The influence of PAMAM dendrimers surface groups on their interaction with porcine pepsin

In this study the ability of three polyamidoamine (PAMAM) dendrimers with different surface charge (positive, neutral and negative) to interact with a negatively charged protein (porcine pepsin) was examined. It was shown that the dendrimer with a positively charged surface (G4 PAMAM-NH₂), as well as the dendrimer with a neutral surface (G4 PAMAM-OH), were able to inhibit enzymatic activity of pepsin. It was also found that these dendrimers act as mixed partially non-competitive pepsin inhibitors. The negatively charged dendrimer (G3.5 PAMAM-COOH) was not able to inhibit the enzymatic activity of pepsin, probably due to the electrostatic repulsion between this dendrimer and the protein. No correlation between changes in enzymatic activity of pepsin and alterations in CD spectrum of the protein was observed. It indicates that the interactions between dendrimers and porcine pepsin are complex, multidirectional and not dependent only on disturbances of the secondary structure.     

Hypoxic condition-selective upconversion via triplet–triplet annihilation based on POSS-core dendrimer complexes

The influence on the efficiencies of the triplet–triplet annihilation (TTA)-supported upconversion by oxygen under biomimetic conditions was investigated. From the solution containing the dendrimer complexes based on polyhedral oligomeric silsesquioxane (POSS)-core dendrimer with the Pt complex of octaethylporphyrin (PtOEP) and anthracene in PBS, the fluorescence emission of anthracene depending on the dissolved oxygen (DO) concentrations via the TTA-supported upconversion was obtained with the excitation light at 540 nm. In particular, we observed strong emission only under hypoxic conditions. In addition, it was found that the emission intensity via TTA-supported upconversion can be reversibly regulated by the DO concentrations in the solution.     

Polyethylene glycol‐based low generation dendrimers functionalized with β‐cyclodextrin as cryo‐ and dehydro‐protectant of catalase formulations

Polyethylene glycol (PEG)‐based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze‐thawing, freeze‐drying, and thermal treatment. A novel functional dendrimer (DG_o‐CD) based on the known PEG's ability as cryo‐protector and β‐CD as supramolecular stabilizing agent is presented. During freeze‐thawing, PEG and β‐CD failed to prevent catalase denaturation, while dendrimers, and especially DG_o‐CD, offered the better protection to the enzyme. During freeze‐drying, trehalose was the best protective additive but DG_o‐CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or β‐CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze‐dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze‐dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with β‐CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer–enzyme supramolecular interactions (host–guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:786–795, 2013     

Functionalization of poly(amidoamine) dendrimer‐based nano‐architectures using a naphthalimide derivative and their fluorescent,dyeing and antimicrobial properties on wool fibers

Novel naphthalimide–poly(amidoamine) dendrimer fluorescent dyes were synthesized, and their structures were identified and confirmed using different characterization methods such as Fourier transform infrared, ¹H NMR, ¹³C NMR, differential scanning calorimetry, elemental analysis and UV–vis spectroscopy. The spectrophotometric studies demonstrated absorption maxima (λ_max) and extinction coefficient (ε_max) values in the ranges of 429–438 nm and 25,635–88,618 L/mol/cm, respectively. The dyeing, fastness and antimicrobial properties of dyed wool fibers were examined. Colorimetric measurements demonstrated a greenish‐yellow hue with remarkable fluorescence intensity on dyed wool. Although the fastness properties of naphthalimide dye on wool fibers were poor/moderate, color fastness was appreciably improved through modification of the dye using dendrimers. The results revealed that the newly synthesized dyes are potent antimicrobial agents on wool fibers. Overall, it was deduced that poly(amidoamine) (PAMAM) dendrimers could be exploited as a promising tool in tailoring the different properties of naphthalimide dyes, being suitable for dyeing and antimicrobial finishing agents for wool fibers. Copyright © 2015 John Wiley & Sons, Ltd.     

Uncoupling the BMP receptor antagonist function from the WNT agonist function of R-spondin 2 using the inhibitory peptide dendrimer RWd

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RW^d) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RW^d in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RW^d with enhanced target selectivity and limited side effects.     

Molecular probes for the A2A adenosine receptor based on a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A_2A adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for ¹⁸F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K_i 15 nM). The potent and A_2AAR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A_2AAR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A_2AAR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A_2AAR.     

Speciation, stability constants and structures of complexes of copper(II), nickel(II), silver(I) and mercury(II) with PAMAM dendrimer and related tetraamide ligands

The acid-base properties and Cu(II), Ni(II), Ag(I) and Hg(II) binding abilities of PAMAM dendrimer, L, and of the simple model compounds, the tetraamides of EDTA and PDTA, L¹, were studied in solution by pH-metric methods and by ¹H NMR and UV-Vis spectroscopy. PAMAM is hexabasic and six pK_a values have been determined and assigned. PAMAM forms five identifiable complexes with copper(II), [CuLH₄]⁶⁺, [CuLH₂]⁴⁺, [CuLH]³⁺, [CuL]²⁺ and [CuLH_-1]⁺ in the pH range 2-11 and three with nickel(II), [NiLH]³⁺, [NiL]²⁺ and [NiLH_-1]⁺ in the pH range 7-11. The complex [CuLH₄]⁶⁺, which contains two tertiary nitrogen and three amide oxygen atoms coordinated to the metal ion, is less stable than the analogous EDTA and PDTA tetraamide complexes [CuL¹]²⁺, which contain two tertiary nitrogen and four amide oxygen atoms, due to ring size and charge effects. With increasing pH, [CuLH₄]⁶⁺ undergoes deprotonation of two coordinated amide groups to give [CuLH₂]⁴⁺ with a concomitant change from O-amide to N-amidate coordination. Surprisingly and in contrast to the tetraamide complexes [CuL¹]²⁺, these two deprotonation steps could not be separated. As expected the nickel(II) complexes are less stable than their copper(II) analogues. The tetra-N-methylamides of EDTA, L¹(b), and PDTA form mononuclear and binuclear complexes with Hg(II). In the case of L¹(b) these have stoichiometries HgL¹(b)Cl₂, [HgL¹(b)H₋₂Cl₂]²⁻, [Hg₂L¹(b)Cl₂]²⁺, Hg₂L¹(b)H₋₂Cl₂ and [Hg₂L¹(b)H₋₅Cl₂]³⁻. Based on ¹H NMR and pH-metric data the proposed structure for HgL¹(b)Cl₂, the main tetraamide ligand containing species in the pH range <3-6.5, contains L¹(b) coordinated to the metal ion through the two tertiary nitrogens and two amide oxygens while the structure of [HgL¹(b)H₋₂Cl₂]²⁻, the main tetraamide ligand species at pH 7.5-9.0, contains the ligand similarly coordinated but through two amidate nitrogen atoms instead of amide oxygens. The proposed structure of [Hg₂L¹(b)Cl₂]²⁺, a minor species at pH 3-6.5, also based on ¹H NMR and pH-metric data, contains each Hg(II) coordinated to a tertiary amino nitrogen, two amide oxygens and a chloride ligand while that of [Hg₂L¹(b)H₋₅Cl₂]³⁻, contains each Hg(II) coordinated to a tertiary amino nitrogen, two amidate nitrogens, a chloride and a hydroxo ligand in the case of one of the Hg(II) ions. The parent EDTA and PDTA amides only form mononuclear complexes. PAMAM also forms dinuclear as well as mononuclear complexes with mercury(II) and silver(I). In the pH range 3-11 six complexes with Hg(II) i.e. [HgLH₄Cl₂]⁴⁺, [HgLH₃Cl₂]³⁺, [Hg₂LCl₂]²⁺, [Hg₂LH₋₁Cl₂]⁺, [HgLH₋₁Cl₂]⁻ and [HgLH₋₂Cl₂]²⁻ were identified and only two with Ag(I), [AgLH₃]⁴⁺ and [Ag₂L]²⁺. Based on stoichiometries, stability constant comparisons and ¹H NMR data, structures are proposed for these species. Hence [HgLH₄Cl₂]⁴⁺ is proposed to have a similar structure to [CuLH₄]⁶⁺ while [Hg₂LCl₂]²⁺has a similar structure to [Hg₂L¹(b)H₋₅Cl₂]³⁻.     

Higher order structure of short collagen model peptides attached to dendrimers and linear polymers

Collagen, which is used as a biomaterial, is the most abundant protein in mammals. We have previously reported that a dendrimer modified with collagen model peptides, (Gly‐Pro‐Pro)₅, formed a collagen‐like triple‐helical structure, showing thermal reversibility. In this study, various collagen‐mimic dendrimers of different generations and at different binding ratios were synthesized, to investigate the relationship between the peptide clustering effect and the higher order structure formation. The formation of the higher order structure was influenced by the binding ratios of the peptide to the dendrimer, but was not influenced by the dendrimer generation. A spacer, placed between the dendrimer terminal group and the peptide, negatively contributed to the formation of the higher order structure. The collagen model peptides were also attached to poly(allylamine) (PAA) and poly‐L ‐lysine (poly(Lys)) to compare them with the collagen‐mimic dendrimers. The PAA‐based collagen‐mimic compound, bearing more collagen model peptides than the dendrimer, exhibited a thermally stable higher order structure. In contrast, this was not observed for the collagen‐mimic polymers based on poly(Lys). Therefore, dendrimers and vinyl polymers act as a scaffold for collagen model peptides and subsequently induce higher order structures. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 640–648, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

应用树状DNA杂交(DDH)对生殖道尖锐湿疣中HPV DNA的分型检测

从手术切除的50例生殖道尖锐湿疣新鲜标本中,以及15例正常人血清中,提取基因组DNA,同时用树状DNA杂交(dendrimer DNA hybridizalion,DDH)技术和PCR进行HPV DNA的分型检测.结果50例尖锐湿疣中,以DDH方法检测,感染HPV6型者20例,感染11型者24例,6/11型混合感染者3例,阴性3例,总检测率达94%;以PCR方法检测,HPV6型感染者21例,11型感染者24例,6/11型混合感染者3例,阴性2例,总检测率为96%.15例正常人血清中,以DDH方法检测,HPV感染的假阳性率为0%;以PCR检测,假阳性率为6.67%.还以HPV阳性标本对DDH方法做了敏感度的测定,结果阳性病例DNA检测最低浓度为97.28pg/ml.研究表明,DDH技术具有较高敏感性和高特异性,且成本较低,操作安全简便,可适用于基层中小医院较大样本量筛查.