Preparation and characterization of novel fluorescent nanocomposite particles: CdSe/ZnS core-shell quantum dots loaded solid lipid nanoparticles

Fluorescent quantum dots (QDs, semiconductor nanocrystals) have gained increasing attention in the past decade due to their unique optical properties. In this work, we synthesized highly luminescent lipophilic CdSe/ZnS core-shell QDs under mild conditions, and encapsulated the QDs into solid lipid nanoparticles (SLNs) to prepare fluorescent nanocomposite particles. The transmission electron microscopy image showed that the QDs were nearly monodispersed and uniform with an average diameter of about 4 nm. The fluorescence spectrum of the QDs was symmetric and narrow with an emission maximum at 556 nm. Characterized by photon correlation spectroscopy (PCS) and zeta potential measurement, the nanocomposite particles (QDs-loaded SLNs) exhibit an average particle size of about 90 nm and zeta potential of about -28 mV. Fluorescence measurements showed that the encapsulated QDs maintain their high fluorescence and narrow/symmetric emission spectra. Assembling many QDs in single nanocomposite particle significantly increases the fluorescence signal and the signal-to-background ratio compared to individual QDs. In vitro and in vivo imaging indicated that QDs-loaded SLNs were stable and slow to photobleach. These fluorescent QDs-loaded SLNs were biocompatible with fluorescence stability and had good potential in biological imaging applications. The approaches could also be used to encapsulate other optical nanocrystals or magnetic nanoparticles, and allow them to be used under aqueous biological conditions.     

Microcontact printing of quantum dot bioconjugate arrays for localized capture and detection of biomolecules

The nanometer size scale of quantum dots (QDs) along with their unique luminescent properties offers great potential as photostable, color-metrically addressable nanoparticle platforms for high-throughput detection and identification of proteins. Here we apply microcontact printing for assembling quantum dot nanoparticle arrays with retained biomolecular capture functionality onto glass surfaces. This method allows the creation of addressable QD arrays on macroscopic glass surfaces. Using fluorescence and AFM imaging, we find that microcontact-printed QDs self-assemble predominantly as monolayers with highly resolved definition. Microcontact-printed streptavidin-conjugated red QDs exhibit retained adsorption onto silane-treated glass and exhibit functionality as demonstrated by the capture of discrete groups of biotin-conjugated red QDs by printed streptavidin-green QD bioconjugates that is at the detection limit of a few discrete protein binding events. These results indicate that microcontact printing of QD bioconjugate arrays serves as a simple technique that allows localized spatial capture and sensitive detection of proteins. This technique may be useful for development of future fluorescent QD-based systems aimed at the parallel capture and detection of trace concentrations of protein.     

Reactive magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles for specific antigen detection using microcontact printing technique

Epoxy-functionalized magnetic poly(divinylbenzene-co-glycidyl methacrylate) colloidal particles (mPDGs) were prepared by co-polymerization of 1,4-divinylbenzene and glycidyl methacrylate monomers. The reaction was conducted by batch emulsion polymerization in the presence of an oil in water magnetic emulsion as a seed. The chemical composition, morphology, iron oxide content, magnetic properties, particle size and colloidal stability of the prepared magnetic polymer particles were characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, thermal gravimetric analysis, vibrating sample magnetometry, dynamic light scattering, and zeta potential determination, respectively. The prepared mPDGs were immobilized on a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES)/octadecyltrichlorosilane (OTS), which were patterned on glass using microcontact printing technique, forming mPDGs–APTES/OTS reactive surface. This construction (mPDGs–APTES/OTS) was used as a solid support for immunoassay. The immobilized magnetic particles were bioconjugated with monoclonal anti-human IL-10 antibody to provide specific and selective recognition sites for the recombinant human IL-10 protein (antigen). Fluorescence microscopic examination was carried out to follow this immunoassay using fluorescently labeled anti-human IL-10 antibody. The results obtained proved the successful use of mPDGs–APTES/OTS microcontact printed surfaces in an immunoassay, which can be exploited and integrated into microsystems in order to elaborate medical devices (e.g. biosensors) which could provide rapid analysis at high sensitivity with low volumes of analyte.     

Fabrication of hydrogel microstructures using polymerization controlled by microcontact printing (PCμCP)

Hydrogels are widely applied as functional biomaterials in the diagnostic and therapeutic fields. For example, intelligent hydrogels containing ionic groups (pH responsive) and poly(ethylene glycol) have promising applications as pH responsive materials in the biomedical and pharmaceutical fields. For potential use of hydrogels in micro- and nano devices, methods are needed to fabricate structures of various geometries at the micro- and nano scale. In this work, polymerization controlled by microcontact printing (PCμCP) is utilized, which is a method that uses microcontact printing to spatially define polymerization zones. Specifically, gold surfaces were modified by a hydrophobic thiol self assembled monolayer via microcontact printing and then a hydrophilic prepolymer solution was applied and only spatially occupied the regions confined by the hydrophobic thiol. Subsequently, polymerization reactions were carried out to create hydrogel microstructures. The patterned hydrogel produced using these methods are highly uniform in size and shape, having potential application in the field of biomedical microdevices.     

Use of near-infrared luminescent gold nanoclusters for detection of macrophages

We determined the effect of aggregation and coating thickness of gold on the luminescence of nanoparticles engulfed by macrophages and in gelatin phantoms. Thin gold-coated iron oxide nanoclusters (nanoroses) have been developed to target macrophages to provide contrast enhancement for near-infrared optical imaging applications. We compare the brightness of nanoroses luminescent emissions in response to 635 nm laser excitation to other nanoparticles including nanoshells, nanorods, and Cy5 conjugated iron oxide nanoparticles. Luminescent properties of all these nanoparticles were investigated in monomeric and aggregated form in gelatin phantoms and primary macrophage cell cultures using confocal microscopy. Aggregation of the gold nanoparticles increased luminescence emission and correlated with increased surface mass of gold per nanoparticle (nanoshells 37 ± 14.30 × 10(-3) brightness with 1.23 × 10(-4) wt of gold (g)/nanoparticle versus original nanorose 1.45 ± 0.37 × 10(-3) with 2.10 × 10(-16) wt of gold/nanoparticle, p<0.05). Nanoshells showed greater luminescent intensity than original nanoroses or Cy5 conjugated iron oxide nanoparticles when compared as nanoparticles per macrophage (38 ± 10 versus 11 ± 2.8 versus 17 ± 6.5, p<0.05, respectively, ANOVA), but showed relatively poor macrophage uptake (1025 ± 128 versus 7549 ± 236 versus 96,000 nanoparticles/cell, p<0.05, student t-test nanoshells versus nanoroses). Enhancement of gold fluorescent emissions by nanoparticles can be achieved by reducing the thickness of the gold coating, by clustering the gold on the surface of the nanoparticles (nanoshells), and by clustering the gold nanoparticles themselves.     

Biological functionalization and surface micropatterning of polyacrylamide hydrogels

Hydrogels are useful for linking proteins to solid surfaces because their hydrophilic nature and porous structure help them to maintain these labile molecules in the native functional state. We have developed a method for creating surface-patterned, biofunctionalized hydrogels on glass or silicon, using polyacrylamide and the disulfide-containing polyacrylamide crosslinker, bis(acryloyl)cystamine. Treatment with a reducing agent created reactive sulfhydryl (-SH) groups throughout these hydrogels that were readily conjugated to iodoacetyl biotin and streptavidin (SA). Immobilization efficiency was approximately 1-2% of the total potential binding capacity of the hydrogel. Porosity of the hydrogel was not a limiting factor for SA immobilization, as determined using fluorescence confocal microscopy. Rather, steric hindrance due to the binding of SA decreased the effective porosity near the surface of the hydrogel, restricting access to the rest of the gel. Using microcontact printing, we indirectly patterned SA on the surface of the hydrogel, generating well-resolved feature sizes of 2 microm in width. Through repeated rounds of microcontact printing, multiple, adjacent protein patterns were generated on the surface of the hydrogel. Biotinylated immune complexes and lipid vesicles readily bound to SA-functionalized hydrogels, demonstrating the feasibility of using this hydrogel system to generate complex biofunctionalized surfaces.     

The role of macrophages in the clearance of inhaled ultrafine titanium dioxide particles

The role of macrophages in the clearance of particles with diameters less than 100 nm (ultrafine or nanoparticles) is not well established, although these particles deposit highly efficiently in peripheral lungs, where particle phagocytosis by macrophages is the primary clearance mechanism. To investigate the uptake of nanoparticles by lung phagocytes, we analyzed the distribution of titanium dioxide particles of 20 nm count median diameter in macrophages obtained by bronchoalveolar lavage at 1 hour and 24 hours after a 1-hour aerosol inhalation. Differential cell counts revealing greater than 96% macrophages and less than 1% neutrophils and lymphocytes excluded inflammatory cell responses. Employing energy-filtering transmission electron microscopy (EFTEM) for elemental microanalysis, we examined 1,594 macrophage profiles in the 1-hour group (n = 6) and 1,609 in the 24-hour group (n = 6). We found 4 particles in 3 macrophage profiles at 1 hour and 47 particles in 27 macrophage profiles at 24 hours. Model-based data analysis revealed an uptake of 0.06 to 0.12% ultrafine titanium-dioxide particles by lung-surface macrophages within 24 hours. Mean (SD) particle diameters were 31 (8) nm at 1 hour and 34 (10) nm at 24 hours. Particles were localized adjacent (within 13-83 nm) to the membrane in vesicles with mean (SD) diameters of 592 (375) nm at 1 hour and 414 (309) nm at 24 hours, containing other material like surfactant. Additional screening of macrophage profiles by conventional TEM revealed no evidence for agglomerated nanoparticles. These results give evidence for a sporadic and rather unspecific uptake of TiO(2)-nanoparticles by lung-surface macrophages within 24 hours after their deposition, and hence for an insufficient role of the key clearance mechanism in peripheral lungs.     

Development of novel dye-doped silica nanoparticles for biomarker application

We report the development of novel luminescent nanoparticles composed of inorganic luminescent dye, Tris(2,2'-bipyridyl) dichlororuthenium (II) hexahydrate, doped inside a silica network. These dye doped silica (DDS) nanoparticles have been synthesized using a water-in-oil microemulsion technique in which controlled hydrolysis of the tetraethyl orthosilicate leads to the formation of monodispersed nanoparticles. They are prepared with a variety of sizes: small (5+/-1 nm), medium (63+/-4 nm), and large (400+/-10 nm), which shows the efficiency of the microemulsion technique for the synthesis of uniform nanoparticles. All these nanoparticles are suitable for biomarker application since they are much smaller than cellular dimension. These nanoparticles are highly photostable in comparison to most commonly used organic dyes. These nanoparticles have been characterized by various microscopic and spectroscopic techniques. The amount of dye content in these nanoparticles has been optimized to eliminate self-quenching. It has been observed that maximum luminescence intensity is achieved when the dye content is around 20 wt%. Silica surface of DDS nanoparticles is available for surface modification and bioconjunction. For demonstration as a biomarker, the DDS nanoparticle's surface has been biochemically modified to attach membrane-anchoring groups and applied successfully to stain human leukemia cells.     

Preparation of Microscale Polymer Janus Particles by Sandwich Microcontact Printing

Sandwich microcontact printing provides a straightforward way to obtain Janus polymer particles by postfunctionalization. In this paper, the synthesis of poly(glycidylmethacrylate‐co‐divinylbenzene) [poly(GMA‐co‐DVB)] particles with a diameter below 3 μm by precipitation polymerization with narrow size distribution and GMA content up to 70% is presented. Infrared spectra show the increasing GMA content of the particles. Successful functionalization of Janus particles with A‐B, A‐B‐A, and A‐B‐C structure by sandwich microcontact printing is demonstrated by printing low‐molecular‐weight fluorescent inks such as dansylcadaverine and rhodamine onto the particles in well‐defined and homogeneous patches. Under optimized conditions, Janus particles with a diameter of 2 μm are accessible in a single printing step.

     

Long-term fate of silica nanoparticles interacting with human dermal fibroblasts

The long-term fate of fluorescent non-porous FITC-SiO(2) nanoparticles of various sizes (10-200 nm) and charge is studied in the presence of human dermal fibroblasts. Particle aggregates are formed in the culture medium and uptaken, at least partially, by macropinocytosis. The smallest particles have a strong impact on cell viability and genotoxic effects can be observed for negatively-charged colloids 10 nm in size. Largest particles do not impact on cellular activity and can be monitored in cellulo via fluorescence and transmission electron microscopy studies over two weeks. These observations reveal a significant decrease in the size of silica particles located in endocytic vesicles. The dissolution process is confirmed by monitoring the cell culture medium that contains both colloidal and soluble silica species. Such dissolution can be explained on the sole basis of silica solubility and has great implication for the use of non-porous silica particles as intra-cellular drug release systems.     

Preparation and characterization of cationic PLGA nanospheres as DNA carriers

Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible co-polyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10mV at pH 7.4 and size under 200nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles.     

Investigation of red blood cell antigens with highly fluorescent and stable semiconductor quantum dots

We report a new methodology for red blood cell antigen expression determination by a simple labeling procedure employing luminescent semiconductor quantum dots. Highly luminescent and stable core shell cadmium sulfide/cadmium hydroxide colloidal particles are obtained, with a predominant size of 9 nm. The core-shell quantum dots are functionalized with glutaraldehyde and conjugated to a monoclonal anti-A antibody to target antigen-A in red blood cell membranes. Erythrocyte samples of blood groups A+, A2+, and O+ are used for this purpose. Confocal microscopy images show that after 30 min of conjugation time, type A+ and A2+ erythrocytes present bright emission, whereas the O+ group cells show no emission. Fluorescence intensity maps show different antigen expressions for the distinct erythrocyte types. The results obtained strongly suggest that this simple labeling procedure may be employed as an efficient tool to investigate quantitatively the distribution and expression of antigens in red blood cell membranes.     

Preparation of ultrafine poly(methyl methacrylate-co-methacrylic acid) biodegradable nanoparticles loaded with ibuprofen

Ibuprofen-loaded polymeric particles with around 9.2 nm in mean diameter, as determined by electron microscopy, dispersed in an aqueous media containing up to 12.8% solids were prepared by semicontinuous heterophase polymerization. The polymeric material is a (2/1 mol/mol) methyl methacrylate-co-methacrylic acid copolymer similar to Eudragit S100, deemed safe for human consumption and used in the manufacturing of drug-loaded pills as well as micro- and nanoparticles. The loading efficiency was 100%, attaining around 10–12% in drug content. Release studies showed that the drug is released from the nanoparticles at a slower rate than that in the case of free IB. Given their size as well as the pH values required for their dissolution, it is believed that this type of particles could be used as a basis for preparing nanosystems loaded with a variety of drugs.     

Biofabrication of discrete spherical gold nanoparticles using the metal-reducing bacterium Shewanella oneidensis

Nanocrystallites have garnered substantial interest due to their various applications, including catalysis and medical research. Consequently important aspects of synthesis related to control of shape and size through economical and non-hazardous means are desirable. Highly efficient bioreduction-based fabrication approaches that utilize microbes and/or plant extracts are poised to meet these needs. Here we show that the γ-proteobacterium Shewanella oneidensis can reduce tetrachloroaurate (III) ions to produce discrete extracellular spherical gold nanocrystallites. The particles were homogeneously shaped with multiple size distributions and produced under ambient conditions at high yield, 88% theoretical maximum. Further characterization revealed that the particles consist of spheres in the size range of ～2-50 nm, with an average size of 12±5 nm. The nanoparticles were hydrophilic and resisted aggregation even after several months. Based on our experiments, the particles are likely fabricated by the aid of reducing agents present in the bacterial cell membrane and are capped by a detachable protein/peptide coat. Ultraviolet-visible and Fourier transform infrared spectroscopy, X-ray diffraction, energy dispersive X-ray spectra and transmission electron microscopy measurements confirmed the formation, surface characteristics and crystalline nature of the nanoparticles. The antibacterial activity of these gold nanoparticles was assessed using Gram-negative (Escherichia coli and S. oneidensis) and Gram-positive (Bacillus subtilis) bacterial species. Toxicity assessments showed that the particles were neither toxic nor inhibitory to any of these bacteria.     

Self-assembled extracellular matrix protein networks by microcontact printing

Physiological patterns of the extracellular matrix protein, laminin-1, were obtained on glass substrates by physisorption-assisted microcontact printing. Besides the well-retained antigenicity confirmed by indirect immunofluorescence assays, we investigated the supramolecular organization of the proteins by atomic force microscopy. We found the characteristic protein self-assembling in polygonal networks with well-defined sub-100 nm quaternary structures of laminin. The formation of these physiological mesh-like protein matrices was obtained by means of one-step soft lithography without any preliminary functionalization of glass, which can be exploited for many possible applications for cell cultures and biomolecular devices.     

Poly(cyclotriphosphazene‐co‐tris(4‐hydroxyphenyl)ethane) Microspheres with Intrinsic Excitation Wavelength Tunable Multicolor Photoluminescence

Functional photoluminescent materials with a tunable multicolor emission without incorporation of metals is the current focus on luminescent materials due to their multifaceted applications. Herein, novel poly(cyclotriphosphazene‐co‐tris(4‐hydroxyphenyl)ethane) microspheres are synthesized with size distribution of ≈0.8–1.6 µm by reacting hexachlorocyclotriphosphazene and tris(4‐hydroxyphenyl)ethane at different molar ratios under facile conditions. The thermogravimetric analysis shows that the microspheres are highly thermally stable with 10% weight loss at around 500 °C. Fluorescence microscopy results show that the microspheres emit three blue, green, and red colors with an emission maximum at 487, 530, and 623 nm under different excitation wavelengths of 365, 420, and 546 nm, respectively. Furthermore, under UV and visible (365 and 420 nm) excitations, microspheres demonstrate extraordinary photobleaching stability and highly intense luminescence for expending time and repeating excitations. The microspheres also demonstrate semiconductor‐like optical absorption at 235–285 nm with a tail extended to 700 nm. This exploration provides an insight for the preparation of intrinsic superior wavelength tunable multicolor emission materials from phosphazene. These microspheres are potential candidates for anti‐counterfeits, solar cells, full spectrum fluorescence emitters, organic light emitting diodes, biological, catalysis applications and unlock new areas of research on multicolor photoluminescence.     

Novel pH responsive polymethacrylic acid-chitosan-polyethylene glycol nanoparticles for oral peptide delivery

In present study, novel pH sensitive polymethacrylic acid-chitosan-polyethylene glycol (PCP) nanoparticles were prepared under mild aqueous conditions via polyelectrolyte complexation. Free radical polymerization of methacrylic acid (MAA) was carried out in presence of chitosan (CS) and polyethylene glycol (PEG) using a water-soluble initiator and particles were obtained spontaneously during polymerization without using organic solvents or surfactants/steric stabilizers. Dried particles were analyzed by scanning electron microscopy (SEM) and particles dispersed in phosphate buffer (pH 7.0) were visualized under transmission electron microscope (TEM). SEM studies indicated that PCP particles have an aggregated and irregular morphology, however, TEM revealed that these aggregated particles were composed of smaller fragments with size less than 1 micron. Insulin and bovine serum albumin (BSA) as model proteins were incorporated into the nanoparticles by diffusion filling method and their in vitro release characteristics were evaluated at pH 1.2 and 7.4. PCP nanoparticles exhibited good protein encapsulation efficiency and pH responsive release profile was observed under in vitro conditions. Trypsin inhibitory effect of these PCP nanoparticles was studied using casein substrate and these particles displayed lesser inhibitory effect than reference polymer carbopol. Preliminary investigation suggests that these particles can serve as good candidate for oral peptide delivery.     

选择性激光熔融打印钛种植体的制备和表面优化处理及其成骨性能的研究

目的　通过表征和体外相关实验分析选择性激光熔融（SLM）3D打印钛种植体试样表面的生物相容性能，为SLM打印种植体最终应用于临床提供理论基础和实验依据。 方法　使用SLM方法制备得圆片形钛种植体试样60枚，经不同处理后分为三组：喷砂酸蚀组（S组）、阳极氧化组（SA组）、阳极氧化+rhBMP-2表面组（SAB组）；场发射扫描电镜（FE-SEM）和原子力显微镜（AFM）观察各组试样的表面形貌和粗糙度，接触角测量仪检测亲水性；SEM观察骨髓间充质干细胞（BMMSCs）在各组试样表面的粘附状态；CCK-8检测BMMSCs在各组试样表面的增殖水平。 结果　SEM观察结果显示阳极氧化后的试样SA组表面可见一层均匀密布的二氧化钛纳米管，直径约为100纳米；AFM观察试样表面粗糙度约为300纳米；接触角测量仪测得经表面处理和改性的SA组，SAB组试样具有超亲水性；BMMSCs粘附和增殖实验显示各组试样表面的促细胞粘附和增殖能力SAB组>SA组>S组。 结论　SLM打印种植体试样未处理组和处理组都具有较好的生物相容性能，阳极氧化与加载BMP-2生长因子处理结合的方法能有效促进BMMSCs的粘附和增殖。     

Fluorescent non-porous silica nanoparticles for long-term cell monitoring: Cytotoxicity and particle functionality

Inorganic nanoparticles such as silica particles offer many exciting possibilities for biomedical applications. However, the possible toxicity of these particles remains an issue of debate that seriously impedes their full exploitation. In the present work, commercially available fluorescent silica nanoparticles 25, 45 and 75 nm in diameter optimized for cell labelling (C-Spec® particles) are evaluated with regard to their effects on cultured cells using a novel multiparametric setup. The particles show clear concentration and size-dependent effects, where toxicity is caused by the number and total surface area of cell-associated particles. Cell-associated particles generate a short burst of oxidative stress that, next to inducing cell death, affects cell signalling and impedes cell functionality. For cell labelling purposes, 45 nm diameter silica particles were found to be optimally suited and no adverse effects were noticeable at concentrations of 50 μg ml^?1 or below. At this safe concentration, the particles were found to still allow fluorescence tracking of cultured cells over longer time periods. In conclusion, the data shown here provide a suitable concentration of silica particles for fluorescent cell labelling and demonstrate that at safe levels, silica particles remain perfectly suitable for fluorescent cell studies.     

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications.