Glycol Spacer Research Articles

Set of Highly Stable Amine- and Carboxylate-Terminated Dendronized Au Nanoparticles with Dense Coating and Nontoxic Mixed-Dendronized Form.

The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug delivery platform is described herein. The AuNP-based platform is composed of three complementary parts: (i) a 15 nm AuNP core, (ii) a heterofunctional thioctic acid-terminated tetraethylene glycol spacer, and (iii) a third-generation PPI dendron with a unique protonation profile and diverse end-group functionalization that allows for further derivatization. The prepared dendronized AuNPs are able to withstand several rounds of lyophilization cycles with no sign of aggregation, are stable in phosphate-buffered saline and Hanks' buffer as well as in serum, and are resistant to degradation by glutathione exchange reactions. This nanocarrier platform displays a dense coating, with >1400 dendrons/AuNPs, which will enable very high payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against the MCF-7 breast cancer cell line from a NP concentration of 1 nM, the mixed monolayer AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of toxicity at concentrations as high as 15 nM, similar to the carboxylate-terminated AuNPs. The described dendronized AuNPs address the current practical need for a stable NP-based drug delivery platform which is scalable and easily conjugable, has long-term stability in solution, and can be conveniently formulated as a powder and redispersed in desired buffer or serum.

Design of novel well-defined oligothiophenes bearing donor-acceptor groups (pyrene-porphyrin): Synthesis, characterization, optical properties and energy transfer

Novel thiophene monomers containing porphyrin units attached via di(ethylene glycol) and tetra(ethylene glycol) spacers were synthesized: 3-methyl-4- (4- (1- (5-phenyl-10,15,20-triphenylporphyrin)) diethoxy) thiophene (M2PPh) and 3-methyl-4- (4- (1- (5-phenyl-10,15,20-triphenylporphyrin)) tetraethoxy) thiophene (M4PPh). These monomers and other co-monomers with pyrene units 3-methyl-4-(diethoxy) thiophene (M2) and 3-methyl-4-(tetraethoxy) thiophene (M4) previously reported by us, were linked to thiophene and bithiophene via a Suzuki coupling reaction to give the corresponding terthiophenes and quaterthiophenes: [3-diethoxypyrene, 3-diethoxyporphyrin, 4,4‴ dimethyl - 2,2: 5′2”- terthiophene (TT2PY-PPh)], [3-tetraethoxypyrene, 3-tetraethoxyporphyrin, 4, 4‴dimethyl - 2,2:5′2”-terthiophene (TT4PY-PPh)], [3′-diethoxypyrene, 3′-diethoxyporphyrin, 4,4‴methyl-2,2′:5′: 2′′:5″,2‴-quaterthiophene (QT2PY-PPh)], and [3′-tetraethoxypyrene, 3′-tetraethoxyporphyrin,4,4‴methyl −2,2′:5′:2′′:5″,2‴-quaterthiophene (QT4PY-PPh)]. The synthesized compounds were characterized by 1H NMR, 13C NMR and MALDI-TOF mass spectrometry; their optical properties were studied by absorption and fluorescence spectroscopy. The emission spectra of the oligothiophenes bearing pyrene-porphyrin units showed a significant decrease in the pyrene monomer emission and the appearance of a new emission band at 650 nm, arising from the porphyrin, which revealed that a FRET phenomenon is occurring from the excited pyrene to the porphyrin. The obtained FRET efficiencies were between 93 and 98% depending on the oligomer structure.

Design, characterization, optical and photophysical properties of novel thiophene monomers and polymers containing pyrene moieties linked via rigid and flexible spacers

Herein, we report the synthesis of three novel thiophene monomers connected to pyrene moieties via a flexible oligo(ethylene glycol) (OEG) spacer with an oxygen α-to-pyrene (T1), a flexible OEG spacer with an oxygen β-to-pyrene (T2), and a rigid acetylene spacer (T3). These monomers were prepared in an easy manner from commercially available 3-bromo-4-methylthiophene and further characterized by NMR spectroscopy and mass spectrometry. Monomer T2 exhibited the absorption band at 344 nm and a strong “monomer” emission band at 375 nm typical of a 1-pyrenemethyl derivative. Moreover, T1 and T3 showed red shifted absorption bands at 352 and 384 nm arising from the pyrene substituents, namely the electron-donating oxygen α−to-pyrene and the extended conjugation length, respectively. These compounds exhibited a broad “monomer” emission band between 360˜480 nm without excimers. Homopolymers (OHP1, OHP2, and OHP3) obtained from these monomers were partially soluble and their photophysical properties such as quantum yield and lifetime were characterized. Emission spectra of the oligomers OHP1 and OHP3 exhibited an emission band at ˜500 nm, which is due to the conjugated polythiophene backbone and does not arise from intramolecular pyrene-pyrene interactions.

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

Ligand Density and Linker Length are Critical Factors for Multivalent Nanoparticle-Receptor Interactions.

Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core-shell structured poly(lactic- co-glycolic acid)/poly(lactic acid)-PEG nanoparticles was evaluated using αvβ3 integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.

Design and Evaluation of Heterobivalent PAR1-PAR2 Ligands as Antagonists of Calcium Mobilization.

A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.

Multivalent Fucosides with Nanomolar Affinity for the Aspergillus fumigatus Lectin FleA Prevent Spore Adhesion to Pneumocytes.

FleA (or AFL), a fucose lectin, was recently identified in the opportunistic mold Aspergillus fumigatus, which causes fatal lung infections in immunocompromised patients. We designed di-, hexa- and octavalent fucosides with various spacer arm lengths to block the hexameric FleA through chelation. Microcalorimetry measurements showed that the ethylene glycol (EG) spacer arm length has a strong influence on the binding affinity of the divalent fucosides. The relationship between the EG length and chelate binding efficiency to FleA was explored according to polymer theory. Hexa- and octavalent compounds based on cyclodextrin and octameric silsesquioxane scaffolds were nanomolar FleA inhibitors, surpassing their monovalent fucose analogue by more than three orders of magnitude. Importantly, some of the fucosides were highly efficient in preventing fungal spore adhesion to bronchoepithelial cells, with half maximal inhibitory concentration values in the micromolar range. We propose that the synergistic antiadhesive effect observed can be ascribed to chelate binding to FleA and to the formation of conidium aggregates, as observed by optical microscopy. These fucosides are promising tools that can be used to better understand the role of FleA in conidia pathogenicity and host defenses against invasive aspergillosis.

Exploring Rigid and Flexible Core Trivalent Sialosides for Influenza Virus Inhibition.

Herein, the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing oligoethylene glycol (OEG) spacers interacting with spike proteins of influenza A virus (IAV) X31 is described. Although the flexible Tris‐based trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well as biophysical analysis, these findings emphasize on the importance of the scaffold rigidity and the challenges associated with the spacer length optimization.

GM1-Binding Conjugates To Improve Intestinal Permeability.

Drugs and proteins with poor intestinal permeability have a limited oral bioavailability. To remediate this problem, a receptor-mediated endocytosis and transcytosis approach was explored. Indeed, the nontoxic β subunit of cholera toxin (CTB) can cross the intestinal barrier by binding to receptor GM1. In this study, we explored the use of GM1-binding peptides and CTB as potential covalent carriers of poorly permeable molecules. GM1-binding peptides (G23, P3) and CTB were conjugated to poorly permeable fluorescent probes such as fluorescein isothiocyanate (FITC) and albumin-FITC using triethylene glycol spacers and click chemistry. The affinity of the peptide conjugates with receptor GM1 was confirmed by isothermal titration calorimetry or microscale thermophoresis, and the results suggested the involvement of nonspecific interactions. Conjugating the model drugs to G23 and P3 improved the internalization into Caco-2 and T84 cells, although the process was not dependent on the amount of GM1 receptor. However, conjugation of bovine serum albumin FITC to CTB increased the internalization in the same cells in a GM1-dependent pathway. Peptide conjugates demonstrated a limited permeability through a Caco-2 monolayer, whereas G23 and CTB conjugates slightly enhanced permeability through a T84 cell monolayer compared to model drugs alone. Since CTB can improve the permeability of large macromolecules such as albumin, it is an interesting carrier for the improvement of oral bioavailability of various other macromolecules such as heparins, proteins, and siRNAs.

Multifunctional Cholesterol-modified Dendrimers for Targeted Drug Delivery to Cancer Cells Expressing Folate Receptors

We present here the development of cholesterol (Chol)-modified dendrimer system for targeted chemotherapy of folate (FA) receptor-expressing cancer cells. In our study, poly(amidoamine) (PAMAM) dendrimers of generation 5 (G5) were functionalized step-by-step with Chol, fluorescein isothiocyanate (FI), and FA via a poly(ethylene glycol) (PEG) spacer (PEG-FA), and then acetamide to shield their remaining surface amines. The synthesized G5.NHAc-Chol-FI-PEG-FA (for short, G5-CFPF) dendrimers were utilized to encapsulate 10-hydroxycamptothecin (HCP), a hydrophobic anticancer drug. We find that each G5-CFPF dendrimer can encapsulate 13.8 HCP molecules. The complexes show a slower release profiles of HCP in a pH-dependent manner than the control complexes formed using the same dendrimers without Chol under the same conditions. Thanks to the targeting role played by FA, the complexes display a specific inhibition efficacy to FA receptor-expressing cervical cancer cells. The designed Chol-modified dendrimers may be adopted as a promising carrier for application in targeted cancer therapy.

“Biowheel-Axle” Assembly of β-Cyclodextrin Fitted onto Bile Acid Units Linked by PEG Spacers through Inclusion Polymerization

A “biowheel-axle” assembly of polyrotaxanes of β-cyclodextrin (β-CD) threaded onto bile acid moieties linked by a poly(ethylene glycol) (PEG) spacer of tunable length was prepared through an “inclusion polymerization” approach. The bile acid-diamine derivatives were allowed to first form inclusion complexes with β-CD and then polymerized with PEG-dicarbonate, followed by end-capping with a stopper of monoamino-β-CD, resulting in “biowheels” of β-CD with PEGylated bile acids as the axle in tandem. All steps were performed without the use of catalysts. The complexation between β-CD and bile acid units of the polymer chain was confirmed by the 2D NOESY NMR technique and powder X-ray diffraction, showing 85% of bile acid units entered the cavity of β-CD. The change of PEG spacer length from 45 to 6 ethylene glycol units led to a flexible–rigid conformation transition of the polyrotaxanes, supported by the random and straight orientation of the “biowheels” shown by scanning tunneling microscopy images. The con...

Effects of bisphosphonate ligands and PEGylation on targeted delivery of gold nanoparticles for contrast-enhanced radiographic detection of breast microcalcifications

A preclinical murine model of hydroxyapatite (HA) breast microcalcifications (µcals), which are an important clinical biomarker for breast cancer detection, was used to investigate the independent effects of high affinity bisphosphonate (BP) ligands and a polyethylene glycol (PEG) spacer on targeted delivery of gold nanoparticles (Au NPs) for contrast-enhanced radiographic detection. The addition of BP ligands to PEGylated Au NPs (BP-PEG-Au NPs) resulted in five-fold greater binding affinity for targeting HA µcals, as expected, due to the strong binding affinity of BP ligands for calcium. Therefore, BP-PEG-Au NPs were able to target HA µcals in vivo after intramammary delivery, which enabled contrast-enhanced radiographic detection of µcals in both normal and radiographically dense mammary tissues similar to previous results for BP-Au NPs, while PEG-Au NPs did not. The addition of a PEG spacer between the BP targeting ligand and Au NP surface enabled improved in vivo clearance. PEG-Au NPs and BP-PEG-Au NPs were cleared from all mammary glands (MGs) and control MGs, respectively, within 24–48 h after intramammary delivery, while BP-Au NPs were not. PEGylated Au NPs were slowly cleared from MGs by lymphatic drainage and accumulated in the spleen. Histopathology revealed uptake of PEG-Au NPs and BP-PEG-Au NPs by macrophages in the spleen, liver, and MGs; there was no evidence of toxicity due to the accumulation of NPs in organs and tissues compared with untreated controls for up to 28 days after delivery. Statement of SignificanceAu NP imaging probes and therapeutics are commonly surface functionalized with PEG and/or high affinity targeting ligands for delivery. However, direct comparisons of PEGylated Au NPs with and without a targeting ligand, or ligand-targeted Au NPs with and without a PEG spacer, on in vivo targeting efficiency, biodistribution, and clearance are limited. Therefore, the results of this study are important for the rationale design of targeted NP imaging probes and therapeutics, including the translation of BP-PEG-Au NPs which enable improved sensitivity and specificity for the radiographic detection of abnormalities (e.g., µcals) in women with dense breast tissue.

Peroxidase-immobilized porous silica particles for in situ formation of peroxidase-free hydrogels with attenuated immune responses

Enzymatic crosslinking chemistry using horseradish peroxidase (HRP) has been widely utilized as an effective approach to fabricating injectable hydrogels with high efficiency under mild reaction conditions. However, their clinical applications are limited by the immunogenicity of the plant-derived enzyme. Herein we report the design, synthesis and characterization of HRP-immobilized porous silica particles (HRP-particles) and their use for in situ formation of HRP-free hydrogels. HRP was immobilized on aminopropyl-modified porous silica particles of 70–140 µm in diameter via poly(ethylene glycol) spacers of different molecular weights by reductive amination reaction. Two different HRP-free hydrogels based on dextran-tyramine and gelatin-hydroxyphenylpropionic acid (GHPA) conjugates were produced by passing a solution containing the conjugates and H2O2 through a syringe packed with HRP-particles. The storage modulus and gelation rate of both hydrogels were tunable by varying the contact time between the polymer solution and HRP-particles. Our in vitro study revealed that HRP-free GHPA hydrogel was less stimulatory to activated mouse macrophages than HRP-containing GHPA hydrogel with the same stiffness. Furthermore, HRP-free GHPA hydrogel exhibited remarkably lower levels of local and systemic inflammation than HRP-containing one upon subcutaneous injection in immunocompetent C57BL/6J mice. The attenuated immunogenicity of HRP-free GHPA hydrogels makes them an attractive platform for tissue engineering applications. Statement of significanceThe immunogenicity of HRP is a significant issue for clinical application of HRP-catalyzed in situ forming hydrogels. HRP-particles are developed to overcome the safety concerns by fabricating HRP-free hydrogels. The porosity of silica particles and molecular weight of poly(ethylene glycol) spacers are discovered as important factors determining the catalytic ability of HRP-particles to induce the in situ crosslinking of polymer-phenol conjugates. Although several articles speculate the potential of HRP to trigger immune responses when administered as a part of hydrogel formulation, no literature has attempted to investigate the immunogenicity of HRP-containing hydrogels in comparison with HRP-free hydrogels. Our findings suggest that the immunogenicity issue should be carefully considered before clinical translation of HRP-containing hydrogels.

Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display

BackgroundExtracellular vesicles (EVs) and exosomes are nano-sized, membrane-bound vesicles shed by most eukaryotic cells studied to date. EVs play key signaling roles in cellular development, cancer metastasis, immune modulation and tissue regeneration. Attempts to modify exosomes to increase their targeting efficiency to specific tissue types are still in their infancy. Here we describe an EV membrane anchoring platform termed “cloaking” to directly embed tissue-specific antibodies or homing peptides on EV membrane surfaces ex vivo for enhanced vesicle uptake in cells of interest. The cloaking system consists of three components: DMPE phospholipid membrane anchor, polyethylene glycol spacer and a conjugated streptavidin platform molecule, to which any biotinylated molecule can be coupled for EV decoration.ResultsWe demonstrate the utility of membrane surface engineering and biodistribution tracking with this technology along with targeting EVs for enhanced uptake in cardiac fibroblasts, myoblasts and ischemic myocardium using combinations of fluorescent tags, tissue-targeting antibodies and homing peptide surface cloaks. We compare cloaking to a complementary approach, surface display, in which parental cells are engineered to secrete EVs with fusion surface targeting proteins.ConclusionsEV targeting can be enhanced both by cloaking and by surface display; the former entails chemical modification of preformed EVs, while the latter requires genetic modification of the parent cells. Reduction to practice of the cloaking approach, using several different EV surface modifications to target distinct cells and tissues, supports the notion of cloaking as a platform technology.

Preparation of photoluminescent grafted polymers, having a polyethylene matrix and pyrene units linked via oligo(ethylene glycol) spacers, using gamma radiation

New polymers were prepared by grafting of low density polyethylene films (PE). Firstly, PE was reacted with acryloyl chloride in the presence of gamma radiation to give the precursor grafted polymers (PE-g-AC). These polymers were further esterified with a new pyrene derivative having a tetra (ethylene glycol) side chain, [2-(2-(2-(2-(pyren-1-yloxy)ethoxy)ethoxy)ethoxy)ethan-1-ol] (PyTEG), leading to the obtainment of a series of luminescent polymers bearing pyrene units (PE-g-AC-PyTEG). The influence of the irradiation dose on the structure and the percentage of grafting of the produced polymers has been studied in detail. These materials have been characterized by FTIR spectroscopy, and their thermal properties have been determined by TGA and DSC. The optical properties of the grafted polymers have been studied by UV–vis and fluorescence spectroscopy.

Synthesis and Avidin Binding of Ruthenium Complexes Functionalized with a Light-Cleavable Free Biotin Moiety.

In this work the synthesis, photochemistry, and streptavidin interaction of new [Ru(tpy)(bpy)(SRR′)](PF6)2 complexes where the R′ group contains a free biotin ligand, are described. Two different ligands SRR′ were investigated: An asymmetric ligand 1 where the Ru‐bound thioether is a N‐acetylmethionine moiety linked to the free biotin fragment via a triethylene glycol spacer and a symmetrical ligand 2 containing two identical biotin moieties. The coordination of these two ligands to the precursor [Ru(tpy)(bpy)Cl]Cl was studied in water at 80 °C. In such conditions the coordination of the asymmetric ligand 1 occurred under thermodynamic control. After the reaction, a mononuclear and a binuclear complex were isolated. In the mononuclear complex, the ratio of methionine‐ {[6](PF6)2} vs. biotin‐bound {[7](PF6)2} regioisomer was 5.3 and the free biotin fragment of [6](PF6)2 allowed to purify it from its isomer [7](PF6)2 at small scales using avidin affinity chromatography. Coordination of the symmetrical ligand 2 afforded [Ru(tpy)(bpy)(2)](PF6)2 {[8](PF6)2} in synthetically useful scales (100 mg), good yield (82 %), and without traces of the binuclear impurity. In this complex, one of the biotin remains free whereas the second one is coordinated to ruthenium. Photochemical release of ligand 2 from [8](PF6)2 occurred upon blue light irradiation (465 nm) with a photosubstitution quantum yield of 0.011 that was independent of the binding of streptavidin to the free biotin ligand.

Mixed Self-Assembled Monolayers with Terminal Deuterated Anchors: Characterization and Probing of Model Lipid Membrane Formation.

We describe herein a series of self-assembled monolayers (SAMs) on gold designed for adjustable tethering of model lipid membrane phases. The SAMs consist of deuterated aliphatic anchors, HS(CH2)15CONH(CH2CH2O)6CH2CONH-X, where X is either -(CD2)7CD3 or -(CD2)15CD3, dispersed in a stable matrix of protein-repellent molecules, HS(CH2)15CONHCH2CH2OH. The mixed SAMs with variable surface densities of the anchors are thoroughly characterized before and after adsorption of phospholipids by means of ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy (IRRAS). In all cases, the bottom portions of the mixed SAMs (i.e., the h-alkyl thiol segments of the molecules) are arranged in a highly ordered all-trans conformation stabilized by a network of lateral hydrogen bonds. The terminal portions of the anchors (the oligo(ethylene glycol) spacer and deuterated alkyl segments, respectively), however, possess less ordered conformations in the mixed composition regime. For the SAMs containing the longer anchors (-(CD2)15CD3), the contact angle and infrared data point toward partial phase segregation. These findings are in excellent agreement with molecular dynamics simulations by Schulze and Stein. Upon analysis in air, the IRRAS data also indicate strong interaction between the adsorbed phospholipid molecules and the d-alkyl tails of the mixed SAM constituents. In such assemblies are the alkyl tails of the phospholipids aligned perpendicularly with respect to the supporting substrate, regardless of the anchor length. We also probed the in situ formation of a tethered bilayer lipid membrane (tBLM) via fusion of small unilamellar vesicles (SUVs) on the characterized SAMs using a quartz crystal microbalance with dissipation monitoring. Our experiments show that SUVs fuse efficiently of the two mixed SAMs with and without a pre-adsorbed lipid layer. Owing to the defined molecular composition and phase behavior, our SAM platform is attractive for detailed studies of tBLM formation and cell mimetic applications.

Enzyme immobilization on protein-resistant PNIPAAm brushes: impact of biotin linker length on enzyme amount and catalytic activity.

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) brushes with terminal click functionality can be used to selectively immobilize enzymes. Exploiting their inherent protein-repellent and thus non-fouling properties, surfaces with specific bioactivity can be created in this way. This report describes the functionalization of alkyne-PNIPAAm brushes with two biotin linkers of different poly(ethylene glycol) (PEG) spacer length via click chemistry and the subsequent immobilization of streptavidin-conjugated horseradish peroxidase (SA-HRP) by the strong interaction between biotin and streptavidin. Spectroscopic ellipsometry is used to quantify the biotin and the SA-HRP amount on the PNIPAAm brushes. Enzyme activities are determined by UV‑vis spectroscopy. A better accessibility to the alkyne-functionalized chain ends for the short biotin-PEG3 linker leads to a higher biotin amount on these PNIPAAm brushes, which in turn results in higher SA‑HRP amounts on biotin-PEG3-modified brushes in the swollen (20 °C) and collapsed state (37 °C) compared to biotin-PEG23-modified ones. For both linkers, an increased immobilization temperature leads to higher SA-HRP amounts due to an enhanced reaction kinetics and mobility. All immobilized SA-HRP amounts are in range of 81-98% monolayer coverage, except for SA-HRP immobilized on biotin-PEG23-modified PNIPAAm brushes at 20 °C, exhibiting only 43% monolayer coverage. A high mobility of the biotin linker combined with a low surface loading of SA-HRP is found to be beneficial for the activity of SA-HRP. Hence, the highest specific activity as measured by HRP-catalyzed 3,3´,5,5´‑tetramethylbenzidine (TMB) oxidation is found for a low SA-HRP amount on biotin-PEG23-modified brushes.

Thermosensitive Amphiphilic Janus Dendrimers with Embedded Metal Binding Sites. Synthesis and Self-Assembly

A family of amphiphilic diblock Janus dendrimers were synthesized by chemoselective “click” coupling of poly(benzyl ether) dendrons, PBE, and poly(ether–ester), PEE, dendrons based on three different ethylene glycol spacers. The well-defined defect-free structure of these dendrimers was proven by MALDI-TOF. In aqueous solutions these dendrimers were able to self-assemble into vesicles with diameter ranging from 140 nm to 3.6 μm or solid micelles (10 to 14 nm diameter). The form of these assemblies was affected by the spacer length between branching points and by the generations of both dendrons. The ethylene glycol moieties in the PEE dendrons induced notable thermosensitivity with sharp transitions observed for all members of the series. The presence of two triazole groups at each branching point was further exploited to fabricate palladium bearing nanomaterials with potential application in catalysis.

Molecular Approaches To Address the Challenges of RNA Analysis in Complex Matrices.

We present on a design change and addition of an internal polyethylene glycol (PEG) spacer to an existing biosensor. There were two reasons for changing the sensor design. The first was to increase the stability of the biosensor to avoid binding off-analytes with single nucleotide polymorphisms. The second was to prevent sensor degradation by nucleases. The biosensor, designed for detection of short noncoding RNA strands, is composed of Reporter and Probe nucleic acid strands that form a partially complementary duplex. The internal PEG was added to the Reporter, and subsequently diminished false negatives that resulted from off-oligonucleotide binding. Furthermore, the PEG eliminated degradation of the sensor by DNase1 endonuclease. Currently, in situ and crude cell lysate RNA analysis is hindered by nonspecific interactions and degradation by endogenous nucleases. Together, the design changes presented here mitigate these matrix effects and allow for robust RNA analysis in complex media.