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1.
The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.  相似文献   

2.
Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.  相似文献   

3.
α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.  相似文献   

4.
The interaction mechanism of lipopeptide antibiotic daptomycin and polyamidoamine (PAMAM) dendrimers was studied using fluorescence spectroscopy. The fluorescence changes observed are associated with daptomycin–dendrimer interactions. The binding isotherms were constructed by plotting the fluorescence difference at 460 nm from kynurenine (Kyn‐13) of daptomycin in the presence and absence of dendrimer. A one‐site and two‐site binding model were quantitatively generated to estimate binding capacity and affinity constants from the isotherms. The shape of the binding isotherm and the dependence of the estimated capacity constants on dendrimer sizes and solvent pH values provide meaningful insight into the mechanism of interactions. A one‐site binding model adequately describes the binding isotherm obtained under a variety of experimental conditions with dendrimers of various sizes in the optimal binding pH region 3.5 to 4.5. Comparing the pH‐dependent binding capacity with the ionization profiles of daptomycin and dendrimer, the ionized aspartic acid residue (Asp‐9) of daptomycin primarily interact with PAMAM cationic surface amine. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

5.
Binding of a poly(amido amine) dendrimer with salmon testes DNA in an aqueous solution of 0.01 M NaCl at pH 6.5 has been investigated. It was shown from the physicochemical experiments of static light scattering and ultra-violet and circular dichroism spectroscopy that at a 0.2 mixing ratio of dendrimer/DNA (number ratio of NH2 groups in dendrimer vs phosphate groups in DNA) significant conformational change of the DNA occurred owing to the binding of dendrimers on the DNA chain. The dendrimer/DNA complexes formed aggregates (nanogels) when the mixing ratio was increased above 0.2. Weight-averaged molecular weight, radius of gyration, and turbidity measurements revealed that the size of the aggregates increased up to a mixing ratio of 0.8. Atomic force microscopic images certified the formation of complexes and the morphology of the nanogels.  相似文献   

6.
We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.  相似文献   

7.
Coles DJ  Yang S  Minchin RF  Toth I 《Biopolymers》2008,90(5):651-654
Understanding the nature of binding of polycationic dendrimers to DNA provides useful information on their role in gene delivery. In the present study, we have characterized the interaction of several peptide-based polycationic dendrimers with salmon sperm DNA using isothermal titration calorimetry. The dendrimers consisted of the cell penetrating peptide TAT, a nuclear localization signal peptide and dendritic polylysine. The binding affinity and thermodynamic parameters were found to increase as the number of positive charges on the dendrimer increased, indicating that ionic interactions were the major binding forces between the two molecules. The effect of acidic pH (3.2) compared to a more neutral pH (7.2) was also examined. The binding affinity was stronger at the lower pH but precipitation of the complex was more prominent at pH 7.2 which was shown by large enthalpies. The results indicate that our dendrimers are forming stable complexes with DNA.  相似文献   

8.
Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K(mPEG-G3) = 1.5 (±0.5) × 10(3) M(-1), K(mPEG-G4) = 3.4 (±0.80) × 10(3) M(-1), and K(PAMAM-G4) = 8.2 (±0.90) × 10(4) M(-1). The order of stability of polymer-DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer-DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations.  相似文献   

9.
Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D350+) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.  相似文献   

10.
Tsai YJ  Hu CC  Chu CC  Imae T 《Biomacromolecules》2011,12(12):4283-4290
This study successfully evaluated gene delivery and transfection toward rat C6 glioma cell lines mediated by intrinsic blue fluorescent poly(amido amine) (PAMAM) dendrimer. We used three antisense oligonucleotides, (AS-ODN) p75, NGF1, and NGF2 for knocking down specific protein expressions. The three oligonucleotides were electrostatically associated with the photoluminescent amino-terminated PAMAM dendrimer to yield fluorescent complexes at various nitrogen-to-phosphorus (N/P) ratios. Compared with pristine PAMAM dendrimer and hyperbranched polyethylenimine (PEI), the fluorescent PAMAM dendrimer revealed lower in vitro cytotoxicity toward C6 cells, allowing us to transfect the cells with the AS-ODN complexes under a higher N/P ratio. Due to the intrinsic fluorescence, cellular uptake behavior could be directly analyzed by fluorescence microscopy and flow cytometry, without additional fluorescence labeling. As expected, the result clearly suggested that the uptake efficiency increased as the N/P value increased. Furthermore, the quantified data obtained from flow cytometry indicated relatively higher uptake efficiency for the p75 complex, which is mainly due to different association patterns between the fluorescent dendrimer and AS-ODNs. At N/P = 20, atomic force microscopic analysis confirmed that the p75 complex formed well-condensed, spherical particles with dimensions less than 200 nm, but that NGF2 AS-ODN associated poorly with the dendrimer. Finally, Western blot analysis indicated that these complexes were capable of knocking down the specific protein expression to a certain level, being comparable to the hyperbranched PEI-mediated gene transfection. Our preliminary results clearly indicated that intrinsic fluorescent PAMAM dendrimers show promise as gene vehicles that can achieve delivery, transfection, and bioimaging at the same time.  相似文献   

11.
The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.  相似文献   

12.
Nucleic acid interaction with nanoscale objects like carbon nanotubes (CNTs) and dendrimers is of fundamental interest because of their potential application in CNT separation, gene therapy and antisense therapy. Combining nucleic acids with CNTs and dendrimers also opens the door towards controllable self-assembly to generate various supra-molecular and nano-structures with desired morphologies. The interaction between these nanoscale objects also serve as a model system for studying DNA compaction, which is a fundamental process in chromatin organization. By using fully atomistic simulations, here we report various aspects of the interactions and binding modes of DNA and small interfering RNA (siRNA) with CNTs, graphene and dendrimers. Our results give a microscopic picture and mechanism of the adsorption of single- and double-strand DNA (ssDNA and dsDNA) on CNT and graphene. The nucleic acid-CNT interaction is dominated by the dispersive van der Waals (vdW) interaction. In contrast, the complexation of DNA (both ssDNA and dsDNA) and siRNA with various generations of poly-amido-amine (PAMAM) dendrimers is governed by electrostatic interactions. Our results reveal that both the DNA and siRNA form stable complex with the PAMAM dendrimer at a physiological pH when the dendrimer is positively charged due to the protonation of the primary amines. The size and binding energy of the complex increase with increase in dendrimer generation. We also give a summary of the current status in these fields and discuss future prospects.  相似文献   

13.
The interactions between dendrimers and different types of drugs are nowadays one of the most actively investigated areas of the pharmaceutical sciences. The interactions between dendrimers and drugs can be divided into: internal encapsulation, external electrostatic interaction, and covalent conjugation. In the present study, we investigated the potential of poly(amidoamine) (PAMAM) dendrimers for solubility of four iminodiacetic acid derivatives. We reported that PAMAM dendrimers contribute to significant solubility enhancement of iminodiacetic acid analogues. The nature of the dendrimer–drug complexes was investigated by 1H NMR and 2D-NOESY spectroscopy. The 1H NMR analysis proved that the water-soluble supramolecular structure of the complex was formed on the basis of ionic interactions between terminal amine groups of dendrimers and carboxyl groups of drug molecules, as well as internal encapsulation. The 2D-NOESY analysis revealed interactions between the primary amine groups of PAMAM dendrimers and the analogues of iminodiacetic acid. The results of solubility studies together with 1H NMR and 2D-NOESY experiments suggest that the interactions between PAMAM dendrimers of generation 1–4 and derivatives of iminodiacetic acid are based on electrostatic interactions and internal encapsulation.  相似文献   

14.
Serum albumins have five sites for binding of cationic dendrimers   总被引:1,自引:0,他引:1  
The detailed analysis of the interaction between PAMAM G4 dendrimer and serum albumins was performed using circular dichroism, isothermal titration calorimetry, capillary electrophoresis, zeta-potential and fluorescence polarization. It was shown that serum albumins and PAMAM G4 dendrimer form the complex with stoichiometry of 4-6:1 for G4:HSA and 4-5:1 for G4:BSA molar ratio. The possible sites of PAMAM G4 dendrimers binding to protein surface were discussed. Also, it has been proposed that dendrimer does not significantly affect the protein secondary structure studied by circular dichroism.  相似文献   

15.
Spatial control over the distribution and the aggregation of arginine-glycine-aspartate (RGD) peptides at the nanoscale significantly affects cell responses. For example, nanoscale clustering of RGD peptides can induce integrins to cluster, thus triggering complete cell signaling. Dendrimers have a unique, highly branched, nearly spherical and symmetrical structure with low polydispersity, nanoscale size, and high functionality. Therefore, dendrimers are a class of ideal scaffold for construction of nanoscale dendritic RGD clusters in which RGD loading degree and cluster size can be finely adjusted. This new type of nanoscale dendritic RGD cluster will aid us to better understand the impact of spatial arrangement of RGD on cellular responses and to engineer RGD to trigger more favorable cellular responses. In this study, nanoscale dendritic RGD clusters were synthesized based on Starburst anionic G3.5 and cationic G4.0 polyamidoamine (PAMAM) dendrimers. The multiple terminal functional groups on the outermost layer of the dendrimer were coupled with RGD tripeptides. Biofunctionalized dendrimer structures were found to be highly dependent on the generation and the extent of peptide modification (ie, number of peptides per PAMAM dendrimer). Fluorescein isothiocyanate (FITC)-conjugated PAMAM dendrimers were utilized to monitor cellular internalization of dendrimers by adherent fibroblasts. Anionic G3.5-based dendritic RGD clusters have been shown to have no negative effect on fibroblast viability and a concentration-dependent effect on lowering cell adhesion on tissue culture polystyrene (TCPS) as that of free RGD. A similar concentration-dependent effect in cell viability and adhesion was also observed for cationic G4.0-based dendritic RGD clusters at lower but not at high concentrations. The results imply that the synthesized nanoscale dendritic RGD clusters have great potential for tissue engineering and drug delivery applications.  相似文献   

16.
PAMAM dendrimers are cationic polymers that have been used for the delivery of genes and oligonucleotides to cells. However, little is known about the behavior of dendrimer–nucleic acid complexes once they reach the cell interior. To pursue this issue, we prepared dendrimers conjugated with the fluorescent dye Oregon green 488. These were used in conjunction with oligonucleotides labeled with a red (TAMRA) fluorophore in order to visualize the sub-cellular distribution of the dendrimer–oligonucleotide complex and of its components by two-color digital fluorescence microscopy. The 2′-O-methyl antisense oligonucleotide sequence used in these studies was designed to correct splicing at an aberrant intron inserted into a luciferase reporter gene; thus effective delivery of the antisense agent results in the expression of the reporter gene product. The dendrimer–oligonucleotide complex remained associated during the process of uptake into vesicular compartments and eventual entry into the nucleus. Since the pharmacological activity of the antisense compound was manifest under these conditions, it suggests that the dendrimer–oligonucleotide complex is functionally active. A surprising result of these studies was that the Oregon green 488-conjugated dendrimer was a much better delivery agent for antisense compounds than unmodified dendrimer. This suggests that coupling of relatively hydrophobic small molecules to PAMAM dendrimers may provide a useful means of enhancing their capabilities as delivery agents for nucleic acids.  相似文献   

17.
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.  相似文献   

18.
The membrane disruption properties of high generation (G4 to G7) poly(amidoamine) (PAMAM) dendrimers are evaluated and compared to linear poly(lysine). The G6 and G7 dendrimers are unusually effective at inducing leaky fusion of anionic, large unilamellar vesicles, as determined by standard fluorescence assays for lipid mixing, leakage, and contents mixing. Both G7 dendrimer and poly(lysine) are able to disrupt sterically stabilized vesicles that are coated with poly(ethylene glycol). A G7 dendrimer/DNA complex with a 1:1 concentration ratio of dendrimer surface amines to DNA phosphate groups is unable to induce leakage of 3:7 POPA-PE vesicles; however, extensive leakage is observed when the surface amine to phosphate stoichiometry is >/=3:1. Thus, the DNA/dendrimer complexes that typically induce high levels of cell transfection are also able to induce high levels of vesicle leakage. The G7 dendrimer does not induce membrane phase separation in 3:7 POPA-PE vesicles, but an inverse hexagonal phase is observed by (31)P NMR. The enhanced membrane disruption is interpreted in terms of a membrane bending model. A rigid, polycationic dendrimer sphere uses electrostatic forces to bend a malleable, anionic membrane and induce bilayer packing stresses. This bending model is biomimetic in the sense that protein-induced membrane bending is currently thought to be an important factor in the fusion mechanism of influenza virus.  相似文献   

19.
Dendrimers represents a highly branched three-dimensional structure that provides a high degree of surface functionality and versatility. PAMAM dendrimers are used as well-defined nanocontainers to conjugate, complex or encapsulate therapeutic drugs or imaging moieties. Star-burst [PAMAM] dendrimers represent a superior carrier platform for drug delivery. The present study was aimed at synthesis of a surface modified dendrimer for cancer targeted drug delivery system. For this 4.0 G PAMAM dendrimer was conjugated with Gallic acid [GA] and characterized through UV, IR, 1H NMR and mass spectroscopy. Cytotoxicity study of dendrimer conjugate was carried out against MCF-7 cell line using MTT assay. The study revealed that the conjugate is active against MCF-7 cell line and might act synergistically with anti-cancer drug and gallic acid-dendrimer conjugate might be a promising nano-platform for cancer targeting and cancer diagnosis.  相似文献   

20.
The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.  相似文献   

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