Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").     

A gaussian model for substrates of entangled cross-linked poly(ethylene glycol) in biomedical applications

Cells usually spread on a synthetic substrate through bonds between receptors and chemical groups on the substrate (ligands). Therefore, it is valuable to study the effects of the average number density of these chemical groups and the average distance between them to model and predict the cell behavior. Poly(ethylene glycol) [PEG] modified with peptide groups has been used widely in biomedical applications as a substrate material. In this study, a coarse-grained model is proposed for PEG to predict the average number density of ligands and the average distance between them. Molecular information such as initial molecular weight distribution, average molecular weight between cross-links, and average molecular weight between entanglements is used as input parameters. Based on simulation results, it is concluded that both entanglement and cross-link densities are required to create a network structure. The results suggest that an average initial molecular weight 2-3 times the average molecular weight between entanglements and a moderate cross-link density are sufficient to create a closed network structure with a high ligand density and a small average distance between them.     

Physicochemical characterization of ultrasmall superparamagnetic iron oxide particles (USPIO) for biomedical application as MRI contrast agents

Ultrasmall superparamagnetic iron oxide (USPIO) particles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging. The coatings surrounding the USPIO inorganic core play a major role in both the in vitro stability and, over all, USPIO's in vivo fate. Different physicochemical properties such as final size, surface charge and coating density are key factors in this respect. Up to now no precise structure--activity relationship has been described to predict entirely the USPIOs stability, as well as their pharmacokinetics and their safety. This review is focused on both the classical and the latest available techniques allowing a better insight in the magnetic core structure and the organic surface of these particles. Concurrently, this work clearly shows the difficulty to obtain a complete physicochemical characterization of USPIOs particles owing to their small dimensions, reaching the analytical resolution limits of many commercial instruments. An extended characterization is therefore necessary to improve the understanding of the properties of USPIOs when dispersed in an aqueous environment and to set the specifications and limits for their conception.     

Adsorption of lipoproteins with the aid of carboxymethylchitosan mircrospheres crosslinked with poly(ethylene glycol) bisglycidyl ether

Carboxymethylchitosan microspheres crosslinked with poly(ethylene glycol) bisglycidyl ether were prepared and then tested as an adsorbent for selective removal of low-density lipoprotein (LDL) in human plasma. The microspheres were formed by a method of electrostatic instillation and crosslinked with poly(ethylene glycol) bisglycidyl ether. FTIR spectral analyses and X-ray photoelectron spectroscopy revealed that carboxymethylchitosan was crosslinked through amino groups to poly(ethylene glycol) bisglycidyl ether. The plasma lipoprotein sorption tests showed that the adsorption properties of the crosslinked microspheres for LDL were dependent on the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether. When the concentrations of carboxymethylchitosan and poly(ethylene glycol) bisglycidyl ether were 3.5% and 6%, respectively, 40% LDL and lower than 10% high density lipoprotein in plasma could be removed and the adsorption could be reach an equilibrium in 30 min.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

Microwave-assisted synthesis of praseodymium (Pr)-doped ZnS QDs such as nanoparticles for optoelectronic applications

Praseodymium (Pr)-doped ZnS nanoparticles were synthesized using a low-cost microwave-assisted technique and investigations on their structure, morphology, optical properties, Raman resonance, dielectric properties, and luminescence were conducted. Broad X-ray diffraction peaks suggested the formation of low-dimensional Pr-doped ZnS nanoparticles with a cubic structure that was validated using transmission electron microscopy (TEM)/high-resolution TEM analysis. The energy gaps were identified using diffuse reflectance spectroscopy and it was found that the values varied between 3.54eV and 3.61eV for different samples. Vibrational experiments on Pr-doped ZnS nanoparticles revealed significant Raman modes at ~270 and ~350 cm⁻¹ that were associated with optical phonon modes that are shifted to lower wavenumbers, indicating phonon confinement in the synthesized products. The photoluminescence (PL) spectra of all samples demonstrated that the pure and Pr-doped ZnS nanoparticles were three-level laser active materials. Energy-dispersive X-ray spectroscopy and mapping study confirmed the homogeneous presence of Pr in ZnS. TEM studies showed that the particles were of very small size and in the cubic phase. The samples had high dielectric constant values between 13 and 24 and low loss values, according to the dielectric analysis. With an increase in frequency and a change in the Pr content of ZnS, an intense peak could be seen in the PL spectra at a wavelength of 360 nm, and some other peaks observed corresponded to the transition of Pr³⁺. The produced nanoparticles were appropriate for optoelectronic applications due to their short dimension, high energy gap, high dielectric constant, and low loss values.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

Biological behavior of the curcumin incorporated chitosan/poly(vinyl alcohol) nanofibers for biomedical applications

Electrospun composite scaffolds show high ability to be used in regenerative medicine and drug delivery, due to the nanofibrous structure and high surface area to volume ratio. In this study, we used nanofibrous scaffolds fabricated by chitosan (CS), poly(vinyl alcohol) (PVA), carbopol, and polycaprolactone using a dual electrospinning technique while curcumin (Cur) incorporated inside of the CS/PVA fibers. Scaffolds were fully characterized via scanning electron microscopy, water contact angle, tensile measurement, hydration, protein adsorption, and wrinkled tests. Furthermore, viability of the buccal fat pad-derived mesenchymal stem cells (BFP-MSCs) was also investigated using MTT assay for up to 14 days while cultured on these scaffolds. Cell cycle assay was also performed to more detailed evaluation of the stem cells growth when grown on scaffolds (with and without Cur) compared with the culture plate. Results demonstrated that Cur loaded nanofibrous scaffold had more suitable capability for water absorption and mechanical properties compared with the scaffold without Cur and it could also support the stem cells viability and proliferation. Cur release profile showed a decreasing effect on BFP-MSCs viability in the initial stage, but it showed a positive effect on stem cell viability in a long-term manner. In general, the results indicated that this nanofibrous scaffold has great potential as a delivery of the Cur and BFP-MSCs simultaneously, and so holds the promising potential for use in various regenerative medicine applications.     

Development and performance of a 3D‐printable poly(ethylene glycol) diacrylate hydrogel suitable for enzyme entrapment and long‐term biocatalytic applications

Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a poly(ethylene glycol) diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s^?1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity is not caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures.     

Systematic investigation of polyamidoamine dendrimers surface-modified with poly(ethylene glycol) for drug delivery applications: synthesis, characterization, and evaluation of cytotoxicity

Surface modification of amine-terminated polyamidoamine (PAMAM) dendrimers by poly(ethylene glycol) (PEG) groups generally enhances water-solubility and biocompatibility for drug delivery applications. In order to provide guidelines for designing appropriate dendritic scaffolds, a series of G3 PAMAM-PEG dendrimer conjugates was synthesized by varying the number of PEG attachments and chain length (shorter PEG 550 and PEG 750 and longer PEG 2000). Each conjugate was purified by size exclusion chromatography (SEC) and the molecular weight (MW) was determined by (1)H NMR integration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). NOESY experiments performed in D 2O on selected structures suggested no penetration of PEG chains to the central PAMAM domain, regardless of chain length and degree of substitution. CHO cell cultures exposed to PAMAM-PEG derivatives (< or =1 microM) showed a relatively high cell viability. Generally, increasing the degree of PEG substitution reduced cytotoxicity. Moreover, compared to G3 PAMAM dendrimers that were N-acetylated to varying degrees, a lower degree of surface substitution with PEG was needed for a similar cell viability. Interestingly, when longer PEG 2000 was fully incorporated on the surface, cell viability was reduced at higher concentrations (32 muM), suggesting increased toxicity potentially by forming intermolecular aggregates. A similar observation was made for anionic carboxylate G5.5 PAMAM dendrimer at the same dendrimer concentration. Our findings suggest that a lower degree of peripheral substitution with shorter PEG chains may suffice for these PAMAM-PEG conjugates to serve as efficient universal scaffolds for drug delivery, particularly valuable in relation to targeting or other ligand-receptor interactions.     

Selective modification of surface-exposed thiol groups in Trigonopsis variabilis D-amino acid oxidase using poly(ethylene glycol) maleimide and its effect on activity and stability of the enzyme

Covalent modification of purified Trigonopsis variabilis D-amino acid oxidase using maleimide-activated poly(ethylene glycol) 5000 yielded a stable bioconjugate in which three surface-exposed cysteine side chains were selectively derivatized. Compared with the native enzyme, the PEGylated variant displayed substantially (approximately 3.3-fold) slowed dissociation rate of FAD cofactor at 50 degrees C, and this caused a twofold thermostabilization of the enzyme activity. The stability under reaction conditions at 30 degrees C was also markedly enhanced in the PEG-oxidase conjugate. PEGylation did not affect steady-state kinetic parameters for oxidative deamination of D-methionine when 2,6-dichloroindophenol replaced dioxygen as the cosubstrate while it caused a ninefold decrease in substrate catalytic efficiency for the dioxygen-dependent reaction.     

Green synthesis of silver nanoparticles from aqueous extract of Scutellaria barbata and coating on the cotton fabric for antimicrobial applications and wound healing activity in fibroblast cells (L929)

Scutellaria barbata is a perennial herb which was vastly prescribed in Chinese medicine to treat inflammations, infections and it is also used a detoxifying agent. We synthesized silver nanoparticles with Scutellaria barbata extract and characterized the nanoparticles with UV–Vis spectroscopic analysis, TEM, AFM, FTIR and XRD. The biofilm inhibiting property of synthesized silver nanoparticles were examined with XTT reduction assay and the antimicrobial property was examined with well diffusion method. The silver nanoparticles were also coated with cotton fabrics and their efficacy against antimicrobials was analyzed to prove its application. The cytotoxic property of synthesized silver nanoparticles was examined with L929 fibroblast cells using MTT assay. Finally we analyzed the wound healing property of synthesized silver nanoparticles with wound scratch assay. The result of our UV–Vis spectroscopic analysis confirms Scutellaria barbata aqueous extract reduced silver ions and synthesized silver nanoparticles. The characterization studies TEM, AFM, FTIR and XRD confirms the synthesized silver nanoparticles are in ideal shape and size to be utilized as a drug. The XTT reduction assay proves silver nanoparticles effectively inhibits the biofilm formation in both resistant and sensitive strains. Antimicrobial sensitivity tests confirms synthesized silver nanoparticles and cotton coated synthesized silver nanoparticles both are effective against gram positive, gram negative and fungal species. Further the results of MTT assay confirms the synthesized silver nanoparticles are non toxic and finally the wound healing potency of the nanoparticles was confirmed with wound scratch assay. Over all our results authentically confirms the silver nanoparticles synthesized with Scutellaria barbata aqueous extract is potent wound healing drug.     

Effect of gemini surfactant (16-6-16) on the synthesis of silver nanoparticles: A facile approach for antibacterial application

In this report, we describe the effect of Gemini surfactants1, 6-Bis (N, N-hexadecyldimethylammonium) adipate (16-6-16) on synthesis, stability and antibacterial activity of silver nanoparticles (AgNPs). The stabilizing effect of Gemini surfactant and aggregation behavior of AgNPs was evaluated by plasmonic property and morphology of the AgNPs were characterized by UV–vis spectroscopy, Dynamic Light Scattering (DLS), X-ray diffraction (XRD), High resolution transmission electron microscopy (HRTEM) and Energy dispersive X-ray analysis (EDX) techniques. Interestingly, the formation of quite mono-dispersed spherical particles was found. Apart from the stabilizing role, the Gemini surfactant has promoted the agglomeration of individual AgNPs in small assemblies whose Plasmon band features differed from those of the individual nanoparticles. The antibacterial activity of the synthesized AgNPs on Gram-negative and Gram-positive bacterium viz., E. coli and S. aureus was carried out by plate count, growth kinetics and cell viability assay. Furthermore, the mechanism of antibacterial activity of AgNPs was tested by Zeta potential and DLS analysis, to conclude that surface charge of AgNPs disrupts the cells causing cell death.     

Germanium tetra(tertiary butoxide): Synthesis, structure and its utility as a precursor for the preparation of europium doped germanium oxide nanoparticles

Germanium tetra(tertiary butoxide), [Ge(O^tBu)₄], has been prepared by the reaction of GeCl₄ with KOBu^t in benzene. It is a monomeric crystalline solid having a distorted tetrahedral configuration, defined by the coordination of four OBu^t groups around germanium atom. The TG analysis showed that the compound is thermally stable and volatilizes at around 130 °C. Europium doped and un-doped germanium oxide nanoparticles were prepared based on the urea hydrolysis of Ge(O^tBu)₄/Eu(OOCCH₃)₃ in ethylene glycol medium at 150 °C followed by heating the resulting product at 750 °C. The nanoparticles were characterized by XRD, TEM and PL measurements. The europium doped nanoparticles, which were nearly monodispersed (∼30 nm), showed luminescence and the Eu³⁺ ions were occupying the surface of the GeO₂ nanoparticles.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016