Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials

Protein engineering to functionalize the self‐assembling enamel matrix protein amelogenin with a cellulose binding domain (CBD) is used. The purpose is to examine the binding of the engineered protein, rh174CBD, to cellulose materials, and the possibility to immobilize self‐assembled amelogenin nanospheres on cellulose. rh174CBD assembled to nanospheres ≈35 nm in hydrodynamic diameter, very similar in size to wild type amelogenin (rh174). Uniform particles are formed at pH 10 for both rh174 and rh174CBD, but only rh174CBD nanospheres showes significant binding to cellulose (Avicel). Cellulose binding of rh174CBD is promoted when the protein is self‐assembled to nanospheres, compared to being in a monomeric form, suggesting a synergistic effect of the multiple CBDs on the nanospheres. The amount of bound rh174CBD nanospheres reached ≈15 mg/g Avicel, which corresponds to 4.2 to 6.3 × 10^?7 mole/m². By mixing rh174 and rh174CBD, and then inducing self‐assembly, composite nanospheres with a high degree of cellulose binding can be formed, despite a lower proportion of rh174CBD. This demonstrates that amelogenin variants like rh174 can be incorporated into the nanospheres, and still retain most of the binding to cellulose. Engineered amelogenin nanoparticles can thus be utilized to construct a range of new cellulose based hybrid materials, e.g. for wound treatment.     

Efficient approach to design stable water-dispersible nanoparticles of hydrophobic cellulose esters

Commercially prepared cellulose acetate, cellulose acetate propionate, -butyrate, and -phthalate as well as cellulose acetates prepared in the laboratory scale with varying degree of substitution (DS) self-assemble into regular nanoparticles, ranging in size from 86 to 368 nm, by using two different techniques of nanoprecipitation. Dialysis of polymers dissolved in N,N-dimethylacetamide results in the formation of regular nanospheres whereas the preparation in acetone by successive adding of water leads to bean-shaped particles in the nanoscale. One criterion for nanoprecipitation is the existence of dilute polymer solutions. Furthermore, the formation of nanoparticles strongly depends on DS and distribution of the substituents. Concerning this issue, quantitative (13)C NMR spectroscopy was applied for detailed structure characterization of selected cellulose acetates. The stability of the nanoparticle suspensions in the physiological pH range was observed by zeta potential measurements.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Lymph node localisation of biodegradable nanospheres surface modified with poloxamer and poloxamine block co-polymers

Studies were performed to develop a sub-100 nm biodegradable colloidal system for the efficient delivery of drugs and diagnostic agents to the lymphatic system. Nanospheres of poly(lactide-co-glycolide) were prepared by interfacial polymer deposition. The nanospheres were coated with block co-polymers in order to modify their surface characteristics. Radiolabelling of the nanospheres for in vivo tracing was achieved by the incorporation of the lipophilic complex ¹¹¹In-oxine during nanosphere preparation. In vitro stability of the radiolabelled nanospheres was determined in rat serum at 37°C. The lymphatic distribution of the nanospheres was determined after subcutaneous administration to the rat. Lymphatic uptake of all coated systems was enhanced compared to the uncoated nanospheres, and a maximal uptake of 17% of the administered dose in the regional lymph nodes was achieved. These observations suggest that the nanospheres are suitable for diagnostic and therapeutic applications in clinical and experimental medicine.     

Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers

A two-step approach is described to chemically camouflage the inert surface of model polystyrene nanospheres of 60 nm in diameter against recognition by the body's defenses. The first step was based on the strong protein adsorbing potency of polystyrene, and the second step utilized the chemical reactivity of the adsorbed proteins for conjugation with cyanuric chloride-activated methoxypoly(ethyleneglycol)5000, mPEG5000. Bovine serum albumin (BSA) and rat IgG were used as models of non-immune and immune proteins, respectively. The maximum adsorbance values for both proteins were near expectation for a close-packed monolayer. Adsorption isotherms studies and analysis of the hydrodynamic thickness of the adsorbed protein layer confirmed the close-packed side-on mode of adsorption for BSA and the end-on mode of adsorption for IgG, respectively. Nucleophiles on the adsorbed proteins were then reacted with cyanuric chloride activated mPEG5000. The average poly(ethyleneglycol) (PEG) content for a 60-nm nanospheres was found to be 13.7+/-0.4 micromol PEG/micromol BSA and 3.6+/-0.3 micromol PEG/micromol IgG. The interaction of both PEG-bearing nanospheres with the hydrophobic column material octyl-agarose indicated surface heterogeneity among the nanospheres. Only nanospheres with the most hydrophilic phenotype (approximately 70% of the total population) exhibited stealth properties after intravenous injection to rats. In contrast to the described approach, incubation of uncoated nanospheres with preformed BSA-mPEG5000 conjugates failed to produce long circulating entities. For design of splenotropic particles cyanuric chloride-activated mPEG5000 was conjugated to BSA-coated polystyrene beads of 225 nm in diameter. Despite their stealth property to hepatic Kupffer cell recognition, these nanospheres were cleared by the splenic red pulp macrophages.     

Evaluation of the permeability of hair growing ingredient encapsulated PLGA nanospheres to hair follicles and their hair growing effects

This paper describes the process of encapsulating hair growing ingredients in the PLGA nanospheres by emulsion solvent diffusion method and investigates the feasibility of using the PLGA nanospheres as the DDS (Drug delivery System) carriers for delivering various hair growing ingredients to hair follicles. In-vitro and in-vivo tests were conducted to verify the performances of encapsulated PLGA nanospheres with three different hair growing ingredients. In the in-vitro tests, the scalp-pore permeability of hair growing ingredient encapsulated PLGA nanospheres (dispersed in the PBS solution) was examined using human scalp biopsies in a modified Bronaugh diffusion chamber in comparison to that of the control samples containing the hair growing ingredient in the PBS solution. Furthermore, the hair growing effect of the encapsulated PLGA nanospheres was evaluated with the C3H mice in the in-vivo tests. By observing the fluorescence intensity of the ingredients, as shown in the cross-section photographs of the human scalp biopsies, it was found that the dispersion liquids containing hair growing ingredient encapsulated PLGA nanospheres exerted a scalp-pore permeability 2.0- to 2.5-fold more marked than that of the control samples. Also, the hair growing activities were enhanced by using the encapsulated PLGA nanospheres, which transformed the hair growth cycle from the resting phase to the growing phase. As a result, the degree of hair growth was improved significantly. These results suggested that the PLGA nanosphere can be a new DDS carrier for delivering hair growing ingredients and drugs to the hair follicles.     

Suspension culture of anchorage-dependent animal cells using nanospheres of the biodegradable polymer, poly(lactic-co-glycolic acid)

A novel method was developed for suspension culture of anchorage-dependent animal cells using biodegradable polymer nanospheres. The addition of poly(lactic-co-glycolic acid) nanospheres (907 nm average diam.) to the culture of human embryonic kidney 293 cells in stirred suspension bioreactors promoted the aggregate formation and cell growth (4.4-fold versus 2.2-fold growth for 7 d), compared to culture without nanospheres. This method may be useful for the culture of various types of anchorage-dependent animal cells in large-scale suspension bioreactors.     

Particle size and temperature effect on the physical stability of PLGA nanospheres and microspheres containing Bodipy

The purpose of this study was to investigate the effect of particle size, storage temperature, and duration of storage on the physical stability and morphology of polylactic-co-glycolic acid (PLGA) nanospheres and microspheres. PLGA nanospheres and microspheres containing the fluorescent dye, Bodipy, were prepared in varying sizes by controlling the method and degree of agitation during the emulsification phase of preparation. Mean diameters of the particles were measured by dynamic light scattering. To evaluate the effect of storage temperature and duration of storage on the extent of aggregation, nanospheres and microspheres were stored at 4°C, 25°C, 37°C, and 50°C for 6 days and then monitored using both confocal and scanning electron microscopy. The mean ±SD diameters of PLGA particles containing Bodipy were: 266.9±2.8, 351.6±1.8, 988.8±14.1, and 1865.9±67.0 nm. The extent of aggregation of the particulate delivery system decreased as the mean diameter increased, and increased as the storage temperature increased. The maximum extent of aggregation was observed with the smallest (266 nm) nanospheres. Microspheres did not aggregate. The aggregation of nanospheres was significantly reduced by introducing an additional evaporation step during preparation, suggesting that migration of residual dichloromethane from within the nanospheres may have dissolved the PLGA on the surface. The extent of aggregation of nanospheres increased as the temperature was increased from 4°C to 50°C, and decreased as particle size increased. To avoid aggregation, PLGA nanospheres should be stored at 4°C.     

Preparation and characterization of antioxidant nanospheres from multiple α-lipoic acid-containing compounds

The purpose of this study was to prepare and characterize antioxidant nanospheres composed of multiple α-lipoic acid-containing compounds (mALAs). It was found that the nanospheres were remarkably stable under physiologic conditions, maintained the antioxidant property of α-lipoic acid, and could be destabilized oxidatively and enzymatically. The preparations were simple and highly reproducible providing a new strategy for the development of nanometer-sized antioxidant biomaterials. The nanospheres may find applications as antioxidant therapeutics and oxidation-responsive antioxidant nanocontainers in drug delivery for pathological conditions characterized by oxidative stress including cancer and neurodegenerative diseases.     

Molecularly imprinted micro and nanospheres for the selective recognition of 17beta-estradiol

A one-step precipitation polymerization procedure for the synthesis of molecularly imprinted polymers selective for 17beta-estradiol yielding imprinted micro and nanospheres was developed in this study and compared to templated materials obtained by conventional bulk polymerization. The polymer particles prepared by precipitation polymerization exhibited a regular spherical shape at the micro and nanoscale with a high degree of monodispersity. Moreover, the influence of the polymerization temperature, and the ratio of functional monomer to cross-linker on the size of the obtained particles was investigated. The selectivity of the imprinted micro and nanospheres was evaluated by HPLC analysis and via radioligand binding assays. HPLC separation experiments revealed that the imprinted microspheres provide higher or similar affinity to the template in contrast to imprinted polymers prepared by conventional bulk polymerization or synthesized by multi-step swelling/polymerization methods. The dimensions of the imprinted nanospheres facilitate suspension in solution rendering them ideal for binding assay applications. Results from saturation and displacement assays prove that the imprinted nanospheres exhibit superior specific affinity to the target molecule in contrast to control materials. The binding properties of the nanospheres including binding isotherms and affinity distribution were studied via Freundlich isotherm affinity distribution (FIAD) analysis. Moreover, release experiments show that 70% of rebound 17beta-estradiol was released from the imprinted nanospheres within the first 2 h, while more intimately bound 17beta-estradiol molecules (approx. 16%) were released in the following 42 h. Fitting Brunnauer-Emmet-Teller (BET) multi-point adsorption isotherms to the obtained results indicated that the micro and nanospheres are characterized by a comparatively homogenous and narrow distribution of mesopores in contrast to the corresponding bulk polymers.     

Therapeutic Use of 3β-[N-(N′,N′-Dimethylaminoethane) Carbamoyl] Cholesterol-Modified PLGA Nanospheres as Gene Delivery Vehicles for Spinal Cord Injury

Gene delivery holds therapeutic promise for the treatment of neurological diseases and spinal cord injury. Although several studies have investigated the use of non-viral vectors, such as polyethylenimine (PEI), their clinical value is limited by their cytotoxicity. Recently, biodegradable poly (lactide-co-glycolide) (PLGA) nanospheres have been explored as non-viral vectors. Here, we show that modification of PLGA nanospheres with 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) enhances gene transfection efficiency. PLGA/DC-Chol nanospheres encapsulating DNA were prepared using a double emulsion-solvent evaporation method. PLGA/DC-Chol nanospheres were less cytotoxic than PEI both in vitro and in vivo. DC-Chol modification improved the uptake of nanospheres, thereby increasing their transfection efficiency in mouse neural stem cells in vitro and rat spinal cord in vivo. Also, transgene expression induced by PLGA nanospheres was higher and longer-lasting than that induced by PEI. In a rat model of spinal cord injury, PLGA/DC-Chol nanospheres loaded with vascular endothelial growth factor gene increased angiogenesis at the injury site, improved tissue regeneration, and resulted in better recovery of locomotor function. These results suggest that DC-Chol-modified PLGA nanospheres could serve as therapeutic gene delivery vehicles for spinal cord injury.     

pH-sensitive polymer nanospheres for use as a potential drug delivery vehicle

We report the development and characterization of pH-sensitive poly(2-tetrahydropyranyl methacrylate) [poly(THPMA)] nanospheres and demonstrate their feasibility as an effective drug delivery vehicle. Poly(THPMA) nanospheres were prepared using either the double emulsion or single emulsion method for the encapsulation of, respectively, water soluble (rhodamine B) or organic soluble (paclitaxel) payloads. The resulting nanospheres showed pH-dependent dissolution behavior, resulting in significant morphologic changes and loss of nanoparticle mass under mild acidic conditions (pH 5.1) with a half-life of 3.3 days, as compared to physiologic condition (pH 7.4) with a half-life of 6.2 days. The in vitro drug release profile of the paclitaxel-loaded poly(THPMA) nanospheres revealed that the rate of drug release in pH 5.1 acetate buffer was relatively faster than that in pH 7.4 HEPES buffer. Furthermore, poly(THPMA) nanospheres showed lower cytotoxicity and higher cellular uptake as compared to the FDA-approved PLGA-based nanospheres currently in clinical practice.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Methacryloylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of ferritin

A new metal-chelate adsorbent utilizing 2-methacryloylamidohistidine (MAH) was prepared as a metalchelating ligand. MAH was synthesized using methacryloly chloride and histidine. Monosize nanospheres with an average diameter of 450 nm were produced by emulsion polymerization of 2-hydroxyetylmethacrylate (HEMA) and MAH. Then, Fe³⁺ ions were chelated directly onto the monosize nanospheres. Mon-poly(HEMA-MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. Fe³⁺ chelated monosize nanospheres were used in ferritin adsorption from an aqueous solution. The maximum ferritin adsorption capacity of Fe³⁺-chelated mon-poly(HEMAMAH) nanospheres was 202 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the monpoly( HEMA-MAH) nanospheres was 20 mg/g. The adsorption behavior of ferritin could be modeled using both Langmuir and Freundlich isotherms. The adsorption capacity decreased with increasing ionic strength of the binding buffer. High desorption ratios (> 95% of the adsorbed ferritin) were achieved with 1.0 M NaCl at pH 7.0. Ferritin could be repeatedly adsorbed and desorbed with the Fe³⁺-chelated mon-poly(HEMA-MAH) nanospheres without significant loss of adsorption capacity.     

The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins

Background

Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins.

Methodology

The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times.

Conclusions

All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.     

Point mutation detection with the sandwich method employing hydrogel nanospheres by the surface plasmon resonance imaging technique

We propose a surface modification procedure to construct DNA arrays for use in surface plasmon resonance (SPR) imaging studies for the highly sensitive detection of a K-ras point mutation, enhanced with hydrogel nanospheres. A homobifunctional alkane dithiol was adsorbed on Au film to obtain the thiol surface, and ethyleneglycol diglycidylether (EGDE) was reacted to insert the ethyleneglycol moiety, which can suppress nonspecific adsorption during SPR analysis. Then streptavidin (SA) was immobilized on EGDE using tosyl chloride activation. Biotinylated DNA ligands were bound to the SA surface via biotin-SA interaction to fabricate DNA arrays. In SPR analysis, the DNA analyte was exposed on the DNA array and hybridized with the immobilized DNA probes. Subsequently, the hydrogel nanospheres conjugated with DNA probes were bound to the DNA analytes in a sandwich configuration. The DNA-carrying nanospheres led to SPR signal enhancement and enabled us to discriminate a K-ras point mutation in the SPR difference image. The application of DNA-carrying hydrogel nanospheres for SPR imaging assays was a promising technique for high throughput and precise detection of point mutations.     

Vectorisation of doxorubicin in nanospheres and reversion of pleiotropic resistance of tumor cells

A multidrug resistance human cell line (Dox-R-MCF7), originating from mammary adenocarcinoma was compared to the drug sensitive parent line (MCF7) to determine whether or not the use of doxorubicin-loaded biodegradable nanospheres (NS-Dox) could circumvent drug resistance. The Dox-R-MCF7 line was shown to resist free doxorubicin (Dox) as the 50% lethal dose for them was 150 times higher than for the sensitive cell-line. Isohexylcyanoacrylate nanospheres, porous matrices of biodegradable innocuous polymer, diameter 300 nm, were loaded with doxorubicin. Free Dox, NS-Dox and Dox-free polymer (NS) were added to the culture medium for 6 hrs. In terms of 50% lethal dose, NS-Dox were cytotoxic for the resistant cell line to an identical extent as for the sensitive parent line. Dox-free nanospheres alone or mixed with free-Dox were noncytotoxic for the resistant line. Drug targetting could be of main importance to overcome multidrug resistance.     

Poly(lactic-co-glycolic acid) nanosphere as a vehicle for gene delivery to human cord blood-derived mesenchymal stem cells: comparison with polyethylenimine

Polyethylenimine (PEI) is one of the most extensively studied non-viral vectors but its cytotoxicity limits its clinical value. PLGA nanospheres are biocompatible and can facilitate sustained release of plasmid DNA. This study compares the cytotoxicity and long-term transgene expression between PLGA nanosphere and PEI. PLGA nanospheres were significantly less cytotoxic than PEI at various concentrations. PLGA nanospheres induced significantly higher transgene expression in vitro for a longer duration (21 days) than PEI. We conclude that PLGA nanospheres have potential as gene delivery vehicles for use in gene therapy for diseases in which a long-term therapeutic gene expression regimen is necessary.     

Assembly of dandelion-like Au/PANI nanocomposites and their application as SERS nanosensors

The monodisperse, uniform dandelion-like Au/polyaniline (PANI) composite nanospheres were synthesized by a simple one-step process without any additives or templates. The nanospheres are really composed of many short nanorods and the average diameter of whole nanospheres is about 180 nm. The morphology of Au/PANI composites could be controlled by adjusting the molar ratio of HAuCl(4) to aniline. The prepared nanocomposite is developed as a wonderful sensor for the detection of Hg(2+) ions, which is based upon the Raman intensity response of PANI to Hg(2+) ions. Results from the morphology-dependent sensitivity investigations show that the dandelion-like nanospheres have an ultra sensitive response (as low as 10(-11)M) compared with other morphologies. The nanosensor also exhibits good reproducibility and greater selectivity for Hg(2+) ions than the other heavy metal ions. And the mechanism was proposed. The proposed nanosensors can be applied for highly sensitive and selective chemical analysis in a variety of environmental detection.     

On-plate digestion of proteins using novel trypsin-immobilized magnetic nanospheres for MALDI-TOF-MS analysis

In this study, a novel method of on-plate digestion using trypsin-immobilized magnetic nanospheres was developed followed by MALDI-TOF-MS for rapid and effective analysis and identification of proteins. We utilized a facile one-pot method for the direct preparation of amine-functionalized magnetic nanospheres with highly magnetic properties and the amino groups on the outer surface. Through the reaction of the aldehyde groups with amine groups, trypsin was simply and stably immobilized onto the magnetic nanospheres. The obtained trypsin-linked magnetic nanospheres were then applied for on-plate digestion of sample proteins (myoglobin and Cytochrome c). Moreover, after digestion, the trypsin-linked nanospheres could be easily removed from the plate due to their magnetic property, which would avoid causing contamination on the ion source chamber in MS. The effects of the temperature and incubation time on the digestion efficiency were characterized. Within only 5 min, proteins could be efficiently digested with the peptide sequence coverage higher than or equal to that of the traditional in-solution digestion for 12 h. Furthermore, RPLC fractions of rat liver extract were also successfully processed using this novel method. These results suggested that our improved on-plate digestion protocol for MALDI-MS may find further application in automated analysis of large sets of proteins.