Glycol Spacer Research Articles

α-Conotoxin dendrimers have enhanced potency and selectivity for homomeric nicotinic acetylcholine receptors.

Covalently attached peptide dendrimers can enhance binding affinity and functional activity. Homogenous di- and tetravalent dendrimers incorporating the α7-nicotinic receptor blocker α-conotoxin ImI (α-ImI) with polyethylene glycol spacers were designed and synthesized via a copper-catalyzed azide-alkyne cycloaddition of azide-modified α-ImI to an alkyne-modified polylysine dendron. NMR and CD structural analysis confirmed that each α-ImI moiety in the dendrimers had the same 3D structure as native α-ImI. The binding of the α-ImI dendrimers to binding protein Ac-AChBP was measured by surface plasmon resonance and revealed enhanced affinity. Quantitative electrophysiology showed that α-ImI dendrimers had ∼100-fold enhanced potency at hα7 nAChRs (IC50 = 4 nM) compared to native α-ImI (IC50 = 440 nM). In contrast, no significant potency enhancement was observed at heteromeric hα3β2 and hα9α10 nAChRs. These findings indicate that multimeric ligands can significantly enhance conotoxin potency and selectivity at homomeric nicotinic ion channels.

Molecular and crystal structure of 2,11,14,17,20,23-hexaoxa-1,12(16,4α)-di-(19-nor-ent-beyerane)tetracosaphane-3,10,13,24-tetraone

The molecular and crystal structure of 2,11,14,17,20,23-hexaoxa-1,12(16,4α)-di-(19-nor-ent-beyerane)tetracosaphane-3,10,13,24-tetraone was determined by X-ray diffraction. This compound has a macrocyclic structure consisting of two molecules of the diterpenoid dihydroisosteviol (16-hydroxy-ent-beyeran-19-oic acid), which are linked by triethylene glycol and hexamethylene spacers. The compound crystallizes in the space group P21 with one independent molecule per asymmetric unit.

A near-infrared two-photon-sensitive peptide-mediated liposomal delivery system

Tumour-oriented nanocarrier drug delivery approaches with photo-sensitivity have been drawing considerable attention over the years. However, due to its low penetrability and ability to induce tissue damage, the use of UV light for triggered nanocarrier release in in vivo applications has been limited. Compared with UV light, near-infrared (NIR) light deeply penetrates tissues and is less damaging to cells. Here, we report on the development of a novel method employing photo-sensitive cell-penetrating peptides (CPPs), which can be used to trigger the transport of liposomes into cells following stimulation, which was irradiation with NIR light in this case. The positive charges of the lysine residues on the CPP were temporarily caged by a NIR two-photon excitation-responsive protective group (PG), thereby forming photo-sensitive peptides (PSPs). The PSP was connected with DSPE via a polyethylene glycol (PEG) spacer to prepare the modified liposomes (PSP-L). Once illuminated by NIR light in tumour tissues, these PGs were cleaved, and the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the liposomes into cancer cells. The PSP-L carrying vinorelbine bitartrate prepared in this work possessed suitable physiochemical properties. In addition, strong cellular uptake and cytotoxic activity of PSP-L in MCF-7 cells were correlated with NIR illumination. Furthermore, triggered NIR activation of PSP-L led to higher antitumour efficacy in the MCF-7 tumour model in nude mice compared with the unmodified liposomes (N-L). In conclusion, the application of PSP modifications to drug-carrying liposomes may provide an approach for the targeted delivery of antitumour agents.

Plasmon waveguide resonance for sensing glycan–lectin interactions

Carbohydrate-modified interfaces have been shown to be valuable tools for the study of protein–glycan recognition events. Label-free approache such as plasmonic based techniques are particularly attractive. This paper describes a new analytical platform for the sensitive and selective screening of carbohydrate–lectin interactions using plasmon waveguide resonance. Planar optical waveguides (POW), consisting of glass prisms coated with silver (50nm) and silica (460nm) layers were derivatized with mannose or lactose moieties. The specific association of the resulting interface with selected lectins was assessed by following the changes in its plasmonic response. The immobilization strategy investigated in this work is based on the formation of a covalent bond between propargyl-functionalized glycans and surface-linked azide groups via a Cu(I) “click” chemistry. Optimization of the surface architecture through the introduction of an oligo(ethylene glycol) spacer between the plasmonic surface and the glycan ligands provided an interface which allowed screening of glycan–lectin interactions in a highly selective manner. The limit of detection (LOD) of this method for this particular application was found to be in the subnanomolar range (0.5nM), showing it to constitute a promising analytical platform for future development and use in a pharmaceutical or biomedical setting.

RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells.

We report the use of arginine-glycine-aspartic (Arg-Gly-Asp, RGD) peptide-modified dendrimer-entrapped gold nanoparticles (Au DENPs) for highly efficient and specific gene delivery to stem cells. In this study, generation 5 poly(amidoamine) dendrimers modified with RGD via a poly(ethylene glycol) (PEG) spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles (AuNPs). The native and the RGD-modified PEGylated dendrimers and the respective well characterized Au DENPs were used as vectors to transfect human mesenchymal stem cells (hMSCs) with plasmid DNA (pDNA) carrying both the enhanced green fluorescent protein and the luciferase (pEGFPLuc) reporter genes, as well as pDNA encoding the human bone morphogenetic protein-2 (hBMP-2) gene. We show that all vectors are capable of transfecting the hMSCs with both pDNAs. Gene transfection using pEGFPLuc was demonstrated by quantitative Luc activity assay and qualitative evaluation by fluorescence microscopy. For the transfection with hBMP-2, the gene delivery efficiency was evaluated by monitoring the hBMP-2 concentration and the level of osteogenic differentiation of the hMSCs via alkaline phosphatase activity, osteocalcin secretion, calcium deposition, and von Kossa staining assays. Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors. The coexistence of RGD and AuNPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers, which is beneficial for highly efficient and specific stem cell gene delivery applications.

Photolabile-caged peptide-conjugated liposomes for siRNA delivery

Light is an ideal general triggered signal, which occurs as a result of its non-invasive nature, desirable controllability and high spatial resolution. However, due to its low penetrability and ability to harm tissues, the use of ultraviolet (UV) light for triggered nanocarrier release in in vivo applications has been limited. Compared with UV light, near-infrared (NIR) light deeply penetrates tissues and is less damaging to cells. In this study, we have devised and tested a strategy for site-specific delivery of small interfering RNA (siRNA) into cancer cells by using liposomes bearing a photolabile-caged peptide (PCP). The positive charges of the lysine residues on the cell-penetrating peptide (CPP) were temporarily caged by the NIR two-photon excitation-responsive protective groups (PG), thereby forming a PCP. Once illuminated by NIR light at tumor tissues, these PGs were cleaved; the positively charged CPP regained its activity and facilitated rapid intracellular delivery of the liposomes into cancer cells. The PCP was connected with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine via a polyethylene glycol (PEG) spacer to prepare the modified liposomes (PCP-LP). Subsequent research demonstrated that the application of the PCP modifications may provide an approach for the selectively targeted delivery of siRNA.

Synthesis and Evaluation of Neoglycoconjugates Based on Adamantyl Scaffolds

AbstractLectin–carbohydrate interactions are important for many biological processes of high pharmaceutical interest. Multivalent carbohydrate conjugates (glycoclusters) have been frequently used to study and manipulate these interactions. In this report, we present the synthesis of trimeric glucose and mannose conjugates that have been assembled with an AB3 scaffold based on adamantane. Several neoglycoconjugates bearing three carbohydrates have been synthesized with different ethylene glycol spacers between the sugar moieties and the adamantyl core. An additional orthogonal functionality at the remaining adamantane bridgehead position allows the conjugation of these trimeric cluster glycosides to other functional molecules such as fluorescent dyes or a solid support for the construction of glycoarrays. We have evaluated the binding properties of trivalent carbohydrate derivatives to ConA as a model lectin in solution by STD‐NMR and on a lectin array by using fluorescent glycoconjugates.

Pinpointing mechanochemical bond rupture by embedding the mechanophore into a macrocycle.

Mechanophores contain a mechanically labile bond that can be broken by an external mechanical force. Quantitative measurement and control of the applied force is possible through atomic force microscopy (AFM). A macrocycle was synthesized that contains both the mechanophore and an aliphatic chain that acts as a "safety line" upon bond breaking. This ring-opening mechanophore unit is linked to poly(ethylene glycol) spacers, which allow investigation by single molecule force spectroscopy. The length increase upon rupture of the mechanophore was measured and compared with quantum chemical calculations.

Incorporation of novel azobenzene dyes bearing oligo(ethylene glycol) spacers into first generation dendrimers

Herein, we report the synthesis and inclusion of a new series of azo-dyes into first generation Fréchet type dendrimers. The incorporated dyes are amino-nitro, amino-methoxy and amino-butyl substituted azobenzenes bearing a well defined oligo(ethylene glycol) side chain. The optical properties of the dendrimers were studied by absorption spectroscopy. Dendrimer bearing amino-nitro substituted azobenzenes (λ = 480 nm in CHCl3) behaved as a pseudostilbene type azobenzene whereas the others (λ = 409 nm in CHCl3) showed the typical behaviour of aminoazobenzenes. Moreover, the trans–cis photoisomerization of the dendrimers was studied by UV–vis spectroscopy by irradiating at two different wavelengths (254 and 365 nm). In chloroform, the appearance of an intense red-shifted band revealed the presence of a photoprotonation effect in the azobenzene moiety. This phenomenon was also studied by absorption spectroscopy in function of time. The results were compared to those predicted by molecular modelling using Density Functional Theory calculations.

Unconventional poly(ionic liquid)s combining motionless main chain 1,2,3-triazolium cations and high ionic conductivity

We report the synthesis of poly(1,2,3-triazolium ionic liquid)s by the polyaddition of α-azide-ω-alkyne monomers with short n-hexyl and diethylene glycol spacers by both copper(I)-catalyzed and thermal Huisgen azide–alkyne 1,3-dipolar cycloaddition.

Targeted CT imaging of human hepatocellular carcinoma using low-generation dendrimer-entrapped gold nanoparticles modified with lactobionic acid.

Development of cost-effective nanoscale contrast agents for targeted tumor computed tomography (CT) imaging remains a great challenge. Here, we report the synthesis of dendrimer-entrapped AuNPs (Au DENPs) using generation 2 poly(amidoamine) dendrimers pre-modified with fluorescein isothiocyanate via a thiourea linkage and lactobionic acid (LA) via a polyethylene glycol spacer as templates. The formed Au DENPs were characterized via different techniques and were used as a nanoprobe for targeted CT imaging of hepatocellular carcinoma (HCC). We show that the LA-modified Au DENPs with a mean Au core size of 1.8 nm are water-dispersible, colloidally stable under different temperatures (4-50 °C) and pH (5-8) conditions, and cytocompatible in the studied concentration range. Flow cytometry results reveal that the LA-Au DENPs are able to specifically target HepG2 cells (a human HCC cell line) overexpressing asialoglycoprotein receptors via a receptor-mediated targeting pathway. Importantly, the developed LA-Au DENPs can be used as a nanoprobe for targeted CT imaging of HepG2 cells in vitro and the xenografted tumor model in vivo. With the demonstrated organ compatibility, the developed LA-Au DENPs using low-generation dendrimers as templates can be a good candidate to be used as a highly efficient and cost-effective nanoprobe for targeted CT imaging of HCC.

“Synthesis and characterization of novel luminescent polythiophenes containing pyrene units and oligo(ethylene glycol) spacers: Thermal and optical properties”

A novel pyrene containing thiophene monomer 3-methyl-4-(4-(pyren-1-yl)butoxy) thiophene was synthesized and fully characterized by FTIR and 1H and 13C NMR spectroscopies. This monomer was polymerized and copolymerized in the presence of 3-methyl-4-(tetraethoxy) thiophene, 3-hexylthiophene, 3-decylthiophene, and 3-dodecylthiophene using FeCl3 as oxidizing agent, to give the corresponding homopolymer HP3 and copolymers (CP1, CP2, CP3, CP4,CP5 and CP6). These polymers exhibited good thermal stability with T10 values between 312 and 414°C, and Tg values varying from −38 to 24°C. CP5 exhibited two absorption bands at 246 and 416nm whereas CP6 absorbed only at 242nm. The emission spectra of both polymers showed a monomer emission at 376–396 followed by an intense excimer emission at 570nm, which reveals the presence of intramolecular non parallel pyrene–pyrene interactions.

Efficacy of dual-functional liposomes containing paclitaxel for treatment of lung cancer.

This study was mainly focused on the development of a dual-ligand liposomal delivery system for targeting the delivery of paclitaxel (PTX) to lung cancer. The specific ligand peptide HAIYPRH (T7) and the cationic cell-penetrating peptide TAT were connected with phospholipid via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (T7/TAT-LP-PTX). Physicochemical characterizations of T7/TAT-LP-PTX, such as particle size, ζ potential, morphology, encapsulation efficiency, and in vitro PTX release, were also evaluated. In the cellular uptake study, the T7/TAT-LP endocytosed by the A549 cells was 2.26-, 3.48- and 8.56-fold higher than TAT-LP, T7-LP and LP, respectively. The IC50 values of TAT-LP-PTX, T7-LP-PTX and LP-PTX were much higher than those of T7/TAT-LP-PTX, respectively. The homing specificity of T7/TAT-LP was evaluated on the tumor spheroids, which revealed that T7/TAT-LP was more efficaciously internalized in tumor cells than TAT-LP, T7-LP and LP, respectively. Compared to LP, TAT-LP and T7-LP, T7/TAT-LP showed the strongest cell uptake property, and the highest accumulation ability in tumor spheroids in vitro. In the in vivo study, the T7/TAT-LP-PTX exhibited the best inhibitory effect of tumor growth for A549-bearing mice. Collectively, these results suggested that T7/TAT-LP-PTX is a promising drug delivery system for the treatment of lung cancer.

Short Peptide Motifs for Long-Lasting Anchoring to the Cell Surface

A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells.

Enhancement of the Superoleophobic Properties of Fluorinated PEDOP Using Polar Glycol Spacers

The formation of superoleophobic materials is extremely important for various potential applications, but these materials are extremely difficult to obtain due to the low surface tension of oils. Here, we synthesized original fluorinated (C4F9, C6F13, and C8F17) 3,4-ethylenedioxypyrrole (EDOP) derivatives with polar glycol spacers (diethylene glycol or triethylene glycol) to enhance the superoleophobic properties by electropolymerization. The mobility of the glycol spacers induces steric hindrance during electropolymerization and affects the presence of surface nanoporosity. However, the superoleophobic properties of fluorinated PEDOP can be improved, even if the presence of nanoporosity decreases, on condition that a high degree of nanostructures is preserved around the surface microstructures. This is possible by controlling the length of the fluorinated chain and the length of the glycol spacer. Using a diethylene glycol spacer and a C8F17 chain it is possible to obtain superoleophobic properties with θdiiodomethane = 153.0°, θsunflower oil = 148.3°, and θhexadecane = 148.0°, and with also low oil adhesion. The micro and nanostructures present on the surface and the high intrinsic oleophobic properties of the polymer allow the stabilization of the Cassie–Baxter state.

Dextran as a generally applicable multivalent scaffold for improving immunoglobulin-binding affinities of peptide and peptidomimetic ligands.

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors.

Efficient synthesis of novel benzylidene barbituric and thiobarbituric acid derivatives containing ethyleneglycol spacers

Knoevenagel condensation reaction of ethylene glycol-based aromatic aldehyde with barbituric acid derivatives in ethanol produced novel benzylidene barbituric and thiobarbituric acid derivatives which contain good yields of ethylene glycol spacers. Structures of the products were deduced from their IR, 1H-NMR, and 13C-NMR spectroscopy and mass spectra.

Clicked and long spaced galactosyl- and lactosylcalix[4]arenes: new multivalent galectin-3 ligands.

Four novel calix[4]arene-based glycoclusters were synthesized by conjugating the saccharide units to the macrocyclic scaffold using the CuAAC reaction and using long and hydrophilic ethylene glycol spacers. Initially, two galactosylcalix[4]arenes were prepared starting from saccharide units and calixarene cores which differ in the relative dispositions of the alkyne and azido groups. Once the most convenient synthetic pathway was selected, two further lactosylcalix[4]arenes were obtained, one in the cone, the other one in the 1,3-alternate structure. Preliminary studies of the interactions of these novel glycocalixarenes with galectin-3 were carried out by using a lectin-functionalized chip and surface plasmon resonance. These studies indicate a higher affinity of lactosyl- over galactosylcalixarenes. Furthermore, we confirmed that in case of this specific lectin binding the presentation of lactose units on a cone calixarene is highly preferred with respect to its isomeric form in the 1,3-alternate structure.

Effect of polyethylene glycol, alkyl, and oligonucleotide spacers on the binding, secondary structure, and self-assembly of fractalkine binding FKN-S2 aptamer-amphiphiles.

Previously we identified an aptamer, named FKN-S2, which binds the cell surface protein fractalkine with high affinity and specificity. In this paper a hydrophobic dialkyl C16 tail was added to the aptamer to create an aptamer-amphiphile. We investigated how the tail and a spacer molecule of varying length and hydrophobicity, inserted between the tail and the aptamer headgroup, affect the binding, structure, and self-assembly properties of the aptamer-amphiphile. We synthesized aptamer-amphiphiles with no spacer (NoSPR), polyethylene glycol (PEG4, PEG8, PEG24), alkyl (C12 and C24), or oligonucleotide (T10 and T5: 10 and 5 thymine, and A10: 10 adenine) spacers. The addition of the tail reduced the binding affinity of the aptamer-amphiphile over 7.5-fold compared to the free aptamer. The hydrophobic alkyl spacers resulted in the greatest loss of affinity, and the hydrophilic PEG spacers improved amphiphile affinity but did not restore it to that of the free aptamer. Interestingly, oligonucleotide spacers produced the highest affinity amphiphiles. Nucleotide composition did not affect affinity, however, as the T10 and A10 spacers had equal affinity. The oligonucleotide spacer amphiphiles had the highest affinity because the oligonucleotide spacer increased the affinity of free aptamer; the FKN-S2 aptamer plus the oligonucleotide spacer had a higher affinity than the free FKN-S2 aptamer. Circular dichroism (CD) spectroscopy and thermal melting studies indicated the aptamer forms a stem-loop and intramolecular G-quadruplex, and the tail strongly stabilized the formation of the G-quadruplex in a buffer. Cryogenic transmission electron microscopy (cryo-TEM) imaging showed the aptamer-amphiphiles, independent of the spacer used, self-assembled into micelles and nanotapes, flat bilayer structures that were often twisted. Finally, liposomes functionalized with the FKN-S2 amphiphile were incubated with fractalkine expressing cells, and the amount of binding was dependent on the concentration of the amphiphile on the liposome surface.