G4 Dendrimer Research Articles

Identification of polyamidoamine dendrimers (PAMAM-NH2) by ESI-Q-TOF method

The method of identification of poly(amidoamine) dendrimers (PAMAM-NH2) G1.0, G2.0 and G3.0 generation with the use of high resolution mass spectrometry and hybrid quadrupole-time-of-flight analyzer with electrospray ionization (ESI-Q-TOF) was developed. The analysis was performed in positive ionization mode in the range m/z 100-3200 where MS spectra (TOF) as well as fragmentation spectra (MS/MS) were recorded simultaneously. The investigated substances were injected to the MS analyzer by the use of UHPLC system in a direct way (with no chromatographic separation). After deconvolution MS spectra were used for accuracy estimation of the mass of the analyzed dendrimers (PAMAM G1.0 - 1430.0153 Da, PAMAM G2.0 - 3256.2696 Da, PAMAM G3.0 - 6908.6850 Da). Registered fragmentation spectra were used for the confirmation of the structure of the analyzed three generation PAMAM-NH2 dendrimers.

Poly(amido)amine dendrimers generation 4.0 (PAMAM G4) reduce blood hyperglycaemia and restore impaired blood–brain barrier permeability in streptozotocin diabetes in rats

We hypothesized that BBB is impaired in rat model of streptozotocin-induced diabetes and can be sealed by poly(amido)amine dendrimers G4.0 (PAMAM G4), which reveal anti-glycation activity. The BBB permeabilization was monitored in rats with the 60-day streptozotocin-diabetes and non-diabetic animals, using three fluorescent dyes (given intraperitoneally) differing in molecular weight: fluorescein, fluorescein isothiocyanate (FITC)-dextran and Evans blue. All animals were administered for 2 months with either PAMAM G4 dendrimer or placebo. The fluorescence intensities of the injected fluorescent markers were recorded in the homogenates of selected brain regions. The highest accumulations of the used fluorescent dyes were observed for fluorescein, predominantly in thalamus, hippocampus, frontal cortex, striatum and cerebellum. FITC-dextran leaked to much smaller extent, however, higher permeabilization for FITC-dextran was revealed in pons-medulla oblongata, frontal and parietal cortex of diabetic compared to control animals. Evans blue leaked very slowly into striatum and pons-medulla oblongata in diabetic rats. The treatment of diabetic animals with PAMAM G4 significantly reduced blood glucose concentration and hallmarks of late diabetic complications, compared to non-treated diabetic animals. PAMAM G4 significantly reduced diabetes-induced permeabilization of BBB, which remained in line with the reduced blood glucose and the amelioration of the biochemical hallmarks of severe hyperglycaemia.

Effects of salt on the size and internal structure of PAMAM dendrimers at different pH

G4 and G5 polyamidoamine dendrimers solvated with explicit water, counterions and NaCl ions (0.15, 1 M) were simulated at two levels of protonation, which mimic their electrostatic charges at pH 5 and 7. We used the previously parametrised coarse-grained dendrimer model which had predicted the experimentally measured and theoretically calculated size and internal structure of the dendrimer at different pH values. In this study, addition of ≤ 1 M NaCl does not significantly modulate the dendrimer size, in agreement with experiments and other theoretical studies. In particular, added salt ions do not change the dense-shell and dense-core structures of dendrimers without salt, respectively, at pH 5 and 7. Besides counterions, only a few excess ions penetrate and occupy the dendrimer interior, leading to unchanged volume of the inner cavity in the dendrimer core. These results indicate that addition of ≤ 1 M NaCl does not modulate the efficiency of encapsulating hydrophobic drugs or compounds.

Novel aldehyde-terminated dendrimers; synthesis and cytotoxicity assay.

Polyamidoamine (PAMAM) dendrimers are a unique family of dendritic polymers with numerous pharmaceutical and biomedical applications. One major problem with these polymers is their cytotoxicity. The purpose of this study was to synthesize novel dendrimers with aldehyde terminal groups and compare their cytotoxicity with that of dendri¬mers containing amine-terminated groups. G1(first generation) and G2 (second generation) dendrimers with amine-terminated groups were synthesized by divergent method and then the amine-terminated groups were converted to the aldehyde groups using surface modification of the functional group inversion (FGI) method. The cytotoxicity of the novel G1 and G2 polyamidoaldehyde (PAMAL) dendrimers together with that of G1 and G2 PAMAM-NH2 dendrimers was investigated by MTT assay using MCF-7 cell line. The results showed that cytotoxicity of dendrimers with aldehyde-terminated groups is much lower than that of G1 and G2 PAMAM-NH2 dendri¬mers. Dendrimers with aldehyde-terminated groups could be used as novel and convenient carriers for drug delivery with low cytotoxic effect compared with the amine-terminated dendrimers. The results revealed that the same generations of the dendri¬mers with aldehyde-terminated groups are far less toxic than the corresponding amine-terminated dendrimers.

DFT study of structure, IR and Raman spectra of the first generation dendrimer built from cyclotriphosphazene core with terminal 4-oxyphenethylamino groups

The FTIR and FT-Raman spectra of the first generation dendrimer G1 built from the cyclotriphosphazene core, six arms OC6H4CHNN(CH3)P(S)< and 12 4-oxyphenethylamino terminal groups OC6H4(CH2)2NH2G1 have been recorded. The structural optimization and normal mode analysis were performed for dendrimer G1 on the basis of the density functional theory (DFT). The calculated geometrical parameters and harmonic vibrational frequencies are predicted in a good agreement with the experimental data. It was found that G1 has a concave lens structure with planar OC6H4CHNN(CH3)P(S)< fragments and slightly non-planar cyclotriphosphazene core. The 4-oxyphenethylamino groups attached to different arms show significant deviations from a symmetrical arrangement relative to the local planes of repeating units. The experimental IR spectra of G1 dendron was interpreted by means of potential energy distributions. Relying on DFT calculations a complete vibrational assignment is proposed.

Efficient in Vitro siRNA Delivery and Intramuscular Gene Silencing Using PEG-Modified PAMAM Dendrimers

Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the results from the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and GFP transgenic mice.

Ambiguous effect of dendrimer PAMAM G3 on rat heart respiration in a model of an experimental diabetes – Objective causes of laboratory misfortune or unpredictable G3 activity?

Poly(amido)amine (PAMAM) dendrimer G3 was investigated for its ability to support the proper functioning of rat heart mitochondria exposed to hyperglycemia, in both the in vitro and in vivo experiments. The main aims of this study were to check whether PAMAM G3 dendrimer improves the efficiency of the impaired respiration of rat heart mitochondria.This study showed that mitochondria isolated from animals studied in different seasons respond to G3 (100μM) exposure to a different extent. Probably, seasonal variations had the impact on rat metabolism and consequently on the received data. The used biological samples formed a heterogenous group and therefore the obtained results were not pooled together but treated separately. Nevertheless, the in vitro part of this study revealed that PAMAM G3 could be successfully used in the protection of heart mitochondria against MG-induced impaired respiratory activity. Despite these promising data, the protective effect of G3 was not confirmed in the in vivo experiment. This study revealed that dendrimer G3 (20mg/kgbw) is toxic and very high mortality among the animals administered with G3 did not allow to perform a reliable data analysis.

Poly (amido amine) dendrimer silences the expression of epidermal growth factor receptor and p53 gene in vitro

To understand more about the lower generations of poly(amido amine) dendrimer (PAMAM) as a non-viral vector for antisense (ANS) therapy, a 21-mer epidermal growth factor receptor (EGFR) ANS was delivered by generation five of PAMAM in T47D breast carcinoma cells in culture. The semi-quantitative polymerase chain reaction (PCR) and western blot analysis were used to quantify the expression of EGFR mRNA and protein, respectively. The results showed that PAMAM G5/ANS nanoparticles were able to decrease the level of EGFR mRNA more than 40% even at the lower dendrimer primary amine to the antisense phosphate groups (N/P) ratio of 0.5. But, only the data of western blot analysis at the higher N/P ratios of 10 and 20 showed a decrease of the protein expression level similar to the mRNA expression level. Moreover, PAMAM dendrimer had a positive effect on the EGFR ANS action to inhibit the EGFR mRNA and protein expression. Further studies revealed that PAMAM G5 dendrimer as such inhibits the expression of EGFR in a concentration-dependent manner. Since PAMAM as such was able to inhibit the mRNA expression of p53 gene, we speculated that the effect of PAMAM G5 on the EGFR is a kind of its non-selective effect on the transcription and/or translation machinery of the cell. Key words: Poly(amido amine) dendrimer (PAMAM) dendrimer, epidermal growth factor receptor (EGFR) antisense, epidermal growth factor, RNAi, polyamidoamine dendrimer, toxicogenomics, gene delivery.

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.

Phosphorus-containing dendrimers against α-synuclein fibril formation

The aim of this work was to study the effect of phosphorus-containing dendrimers (generations G3 and G4) on the fibrillation of α-synuclein (ASN). The inhibition of fibril formation (filamentous and aggregates) is a potential therapeutic strategy for neurodegenerative disorders such as Parkinson's and other motor disorder neurodegenerative diseases. The interaction between phosphorus-containing dendrimers and ASN was studied by fluorescence spectroscopy. The decrease in the fluorescence intensity of intrisinic tyrosine was the most marked change in the fluorescence intensity observed upon addition of dendrimers. Furthermore, the effect of dendrimers on ASN fibril formation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by fluorescence assays using the dye thioflavin T (ThT). The results showed that phosphorus-containing dendrimers G3 and G4 inhibited fibril formation, when they were used in the ASN/dendrimer ratios 1:0.1 and 1:0.5. However, the higher concentrations of dendrimers did not show this effect.

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 Tmax, Cmax, and AUC0–8 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.

DFT study of structure, IR and Raman spectra of the first generation dendron built from cyclotriphosphazene core with terminal carbamate and ester groups

The FTIR and FT-Raman spectra of the first generation dendron built from the cyclotriphosphazene core, five arms OC6H4CHNN(CH3)P(S) with ten carbamate terminal groups and one ester function Gv1 have been recorded. The IR and Raman spectra of the zero generation dendron Gv0 and first generation dendrimer G1 with the same core and terminal groups were also examined. The structural optimization and normal mode analysis were performed for dendron Gv1 on the basis of the density functional theory (DFT). The calculated geometrical parameters and harmonic vibrational frequencies are predicted in a good agreement with the experimental data. It was found that Gv1 has a concave lens structure with planar OC6H4CHNN(CH3)P(S) fragments and slightly non-planar cyclotriphosphazene core. The carbamate groups attached to different arms show significant deviations from a symmetrical arrangement relative to the local planes of repeating units. The experimental IR spectrum of Gv1 dendron was interpreted by means of potential energy distributions. The strong band 1604cm−1 shows marked changes of the optical density in dependence of the carbamate, ester or azomethyne substituents in the aromatic ring. The frequencies of ν(NH) and ν(CO) bands in the IR spectra reveal the presence of the different types of H-bonds in the studied dendrimers.

Synthesis and characterization of G5 PAMAM dendrimer containing daunorubicin for targeting cancer cells

A multifunctional dendritic nanodevice containing folate (FA) as the targeting molecule and daunorubicin (DNR) as the chemotherapeutic drug has been designed and synthesized. The primary amino groups on the surface of the generation 5 poly(amidoamine) dendrimer (G5 dendrimer) were acetylated partially. FA and DNR were conjugated to the remaining primary amino groups on the G5 dendrimer through amide and ester linkage, respectively, to generate the difunctional dendritic device. The synthetic approach of G5 PAMAM dendrimer containing daunorubicin and folic acid would be useful for targeting the overexpressed membrance-associated folate receptors of KB human carcinoma cell line and discreating them effectively.

Effect of polyamidoamine dendrimer G3 and G4 on skin permeation of 8-methoxypsoralene—In vivo study

In the present study we have assessed the ability of (PAMAM) dendrimers G3 and G4 to facilitate transdermal delivery of 8-methoxypsoralen (8-MOP) in vivo. In vitro study using Franz diffusion cell revealed an enhanced transdermal flux for 8-MOP in complex with G3 and G4 dendrimer in relation to standard 8-MOP solution. In present study in vivo skin permeation potential of 8-MOP complex with G3 and G4 PAMAM dendrimer was assessed using confocal laser scanning microscopy (CLSM), which revealed an enhanced permeation of the 8-MOP to the deeper layers of the skin and significantly higher concentration in comparison with standard 8-MOP solution. Skin tissue 8-MOP concentration, evaluated by HPLC indicates that G3 and G4 PAMAM application significantly increase 8-MOP skin deposition in comparison with standard 8-MOP solutions after 1 and 2h. G4 appeared to be a more effective 8-MOP penetration enhancer than G3 PAMAM. Our results suggest the feasibility of G3 and G4 PAMAM dendrimers for transdermal delivery of 8-MOP resulting in better skin permeation and higher concentration of 8-MOP in epidermis and dermis of the drug that could help to improve effectiveness and safety of PUVA therapy.

Patterning Carbazole–Phenylazomethine Dendrimer Films

The synthesis of double-layer-type dendrimers with carbazole and phenylazomethine as the dendron with a symmetric tetraphenylmethane core is reported. Structural modeling studies showed that the G3 dendrimer has a rigid and spherical structure. These dendrimers were thermally stable (Td10% over 500 °C) with the TGA-MS study revealing a degradation mechanism occurring first at the inner-layer phenylazomethine group. The metal (Lewis acid) complexation property of these dendrimers was also studied. Electrochemical measurements showed that these dendrimers have the appropriate HOMO level as a hole-transporting material with electropolymerizability on the peripheral carbazole groups. A photo-cross-linking property of the dendrimer film was also observed. Finally, electro-nanopatterning with conducting AFM and photopatterning of the dendrimer film were demonstrated. Thus, the new dendrimer is a potential hole-transporting material that is patternable through oxidation of the peripheral carbazole units by either photochemical or electrochemical methods.

Structure of DNA-functionalized dendrimer nanoparticles

Atomistic molecular dynamics simulations have been carried out to reveal the characteristic features of ethylenediamine (EDA) cored protonated (corresponding to neutral pH) poly amido amine (PAMAM) dendrimers of generation 3 (G3) and 4 (G4) that are functionalized with single strand DNAs (ssDNAs). The four ssDNA strands that are attached via an alkythiolate [–S(CH2)6–] linker molecule to the free amine groups on the surface of the PAMAM dendrimers are observed to undergo a rapid conformational change during the 25 ns long simulation period. From the RMSD values of ssDNAs, we find relative stability in the case of purine rich (having more adenine and guanine) ssDNA strands than pyrimidine rich (thymine and cytosine) ssDNA strands. The degree of wrapping of ssDNA strands on the dendrimer molecule was found to be influenced by the charge ratio of DNA and the dendrimer. As the G4 dendrimer contains relatively more positive charge than G3 dendrimer, we observe extensive wrapping of ssDNAs on the G4 dendrimer than G3 dendrimer. This might indicate that DNA functionalized G3 dendrimer is more suitable to construct higher order nanostructures. The linker molecule was also found to undergo drastic conformational change during the simulation. During nanosecond long simulation some portion of the linker molecule was found to be lying nearly flat on the surface of the dendrimer molecule. The ssDNA strands along with the linkers are seen to penetrate the surface of the dendrimer molecule and approach closer to the center of the dendrimer indicating the soft sphere nature of the dendrimer molecule. The effective radius of DNA-functionalized dendrimer nanoparticles was found to be independent of base composition of ssDNAs and was observed to be around 19.5 Å and 22.4 Å when we used G3 and G4 PAMAM dendrimers as the core of the nanoparticle respectively. The observed effective radius of DNA-functionalized dendrimer molecules apparently indicates the significant shrinkage in the structure that has taken place in dendrimer, linker and DNA strands. As a whole our results describe the characteristic features of DNA-functionalized dendrimer nanoparticles and can be used as strong inputs to design effectively the DNA–dendrimer nanoparticle self-assembly for their active biological applications.

Interactions between oppositely charged dendrimers

The interactions between cationic (G3, G4, and G5) and anionic (G4.5) poly(amidoamine) (PAMAM) dendrimers were investigated by isothermal titration calorimetry (ITC), photometric titration, nuclear magnetic resonance (NMR), and dynamic light scattering (DLS) studies. The results suggest that the formation of anionic–cationic dendrimer aggregates is an enthalpy-driven process. Ionic interactions occur between the cationic and anionic dendrimers, followed by the formation of supramolecular aggregates and precipitates. The phase separation of the resulting aggregate depends on dendrimer generation, surface charge density, anionic–cationic dendrimer molar ratio, and solution pH values. G5, with a relatively rigid structure, displayed a different binding behavior with G4.5 as compared to the G3 and G4 dendrimers with more flexible scaffolds. G4.5 and G5 with a molar ratio of 0.5 : 1 form the largest aggregates among the investigated G4.5–G5 molar ratios. G4.5 and G5 form aggregates in a size range of 300–3000 nm depending on the G4.5–G5 molar ratio. The G4.5–G5 supramolecular aggregates are not stable and will disassemble after several days of incubation. The results help us to understand the interactions between globular polyelectrodes as well as oppositely charged biomacromolecules.

Bioconjugates of PAMAM dendrimers with trans-retinal, pyridoxal, and pyridoxal phosphate

BackgroundBioconjugates of a polyamidoamine (PAMAM) G3 dendrimer and an aldehyde were synthesized as carriers for vitamins A and B6, and the bioavailability of these vitamins for skin nutrition was investigated.MethodsNuclear magnetic resonance (NMR) and ultraviolet-visible methods were used to characterize the structure of the bioconjugates and for monitoring release of pyridoxal (Pyr) and pyridoxal phosphate (PLP) from these bioconjugates in vitro. A skin model permeation of bioconjugates was also studied in a Franz chamber.ResultsA transdermal G3 PAMAM dendrimer was used to synthesize bioconjugates with trans-retinal (Ret), pyridoxal (Pyr), or PLP. These nanomolecules, containing up to four covalently linked Ret, Pyr, or PLP (G34Ret, G34Pyr, and G34PLP), were able to permeate the skin, as demonstrated in vitro using a model skin membrane. PLP and Pyr bound to a macromolecular vehicle were active cofactors for glutamic pyruvic transaminase, as shown by 1H NMR spectral monitoring of the progress of the L-alanine + α-ketoglutarate → glutamic acid + pyruvic acid reaction.ConclusionPAMAM-PLP, PAMAM-Pyr, and PAMAM-Ret bioconjugates are able to permeate the skin. PLP and Pyr are available as cofactors for glutamic pyruvic transaminase.

Characteristics of complexes between poly(propylene imine) dendrimers and nucleotides

The aim of our work was to study the G4 and the G5 generations of cationic poly(propylene imine) dendrimers (PPI) as potential anticancer drug carriers. PPI dendrimers with 50% of primary amino surface groups modified with maltose were applied (PPI-m). Adenosine 5′-phosphates (AMP, ADP and ATP)—structural analogues of anticancer drug (fludarabine) were chosen as model molecules. Using Fast Performance Liquid Chromatography (FPLC) it has been shown that nucleotide analogues form stable complexes with the dendrimers. The number of nucleotides complexed with PPI-m G4 and G5 dendrimers depends on (1) nucleotide form, (2) time, (3) concentration of NaCl in the solution and (4) pH of solution. Obtained results are promising enough to further study PPI-m dendrimers as nanosized carrier systems for anticancer drugs.

Antimicrobial activity of poly(propylene imine) dendrimers

Poly(propylene imine) dendrimers have been investigated for their biological applications but their antibacterial activity has not been extensively explored. Thus, the fourth generation of poly(propylene imine) dendrimers (PPI-G4) and PPI dendrimers with a surface modified by attaching maltose in 25% and 100% (PPI-25%mG4 and PPI-100%mG4, respectively) was evaluated for the antibacterial activity against Gram-positive bacteria: Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Gram-negative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 15442 and yeast Candida albicans ATCC 10231. Cytotoxicity of all tested dendrimers was checked on a Chinese hamster fibroblast cell line (B14), human liver hepatocellular carcinoma cell line (HepG2), mouse neuroblastoma cell line (N2a) and rat liver cell line (BRL-3A). The obtained results indicate that studied nano-sized macromolecules possess the greatest antimicrobial activity against S. aureus. PPI G4 dendrimers modified with 25% of maltose display antibacterial activity and a striking selectivity toward S. aureus, at the concentrations, which are at the same time harmless for the eukaryotic cell lines. Moreover, at the higher concentrations of unmodified dendrimers efficient growth inhibition of S. epidermidis and C. albicans has been observed.