly (p < 0.05) induces ΔF508-CFTR’s forward-trafficking to the plasma membrane. Furthermore, cysteamine’s known antibacterial and anti-biofilm properties against Pa were enhanced as our findings demonstrate that both G4-CYS and its control DAB-core dendrimer, G4-DAB, exhibited significant (p < 0.05) bactericidal-activity against Pa. We also found that both G4-CYS and G4-DAB exhibit marked mucolytic-activity against porcine-mucus (p < 0.05). Finally, we demonstrate that G4-CYS not only corrects the autophagy-impairment by rescuing ΔF508-CFTR in CFBE41o-cells but also corrects the intrinsic phagocytosis defect (p < 0.05).

Conclusions: Overall, our data demonstrates the efficacy of novel cystamine-dendrimer formulation in rescuing ΔF508-CFTR to the plasma membrane and inhibiting Pa bacterial-infection by augmenting autophagy.     

Scaling Behaviour of PAMAM Dendrimers Determined by Diffusion NMR

Summary: The hydrodynamic radius of PAMAM dendrimers as a function of molar mass is investigated by diffusion NMR. As a characteristic length, the hydrodynamic radius is calculated by Stokes‐Einstein equation. Poly(amidoamine) (PAMAM) dendrimers of generations 0 to 7 of two different terminal groups (NH₂, COONa) have been investigated. This dependence of the hydrodynamic radius from molar mass is compared with a scaling model and statistical model. A scaling exponent of 3.7 has been found in both cases. The scaling exponent found exceeds the dimension of the embedding space, thus the possible growth of dendrimers of this structure is limited.

Hydrodynamic radius of PAMAM‐NH₂ □, PAMAM‐COONa ○ as a function of molar mass.     

Dendrimer-mediated approaches for the treatment of brain tumor

Worldwide, the cancer appeared as one of the most leading cause of morbidity and mortality. Among the various cancer types, brain tumors are most life threatening with low survival rate. Every year approximately 238,000 new cases of brain and other central nervous system tumors are diagnosed. The dendrimeric approaches have a huge potential for diagnosis and treatment of brain tumor with targeting abilities of molecular cargoes to the tumor sites and the efficiency of crossing the blood brain barrier and penetration to brain after systemic administration. The various generations of dendrimers have been designed as novel targeted drug delivery tools for new therapies including sustained drug release, gene therapy, and antiangiogenic activities. At present era, various types of dendrimers like PAMAM, PPI, and PLL dendrimers validated them as milestones for the treatment and diagnosis of brain tumor as well as other cancers. This review highlights the recent research, opportunities, advantages, and challenges involved in development of novel dendrimeric complex for the therapy of brain tumor.     

Determination of the structure factor of polymeric systems in solution by small‐angle scattering: A SANS‐study of a dendrimer of fourth generation

The analysis of concentrated solutions of a dendrimer G4 of fourth generation by small‐angle neutron scattering (SANS) is presented. The determination of the structure factor S(q) (q = (4π/λ)sin(θ/2); λ: wavelength of radiation; θ: scattering angle) from experimental data is discussed in detail. It is shown that the incoherent contribution I_incoh to the measured scattering intensity may profoundly disturb the determination of S(q), in particular at high concentrations of the solute molecules. Contrast variation is used to determine I_incoh which is subtracted subsequently from the measured intensities. Moreover, a general method for the check of the data of S(q) for internal consistency is presented. It is based on the expansion of S(q) into powers of q². The structure factor S(q) obtained from the corrected data is shown to be consistent except at the highest concentration of the dendrimer G4 under consideration here. This finding is explained by a change of the form factor of the dendrimers due to overlap of the particles. The method of analysis presented here is general and can be applied to SANS‐data of other structures as well.

Chemical structure of dendrimer G4.     

Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems

This study aimed to identify suitable siRNA delivery systems based on flexible generation 2-4 triazine dendrimers by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with thermodynamic parameters calculated using molecular modeling. The siRNA binding properties of the dendrimers and PEI 25 kDa were simulated, binding and stability were measured in SYBR Gold assays, and hydrodynamic diameters, zeta potentials, and cytotoxicity were quantified. These parameters were compared with cellular uptake of the complexes and their ability to mediate RNAi. Radiolabeled complexes were administered intravenously, and pharmacokinetic profiles and biodistribution of these polyplexes were assessed both invasively and non-invasively. All flexible triazine dendrimers formed thermodynamically more stable complexes than PEI. While PEI and the generation 4 dendrimer interacted more superficially with siRNA, generation 2 and 3 virtually coalesced with siRNA, forming a tightly intertwined structure. These dendriplexes were therefore more efficiently charge-neutralized than PEI complexes, reducing agglomeration. This behavior was confirmed by results of hydrodynamic diameters (72.0 nm-153.5 nm) and zeta potentials (4.9 mV-21.8 mV in 10 mM HEPES) of the dendriplexes in comparison to PEI complexes (312.8 nm-480.0 nm and 13.7 mV-17.4 mV in 10 mM HEPES). All dendrimers, even generation 3 and 4, were less toxic than PEI. All dendriplexes were efficiently endocytosed and showed significant and specific luciferase knockdown in HeLa/Luc cells. Scintillation counting confirmed that the generation 2 triazine complexes showed more than twofold prolonged circulation times as a result of their good thermodynamic stability. Conversely, generation 3 complexes dissociated in vivo, and generation 4 complexes were captured by the reticulo-endothelial system due to their increased surface charge. Molecular modeling proves very valuable for rationalizing experimental parameters based on the dendrimers' structural properties. Non-invasive molecular imaging predicted the in vivo fate of the complexes. Therefore, both techniques effectively promote the rapid development of safe and efficient siRNA formulations that are stable in vivo.     

Dendrimeric Structures in the Synthesis of Fine Chemicals

Dendrimers are highly branched structures with a defined shape, dimension, and molecular weight. They consist of three major components: the central core, branches, and terminal groups. In recent years, dendrimers have received great attention in medicinal chemistry, diagnostic field, science of materials, electrochemistry, and catalysis. In addition, they are largely applied for the functionalization of biocompatible semiconductors, in gene transfection processes, as well as in the preparation of nano-devices, including heterogeneous catalysts. Here, we describe recent advances in the design and application of dendrimers in catalytic organic and inorganic processes, sustainable and low environmental impact, photosensitive materials, nano-delivery systems, and antiviral agents’ dendrimers.