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.     

Methylation of poloxamine for enhanced cell adhesion

Aiming at producing a synthetic collagen-mimetic material that is stiffer than collagen but that like collagen allows both cell encapsulation and cell growth on the surface, a positively charged poloxamine hydrogel was prepared by methylating the tertiary amine groups of a four-arm poly(ethylene oxide)-poly(propylene oxide) block copolymer derivative (Tetronic 1107). This derivative was subsequently reacted with methacryloyl isocyanate, rendering positively charged materials that are further cross-linkable by a photointiated free radical polymerization. Different hydrogels containing methylated poloxamine methacrylate concentrations between 6% and 18% were produced and characterized by means of water uptake and viscoelastic properties. A sharp increase in water content was observed in distilled water during the first week; some of the gels showed water uptakes as high as 2 times the initial wet weight. In PBS, this effect was less prominent due to the decrease in the osmotic gradient. Also, a gradual increase of both the storage modulus (G') and the loss modulus (G") resulted from increasing the polymer concentration: for example, G' values ranged between 70 and 23000 Pa for 6% and 18% methylated poloxamine methacrylate hydrogels (at 1 Hz, 100 Pa of oscillatory stress). HepG2 cells embedded in different compositions and exposed to UV light displayed good viability levels after the cross-linking, unlike a previously reported attempt at creating a synthetic collagen-mimetic material. A well-spread endothelial cell morphology was apparent on methylated poloxamine films after preincubation in serum-containing medium, while on unmodified poloxamine methacrylate hydrogels cells attached poorly. However, EC did not attach well to the same material when fabricated not as films but as cylindrical modules as needed for the modular construct for which this material was intended. Thus, for this apparently more challenging geometry, it was necessary to combine collagen with the methylated poloxamine to have good attachment of EC on the surface of modules as well as films. While the challenge of creating a synthetic alternative to collagen as a stiffer cell-compatible substrate remains, methylated poloxamine displays many of the attributes that make it a useful material for tissue engineering.     

Development of a localized surface plasmon resonance-based gold nanobiosensor for the determination of prolactin hormone in human serum

A localized surface plasmon resonance immunoassay has been developed to determine prolactin hormone in human serum samples. Gold nanoparticles were synthesized, and the probe was prepared by electrostatic adsorption of antibody on the surfaces of gold nanoparticles. The pH and the antibody-to-gold nanoparticle ratio, as the factors affecting the probe functions, were optimized. The constructed nanobiosensor was characterized by dynamic light scattering. The sensor was applied for the determination of prolactin antigen concentration based on the amount of localized surface plasmon resonance peak shift. A linear dynamic range of 1–40 ng ml⁻¹, a detection limit of 0.8 ng ml⁻¹, and sensitivity of 10 pg ml⁻¹ were obtained. Finally, the nanobiosensor was applied for the determination of prolactin in human control serum sample.     

Nanostructuring of Continuous Gold Film by Laser Radiation Under Surface Plasmon Polariton Resonance Conditions

The physical mechanisms of metallic nanoparticles formation by laser technology were studied. The system air/Au film/glass was irradiated by laser at the conditions of surface plasmon resonance. A surface electromagnetic wave was excited in Kretchmann configuration by the fundamental and second harmonics of the Q-switched YAG/Nd⁺³ laser with pulse power density close to the threshold of melting. Nanostructuring of Au film was observed only for the second harmonic (λ = 0.532 μm) irradiation at the surface plasmon polariton resonance (SPR) conditions. Estimations were done using the interference model of the differently directed plasmon polariton waves excited by a surface electromagnetic wave on the metal surface. It was shown that a regular pattern of locally heated spots can be formed in a metallic film by pulsed laser irradiation. The spatial distribution of this pattern is close to the period of interference. The observed effect of laser nanofragmentation is explained by the self-organization of plasmon polariton subsystem in the process of Au nanoparticles formation at high laser intensity levels. These methods open new possibilities for nanostructured surfaces formation utilizing simple self-organization processes.     

The effect of surface coverage and conformation of poly(ethylene oxide) (PEO) chains of poloxamer 407 on the biological fate of model colloidal drug carriers

Poloxamer 407 was adsorbed onto the surface of model colloidal drug carriers, polystyrene nanoparticles of 40, 70 and 137 nm in diameter, and the effect of the degree of surface coverage and the conformation of the poly(ethylene oxide) (PEO) chains on biological fate was studied. The relationship between the physicochemical and the biological properties of the nanoparticle systems was also investigated. The adsorbed layer of poloxamer 407 was characterised in terms of percentage surface coverage, thickness of the adsorbed layer and average surface area per PEO chain. Computer modelling of the adsorbed layer was performed (applying the self-consistent field technique), to obtain the structural information of the PEO chains in the layer. The in vitro interaction of the nanoparticles with different degrees of poloxamer 407 surface coverage with serum components and the in vivo biodistribution in the rat model were assessed. The results demonstrated that an increase in the surface coverage with poloxamer 407 resulted in an increased volume fraction of the PEO in the adsorbed layer, further extension of the PEO chains from the surface and closer packing of the chains at the surface. With regard to the interaction with the serum components, an increased surface coverage resulted in a reduction of the amount of serum proteins adsorbed, and, importantly, affected the type of proteins adsorbed. High molecular weight proteins were not adsorbed onto the nanoparticles with a surface coverage above approx. 25%. Following the intravenous administration to rats, even the nanoparticles with the lowest degree of surface coverage (approx. 5%) showed improved circulation profiles relative to the uncoated nanoparticles. The effect was more pronounced for the 40 nm nanoparticles. A further increase in the surface coverage to approx. 25% resulted in a significant increase in circulation time, as compared to uncoated and 5% coated systems, for all sizes of nanoparticles. Importantly, it was found that a long in vivo blood circulation time could be achieved for nanoparticles with a relatively low degree of surface coverage with PEO chains.     

Development and estimation of nanosomal rifampicin]

A lab-scale method for preparation of rifampicin-loaded polybutylcyanoacrylate nanoparticles (nanosames) was developed. The biodistribution of the nanosome-entrapped rifampicin after its intravenous administration was studied on healthy mice. The nanoparticles provided significant liver and spleen accumulation of rifampicin. Modification of the nanoparticles surface with poloxamer 407 or poloxamine 908 led to optimization of the biodistribution: the concentrations of rifampicin in the lungs and plasma increased, whereas the liver accumulation decreased vs. the unmodified nanoparticles. The increased lung accumulation of rifampicin enhanced bacterial clearance in the lungs of the mice infected with M. tuberculosis. The results showed that the use of the nanoparticles for optimization of the drug biodistribution was effective for increasing the efficacy of antiinfective chemotherapy.     

Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector-DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.     

Enhanced Sensitivity of Surface Plasmon Resonance Phase-Interrogation Biosensor by Using Silver Nanoparticles

It is demonstrated that the sensitivity of surface plasmon resonance phase-interrogation biosensor can be enhanced by using silver nanoparticles. Silver nanoparticles were fabricated on silver films by using thermal evaporation. Sizes of silver nanoparticles on silver thin film can be tuned by controlling the deposition parameters of thermal evaporation. By using surface plasmon resonance heterodyne interferometey to measure the phase difference between the p and s polarization of incident light, we have demonstrated that sensitivity of glucose detection down to the order of 10⁻⁸ refractive index units can be obtained.     

Blueshift and Narrowing of Localized Surface Plasmon Resonance of Silver Nanoparticles Exposed to Plasma

We have demonstrated that plasma treatments of silver nanoparticles bring about blueshift and narrowing in their localized surface plasmon resonance. Surface-enhanced Raman scattering analysis revealed that hydrocarbons adsorbed on silver surfaces were removed effectively by plasma exposure. It was found that the decrease in Raman line intensity for hydrocarbons was correlated well with the blueshift. Our findings indicate that one of the most important factors for remarkable differences in plasmon resonance wavelengths and line widths reported for the silver nanoparticles supported on substrates between most of the experimental data and calculations by Mie’s theory is due to the impurity adsorption on silver surfaces.     

Size and Temperature Effects on the Surface Plasmon Resonance in Silver Nanoparticles

The surface plasmon energy in spherical silver nanoparticles embedded in silica host matrix depends on the size and temperature of the nanoparticles. The dependences of the surface plasmon energy were studied for silver nanoparticles in the size range 11?C30?nm and in the temperature interval 293?C650?K. As the size of the nanoparticles decreases or the temperature increases, the surface plasmon resonance shifts to red. When the size of the nanoparticles decreases, the scattering rate of the conduction electrons increases, which results in the nonlinear red shift of the surface plasmon resonance. The red shift with temperature is linear for larger nanoparticles and becomes nonlinear for smaller ones. As the temperature of the nanoparticles increases, the volume thermal expansion of the nanoparticles leads to the red shift of the surface plasmon resonance. The thermal volume expansion coefficient depends on the size and temperature. It increases with a decrease of the nanoparticle size and an increase of the temperature.     

Surface Plasmon as a Probe of Local Field Enhancement

New method of experimental determination of local field enhancement at metal nanoparticles is suggested. It uses surface plasmon as a probe. Alternating-sign shift of surface plasmon resonance in copper nanoparticles incorporated in silica matrix has been observed under irradiation by intense femtosecond laser pulse. The red shift of plasmon observed during the action of pump pulse is interpreted as a result of change of dielectric constant of silica matrix due to optical Kerr effect in electric field of pump pulse enhanced in a vicinity of metal nanoparticles. The field enhancement factor is estimated from the value of the observed red shift of plasmon resonance.     

Enhancing the Surface-State Emission in Trap-Rich CdS Nanocrystals by Silver Nanoparticles

It is well known that surface plasmon resonance (SPR) can selectively enhance the photoluminescence (PL) from nearby chromophores with a single emission peak at an appropriate distance. Here, we combine white light-emitting CdS quantum dot nanocrystals containing band-edge and surface-state emissions simultaneously with Ag nanoparticles and study the interaction between them. It is found that the surface-state emission is always enhanced while the band-edge emission quenched regardless of the SPR wavelength of Ag nanoparticles. This phenomenon reveals that the SPR of Ag nanoparticles is not enhancing the emission from a wavelength-matched state. We propose that the surface plasmon of Ag nanoparticles is first excited by the energy of the band-edge emission and then the excited energetic electrons transfer to the surface-state of CdS. Through this energy transfer process, the surface-state emission is enhanced and band-edge emission quenched. This investigation can not only deliver understanding of the complicated interaction between metallic nanoparticles and nearby multi-emission-peak contained chromophores, but it also has potential applications in tuning the color temperature of white light-emitting materials.     

A label-free visual immunoassay on solid support with silver nanoparticles as plasmon resonance scattering indicator

By taking silver nanoparticles (Ag-NPs) as plasmon resonance scattering (PRS) indicator considering that Ag-NPs have strong plasmon resonance light scattering signals corresponding to their plasmon resonance absorption (PRA), we propose a label-free visual immunoassay on the solid support of glass slides. Our investigations showed that Ag-NPs could be adsorbed on the surface of glass slides where immunoreactions between a previously immobilized antigen and its antibody have occurred if the glass slides were immersed in an Ag-NP suspension whose pH value has been carefully adjusted. The optimal pH of the Ag-NP suspension depends on the nature of previously immobilized antigen and its antibody. It was found that the adsorption of negative-charged Ag-NPs on the surface of glass slides depends only on the content of antibody under optimal conditions. With a common spectrofluorometer to measure the PRS signals of the Ag-NPs adsorbed on the surface, we could detect antibody in the range of 10 to 160 ng ml⁻¹. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye.     

Human serum albumin interaction,in silico and anticancer evaluation of Pine-Gold nanoparticles

Unique characteristics displayed by phytoconstituent conjugated nanoparticles and their crucial interactions with proteins serve to develop nanoparticle-bio-interface platform. Gold nanoparticles of 16 nm in size were generated using aqueous extracts of pine bark and further conjugated to human serum albumin. The gold nanoparticles-protein complex was characterized by surface plasmon resonance, UV–vis and emission spectroscopy techniques. Further, it was characterized for surface morphology and elemental composition, crystallographic quality, nanoparticles size, shape, stability, structural determination and the identification of capping agent. Moreover, the interaction of gold nanoparticles with human serum albumin was investigated using conventional spectroscopy techniques. Fluorescence quenching and absorption studies demonstrated an effective binding of human serum albumin with oleamide capped gold nanoparticles. The molecular docking study showed a binding affinity of -6.1 kcal/mol whereas the molecular dynamics simulation indicated that the binding of oleamide to human serum albumin. A biological evaluation of pine bark extract-gold nanoparticles showed cytotoxicity with increased cell mortality in lung cancer cells and minimal toxicity on non-cancerous human embryonic kidney cells, respectively.     

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.     

Growth and fragmentation of silver nanoparticles in their synthesis with a fs laser and CW light by photo-sensitization with benzophenone.

The photo-sensitization synthetic technique of making silver nanoparticles using benzophenone is studied using both a laser and a mercury lamp as light sources. The power and irradiation time dependence of the synthesized nanoparticle absorption spectra and their size distribution [as determined by transmission electron microscopy (TEM)] are studied in each method and compared. In the laser synthesis, as either the laser power or the irradiation time increases, the intensity of the surface plasmon resonance absorption at 400 nm is found to increase linearly first, followed by a reduction of the red edge of the plasmon resonance absorption band. The TEM results showed that in the laser synthesis low powers and short irradiation times produce nanoparticles around 20 nm in diameter. Increasing the power or irradiation time produces a second population of nanoparticles with average size of 5 nm in diameter. These small particles are believed to be formed from the surface ablation of the large particles. The surface plasmon absorption band is found to be narrower when the nanoparticles are produced with laser irradiation. Throughout the exposure time with the CW lamp, the plasmon resonance absorption band of the particles formed first grows in intensity, then blue shifts and narrows, and finally red shifts while decreasing in intensity. The TEM results for lamp samples showed particle formation and growth, followed by small nanoparticle formation. The above results are discussed in terms of a mechanism in which, the excited benzophenone forms the ketal radical, which reduces Ag+ in solution and on the Ag nanoparticle surface. As the time of irradiation or the light energy increases the benzophenone is consumed, which is found to be the limiting reagent. This stops the formation of the normal large nanoparticles while their photo-ablation continues to make the small particles.     

Plasmonic and Energy Studies of Ag Nanoparticles in Silica-Titania Hosts

The present work reports an investigation of surface plasmon resonance (SPR) of silver nanoparticles in SiO₂–TiO₂ hosts. The surface plasmon resonance of silver nanoparticles was observed in the wavelength range 300–400 nm. Numerical calculation of SPR of silver nanoparticles with spherical morphology was done on the basis of discrete dipole approximation (DDA) method. The observed fluorescence spectrum fits well with the theoretically calculated one. The luminescence enhancement is attributed to the strong local electric field which increases the exciting and emitting photons coupled to SPR. An effort has been made to study the surface plasmon mediated excitation energy transfer (EET) between two spherical metal nanoparticles. The van der Waals (vdW) energy between plasmonic silver nanoparticles in the present hosts has been estimated.     

Gold Nanoparticle Ring and Hole Structures for Sensing Proteins and Antigen–Antibody Interactions

A new experimentally simple nanosphere lithography method was used to fabricate gold ring and nanohole structures. The method is based on the simultaneous self-assembly of polystyrene microspheres and gold colloids in multilayers, by a vertical deposition method. Dissolution of polystyrene microspheres resulted in the formation of a monolayer, where holes are surrounded by gold nanoparticles. The dependency of the sensitivity of the sensor platform on the size of the holes and their density has been demonstrated. Furthermore, sensing experiments have shown a high sensitivity of the hole structure toward fibrinogen, amyloid-derived diffusible ligands, and a plant protein (AT5G07010.1). It was found that the position and shape of the localized surface plasmon resonance band changed significantly as a result of the antigen–antibody recognition event.     

Trapping Proteins within Gold Nanoparticle Assemblies: Dynamically Tunable Hot-spots for Nanobiosensing

The combination of stimuli-responsive materials with localized surface plasmon resonance nanotransducers provides new leverages in hot spot-based nanosensing. We introduce a simple and effective biodetection method based on the hydro-responsive property of (3-aminopropyl)-triethoxysilane (APTES). Gold nanoparticles were adsorbed onto hydro-responsive APTES thin film. The exposure of the film surface to an aqueous solution results in opening inter-particle gaps, allowing analyte binding. A subsequent drying of the sensor surface closes the gap by bringing the nanoparticles to the initial position, thereby trapping the analyte in the most sensitive regions (electromagnetic hot spots). In this reversible configuration, the generation and tuning of the hot spots are independent from both the presence of the analyte and the functionalization of the nanoparticles, which yields highly resolved coupled plasmon bands and provide a general and flexible nanosensing modality. Furthermore, the intensity of the hot spots can be easily and reversibly tuned to obtain picomolar sensitivity.     

Role of Stopband and Localized Surface Plasmon Resonance in Raman Scattering from Metallo-Dielectric Photonic Crystals

Self-assembled photonic crystals grown from different colloidal sizes are coated with gold nanoparticles preferentially on their surface. The effect of localized surface plasmon resonance and the photonic stopband on the Raman scattering from these crystals is analyzed from the angle-dependent scattering measurements. The coupling of photonic and plasmonic modes at the surface of the photonic crystal is verified by measuring the increment in Raman scattering from the crystals containing the gold nanoparticles, and this increment is found to follow the spectral trend of localized surface plasmon resonance.