Induced Particle Formation Research Articles

A Potent Cancer Vaccine Adjuvant System for Particleization of Short, Synthetic CD8+ T Cell Epitopes.

Short major histocompatibility complex (MHC) class I (MHC-I)-restricted peptides contain the minimal biochemical information to induce antigen (Ag)-specific CD8+ cytotoxic T cell responses but are generally ineffective in doing so. To address this, we developed a cobalt-porphyrin (CoPoP) liposome vaccine adjuvant system that induces rapid particleization of conventional, short synthetic MHC-I epitopes, leading to strong cellular immune responses at nanogram dosing. Along with CoPoP (to induce particle formation of peptides), synthetic monophosphoryl lipid A (PHAD) and QS-21 immunostimulatory molecules were included in the liposome bilayer to generate the "CPQ" adjuvant system. In mice, immunization with a short MHC-I-restricted peptide, derived from glycoprotein 70 (gp70), admixed with CPQ safely generated functional, Ag-specific CD8+ T cells, resulting in the rejection of multiple tumor cell lines, with durable immunity. When cobalt was omitted, the otherwise identical peptide and adjuvant components did not result in peptide binding and were incapable of inducing immune responses, demonstrating the importance of stable particle formation. Immunization with the liposomal vaccine was well-tolerated and could control local and metastatic disease in a therapeutic setting. Mechanistic studies showed that particle-based peptides were better taken up by antigen-presenting cells, where they were putatively released within endosomes and phagosomes for display on MHC-I surfaces. On the basis of the potency of the approach, the platform was demonstrated as a tool for in vivo epitope screening of peptide microlibraries comprising a hundred peptides.

Antibody response of a particle-inducing, liposome vaccine adjuvant admixed with a Pfs230 fragment

Pfs230 is a malaria transmission-blocking antigen candidate, expressed on the surface of Plasmodium falciparum gametocytes. A recombinant, his-tagged Pfs230 fragment (Pfs230C1; amino acids 443–731) formed serum-stable particles upon incubation with liposomes containing cobalt-porphyrin-phospholipid (CoPoP). In mice, immunization with Pfs230C1, admixed with the adjuvants Alum, Montanide ISA720 or CoPoP liposomes (also containing synthetic monophosphoryl lipid A; PHAD), resulted in elicitation of IgG antibodies, but only those induced with CoPoP/PHAD or ISA720 strongly reduced parasite transmission. Immunization with micrograms of Pfs230C1 adjuvanted with identical liposomes lacking cobalt (that did not induce particle formation) or Alum was less effective than immunization with nanograms of Pfs230C1 with CoPoP/PHAD. CoPoP/PHAD and ISA720 adjuvants induced antibodies with similar Pfs230C1 avidity but higher IgG2-to-IgG1 ratios than Alum, which likely contributed to enhanced functional activity. Unlike prior work with another transmission-blocking antigen (Pfs25), Pfs230C1 was found to be effectively taken up by antigen-presenting cells without particle formation. The anti-Pfs230C1 IgG response was durable in mice for 250 days following immunization with CoPoP/PHAD, as were antibody avidity and elevated IgG2-to-IgG1 ratios. Immunization of rabbits with 20 µg Pfs230C1 admixed with CoPoP/PHAD elicited antibodies that inhibited parasite transmission. Taken together, these results show that liposomes containing CoPoP and PHAD are an effective vaccine adjuvant platform for recombinant malaria transmission blocking antigens.

Microparticles formed by heating potato protein—polysaccharide electrostatic complexes

Thermal treatment of electrostatic complexes, above the denaturation temperature of the protein, is one possible method to induce particle formation and improve their stability towards changes in environmental conditions. The purpose of this study was to examine the effect of thermal treatment on mixtures of commercial, food-grade potato protein (PP) and anionic polysaccharides. Mixed solutions (1 wt%) of PP and gum arabic (GA) or carboxymethylcellulose (CMC) were acidified (pH 9.5 to 3.5), homogenized (200/50 bar) and pasteurized (90 °C, 10 min). The physicochemical properties of the acidified and heated samples were compared on particle size, charge, composition, turbidity and stability. It was found that the acidification in itself initiated formation of PP—GA and PP—CMC microparticles (MP). Upon heating, PP–GA MPs became smaller (d4,3 from 2.5 to 1.3 μm), uniformer, and the amount of PP incorporated was increased with 33%, causing more than a doubling of the turbidity. Oppositely, PP–CMC MPs did not change upon heating; the particles remained small (d4,3 0.8 μm) and polydisperse, consisting of 2.5:1 PP:CMC. It was hypothesized that the heat resistance of PP—CMC MPs was caused by stronger electrostatic forces between PP and CMC, while the heat-induced salt and pH resistance of PP—GA particles indicated the formation of other types of interactions preserving the MPs. Negative zeta potentials suggested PS was present at the exterior of the MPs. The obtained results demonstrated PP's ability to create micron-sized particles with polysaccharides, which could potentially replace dairy proteins in various applications.

Femtosecond filamentation induced particles and their cloud condensation nuclei activity

Laboratory experiments were conducted to investigate femtosecond filamentation induced particle formation and growth in particle-free air as well as in the presence of pre-existing ammonium sulfate aerosols. The ability of laser induced particles to act as cloud condensation nuclei (CCN) was explored. The influence of water vapor and the pulse energy on the production efficiency and activation properties of particles were also examined. In particle-free air, laser filaments could lead to a significant production of new particles, the number concentration of which was comparable to or larger than that in polluted ambient air, depending upon the pulse energy and humidity. Total particle number and mass concentrations within 20–708 nm were predominantly contributed by particles with a diameter smaller than 100 nm. Remarkably high NOx up to an order of ∼101 ppm was generated in the chamber, allowing sufficient production of HNO3 and probably subsequent HNO3-H2O binary nucleation. In the presence of pre-existing particles, the yield of particle number was largely enhanced in laser filaments, especially in humid air. Particle number size distribution shifted towards smaller particle size, leading to a slight increase in particle mass. It was found that a considerable portion of laser induced particles, no matter generated with or without pre-existing particles, could act as CCN. The amount of CCN at 0.80% supersaturation was in the magnitude of 104∼105 cm−3 in all experiments, nearly one order larger than the average ambient level even in severely polluted regions. Overall, increasing the pulse energy and water vapor would favor particle formation and growth as well as the production of CCN. Raising the pulse energy also resulted in a decline of the hygroscopic parameter κ, implying the modification of aerosol chemical composition. Though more investigative studies are still needed to explore mechanisms underlying some of these experimental results, laser filaments are undoubtedly capable of inducing substantial particle formation and modifying microphysical/chemical properties of pre-existing particles. By influencing these crucial properties and thereby aerosol direct/indirect climatic and environmental effects, laser filamentation would provide a promising potential for related atmospheric applications.

Characterization of Inherent Particles and Mechanism of Thermal Stress Induced Particle Formation in HSV-2 Viral Vaccine Candidate.

Vaccine formulations may contain visible and/or subvisible particles, which can vary in both size and morphology. Extrinsic particles, which are particles not part of the product such as foreign contaminants, are generally considered undesirable and should be eliminated or controlled in injectable products. However, biological products, in particular vaccines, may also contain particles that are inherent to the product. Here we focus on the characterization of visible and subvisible particles in a live, replication-deficient viral vaccine candidate against HSV genital herpes in an early developmental stage. HSV-2 viral vaccine was characterized using a panel of analytical methods, including Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, liquid chromatography-mass spectrometry (LC-MS), light microscopy, transmission electron microscopy (TEM), micro-flow imaging (MFI), dynamic light scattering (DLS), right angle light scattering (RALS), and intrinsic fluorescence. Particles in HSV-2 vaccine typically ranged from hundreds of nanometers to hundreds of micrometers in size and were determined to be inherent to the product. The infectious titer did not correlate with any trend in subvisible particle concentration and size distribution as shown by DLS, MFI, and TEM under stressed conditions. This suggested that particle changes in the submicron range were related to HSV-2 virion structure and had direct impact on biological activity. It was also observed that subvisible and visible particles could induce aggregation in the viral product. The temperature induced aggregation was observed by RALS, intrinsic fluorescence, and DLS. The increase of subvisible particle size with temperature could be fitted to a two-step thermokinetic model. Visible and subvisible particles were found to be inherent to the HSV-2 viral vaccine product. The mechanism of protein aggregation was discussed and a two-step thermokinetic aggregation profile was proposed. The approaches reported in this study may be applied to a variety of vaccines and other biological products, as a way to assess the consistency of the manufacturing process and identify key product quality attributes.

Sonication induced particle formation in yogurt: Influence of the dry matter content on the physical properties

Yogurt texture can be adjusted by various compositional and processing factors. The factors protein content and type, heat treatment, and fermentation temperature are well-known and studied. In a previous study we were able to show sonication as an additional parameter affecting texture. The aim of this study was to test the hypothesis that sonication during fermentation induces large particles which, thus, have an impact on the texture perception of stirred yogurt. 26 stirred yogurt systems, differing in the fat (0.1−3.5%) and protein content (3.8−5.2%) were produced and a short sonication (35 kHz, 5 min) was applied within the pH range 5.1–5.2. Image analysis was performed to quantify the number and size of visually detectable particles/grains. Furthermore, the physical properties were studied by means of static light scattering and rheological measurements. Multivariate data analysis was used to discriminate between the effects of sonication and dry matter. Sonication resulted in new larger particles whereas an increase in dry matter content mainly affected the rheological properties. Above a dry matter content of 14.2%, no significant effect of the sonication was distinguishable.

Controlling the size and morphology of supramolecular assemblies of viologen-resorcin[4]arene cavitands.

A novel class of self-assembling nanoparticles is formed with viologen-resorcin[4]arene cavitands; the association model is strongly controlled by their hydrophobicity. Interestingly, the cavitand assemblies are designed through click chemistry to form self-assembled noncovalently connected aggregates through counterion displacement. The iodide and benzoate ions are utilized as strongly polarizable counterions to induce cavitand self-assembly. The counterion-mediated decrease in hydrophilicity of the viologen-resorcin[4]arenes is the underlying trigger to induce particle formation. These particles can be used as nanocontainers and find their applications in delivery systems.

Tuning the morphology of lead zirconate titanate (PZT) nanostructures

We describe the synthesis of PZT nanoparticles using a hydrothermal method. We demonstrate that the morphology of the PZT nanoparticles can be controlled by tuning the concentration of the mineraliser, KOH. The experimental results show that for low concentrations of KOH (2M) the reaction kinetics favor a high yield of spherical particles with an average diameter of 5.3µm. However, higher concentrations of KOH lead to the formation of branched nanostructures containing up to 8µm long nanorods. TEM and XRD investigations provide evidence that the branches are single crystalline. The influence of KOH concentration on polymer induced particle formation is discussed.

Irradiation induced particle formation in Ag-doped sulfophosphate glass

Silver-doped sulfophosphate glasses show irreversible Agmx+ silver cluster formation upon X-ray irradiation. Reversible cluster formation occurs upon UV irradiation in the presence of Ce3+ as electron reservoir. Additional intrinsic irradiation induced defects such as POHC, PEC, OHC, SEC and Zn*-HC recover at room temperature. The presence of silver clusters results in the occurrence of an optical absorption band at λ ~330nm. The formation and decay kinetics of irradiation induced defects and clusters are followed by photoluminescence and UV–VIS spectroscopy as well as by small-angle X-ray scattering.

Laser filament-induced aerosol formation

Abstract. Using the aerosol and cloud simulation chamber AIDA, we investigated the laser filament induced particle formation in ambient air, humid synthetic air, humid nitrogen, argon–oxygen mixture, and pure argon in order to simulate the particle formation under realistic atmospheric conditions as well as to investigate the influence of typical gas-phase atmospheric constituents on the particle formation. Terawatt laser plasma filaments generated new particles in the size range 3 to 130 nm with particle production rates ranging from 1 × 107 to 5 × 109 cm−3 plasma s−1 for the given experimental conditions. In all cases the particle formation rates increased exponentially with the water content of the gas mixture. Furthermore, the presence of a few ppb of trace gases like SO2 and α-pinene clearly enhanced the particle yield by number, the latter also by mass. Our findings suggest that new particle formation is efficiently supported by oxidized species like acids generated by the photoionization of both major and minor components of the air, including N2, NH3, SO2 and organics.

Sulfur dioxide oxidation induced mechanistic branching and particle formation during the ozonolysis of β-pinene and 2-butene

Recent studies have suggested that the reaction of stabilised Criegee Intermediates (CIs) with sulfur dioxide (SO(2)), leading to the formation of a carbonyl compound and sulfur trioxide, is a relevant atmospheric source of sulfuric acid. Here, the significance of this pathway has been examined by studying the formation of gas phase products and aerosol during the ozonolysis of β-pinene and 2-butene in the presence of SO(2) in the pressure range of 10 to 1000 mbar. For β-pinene at atmospheric pressure, the addition of SO(2) suppresses the formation of the secondary ozonide and leads to highly increased nopinone yields. A complete consumption of SO(2) is observed at initial SO(2) concentrations below the yield of stabilised CIs. In experiments using 2-butene a significant consumption of SO(2) and additional formation of acetaldehyde are observed at 1 bar. A consistent kinetic simulation of the experimental findings is possible when a fast CI + SO(2) reaction rate in the range of recent direct measurements [Welz et al., Science, 2012, 335, 204] is used. For 2-butene the addition of SO(2) drastically increases the observed aerosol yields at higher pressures. Below 60 mbar the SO(2) oxidation induced particle formation becomes inefficient pointing to the critical role of collisional stabilisation for sulfuric acid controlled nucleation at low pressures.

Ebola virus VP40-induced particle formation and association with the lipid bilayer.

Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.

Water induced particle formation in the ion chemistry of silane

Potential particle-forming reactions of hydrogenated silicon cations with silane and disilane, in the presence and absence of water, are summarized. The reactions are studied in the ion trap of a Fourier transform mass spectrometer at 10-7-10-5 Torr. Cations react sequentially with silane to produce larger silicon-containing ions. In general, reaction rates decrease with increasing number of silicon atoms in the ion; no ions containing more than six silicon atoms are formed in reactions with pure silane. Reaction with disilane produces larger silicon-containing ions; reaction rates decrease with increasing number of silicon atoms in the cation. The largest ion produced is larger than for the silane system but still contains less than nine silicon atoms. There is insufficient reactivity for hydrogenated silicon cations in pure silane or disilane to produce macroscopic particles. Mixtures of silane or disilane containing 7% water, however, significantly enhance the growth of ions. In the limit of our measurements, the reactions in both silane and disilane do not terminate.

Incorporation of chimeric gag protein into retroviral particles

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.