Sandra S. Soares

Purification of Bionanoparticles

Abstract The recent demand for nanoparticulate products such as viruses, plasmids, protein nanoparticles, and drug delivery systems have resulted in the requirement for predictable and controllable production processes. Protein nanoparticles are an attractive candidate for gene and molecular therapy due to their relatively easy production and manipulation. These particles combine the advantages of both viral and non‐viral vectors while minimizing the disadvantages. However, their successful application depends on the availability of selective and scalable methodologies for product recovery and purification. Downstream processing of nanoparticles depends on the production process, producer system, culture media and on the structural nature of the assembled nanoparticle, i.e., mainly size, shape and architecture. In this paper, the most common processes currently used for the purification of nanoparticles, are reviewed.

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly‐D‐lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL‐coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)‐binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly‐Arg‐Gly‐Asp‐Ser (GRGDS) peptides were used to interfere with cell‐ECM binding motifs onto FN‐coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.

Molecular determinants of virus-like nanoparticle assembly in vitro and in animal cell culture

Protein nanoparticles, such as virus-like particles (VLPs), consist only of the virus shell without any viral genetic information packaged inside. Similarly to viruses, the structural proteins that comprise a VLP can spontaneously self-assemble to form the particle or can assemble through several intermediate steps. This work addresses the characterization, manipulation and purification of a chimeric simian-human immunodeficiency VLP constructed by fusion of SIV matrix protein (p17) and HIV-1 p6 accessory protein. This fusion protein assembles as spherical nanoparticles of about 80 nm in diameter that are released to the culture media when expressed in HEK 293T cells. A simple two-step purification process was used to purify these nanoparticles. Also, different approaches - multiple-transfections or chemical coupling - were performed to target manipulation. Finally, a new approach for the production of these virus-like particles is described where the structural protein subunits are used and their assembly is promoted in vitro.

Thermal and Detergent Tolerance for a Chimeric Bionanoparticle

Protein nanoparticles, such as virus-like particles (VLPs), are becoming the most attractive candidate for prophylactic vaccination, genetic and molecular therapies, since they can be engineered in order to encapsulate therapeutics, to target specific cells or tissues, and/or to stimulate humoral or cytotoxic responses (Kang et al. 1999; Schaffer and Lauffenburger 2000; Yamada et al. 2003). Nevertheless their successful application depends on a larger number of factors, in which their stability plays one of the most important roles. Moreover these bionanoparticles have to guaranty delivery of the therapeutical agent to the target cell overcoming different biological barriers in vivo (Kang et al. 1999). In this communication we studied the thermal stability and detergent tolerance of the viral bionanoparticles produced in our laboratory, based on a minimal construction by fusion of the SIV (Simian Immunodeficiency Virus) p17 matrix protein with the HIV-1 (Human Immunodeficiency Virus Type 1) p6 protein (Costa et al. 2007). The lipid membrane surrounding these VLPs confers them stability against proteolysis and may contribute for the thermal stability of the bionanoparticles. Stability studies have shown that bionanoparticles are stable at 37oC for 96 h. Also the matrix core has shown to be highly stable even at detergent concentrations as high as 20% (v/v) of Triton X-100.