Novel encapsulation of partially dehydrated protein microparticles in PLGA microspheres

Abstracts / Journal of Biotechnology 131S (2007) S48–S52

6. Time-resolved gene expression experiments by using a single-cell electroporation microarray Lisbet Santoni 1,∗ , Cinzia Bersani 2 , Mauro Borgo 2 , Leonardo Bandiera 1 , Giorgio Cellere 3 , Stefano Vassanelli 4 , Samuele Chiaramello 2 1 BioSiLab

s.r.l. Rovereto, Trento, Italy s.r.l. Collereto Giacosa, Torino, Italy 3 Department of Information Engineering (DEI), University of Padova, Padova, Italy 4 Department of Human Anatomy and Physiology, University of Padova, Padova, Italy 2 Narvalus

The availability of vast gene and short interfering RNAs repertoires demands for in vitro functional screening and offer new perspectives to investigate genetic mammalian signaling pathways, causing the need for large-scale and efficient delivery methods of genetic constructs to cell lines and primary cultures. Following the introduction of systems biology approaches, animal and human cell-based assays are becoming crucial in drug discovery and to gain a fine access to intracellular molecular pathways by non-invasive and versatile delivery of biological compounds would be most valuable in these complex systems. So far, most delivery techniques act randomly on cell populations, including those applications where individual cells among those transfected need to be searched and studied. In this framework, targeting single cells represents an alternative that, when implemented at large-scale, can be translated into multipurpose and extremely versatile delivery systems. We devised and developed a semiconductor chip featuring an array of 61 cell-sized planar microelectrodes used to electroporate single cells in culture, thereby allowing an efficient and finely tunable delivery of several compounds, including plasmid vectors for gene expression and siRNAs for silencing. By finely tuning the electroporation protocols and by addressing each single electrode via a computer-driven control system, molecules could be delivered to pre-selected target cells both in cell lines and primary cultures, thus providing a new platform for versatile and easy-to-use assay systems based on living cells. Further, by acting in situ, without the need to remove the cells under study from their environment, this method allows following the behaviour of the targeted cell(s) through time. Different nucleic acids ranging from ODNs, to siRNAs, and DNA plasmid vectors, were successfully delivered to different pre-selected adherent mammalian cells on the same chip (spaceresolved), at arbitrarily chosen time points (time-resolved) and even sequentially (serial delivery) to the same cell. We firstly proved the method by efficiently deliver simple fluorescent probes such as Lucifer Yellow (LY) into CHO (Chinese Hamster Ovary) cells. Delivery of ODNs and plasmid vectors to NIH/3T3, BAEC (Bovine Aortic Endothelial Cells), NHEK16 (Normal Human Epidermal Keratinocytes; human papillomavirus 16 HPV-16- E6/E7 transformed), HDF (Human Dermal Fibroblast) and neuroblastoma cells supported the results obtained with CHO cells.

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In conclusion this method will provide a cell-based assay to get further insights not only into gene expression/silencing, but also into other areas of research such as single-cell analysis, single-cell biophysics, cell–cell signaling and microkinetics studies. For example, it will provide a useful tool for studying gene expression by simply applying the electric pulse in the presence of various stimuli (structural proteins, genetic construct, transcription factors and cell-cycle regulators) and observing the cell reaction directly under microscope revealing cellular and molecular events of biology in real time. doi:10.1016/j.jbiotec.2007.07.083 7. Novel encapsulation of partially dehydrated protein microparticles in PLGA microspheres Brenda Liz Montalvo-Ortiz 1,∗ , Brian Sosa 1 , Denisse Velez 1 , David Needham 2 , Kai Griebenow 1 1 University

of Puerto Rico Rio Piedras Campus San Juan, Puerto Rico, United States 2 Duke University Durham, North Carolina, United States Proteins and peptides have an enormous potential to be used as therapeutic drugs. However, the formulation of sustained release systems is still problematic. One approach is the use of biocompatible and biodegradable polyesters, such as, PLGA. The solid-in-oil-in-water (s/o/w) encapsulation method has been used to incorporate proteins in polymer microspheres. In this method, the protein is lyophilized to obtain a more rigid conformation and thus potentially reduce aggregation when the protein is exposed to organic solvents. However, some structural changes occur when the protein is lyophilized and these changes could lead to protein aggregation. We developed a new technology of creating partially dehydrated protein micro- and nano-spheres to replace the need of lyophilization. The protein is dehydrated with organic solvents, a process called glassification, where the protein is solidified forming glass beads. In this study, horseradish peroxidase (HRP) was encapsulated as glass beads (created using ethyl acetate) into PLGA microspheres by a s/o/w technique. An encapsulation efficiency (EE) of 96% was obtained when HRP was formulated as glassified beads, for the lyophilized HRP we got 73%. The protein activity upon encapsulation was also greater for the HRP glassified beads (91%) than for the lyophilized protein (73%). However, the percent of insoluble aggregates was higher for the HRP glass beads (24%) than for the lyophilized protein (10%). To decrease the amount of insoluble aggregates we changed the polymer solvent using during encapsulation from dichloromethane to ethyl acetate. For such formulation, we obtained an EE of 63 and 8% aggregates for the glassified HRP beads. For the lyophilized protein we obtained 34 and 10% for the encapsulation efficiency and aggregates, respectively. We also measured the size of the beads formed using a particle size analyzer and obtained protein nano- and micro-particles (500 nm – 38 ␮m). In the release profiles, less burst release (<10%) and a better sustained release was

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Abstracts / Journal of Biotechnology 131S (2007) S48–S52

observed for HRP glassified beads than for the lyophilized protein (25%). We also achieved higher stability during release for HRP glassified beads. The polymer microspheres obtained were more homogeneous and smooth for the HRP glass beads than for the lyophilized HRP. Glassification of proteins can be an alternative dehydration method that can be used for protein encapsulation in polymer microspheres for controlled delivery systems.

References Costantino, H.R., Langer, R., Klibanov, A.M., 1994. Pharm. Res. 11, 21–29. Griebenow, K., Klibanov, A.M., 1995. Proc. Natl. Acad. Sci. U.S.A. 92, 10969–10976. Hageman, M.J., 1988. Drug Dev. Ind. Pharm. 14, 2047–2070. Pearlman, R., Nguyen, T., 1992. J. Pharm. Pharmacol. 44 (Suppl. 1), 178–185.

doi:10.1016/j.jbiotec.2007.07.085 doi:10.1016/j.jbiotec.2007.07.084 8. Mechanism of moisture induced solid phase aggregation of pharmaceutical proteins

9. The interaction of cardanol derivatives on the activity of PDR pumps in Saccharomyces cerevisiae Bartosiewicz ∗ , Anna

Giselle Flores ∗ , Myriam Diaz, Jose Rodriguez, Kai Griebenow

Dominika Lukaszewicz

University of Puerto Rico Rio Piedras, Puerto Rico, United States

Wroclaw University Faculty of Biotrchnology, Wroclaw, Poland

Solid protein pharmaceuticals often suffer from instability problems hampering their successful therapeutic application. Upon storage, proteins may become partially hydrated and detrimental moisture induced aggregation is observed (Costantino et al., 1994; Pearlman and Nguyen, 1992; Hageman, 1988). The extent of protein aggregation has been related to the amount of water sorbed and often a bell-shaped curve is observed when plotting the amount of aggregates formed versus the relative humidity used to incubate the protein. One frequently formulated hypothesis in this context is that partial hydration influences protein dynamics and thus unfolding in the solid state (Costantino et al., 1994; Griebenow and Klibanov, 1995). Simply put, increased hydration leads to increased protein dynamics allowing for unfolding and aggregation. The decrease in protein aggregation at very high relative humidity in this model is explained by protein refolding. However, thus far, no solid data on protein structure in partially hydrated states after incubation under defined humidity conditions have been published and thus the hypothesis has not verified. To test the hypothesis, lyophilized insulin powder was incubated in humidity chambers at various relative humidities. Formation of buffer insoluble aggregates was monitored for these samples for weeks. To detect unfolding/refolding events occurring to the solid protein powder, these were subjected to analysis by Fourier-transform infrared spectroscopy and circular dichroism (CD) after 48 h of incubation (after this time the system is in complete equilibrium). We employed an FTIRmicroscope using a diamond cell to avoid exposing the moist protein powders to the high pressure encountered in the formation of a KBr pellets usually employed in such studies on solid protein powders. Furthermore, to avoid artifacts by water vapor and sorbed water, the amide III region (1220–1330 cm−1 ) was used. Results obtained show that insulin forms aggregates upon storage in humidity chambers. Preliminary analysis of the FTIR data currently point in the direction that the hypothesis explaining the bell-shaped curve is correct, but further experiments are currently ongoing to verify this including experiments such as H/D exchange.

Krasowska, Marcin

Phenolic lipids are natural amphiphilic long-chain homologues of phenol, resorcinol and catechol occuring in numerous plants and microorganisms. They are non-isoprenoid compounds that consist of a phenolic structure and a long aliphatic hydrocarbon chain (of varying length and saturation) attached to it. They exhibit high affinity for lipid bilayers and biological membranes and are able to modify the activity of membrane enzymes (AChE, PLA2, or Ca2+ -ATPase). Differences in the chemical structure of phenolic lipids are reflected in different properties of these compounds. Efflux pumps are present in all living cells and participate in the detoxifying process by expelling various harmful compounds and xenobiotics. The function of active efflux systems is to transport drugs through the membrane and limit the intracellular accumulation of toxic compounds. This export is energized by ATP hydrolysis or by an ion antiport mechanism that contributes to the membrane energy state. To prevent this resistance, effective inhibitors of efflux pumps are sought. These compounds either specifically interact with MDR proteins while others influence the supply of energy for MDR pumps by affecting cellular ATP production or the ion-motive force driving the pumps what inhibits pumps activity, e.g. by influencing membrane phospholipids and increasing the permeability of the membrane for small non-electrolytes and cations. The properties of semisynthetic derivatives of a phenolic lipid, cardanol, and their effect on ABC transporters in S. cerevisiae are presented. The influence of semisynthetic derivatives of cardanol on yeast PDR proteins activity was studied by spectrofluorimetric method using the potentiometric fluorescence probe diS-C3(3). The probe is expelled from S. cerevisiae by ABC pumps and can conveniently be used for studying their performance. Two pump-competent S. cerevisiae strains and different pump-free mutant strains were used to check the effect of the studied compounds on the activity of major ABC transporters. Luciferase assay was used to studied the changing of ATP cellular level under the influence of semisynthetic compounds. doi:10.1016/j.jbiotec.2007.07.086