Unmodified Complexes Research Articles

Effect of ligand methylation on the spin-switching properties of surface-supported spin-crossover molecules

X-ray absorption spectroscopy investigations of the spin-state switching of spin-crossover (SCO) complexes adsorbed on a highly-oriented pyrolytic graphite (HOPG) surface have shown so far that HOPG is a promising candidate to realize applications such as spintronic devices because of the stability of SCO complexes on HOPG and the possibility of highly efficient thermal and light-induced spin-state switching. Herein, we present the spin switching of several Fe(II) SCO complexes adsorbed on an HOPG surface with particular emphasis on the thermally induced spin transition behaviour with respect to different structural modifications. The complexes of the type [Fe(bpz)2(L)] (bpz = dihydrobis(pyrazolyl)borate, L = 1,10-phenanthroline, 2,2′-bipyridine) and their methylated derivatives exhibit SCO in the solid state with some differences regarding cooperative effects. However, in the vacuum-deposited thick films on quartz, complete and more gradual spin transition behavior is observable via UV/vis spectroscopy. In contrast to that, all complexes show large differences upon direct contact with HOPG. Whereas the unmodified complexes show thermal and light-induced SCO, the addition of e.g. two or four methyl groups leads to a partial or a complete loss of the SCO on the surface. The angle-dependent measurement of the N K-edge compared to calculations indicates that the complete SCO and HS-locked molecules on the surface exhibit a similar preferential orientation, whereas complexes undergoing an incomplete SCO exhibit a random orientation on the surface. These results are discussed in the light of molecule-substrate interactions.

A Dual Targeting Drug Delivery System for Penetrating Blood-Brain Barrier and Selectively Delivering siRNA to Neurons for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is one of most serious threats to human beings, however, the treatment is hindered by blood-brain barrier and poor intra-brain cell selectivity. In this study, we developed a novel dual targeting drug delivery system by modification of NL4 peptide and apolipoprotein A-I (ApoA-I) onto dendrimer particles that may efficiently deliver siRNA into neuron cells to down-regulate BACE1 and inhibit Aβ formation. The constructed ANNP/ siRNA was approximately 79.26 nm with a spherical structure and a zeta potential of 3.53 mV. At N/P ratio of 10, the siRNA could be completely packaged into particles to avoid degradation by RNAase. In vitro, the modification with ApoA-I considerably increased bEnd.3 cell uptake and NL-4 considerably increased PC12 cell uptake. As a result, ANNP/siRNA showed higher uptake in both the cells. In addition, ANNP/siRNA could efficiently penetrate through bEnd.3 monolayers, which was 2.4-fold higher than unmodified complexes. In PC12 cells, the ANNP/siRNA could escape from endosomes and transport into cytoplasm after 8 h incubation, resulting in 87.5% BACE1 gene knockdown capacity, which was better than PEI. Additionally, the particles showed low cytotoxicity to both bEnd.3 and PC12 cells. In conclusion, this study preliminarily demonstrated that ApoA-I and NL4 dual modified dendrimer nanoparticles were efficient carriers for siRNA delivery to AD bearing brain.

Ion Mobility-Mass Spectrometry Analysis of Cross-Linked Intact Multiprotein Complexes: Enhanced Gas-Phase Stabilities and Altered Dissociation Pathways.

Analysis of protein complexes by ion mobility-mass spectrometry is a valuable method for the rapid assessment of complex composition, binding stoichiometries, and structures. However, capturing labile, unknown protein assemblies directly from cells remains a challenge for the technology. Furthermore, ion mobility-mass spectrometry measurements of complexes, subcomplexes, and subunits are necessary to build complete models of intact assemblies, and such data can be difficult to acquire in a comprehensive fashion. Here, we present the use of novel mass spectrometry cleavable cross-linkers and tags to stabilize intact protein complexes for ion mobility-mass spectrometry. Our data reveal that tags and linkers bearing permanent charges are superior stabilizers relative to neutral cross-linkers, especially in the context of retaining compact forms of the assembly under a wide array of activating conditions. In addition, when cross-linked protein complexes are collisionally activated in the gas phase, a larger proportion of the product ions produced are often more compact and reflect native protein subcomplexes when compared with unmodified complexes activated in the same fashion, greatly enabling applications in structural biology.

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Abstract NG06: The Trojan Horse Strategy: Packaging chemotherapeutics can help alleviate toxicity

Nuclear location signal peptide–modified poly (ethyleneimine)/DNA complexes: An efficient gene delivery vector in vitro and in vivo

The low transfection efficiency of nonviral gene delivery systems limits their applications. In this study, we demonstrated a simple method to modify poly(ethyleneimine)/DNA complexes with a nuclear location signal peptide via bis(succinimidyl) penta(ethylene glycol) coupling. The amount of grafted nuclear location signal peptide was controlled within a range of 0–9 µg for poly(ethyleneimine)/DNA complexes containing 10 µg DNA and 100 µg poly(ethyleneimine) by adjusting the grafting agent and peptide feeds. The particle size and surface zeta-potential of the complexes were largely retained after nuclear location signal immobilization. Based on the results of the flow cytometry measurements, the nuclear location signal–modified poly(ethyleneimine)/DNA complexes were internalized into at bone marrow stem cells at a significantly faster rate and a higher amount than the unmodified complexes. In vitro transfection experiments, using plasmid DNA encoding bone morphogenetic protein 2, indicated that the nuclear location signal peptide–modified poly(ethyleneimine)/DNA complexes have significantly higher gene transfection ability toward bone marrow stem cells than unmodified complexes. The porous collagen scaffolds loaded with nuclear location signal–modified poly(ethyleneimine)/plasmid DNA encoding bone morphogenetic protein 2 complexes successfully transfected tissue cells and induced the human bone morphogenetic protein 2 expression in a rat. The modification of the poly(ethyleneimine)/DNA complexes with nuclear location signal peptide was effective in enhancing gene transfection of complexes in vitro and in vivo, thus indicating potential applications for bioactive scaffolds with enhanced tissue regeneration performance.

Nanocrystalline carbonate-apatites: role of Ca/P ratio on the upload and release of anticancer platinum bisphosphonates

In the present study two nanocrystalline apatites have been investigated as bone-specific drug delivery devices to be used for treatment of bone tumors either by local implantation or by injection. In order to assess how the Ca/P ratio can influence the adsorption and release of anticancer platinum-bisphosphonate complexes, two kinds of apatite nanocrystals having different Ca/P ratios but similar morphologies, degree of crystallinity, and surface areas have been synthesized and characterized. The two platinum-bisphosphonate complexes considered were the bis-{ethylenediamineplatinum(ii)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and the bis-{ethylenediamineplatinum(ii)}medronate. The Ca/P ratio plays an important role in the adsorption as well as in the release of the two drugs. In fact, the apatite with a higher Ca/P ratio showed greater affinity for both platinum complexes. Also the chemical structure of the two Pt complexes appreciably affects their affinity towards as well as their release from the two kinds of apatites. In particular, the platinum complex whose bisphosphonate contains a free aminic group showed greater upload and smaller release. The cytotoxicity of the Pt complexes released from the apatite was tested against human cervical, colon, and lung cancer cells as well as against osteosarcoma cells. In agreement with previous work, the Pt complexes released were found to be more cytotoxic than the unmodified complexes.

A facile preparation of novel multifunctional vectors by non-covalent bonds for co-delivery of doxorubicin and gene

In this study, novel multifunctional ternary complexes of biotinylated transferrin–avidin–biotin–poly(ethylene glycol)–poly( l-glutamate acid)/poly(2-(2-aminoethylamino) ethyl methacrylate)/doxorubicin–poly( l-aspartic acid)/pDNA (TAB/PIC–D/pDNA complexes) were prepared based on polyion complex micelles (PIC) and the avidin–biotin system, which aimed to target co-delivery of anti-cancer doxorubicin and gene. Cytotoxicity studies revealed that PIC–D could have anti-tumor effect on HeLa cells and HepG2 cells; TAB coating could increase the biocompatibility of PIC–D/pDNA complexes and the targeting delivery efficiency of doxorubicin. TAB/PIC–D/pDNA complexes possessed higher transfection efficiency than the unmodified complexes in serum, and transferrin could enhance luciferase expression in HeLa cells and HepG2 cells. Furthermore, confocal laser scanning microscopy showed that doxorubicin and gene could be delivered into HepG2 cells simultaneously by TAB/PIC–D/pDNA complexes. The formation of the ternary complexes provides a facile approach to constructing a multifunctional delivery system for targeted co-delivery of anticancer drugs and gene.

How does modification of adenine by hydroxyl radical influence the stability and the nature of stacking interactions in adenine-cytosine complex?

This study reports on ab initio calculations of adenine-cytosine complexes in two different context alignments appearing in B-DNA. The influence of adenine modification by hydroxyl radical on the stability of the complexes is also discussed. The analysis was performed on over 40 crystallographic structures for each of the sequence contexts. In most cases, modification of adenine by hydroxyl radical leads to less negative intermolecular interaction energies. The issue of the influence of alteration of structural base step parameters on the stability of modified and unmodified adenine-cytosine complexes is also addressed. Analysis of the dependence of intermolecular interaction energy on base step parameters reveals that for twist and shift modification of adenine by hydroxyl radical leads to quite different interaction energy profiles in comparison with unmodified complexes. In order to elucidate the physical origins of this phenomenon, i.e. to analyze how the modification of adenine by hydroxyl radical is reflected in the change of intermolecular interaction energy components, a variational-perturbational decomposition scheme was applied at the MP2/aug-cc-pVDZ level of theory.

Smart delivery of antitumoral platinum complexes from biomimetic hydroxyapatite nanocrystals

This study widens the role of biomimetic hydroxyapatite (HA) nanocrystals as bone substitutes and describes how they can be used as bone-specific drug delivery devices for in situ treatment of bone tumors upon local implantation. The adsorption and release kinetics of bis-{ethylenediamineplatinum(II)}-2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate and bis-{ethylenediamineplatinum(II)}medronate on two kinds of HA nanocrystals having different morphologies, crystallinity degrees and surface areas have been investigated. The different chemical structures of the two Pt complexes appreciably affect not only the affinity towards the two kinds of HA, but also their release. The Pt complex loading is slightly greater for the HA characterized by lower crystallinity and higher surface area, with respect to the more crystalline one. The cytotoxicity of Pt complexes released from the HA were tested against human cervix carcinoma cells and, interestingly, were found to be more cytotoxic than the unmodified complexes. The released Pt species are therefore the active dichloridoethylenediamineplatinum(II) or related solvato species formed by Pt-bisphosphonate bond breaking.

A convenient and adjustable surface-modified complex containing poly-L-glutamic acid conjugates as a vector for gene delivery

In order to quantify the amount of ligands or poly(ethylene glycol) (PEG) on each vector, here we developed a system in which poly-L-glutamic acid (PLG) was used as surface modification loading backbone, to which one PEG (MW 5000, 10000, 20000) or epidermal growth factor (EGF) was linked. The PLG conjugates can electro-statically adsorb upon DNA/ polycation complex with positive charge, and, the amount of EGF or PEG on the surface of complexes could be varied. We have made a series of complexes containing the various PLG conjugates and examined their physicochemical properties, and made a comparison of properties and transfection efficiency between these complexes. EGF- and PEG-modified complexes showed 10-25-folds higher cell transfection efficiency than unmodified complexes in medium with or without serum.

Targeting of polyamidoamine–DNA nanoparticles using the Staudinger ligation: Attachment of an RGD motif either before or after complexation

Two new methods for the modular synthesis of targeted gene delivery systems are reported. The PEGylated polyamidoamine DMEDA-PEG-DMEDA-(MBA-DMEDA) n+1 -PEG-DMEDA 3 was sequentially modified to contain an integrin-binding peptide ligand via the Staudinger ligation. The conjugation of the ligand was achieved either before particle complexation (precomplexation) or after particle complexation (postcomplexation). Comparison of the two systems showed that postcomplexation strategy led to small and discrete toroidal nanoparticles whilst the precomplexation particles showed loose complexes. The targeted particles showed an increased uptake into cells compared to unmodified complexes however no significant increase in transfection was seen.

Recognition of Base Pair Inversions in Duplex by Chimeric (α,β) Triplex-Forming Oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10°C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in Tm values as compared with unmodified complexes.

Structure−Optical Property Relationships in Organometallic Sydnones

As part of an effort to develop a spectroscopic structure-property relationship in platinum acetylide oligomers, we have prepared a series of mesoionic bidentate Pt(PBu3)2L2 compounds containing sydnone groups. The ligand is the series o-Syd-(C6H4-C[triple bond]C)n-H, where n = 1-3, designated as Syd-PEn-H. The terminal oligomer unit consists of a sydnone group ortho to the acetylene carbon. We synthesized the platinum complex (Syd-PEn-Pt), the unmodified ligands (PEn-H), and the unmodified platinum complexes (PEn-Pt). The compounds were characterized by various methods, including X-ray diffraction, 13C NMR, ground-state absorption, fluorescence, phosphorescence, and laser flash photolysis. From solving the structure of Syd-PE1-Pt, we find the angle between the sydnone group and the phenyl group is 45 degrees . By comparison of the 13C NMR spectra of the sydnone-containing ligands, the sydnone complexes with the corresponding unmodified ligands and complexes not containing the sydnone group, the sydnone group is shown to polarize the nearest acetylenes and have a charge-transfer interaction with the platinum center. Ground-state absorption spectra of the complexes in various solvents give evidence that the Syd-PE1-Pt complex has an excited state less polar than the ground state, while the PE1-Pt complex has an excited state more polar than the ground state. In all the higher complexes the excited state is more polar than the ground state. The phosphorescence spectrum of the Syd-PE1-Pt complex has an intense vibronic progression distinctly different from the PE1-Pt complex. The sydnone effect is small in Syd-PE2-Pt and negligible in Syd-PE3-Pt. From absorption and emission spectra, we measured the singlet-state energy E(S), the triplet-state energy E(T), and the singlet-triplet splitting Delta E(ST). By comparison with energies obtained from the unmodified complexes, attachment of the sydnone lowers E(S) by approximately 0.1 eV and raises E(T) by approximately 0.1 eV. As a result, the sydnone group lowers Delta E(ST) by approximately 0.2 eV. The trends suggest one of the triplet-state singly occupied molecular orbitals (SOMOs) is localized on the sydnone group, while the other SOMO resides on the rest of the ligand.

Cisplatin-DNA Adducts by Vibrational Circular Dichroism Spectroscopy: Structure and Isomerization of d(CCTG*G*TCC)·d(GGACCAGG) Intrastrand Cross-Linked by Cisplatin

The intrastrand cisplatin adduct of the duplex d(CCTG*G*TCC)‚d(GGACCAGG), where the asterisks denote the 1,2-intrastrand crosslink, was synthesized and studied in solution in D 2O by vibrational circular dichroism (VCD) and infrared absorption spectroscopy. Comparison of the spectra of the platinated and unmodified complexes confirmed that the entire structure is considerably distorted, as a result of the platinum coordination to the octamer duplex. The major changes in the VCD spectra were detected in the fourth base step, where the cisplatin cross-links the adjacent guanine bases G4 and G5. Platinum coordination shifts the G4-C13 and G5-C12 base pairs apart, with a concomitant disruption of the stacking between the neighboring bases T3-G4 and G5-T6. The resultant new VCD couplets were assigned to stacking interactions between A11 and A14, which implies a substantially distorted structure. Slow isomerization is indicated by diagnostic changes in VCD, converting the intrastrand adduct to other adducts. The corresponding absorption spectra remain essentially unchanged. This DNA octamer was previously studied by NMR in great detail when the isomerization was first detected after one day. Our measurements show that the conversion began soon after both strands were annealed and the first signs were detected in 2 h. This investigation again demonstrated convincingly that VCD is very sensitive to DNA structure modified by complex formation with cisplatin and may become more widely applicable for other similar applications.

Modified DTPA, EDTA ligands and their paramagnetic metal complexes as contrast agents for MRI

Six new aminocarboxylic ligands were synthesized by reactions of EDTA (ethylenediaminetetraacetic acid) dianhydride or DTPA (diethylenetriaminepenta—acetic acid) dianhydride and ethyl ester of serine, threonine and tyrosine. Their paramagnetic metal complexes were also synthesized. All ligands and paramagnetic metal complexes were characterized by IR spectra,1H NMR and elemental analyses. Relaxivity study showed that the metal complexes had higher relaxation effectiveness as compared to corresponding unmodified metal complexes. Imaging study of gadolinium complex of tyrosine ethyl ester modified DTPA in mice demonstrated that this metal complex could apparently increase the signal intensity of MR images.

Interactions of poly-N6-methyladenylic acid and N6-substituted-9-methyladenines with poly-5-bromouridylic acid: a novel base-pairing.

The interaction of poly-N6-methyladenylic acid (poly(m6A) with poly-5-bromouridylic acid (poly(BU) was studied by the mixing curve method. A.1 m6A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m6A) to poly(U) is known to occur with 1 m6A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N6-methyl-9-methyladenine (m6m9A), a corresponding monomer of poly(m6A), with poly(BU) was investigated. Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m6A:2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i.r. spectroscopy. Poly (m6A).2poly(BU) and m6m9A.2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N6-(delta 2-isopentenyl)- and N6-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m6m9A.2poly(BU). The melting experiments indicated the Tms to be substantially decreased, compared to the parent unmodified complexes, even though the Tm dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.

Structural studies of iron and cobalt phthalocyanine-substituted cytochromes c: Carboxymethylation and peptide fragments investigations

In order to obtain information on a way of prosthetic group binding by apocytochrome c in phthalocyanine-substituted cytochrome c, modification of these complexes by carboxymethylation at methionines, as well as cleavage with cyanogen bromide, has been performed. Properties of the reaction products have been investigated by spectroscopic methods, gel electrophoresis, column chromatogrdphy, peptide mapping, and amino acid analysis. Spectrophotometric data indicate that carboxymethylation results in a complete loss of the S-Fe sensitive 695 nm absorption band that appears in absorption spectra of the phthalocyanine-substituted cytochromes c. Unlike unmodified complexes, carboxymethylated derivatives are capable of ligating with typical iron ligands, in both oxidation states. Studies of iron and cobalt tetrasulfonated phthalocyanine interactions with fragments of cytochrome c demonstrate complex formation solely with the heme fragment apopeptides. These complexes do not exhibit a 695 nm absorption band. The results of the chemical examination suggest that the metal phthalocyanine of the model complexes is coordinated axially with histidine-18 and methionine-80 of apocytochrome c, in spite of the weakening of its heme crevice. Identification of these ligands by nuclear magnetic resonance method is difficult due to low solubility of the model complexes.